How do biological multicellular complexity and a spatially organized body plans emerge ?

How do biological multicellular complexity and a spatially organized body plans emerge?

https://reasonandscience.catsboard.com/t2990-how-does-biological-multicellular-complexity-and-a-spatially-organized-body-plan-emerge

E. B. Wilson (1856-1939), Cell biologist:  'The key to every biological problem must finally be sought in the cell, for every living organism is, or at some time has been, a cell. '

Biochemistry is basically about instructional complex Information and specification directing the make and control of complex molecular physiological structures, which dictate function.
Molecular machines work with exquisite precision, accuracy, and specificity, composed of an intricately ordered assembly of subunits each with a clearly defined role—which mechanically interlocks with one another in a particular temporal sequence into a unique configuration that allows it to perform its function in an effective and predictable way. Protein assemblies contain highly coordinated moving parts. Within each protein assembly, intermolecular collisions are not only restricted to a small set of possibilities, but reaction C depends on reaction B, which in turn depends on reaction A—just as it would in a machine of our common experience. Proteins are highly dynamic entities that display very high degrees of flexibility, ranging from simple side-chain rotations to complete rearrangements of their secondary structure.

J. A. Shapiro: Evolution: A View from the 21st Century 2011
How do cells become different from each other? How do tissues composed of specialized cell types form? What principles drive tissue formation and morphogenesis down well-defined paths during embryonic development?

M. Lynch (2020): All organismal features are functions of structures and processes that develop at the cellular level. Thus, given that most of the prominent advances in the life sciences over the past fifty years have emerged from the field of cell biology, the incorporation of cell-biological issues into the mainstream of evolutionary science is long overdue.
https://www.sciencedirect.com/science/article/abs/pii/S002228362030156X

Explaining organismal form depends on explaining how organs, tissues, and cells form and gain shape. On the lowest level of the hierarchy, the formation of cells in a multicellular organism depends on the specification of: 
1. Morphogenesis of eukaryotic cells, structure, and shape
2. Cell fate determination and differentiation ( phenotype, or what cell type each one will become )
3. Cell growth and size
4. Development and cell division counting: cells need to be programmed  to stop self-replicating after the right number of cell divisions
5. Mechanisms of pattern formation
6. Hox genes
7. Position and place in the body. This is crucial. Limbs like legs, fins, eyes etc. must all be placed at the right place.
8. What communication it requires to communicate with other cells, and the setup of the communication channels
9. Sensory and stimuli functions of cells
10. What specific new regulatory functions do cells have to acquire
11. When will the development program of the organism express the genes to grow the new cells during development?
12. Change regulation in the composition of the cell membrane and/or secreted products.
13. Specification of the cell-cell adhesion proteins and which ones will be used in each cell to adhere to the neighbor cells ( there are 4 classes )
14. Apoptosis: programming of the time period the cell keeps alive in the body, and when is it time to self-destruct and be replaced by newly produced cells of the same kind
15. Set up each cells specific nutrition demands
16. Cell shape changes
17. Cell proliferation is the process that results in an increase in the number of cells, and is defined by the balance between cell divisions and cell loss through cell death or differentiation.
18. Differences in Regulatory DNA

Each cell in an embryo receives molecular signals from neighboring cells in the form of proteins, RNAs and even surface interactions. Almost all animals undergo a similar sequence of events during very early development, a conserved process known as embryogenesis. During embryogenesis, cells exist in three germ layers, and undergo gastrulation. A basic set of the same proteins and mRNAs are involved in embryogenesis. 

How did complex multicellular organisms emerge, and how is genetic and epigenetic information translated into a spatially organized body plan?  To allow the building of complex organs with intricate patterns of cellular specialization, such decisions are taken on a supra-cellular level in a cell-nonautonomous manner. Organisms do not only require mechanisms of development but also strategies for arranging and differentiating their cells for survival and reproduction.

A body is more than a collection of randomly distributed cell types. Development involves not only the differentiation of cells but also their organization into multicellular arrangements such as tissues and organs. When we observe the detailed anatomy of a tissue such as the neural retina of the eye, we see an intricate and precise arrangement of many types of cells. How can matter organize itself so as to create a complex structure such as a limb or an eye?

There are five major questions for embryologists who study morphogenesis:
1. How are tissues formed from populations of cells? For example, how do neural retina cells stick to other neural retina cells and not become integrated into the pigmented retina or iris cells next to them? How are the various cell types within the retina (the three distinct layers of photoreceptors, bipolar neurons, and ganglion cells) arranged so that the retina is functional?
2. How are organs constructed from tissues? The retina of the eye forms at a precise distance behind the cornea and the lens. The retina would be useless if it developed behind a bone or in the middle of the kidney. Moreover, neurons from the retina must enter the brain to innervate the regions of the brain cortex that analyze visual information. All these connections must be precisely ordered.
3. How do organs form in particular locations, and how do migrating cells reach their destinations? Eyes develop only in the head and nowhere else. What stops an eye from forming in some other area of the body? Some cells for instance, the precursors of our pigment cells, germ cells, and blood cells must travel long distances to reach their final destinations. How are cells instructed to travel along certain routes in our embryonic bodies, and how are they told to stop once they have reached their appropriate destinations?
4. How do organs and their cells grow, and how is their growth coordinated throughout development? The cells of all the tissues in the eye must grow in a coordinated fashion if one is to see. Some cells, including most neurons, do not divide after birth. In contrast, the intestine is constantly shedding cells, and new intestinal cells are regenerated each day. The mitotic rate of this tissue must be carefully regulated. If the intestine generated more cells than it sloughed off, it could produce tumorous outgrowths. If it produced fewer cells than it sloughed off, it would soon become nonfunctional. What controls the rate of mitosis in the intestine?
5. How do organs achieve polarity? If one were to look at a cross-section of the fingers, one would see a certain organized collection of tissue bone, cartilage, muscle, fat, dermis, epidermis, blood, and neurons. Looking at a cross-section of the forearm, one would find the same collection of tissues. But they are arranged very differently in different parts of the arm. How is it that the same cell types can be arranged in different ways in different parts of the same structure? All these questions concern aspects of cell behavior. There are two major types of cell arrangements in the embryo: epithelial cells, which are tightly connected to one another in sheets or tubes, and mesenchymal cells, which are unconnected to one another and which operate as independent units. Morphogenesis is brought about through a limited repertoire of variations in cellular processes within these two types of arrangements:

Answering the questions about how cells, tissues, and organisms form, precedes the question of how they can eventually diversity, evolve, change and morph from one species to another through a macroevolutionary primary speciation transition zone, where novel organismal features arise, like wings, eyes, ears, legs, arms, and so forth. The fact is, that science is still FAR from being able to answer that question in an exhaustive manner.  At least 43 epigenetic codes and languages are scientifically known. They all contribute to organismal development in a decisive way. A Google search will demonstrate, that there are no scientific papers that show the breaking of just one of those codes. Science has NO CLUE about how and where most of them are stored, nor the meaning of the codification, the assignment, and rules, the language, nor the cipher. Science knows that they exist through experimental tests, but deeper details are not known.
 
The naive view by uninformed people is that evolution is a fact and that microevolution leads to macroevolution, and that genes are enough to explain phenotype and physiological form. That is, as above demonstrates, false. Variation within species on a second-degree level, that leads even to speciation where inbreeding is not further possible, is not evidence of evolution, but pre-programmed adaptation, which is life essential. When life started, adaptation had to be fully operational, otherwise, the organism would soon die. That explains why very varied organismal forms within species can arise within a few generations. Why artificial breeding can produce a danish dog, and a chihuahua, I outline the mechanisms involved here:

Claim: In biochemistry, there are just chemical reactions.
Reply: Bruce Alberts: “The Cell as a Collection of Protein Machines: Preparing the Next Generation of Molecular Biologists,” Cell, 92 (February 6, 1998): 291-294)

“We have always underestimated cells. Undoubtedly we still do today. But at least we are no longer as naïve as we were when I was a graduate student in the 1960s. Then, most of us viewed cells as containing a giant set of second-order reactions: molecules A and B were thought to diffuse freely, randomly colliding with each other to produce molecule AB — and likewise for the many other molecules that interact with each other inside a cell.,,,

But instead of a cell dominated by randomly colliding individual protein molecules, we now know that nearly every major process in a cell is carried out by assemblies of 10 or more protein molecules. And, as it carries out its biological functions, each of these protein assemblies interacts with several other large complexes of proteins. Indeed, the entire cell can be viewed as a factory that contains an elaborate network of interlocking assembly lines, each of which is composed of a set of large protein machines.”

https://brucealberts.ucsf.edu/publications/BAPub157.pdf

Why Darwin's theory of evolution does not explain biodiversity
https://reasonandscience.catsboard.com/t2623-why-darwins-theory-of-evolution-does-not-explain-biodiversity

We know as a fact, that common descent is a failed hypothesis:

Common descent, the tree of life, a failed hypothesis
https://reasonandscience.catsboard.com/t2239-evolution-common-descent-the-tree-of-life-a-failed-hypothesis

That, however, does not explain BY FAR, the rise of multicellular complexity, and biological variation of species. For that, the molecular landscape has to be fare more complex and involves many different mechanisms on an intra and extracellular level as exposed above, and here:

Where Do Complex Organisms Come From?
https://reasonandscience.catsboard.com/t2316-evolution-where-do-complex-organisms-come-from

1. Morphogenesis of eukaryotic cells, structure, and shape

1. The grand overarching scheme of the morphogenesis and formation of single eukaryotic cell form, shape, architecture, function, and structure, following the formation of tissues and higher order of biological structures is that information conveyed through codified signaling directs intracellular molecular actors. It is due to at least 36 different signaling molecules & mechanisms that direct at least 15 force-generating molecules, through at least 38 different mechanisms. They are classified into two groups: 
a) Semantophoretic molecules ( carry genetic and epigenetic information) that provide complex instructional cues of action through signaling which is fundamental to coordinate any common behavior and organize the division of labor. The following activities are performed in an integrated, orchestrated, interdependent manner by at least 36 different signaling molecules & mechanisms in the following different ways: 
8 molecules signal, 4 orient, 5 activate,  2 direct, 3 promote, 2 regulate, 1 guide, 3 organize, 1 inform, 1 coordinate, 1 specify, 1 modulate, 1 provide position cues, 2 mediate, 1 provoke change.
b) At least 15 force-generating actors, molecules that are directed through those signals and instructions, are responsible for cell morphogenesis, that is: Filaments, microtubules, lamellopodia, crosslinkers, motors, protein complexes, the centrosome, adhesion proteins, force generators, the extracellular matrix, intra-cellular modules, key regulators, protein gradients,  the mitotic spindle, mechanical signals which act in at least 38 different ways, to name: activating, binding, breaking, coordinating, conferring positional information, directing, forcing transmission, generating, guiding, helping to organize, inducing, informing, mediating, modulating, organizing, orienting, providing positioning rules, provoking changes, promoting, regulating, signaling, stretching, specifying. 
2. The molecules, acting upon the instructional signals received,  form patterns, force change, stretch, change and induce cell shape, move positions, orient, provoke ingress, express, align, deform, accumulate molecules, invaginate, extend, form a web, concentrate, hold together, give mechanical support, stiffen, promote stabilization, form geometry, polarity, form division-plane positioning, phosphorylate, dephosphorylate, force transmission,  couple molecules physically, form gradients, deform, pull forces, promote cell rounding, transduce, specify cell-fate, extend, provide structural links, form aspect ratio, and size, form filament ends, etc.
3. Codified signals and instructional assembly information directing the making and operating of complex irreducible machines ( proteins) and factories full of interdependent comparments ( cells)  have only been observed to be instantiated by intelligent agents with foresight for specific purposes.  Semiotic functional information is not a tangible entity, and as such, it is beyond the reach of, and cannot be created by any undirected physical process.
This is not an argument about probability. Conceptual semiotic information is simply beyond the sphere of influence of any undirected physical process. To suggest that a physical process can create semiotic codes, languages, and words, and upon it, generating instructional information, is like suggesting that a rainbow can write poetry... it is never going to happen!  Physics and chemistry alone do not possess the tools to create a concept. The only cause capable of creating conceptual semiotic information is a conscious intelligent agent. Life is no accident, the vast quantity of semiotic information in life provides powerful positive evidence that we have been designed.

1. At least 15 cellular actors, that is: Filaments, microtubules, lamellopodia, crosslinkers, motors, protein complexes, the centrosome, adhesion proteins, force generators, the extracellular matrix, intracellular modules, key regulators, protein gradients,  the mitotic spindle, mechanical signals act through at least 38 different mechanisms, to name: activating, binding, breaking, coordinating, conferring positional information, directing, forcing transmission, generating, guiding, helping to organize, inducing, informing, mediating, modulating, organizing, orienting, providing positioning rules, provoking changes, promoting, regulating, signaling, stretching, specifying, in the formation of morphogenesis of single eukaryotic cells, structure, and shape, and tissues. 
2. The overarching scheme is that information conveyed through codified signaling directs intracellular molecular actors contributing to the grand scheme or the formation of cells form, shape, architecture, and function. Codified signals and information have only been observed to come from intelligent minds.  
3. Organismal form, architecture, and variety are therefore best explained by the direct input of the information by an intelligent designer. 

36 different signaling molecules & mechanisms direct (at least) 15 force-generating molecules, through (at least) 39 different mechanisms:
1. Actomyosin mediates apical constriction which drives a wide range of morphogenetic processes.
2. Actin filaments, actin crosslinkers, and myosin motors form an intracellular-contractile network which provokes changes in cell shape.
3. Actin filaments in animal cells are organized into several types of arrays: dendritic networks, bundles, and weblike (gel-like) networks which permit the formation of different cell structures. The Arp 2/3 complex and formins contribute to these formations.
4. Arp2/3 complex is an essential organizer of treadmilling actin filament arrays.
5. Biochemical signaling directs the graded distribution of a morphogen across a tissue, which permits cells to interpret their position to make fate choices. The morphogen concentration, therefore, provides positional coordinates along the embryonic axis.
6. Centrosomes provide precise cell division positioning rules
7. Chemical signaling cascades bridge the plasma membrane and the nucleus. Focal adhesion proteins such as zyxin and paxillin shuttle between the cytoplasm and nucleus to modulate transcriptional activity.
8. Conserved geometrical features provided by cleavage patterns. The geometry of these patterns may specify developmental axes, germ layers, and cell fates. 
9. Cortical force generators interact with spindle microtubules and are activated by cortical cues
10. Diverse sets of signaling pathways perform spatial-temporal coordination, regulation and control of actomyosin networks, co-responsible for morphogenesis, and cell shape change to get proper size and shape
11. Dynein force generators are employed to orient cell division axes at a specific angle. They work at the cell cortex and the cytoplasm. 
12. Ezrin, radixin, and moesin proteins (ERM) help organize membrane domains through their ability to interact with transmembrane proteins and the underlying cytoskeleton. In so doing, they not only provide structural links to strengthen the cell cortex, but also regulate the activities of signal transduction pathways. 
13. Extracellular matrix ECM stiffness directs differentiation or self-renewal of embryonic or somatic stem cells, and the ECM viscosity can also influence cell-fate specification. [/size
14. Filamins function as signaling scaffolds by connecting and coordinating a wide variety of cellular processes with the actin cytoskeleton. 
15. Ga/LGN/NuMA complex binds to dynein, which then generates microtubule pulling forces toward the cell cortex
16. Gelsolin proteins break an actin filament into many smaller filaments, thereby generating a large number of new filament ends.
17. Intracellular modules  such as the cytoskeleton inform and provide global and local cell geometry features, such as aspect ratio, size, or membrane curvature
18. Key regulator Rac activates the WAVE complex through coincident signals.
19. Lamellipodia permit the extension of sheetlike membrane projections that help cells to crawl across solid surfaces which is important for migration during early embryonic development.
20. Lamin A is a nuclear lamina component. In stiff tissue, tension from the ECM transmitted via the cytoskeleton to the nucleus can enhance lamin A transcription and stability of lamin A/C, leading to activation of Yap, serum response factor (SRF) and retinoic acid signaling pathways. 
21. Mechanosensing and transduction employed by cells sense forces and mechanical cues, and subsequently, control cell differentiation and morphogenesis.  How genetic cascades link cell-fate specification to tissue morphogenesis remains unclear.
22. Moesin also increases cortical stiffness to promote cell rounding during mitosis.  ERM proteins are thought to bind to and organize the cortical actin cytoskeleton in a variety of contexts, thereby affecting the shape and stiffness of the membrane as well as the localization and activity of signaling molecules.
23. Morphogen-mediated chemical signaling induces cell shape, cell geometry, deformation,  pulling forces of the extracellular matrix (ECM), and in the end, the architectural form of tissue. 
24. Physical mechanotransduction relies on direct force transmission from the cell surface to the nucleus through physical coupling between the nuclear membrane and the extracellular space by cytoskeletal components.
25. Protein gradients that are mostly independent of DNA or any cytoskeletal structure
26. RhoGEFs of the small GTPase Rho1 activates ROCK,  formin and formin-related proteins, such as Daam proteins, and they direct and regulate Myosin II motor protein phosphorylation and dephosphorylation
27. Signaling provides spatial information,  guiding cellular geometry, conveys polarity, cell morphogenesis, and division-plane positioning.
28. Spectrins are proteins that form a web. Spectrin is a long, flexible protein made out of four elongated polypeptide chains.  In the red blood cell, spectrin is concentrated just beneath the plasma membrane, where it forms a two-dimensional weblike network held together by short actin filaments whose precise lengths are tightly regulated by capping proteins at each end. The resulting network creates a strong, yet flexible cell cortex that provides mechanical support for the overlying plasma membrane, allowing the red blood cell to spring back to its original shape after squeezing through a capillary. Close relatives of spectrin are found in the cortex of most other vertebrate cell types, where they also help to shape and stiffen the surface membrane. A particularly striking example of spectrin’s role in promoting mechanical stability is the long, thin axon of neurons
29. Static and pulsed mechanical deformations of mesodermal cells have been shown to promote Folded gastrulation (Fog) signaling and apical myosin II accumulation, which is required for mesoderm invagination prior to germband extension.
30. Talin signaling is able to orchestrate multiple cytoskeletal systems in the cell to influence cell shape, dynamics and signaling outputs.
31. The mitotic spindle directs spatial cues for cytokinesis (  the physical process of cell division ). Septum ingression is pre-specified from the position of the nucleus, and the spindle usually aligns orthogonal to the septum. 
32. The nucleus acts as a mechanosensor, whereby changes in nuclear shape can evoke transcriptional changes by locally altering the spatial accessibility of chromatin to transcriptional regulators. The physical links between the cytoskeleton and nuclear membrane proteins allow the entire cell to function as a single mechanically coupled system.
33. The dynamic orientation of nuclei and spindles, which are moved and oriented from the forces exerted by microtubules (MTs) and associated motors such as dynein. 
34. Tissue-scale mechanical signals can confer positional information and control gene expression and fate at the cellular level, thereby allowing cells to coordinate embryonic patterning.
35. Tropomodulin proteins regulate actin filament length and stability in many cell types.
36. Twist, a transcription factor, and activation of Rho signaling direct ratcheting which is critical for the generation of tissue‐level tension and changes in tissue shape. That is provided by βH‐spectrin which forms a network of filaments which co‐localize with Medio‐apical actomyosin fibers. 
37. Upstream developmental cues,  downstream force generators that orient cell division and cue-dependent spatial control of the force generators generate diversity in division axis orientation
38. WAVE complex mediates the activation of the Arp2/3 complex.  
39. Yap/Taz signaling, where mechanical tension arising from physical stretching of cells or increased stiffness of the extracellular matrix (ECM) activates F-actin remodeling and Yap/Taz nuclear localization.

Understanding the molecular mechanisms that specify and maintain the identities of more than 200 cell types of the human body is arguably one of the most fundamental problems in molecular and cellular biology, with critical implications for the treatment of human diseases. Central to the cell fate decision process are stem cells residing within each tissue of the body.
https://evolutionnews.org/2022/05/cell-fate-another-hurdle-for-evolution/

Morphogenesis of eukaryotic cells, structure, and shape: by random chance, evolution, or design?
https://www.youtube.com/watch?v=BP8lJjzQm-c

https://reasonandscience.catsboard.com/t3003-morphogenesis-of-eukaryotic-cells-structure-and-shape-by-random-chance-or-design

Morphogenesis is important in the context of developing tissues, but also on events at the cellular scale. This is because the cell is the unit of life and because tissue movements are driven by changes in the shape and mechanical properties of individual cells, which in turn depend on forces generated by a small number of cytoskeletal elements within these cells. 15

In the context of animal or plant development, we tend to think of cells as small, simple, building blocks, such that complex patterns or shapes can only be constructed from large numbers of cells, with cells in different parts of the organism taking on different fates. However, cells themselves are far from simple and often take on complex shapes with a remarkable degree of intracellular patterning. How do these patterns arise? As in embryogenesis, the development of structure inside a cell can be broken down into a number of basic processes. For each part of the cell, morphogenetic processes create internal structures such as organelles, which might correspond to organs at the level of a whole organism. Given that mechanisms exist to generate parts, patterning processes are required to ensure that the parts are distributed in the correct arrangement relative to the rest of the cell. Such patterning processes make reference to global polarity axes, requiring mechanisms for axiation which, in turn, require processes to break symmetry. 

The shape of biological structures ultimately must arise from physical forces. 16

The most extraordinary and fundamental of all morphogenetic processes is cell division: the process by which one cell becomes two. During division, in the space of a few minutes, all the component parts of the cell must be moved apart and partitioned into two daughter cells. Moreover, the process must be precise, since errors are associated with diseases like cancer in humans, and frequently cause cell death.

My comment: The first event of cell division of a primordial cell would have had to be as precise as in "modern" cells. With all the 16 cell-cycle regulators in place since they are essential. If one is missing, the cell-cycle is not completed. That is another intractable abiogenesis challenge. 



It is one of three fundamental aspects of developmental biology along with the control of tissue growth and patterning of cellular differentiation. 1  Centrosomes have an important role in cell physiology. The cytoskeleton and reaction-diffusion systems have self-organizing properties 2 

Cells use mechanisms for sensing their shape. Morphogenesis is the biological process that causes a cell, tissue, or organism to develop its shape. Signaling and spatial information guide cellular geometry, convey polarity, cell morphogenesis, and division-plane positioning. Global and local cell geometry features, such as aspect ratio, size, or membrane curvature, may be probed by intracellular modules, such as the cytoskeleton, reaction-diffusion systems or molecular complexes. Cell shape emerges as an important means to transduce tissue-inherent chemical and mechanical cues into intracellular organization. Cellular geometry is conveyed into spatial information to guide processes, such as signaling, morphogenesis, division-plane positioning, and polarity ( Cell polarity is the asymmetric organization of several cellular components, including its plasma membrane, cytoskeleton or organelles.)

In animal cells, the position at which cytokinesis (  the physical process of cell division ) occurs depends upon spatial cues ( specific line or action ) that are provided by the mitotic spindle towards anaphase.  In most fungi, cytokinesis and septum ingression are pre-specified from the position of the nucleus, and the spindle usually aligns orthogonal to the septum. (At cell division, a cross-wall, the septum, is made between the mother and daughter cell to permit their separation 3 )

These considerations raise the question of what kind of evolutionary pressure could have driven cells of various sizes and shapes, and within different environments, to divide along their long axes.

Question: Had this functional arrangement, the pre-specification from the position of the nucleus, and the spindle alignment not to be correct right from the start, otherwise, misformed, non-functional daughter cells would be the result after the first cell replication event? If it were not so, how could cell-replication even take off?

One possibility is that this geometrical design provides the largest cytoplasmic space for DNA segregation

My comment: That observation does not explain or give a hint if evolutionary mechanisms would explain the right specification adequately. Since the right cues mean either a successful cell division or death, this is an all or nothing business, and this specification had to be right from the start of the first cell replication event, the origin is better explained by the injection of the right specification by an intelligent designer, rather than random mutations and natural selection, which would require several attempts and mutations until getting the right geometrical design, that means, self-replication would probably never take off, but immediately cause cell death and not the perpetuation of life.

In bacteria and other prokaryotes, septum positional information arises from protein gradients that are mostly independent of DNA or any cytoskeletal structure. Cell division along the long axis is geometry-sensed by division-positioned machineries that organize themselves along the longest axis 4

Question: Had this division positioning not have to be just right from the beginning to generate daughter cells with the right shape, otherwise, if evolution would have been tinkering with mutations, aberrant and nonfunctional cells would have been the result, and consequently, in case of the first self-replicating cells, immediate death and the non-perpetuation of life?

Microtubules MT exert forces depend on the length of astral MTs. ( Astral microtubules are a subpopulation of microtubules, which only exist during and immediately before mitosis.  mitosis is a part of the cell cycle when replicated chromosomes are separated into two new nuclei. 5 6)  

The combination of aster-like geometry with the dynamic growth and shrinkage properties of MTs, and their length-dependent forces provides a simple, yet extremely robust, design for the cell to probe the cellular volume and center the centrosome


Cell-shape sensing for division positioning.
(A) Astral microtubules (MTs) exert length-dependent forces to center nuclei and centrosomes. Length dependency may arise when MTs polymerize and buckle, or when they are pulled in the cytoplasm through dynein-cargo motions. 
(B) Aster growth and length-dependent forces provide dynamic shape-sensing abilities to MT asters 7, and result in aster-centering paths that depend on cell shape. 
(C) The cellular response to external forces on centered asters suggests that asters behave like elastic springs to maintain the spindle in the cell center. Red curve: Upon force application, the distance to the cell center (d) increases over time, reaching a plateau value at which aster forces balance the external forces. When the force is released, relaxes exponentially to 0. Blue curve: By varying externally applied forces, the stiffness of the ‘centering spring’ can be computed. 
(D) When an egg is shaped into a rectangular microwell, the torques and forces generated through length-dependent MTs align nuclei and spindles along the long axis of a cell. 
(E) Cleavage patterns of zebrafish embryos exemplify the iterative influence of cell shape on division orientation and vice versa. 
(F) Cells in tissues can be exposed to external mechanical forces, such as tissue tension or compression from neighboring cells, which may influence cell shape and resulting spindle orientation with respect to external forces (left). As a consequence cells will divide according to those mechanical forces; which could in turn relax tissue stress or influence the topology of cell-cell contacts (right).

The endomembrane system is composed of the different membranes that are suspended in the cytoplasm within a eukaryotic cell. These membranes divide the cell into functional and structural compartments or organelles. Centrosomes moved away from the cell center to stop at a position that depended on the applied force and rapidly repositioned to the center after force cessation, much like a spring (Fig. C). The stiffness value of this spring is of key biological significance. A low stiffness value would permit aster fluctuations, rotations or displacement owing to other cellular cues (e.g. an asymmetric cortical domain), whereas a high stiffness value would tend to ‘freeze’ the aster at the cell center

My remark: The " spring-stiffness " raises the question: How was the right stiffness value achieved? By trial and error? Let us suppose, genetic mutations would have produced asters and microtubules. if they were not equipped right from the beginning with the right forces and stiffness, the cell center would not be encountered, and subsequently, correct cell-division would not occur, leading, as the author observes, to aster fluctuations, rotations or displacement owing to other cellular cues, and subsequently, incorrect cell division, and in the end, cell death. 

Finally, it is worth noting that some cells may promote nuclear or centrosome centration relative to cell shape by using systems that do not solely rely on MT forces. Actomyosin contraction might produce forces that directly influence MT dynamics, aster motion, and position.

In all organisms that use a contractile ring for cell division, the process of cytokinesis can be divided into four distinct stages. Firstly, the cell needs to be preprogrammed to specify correctly a location at which to place the cell division ring to ensure proper separation of the cell contents into two daughter cells. Secondly, the cell needs to be able to transport all the necessary components to this region, and construct the cell division ring reliably and efficiently. Thirdly, the cell division ring needs to generate contractile stress in a regulated manner, to physically cleave the mother cell into two daughter cells. Finally, the ring must be disassembled to allow for the final abscission and separation of the daughter cells.

My comment: This is clearly an engineered process, which must be just right in all its unfolding sequence of events, leading to the separation of the daughter cells. If one of the four steps is not performed right and with precision, cell separation cannot occur correctly, and failure and death is the consequence. 

Life for all animals starts with a precise 3D choreography of reductive divisions of the fertilized egg, known as cleavage patterns. These patterns exhibit conserved geometrical features and striking interspecies invariance within certain animal classes. 9  A geometrical system based on length-dependent microtubule forces that probe blastomere a) shape and yolk gradients, biased by cortical polarity domains, may dictate division patterns and overall embryo morphogenesis. After fertilization, animal eggs undergo a precise series of subsequent reductive blastomere divisions called cleavage patterns. The geometry of these patterns may specify developmental axes, germ layers, and cell fates.  Division positioning is now known to involve the dynamic orientation of nuclei and spindles, which are moved and oriented from the forces exerted by microtubules (MTs) and associated motors such as dynein. 


We now know that specific molecular modules participate in positioning nuclei, spindles and subsequent division planes in animals and plants and that regulatory pathways may vary largely between cell types and situations. Nuclear and spindle positioning is a dynamic event that depends on the detailed forces and torques applied by the cytoskeleton and molecular motors. Experimental and computational studies in plant and animal cells show refined division positioning rules 11The position and the orientation of the spindle are often set by forces exerted onto astral microtubules extending out from the two spindle poles (centrosomes) towards the cell cortex. Force generators, which may include microtubule minus-end motors such as dynein, are differentially recruited or activated at the anterior or posterior pole by specific polarity pathways. More force is exerted on posterior than on the anterior astral microtubules, which yields net pulling forces and spindle displacement for asymmetric positioning. The specific localization of force generators, or force generation events, around cells is key in defining mitotic spindle position in animal cells.

Observation: While the author does not point to that, it can be inferred that the quantification of these forces and localization arrangement must be preprogrammed and informed in advance. 

When rounding up, cells maintain a memory of this adhesion pattern through actin retraction fibers (Figure 2b). Recent data suggestthat these actin fibers exert local mechanical stress on the cortex and recruit polarity components such as intracellular actin through putative mechanosensing mechanisms


New rules can predict full distribution of division plane positions. 
(a) The case of plant cells. 

(i) Divided cells of the leaf of the fern Microsorum punctatum. 
(ii) Four possible organizations of microtubule bundles around the nucleus mark potential sites of cell plate positioning. Site 1 is the shortest possible plane, 2 is the second shortest, etc. 
(iii) Schematic energy profile for plane positioning in geometries of (ii). Each local minimum corresponds to a possible plane positioning, and the depth of the minima informs on the likelihood of observing this position. 
(iv) Proportion of observed division plane positioning (colors correspond to those in ii) as a function of the relative plane length difference between mode 1 and mode 2. 
(b) The case of adherent mammalian cells. 
(i) Fibronectin micropattern used to control the geometry of cell adhesion. 
(ii) Immunostaining of actin and DNA in a rounded HeLa mitotic cell on this adhesive pattern. Actin retraction fibers that connect the cell to the adhesive pattern are clearly visible. 
(iii) Theoretical energy profile of spindle orientation. 
(iv) Theoretical probability density (red) computed from the energy profile, compared with observed experimental distribution (gray). 
(c) The case of nonadherent animal cells. 
(i) Sea urchin zygote divided in a rectangular microchamber. 
(ii) Immunostaining of microtubules and DNA in an interphase egg shaped in the same geometry as in (i). 
(iii) Theoretical energy profile of cleavage plane orientation in the same geometry as above. 
(iv) Theoretical probability density (red) computed from the energy profile, compared with observed experimental distribution (gray).

Retraction fibers connected to the cortex define local landmarks at the surface of the mitotic rounded cell, where force generators that pull on astral microtubules are recruited or stabilized.

Spindle orientation can be understood as the result of the action of cortical force generators, which interact with spindle microtubules and are activated by cortical cues. 12
Cell division axes are arranged in different orientations during embryogenesis, stem cell division, and organogenesis. Oriented divisions are critical for development as they contribute to both spatial cellular patterning and cell fate specification, and mutations in genes required for oriented cell division are associated with human diseases, including microcephaly, leukemia, and multiple cancers. 13 


My comment: This is a remarkable observation. Mutations do not improve the complexity and organismal architecture, in this case, but diseases! 

To understand the mechanisms that generate diversity in division axis orientation, three different regulatory layers should be considered: 

upstream developmental cues, 
downstream force generators that orient cell division, 
cue-dependent spatial control of the force generators

(Figure A, below, left).



Oriented AB Cell Division during D-V Body Axis Establishment Does Not Require Microtubule-Pulling Forces 
(A) General principle of cell division orientation mechanism (left) and known cell division orientation pathways (right). 
(B) Oriented AB and P1 divisions at two-cell stage that precede establishment of the dorsal and ventral body axis. 
(C) Orientation of AB cell division does not require cortical dynein recruiter LGN. Centrosomes (green), histone H2B (magenta), and cell outlines (white dotted line) are shown. 
(D) Cell long axis does not dictate AB cell division orientation. Values at bottom are cellular aspect ratios. 
(E) Mild nocodazole treatment (12.5 ng/mL) disrupted P1 but not AB division orientation. 
(F) Cleavage furrow orientation is not affected after strong nocodazole treatment (20 mg/mL). Non-muscle myosin II (green), centrosomes (green; asterisks), histones (magenta), cell-cell boundary (white dotted line), and cleavage furrow position (arrowheads). 
(G) Distributions of mitotic spindle orientations relative to the cell contact plane. 
(H) Distributions of cleavage furrow orientations relative to the cell contact plane. Scale bars, 10 mm.

For cell division axes to be oriented in a specific angle, cells need to employ force generation systems that move the division apparatus. Thus far, the microtubule motor protein dynein is the only known force generator. Dynein works at two different cellular locations: the cell cortex and the cytoplasm. ( The cell cortex, also known as the actin cortex or actomyosin cortex, is a specialized layer of cytoplasmic proteins on the inner face of the cell membrane. It functions as a modulator of membrane behavior and cell surface properties 14) tricellular junctions, and mechanical forces localize an evolutionarily conserved protein complex composed of Ga, LGN, and NuMA. The Ga/LGN/
NuMA complex binds to dynein, which then generates microtubule pulling forces toward the cell cortex through minus-end-directed dynein movement in association with depolymerizing microtubules (Figure A, above, middle).

Actins and spectrins
Actomyosin‐mediated b) apical constriction drives a wide range of morphogenetic processes. Activation of myosin‐II c) initiates pulsatile cycles of apical constrictions followed by either relaxation or stabilization (ratcheting) of the apical surface.d) While relaxation leads to dissipation of contractile forces, ratcheting is critical for the generation of tissue‐level tension and changes in tissue shape. βH‐spectrin forms a network of filaments which co‐localize with medio‐apical actomyosin fibers, in a process that depends on the mesoderm‐transcription factor Twist and activation of Rho signaling.

Tissue morphogenesis is driven by coordinated cellular deformations. Recent studies have shown that these changes in cell shape are powered by intracellular-contractile networks comprising actin filaments, actin crosslinkers, and myosin motors. The subcellular forces generated by such actomyosin networks are precisely regulated and are transmitted to the cell cortex of adjacent cells and to the extracellular environment by adhesive clusters comprising cadherins or integrins. Here, and in the accompanying poster, we provide an overview of the mechanics, principles, and regulation of actomyosin-driven cellular tension driving tissue morphogenesis.22 A developing tissue can undergo a diverse set of changes, such as bending, lengthening, narrowing, branching and folding, and during these processes, it faces a number of challenges. The cell behaviors that drive morphogenesis cannot simply stem from
adhesion alone but must also depend on active cytoskeletal elements, mainly actin filaments and myosin motors. The actin filaments alone can impart rigidity to the plasma membrane, whereas active contractile tension (also called contractility) is generated by myosin motors that use ATP hydrolysis to pull on the actin filaments. Tissue dynamics require active contributions from actomyosin networks that change cell shape and cell contacts.

Myosins are motor proteins that hydrolyze ATP to move along actin filaments.
The myosin superfamily is a diverse family of proteins, each containing a conserved head domain and a divergent tail domain harboring properties that are unique to each family member. The features that are important for force production, i.e. the actin-binding sites and the ATP-hydrolysis sites, are conserved in the head domain. Although most myosins are monomeric, members of the myosin II sub-class form hexamers consisting of two heavy chains, two essential light chains (ELCs) and two regulatory light chains (RLCs). The heavy chain folds into an N-terminal globular head that mediates motor activity. The C-terminal part of the heavy chain folds into an alpha-helical coiled-coiled tail domain that is required for the formation of tail-to-tail homodimers. Monomeric myosin II is poorly processive and exhibits little activity, but once assembled into bipolar filaments made from several homodimers it can act as a processive motor complex that pulls on flexible actin filaments. Although studied extensively in the context of muscle cells, myosin II is also found in non-muscle cells, and recent studies have shown that non-muscle myosin-II performs a number of functions, such as generating cortical tension, mediating cytokinesis and, most importantly, mediating cell shape changes during development. Unlike muscle myosin II, non-muscle myosin II (referred to hereafter simply as Myo II) can undergo dynamic assembly and disassembly, allowing its spatial and temporal regulation.

The mechanical properties of actomyosin networks are also dependent on the organization of actin filaments. The dynamics of cellular behaviors that drive morphogenesis are crucially dependent on such mechanical properties. For instance, in order to resist compression or stretch, a network needs to be stiff. Actin filaments in a loose network, by contrast, can be pulled and reorganized. The forces generated by such networks, however, cannot result in any cellular behaviors, such as migration or cell deformation, unless they are coupled to the plasma membrane or the extracellular matrix (ECM) via adhesion complexes. This anchorage is crucial not only for force transmission but also for force integration between many cells in a tissue. There are two important classes of adhesion molecules. The first comprises cadherins, which mediate intercellular adhesion. In many epithelial tissues, the contractile machinery shrinks the apical surface of the cells and transmits forces through a cadherin-containing adherens junction belt, thereby bending the whole tissue. The second class of adhesion molecules comprises integrins, which mediate anchorage of the cell surface to the ECM. Such coupling with the ECM not only provides a substrate for tissue migration, but can also lead to active remodeling of the ECM during many morphogenetic processes.

The total mechanical tension in the cellular cortex thus comprises contributions from active stresses (the contractility generated by the actomyosin network), elastic stresses that depend on the organization of the actin network and hence the extent of network deformation, and viscous stresses that are proportional to the rate of network deformation. The emergence of contractility in actomyosin networks requires motor-dependent filament buckling and the presence of actin cross-linkers that dynamically stabilize actomyosin networks under stress (Gardel et al., 2004). Finally, the forces generated by such networks are transmitted to the plasma membrane or the ECM via adhesion complexes, giving rise to a repertoire of cellular morphogenetic outcomes, such as migration, cell shape changes and cell contact remodeling.

The waves and tides of cellular morphogenesis
Live imaging of actomyosin networks and membrane markers has revealed an unexpectedly high degree of network dynamics during cellular morphogenesis. Although the actomyosin intensity levels are sometimes steady, for example during cytokinesis, there exist other situations in which they fluctuate dramatically over a few tens of seconds. These fluctuations are characterized by cycles of recruitment of Myo II and its coalescence to form bigger aggregates, causing spatial deformation, followed by disassembly. These pulsed contractions of actomyosin networks are conserved across species and, depending on the context, can result in completely different outcomes at the cellular level. For instance, the Myo II contractions in the medial apical plane of the Drosophila mesoderm correlate with constriction of the apical cell surface. Similar pulses were reported in the nematode endoderm. Myo II pulses in the Drosophila ectoderm control steps of junction shrinkage, which facilitates cell intercalation thereby extending the tissue. Deformations caused by the pulsatile activity of Myo II are often interspersed with stabilization phases to create a step-wise unidirectional process that allows irreversible shape changes, a phenomenon analogous to a mechanical ratchet. However, what regulates Myo II spatially and temporally to generate these pulsed contractions during morphogenesis still remains unclear. It is possible that the localization, amplitude, and frequency of the pulses are under the control of separate regulatory mechanisms, each controlling the type, range and speed of deformation, respectively.

Spatial control of actomyosin networks by signaling pathways
In a framework in which the local modulation of actomyosin network contraction can emerge into varied cell behaviors and tissue outcomes, spatial and temporal regulation is essential. Contractility is indeed regulated at both cellular and tissue levels by diverse sets of signaling pathways that are conserved across species. We can delineate three tiers in the regulation of actomyosin contractility. First, a conserved subcellular pathway is responsible for regulating Myo II phosphorylation and dephosphorylation. It involves activation by RhoGEFs of the small GTPase Rho1, which in turn activates ROCK as well as formin and formin-related proteins, such as Daam proteins, and inactivates myosin phosphatase. The actin-binding protein Shroom binds ROCK and is also required for ROCK and Myo II distribution. Second, this core subcellular pathway is activated by membrane signaling modules such as those involving the core planar cell polarity protein Celsr1 in the vertebrate neural tube and G protein-coupled receptor (GPCR) signaling in Drosophila embryos. Finally, membrane signaling can be induced by unknown developmental signals downstream of tissue-specific transcription factors. For instance, in the Drosophila mesoderm, the apical localization of Myo II activity is regulated by the mesoderm-specific transcription factors Twist and Snail. Twist and Snail activate the expression of an extracellular ligand named Fog that, through an unknown GPCR and a transmembrane protein called T48, leads to the recruitment of RhoGEF2 and activation of Myo II contractility via ROCK (Rok – FlyBase).

In some cases, the regulation of Myo II by phosphorylation can be specific such that mono- and bi-phosphorylated Myo II serve distinct functions, as seen in the case of endoderm invagination in ascidians. In this case, the invagination of the endoderm is initiated by apical constriction followed by apicobasal shortening. Whereas apical constriction relies on apical recruitment of ROCK-dependent monophosphorylated Myo II, apicobasal shortening is driven by ROCK-independent basolateral enrichment of monophosphorylated Myo II and ROCK-dependent apical enrichment of bi-phosphorylated Myo II to prevent apical expansion and to mediate deep invagination.

Coordinating contractility in a tissue
The existence of subcellular forces generated by actomyosin networks and their transmission at cell contacts by adhesive systems begs the question of whether cells coordinate their local mechanics to yield tissue-level deformations. Coordinated cell shape changes require interactions between cells through cell-cell adherens junctions that transmit subcellular tensions. In addition, supracellular actomyosin cables have been reported in diverse morphogenetic processes and contexts, such as at compartment boundaries, in tissue wound healing, and during developmental closure events, such as dorsal closure and epiboly. It is possible that such cables are part of a tissue level network that coordinates contractility across cells. To what extent such tissue level actomyosin networks result from local biochemical control of Myo II by signaling pathways or mechanical coupling between cells is unclear.

Recent studies suggest that the partitioning of tissues into compartments to prevent intermixing of cells does indeed require the formation of large-scale actomyosin networks. As an alternative to the DAH described above, another hypothesis called the ‘fence model’ attributes compartmentalization to the formation of stiff mechanical barriers that prevent crossing of cells across boundaries. One of the oldest examples of compartment boundaries is in the Drosophila wing imaginal disc (the sac of epithelial cells from which the adult wing is formed). Although there is no detectable E-cadherin increase at compartment boundaries, F-actin and Myo II accumulate there, control the local increase in intercellular tension, and prevent cell mixing between compartments.

The network is a highly adaptable biomechanical system that can adjust to external and internal stresses and subtle changes without involving complex genetic circuits.

Besides driving changes through force production, actomyosin networks orchestrate intrinsic forces to coordinate tissue movements and shape changes. This coordination is crucial for integrating incoherent or stochastic local deformations into ordered global changes, although the underlying biomechanical signals are still poorly understood.



Spectrins
Cell membranes have a skeleton based on spectrins, which is located at the inner surface of the plasma membranes, linked to a number of integral membrane proteins. 20 D


A schematic diagram of spectrin and other cytoskeletal molecules

Spectrin is a cytoskeletal protein that lines the intracellular side of the plasma membrane in eukaryotic cells. Spectrin forms pentagonal or hexagonal arrangements, forming a scaffold and playing an important role in maintenance of plasma membrane integrity and cytoskeletal structure. The hexagonal arrangements are formed by tetramers of spectrin subunits associating with short actin filaments at either end of the tetramer. These short actin filaments act as junctional complexes allowing the formation of the hexagonal mesh.23

In certain types of brain injury such as diffuse axonal injury, spectrin is irreversibly cleaved by the proteolytic enzyme calpain, destroying the cytoskeleton. Spectrin cleavage causes the membrane to form blebs and ultimately to be degraded, usually leading to the death of the cell. Spectrin subunits may also be cleaved by caspase family enzymes, and calpain and caspase produce different spectrin breakdown products. Rather than the one α and two β genes in invertebrates, there are two α spectrins (αI and αII) and five β spectrins (βI to V), named in the order of discovery.

During tissue morphogenesis, the spatial-temporal coordination between cell proliferation and cell shape change produces organs of proper size and shape, and disruption of this coordination is a common characteristic of developmental anomalies. This process is mediated not only by morphogen-mediated chemical signaling but also by mechanical signals such as cell shape, cell geometry, deformation caused by the pulling forces of the extracellular matrix (ECM) and of neighboring cells, and the associated changes in cytoskeleton organization and tension, which together represent the architectural signal of a tissue 21 

cell shape regulation in tissue morphogenesis is largely governed by two antagonistic forces: an E-cadherin–mediated adhesion force that stabilizes cell contacts, and cell cortical tension exerted by the actomyosin network that tends to reduce cell–cell interface (Fig.A). 


Spectrin couples cell shape and Hippo signaling in Drosophila retina development.
(A) A cartoon showing side view (cross-section) and top view (tangential section around AJs) of imaginal disc epithelial cells. Left (side view): The apical membrane domain includes the membrane region above the AJs; the lateral membrane domain includes the membrane region below the AJs but not the basal membrane that faces the ECM; junctional membrane domain refers to AJ-associated membrane region. Note the AJ-associated actomyosin cytoskeleton attached to membrane through AJs, and the apical and lateral cortical actomyosin cytoskeleton attached to the membrane. The septate junctions (SJs) containing Dlg are located laterally to AJs. Right (top view): Cell shape is dictated by cell surface tension, which is determined by the antagonistic cortical tension and adhesion force.

The antagonistic interplay of these two forces produces the intercellular surface tension that dictates cell-cell interaction according to the principle of energy minimization. Mechanistically, it is believed that this interplay is mediated by adherens junctions (AJs) where E-cadherin organizes the cortical actomyosin network through α-catenin and its associated proteins, (Fig.  A). According to this model, contraction force generated by actomyosin is transferred to cell plasma membrane through adherens junctions AJs to effect cell shape change. This model was recently challenged by findings that in embryonic cells undergoing apical constriction during Drosophila melanogaster gastrulation, the cell shape changes follow the contraction pulse of the apical-medial cortical actomyosin, not the actomyosin belt linked with AJs. These findings raised two interesting questions: does non–AJ-associated cortical actomyosin regulate cell shape in other epithelia such as imaginal discs? How is non–AJ-associated cortical actomyosin linked to plasma membrane?

Spectrin is a large, springlike protein that forms the spectrin-based membrane skeleton SBMS beneath the plasma membrane by cross-linking short F-actin and binding integral membrane proteins.



a) A Blastomere is a type of cell produced by cleavage (cell division) of the zygote after fertilization and is an essential part of blastula formation 10
b) Actomyosin contractile ring is a prominent structure during cytokinesis.^[1] It forms perpendicular to the axis of the spindle apparatus^[2] towards the end of telophase, in which sister chromatids are identically separated at the opposite sides of the spindle forming nuclei 17
c) Myosins are a superfamily of motor proteins best known for their roles in muscle contraction and in a wide range of other motility processes in eukaryotes.18
d) Epithelial cells are polarized with an apical surface that faces the lumen of a tube or the external environment and a basal surface that attaches to the basement membrane. The apical and basal surfaces perform different functions and have unique biochemical compositions.[url=http://medcell.med.yale.edu/lectures/epithelial_structure.php#:~:text=Epithelia cells are polarized with,Epithelial cells are continuously renewed.]19[/url]

1. https://en.wikipedia.org/wiki/Morphogenesis
2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6678760/
3. https://www.jbc.org/content/276/23/19679
4. https://reasonandscience.catsboard.com/t1992-mitosis-and-cell-division
5. https://en.wikipedia.org/wiki/Astral_microtubules
6. https://en.wikipedia.org/wiki/Mitosis
7. https://www.thoughtco.com/asters-373536
8. https://sci-hub.ren/https://www.sciencedirect.com/science/article/pii/S096098221630745X
9. https://sci-hub.ren/https://www.sciencedirect.com/science/article/pii/S1534580716308309
10. https://en.wikipedia.org/wiki/Blastomere
11. https://sci-hub.ren/https://www.sciencedirect.com/science/article/abs/pii/S0962892412000128
12. https://sci-hub.ren/https://www.nature.com/articles/nature05786?proof=true1
13. https://sci-hub.ren/https://www.sciencedirect.com/science/article/pii/S1534580718305057
14. https://en.wikipedia.org/wiki/Cell_cortex
15. https://www2.mrc-lmb.cam.ac.uk/group-leaders/a-to-g/buzz-baum/
16. https://sci-hub.ren/https://www.sciencedirect.com/science/article/abs/pii/S0960982220304917
17. https://en.wikipedia.org/wiki/Actomyosin_ring
18. https://en.wikipedia.org/wiki/Myosin
19. http://medcell.med.yale.edu/lectures/epithelial_structure.php#:~:text=Epithelia%20cells%20are%20polarized%20with,Epithelial%20cells%20are%20continuously%20renewed.
20. https://sci-hub.ren/https://www.sciencedirect.com/science/article/pii/S000527361300148X
21. https://rupress.org/jcb/article/219/4/e201907018/133846/Spectrin-couples-cell-shape-cortical-tension-and
22. https://dev.biologists.org/content/141/9/1789
23. https://en.wikipedia.org/wiki/Spectrin

The role of spectrin in cell adhesion and cell–cell contact
https://journals.sagepub.com/doi/full/10.1177/1535370219859003[/b]

Multicellular morphogenetic events and their underlying mechanical forces can feedback into gene regulatory pathways to specify cell fate. Mechanical forces play an integral role in tissue morphogenesis. Mechanical cues, either extrinsically induced by the cellular microenvironment or intracellularly generated, can be transduced into biochemical signals that regulate cell proliferation, migration, and differentiation. Understanding the crosstalk between tissue-scale mechanics and cell-fate specification is essential to uncover the key design principles that regulate robust tissue patterning during development. How genetic cascades link cell-fate specification to tissue morphogenesis remains unclear..

Impact of Tissue Stress on Cell-Fate Specification
Cells can ‘sense’ forces through mechanosensing and mechanotransduction, and subsequently, control their differentiation. The terms ‘mechanosensation’ and ‘mechanotransduction’ have been used extensively, and sometimes interchangeably, in different biological contexts. Mechanosensation is the physical mechanism by which a cell senses mechanical cues, such as forces or stiffness, from its microenvironment. Meanwhile, mechanotransduction involves the conversion of these mechanical cues to biochemical signals downstream of mechanosensation. Such transduction may affect various cellular components at a post-translational level, and it triggers gene expression changes to impact fate specification.

Cellular Mechanisms of Mechanotransduction
In general, cellular mechanotransduction can be classified into chemical or physical transduction. Chemical transduction relies on a series of chemical signaling cascades bridging the plasma membrane and the nucleus. When the cells experience mechanical strain, focal adhesion proteins such as zyxin and paxillin can alter their binding kinetics and shuttle between the cytoplasm and nucleus to modulate transcriptional activity. Force-induced phosphorylation of E-cadherin-bound b-catenin at intercellular junctions results in its translocation to the nucleus and activation of transcription of downstream effectors. Mechanotransduction can also involve the nuclear translocation of transcription factors from the cytoplasm, such as NF-kB or MAL, in response to fluid shear and mechanical stress, respectively. Recently, the stretch-activated channels Piezo1 and Piezo2 were identified as key mechanosensors regulating calcium signaling by sensing changes in membrane tension and they can potentially play a role in neural stem cell differentiation. Another well-studied signaling cascade involved in mechanotransduction is Yap/Taz signaling, where mechanical tension arising from physical stretching of cells or increased stiffness of the extracellular matrix (ECM) activates F-actin remodeling and Yap/Taz nuclear localization. While ECM stiffness is known to direct differentiation or self-renewal of embryonic or somatic stem cells, ECM viscosity can also influence cell-fate specification, for example during the osteogenic differentiation of mesenchymal stem cells.  There is a diverse set of mechanical cues that cells can sense and biochemically transduce to regulate differentiation. Physical mechanotransduction relies on direct force transmission from the cell surface to the nucleus through physical coupling between the nuclear membrane and the extracellular space by cytoskeletal components. The nucleus has been proposed to act as a mechanosensor, whereby changes in nuclear shape can evoke transcriptional changes by locally altering the spatial accessibility of chromatin to transcriptional regulators. Importantly, the physical links between the cytoskeleton and nuclear membrane proteins allow the entire cell to function as a single mechanically coupled system. A key molecular player involved in such physical transduction is the nuclear lamina component lamin A. In stiff tissue, tension from the ECM transmitted via the cytoskeleton to the nucleus can enhance lamin A transcription and stability of lamin A/C, leading to activation of Yap, serum response factor (SRF) and retinoic acid signaling pathways. 

Mechanotransduction in Development
During embryonic development, forces are generated and transmitted across multiple scales. The interplay between tissue mechanics and biochemical signaling involves multiple feedback interactions, rendering the assessment of mechanotransduction in vivo at the organismal level non-trivial. To date, mechanotransduction has been identified in only a few developmental contexts where the underlying genetics, cell-fate changes and mechanics are sufficiently well characterized. In Drosophila embryos, tissue deformations caused by germband extension upregulate the expression of the transcription factor Twist via nuclear translocation of b-catenin (Figure A). 


Examples of mechanotransduction during embryonic and adult organ development.
(A) Tissue-scale compression in Drosophila embryos caused by germ-band extension triggers Src42A-dependent nuclear translocation of b-catenin in anterior stomodeal cells, thereby activating the transcription of Twist, which is crucial for subsequent midgut differentiation. 
(B) In early mouse embryonic development, asymmetrical inheritance of the apical domain results in a polarized daughter cell that exhibits lower cortical contractility than its apolar sister cell. The difference in contractility leads to internalization of the apolar cell. The outer polar cell adopts the trophectoderm fate with nuclear Yap localization while the apolar cell adopts the inner cell mass fate with cytosolic Yap localization. 
(C) During the formation of the hematopoietic system, shear stress can induce nitric oxide production and upregulation of Runx1 transcriptional factor, thereby inducing endothelial-to-hematopoietic cell-fate switch. 
(D) Mechanical stretching of the lung drives myogenesis in airway smooth muscle, which is crucial for bronchial development. This process is mediated by the activation of the transcription factor SRF. 
(E) In Arabidopsis, strong mechanical stresses arising from tissue folding at the shoot apical meristem can induce the expression of STM, a master regulator of meristematic identity.


Furthermore, both static and pulsed mechanical deformations of mesodermal cells have been shown to promote Folded gastrulation (Fog) signaling and apical myosin II accumulation, which is required for mesoderm invagination prior to germband extension.

Impact of Geometry and Cytoskeletal Forces on Tissue Patterning
Establishment of well-defined forms and patterns during development requires that cells recognize their position within the embryo and differentiate accordingly. Two current pre-eminent concepts for tissue patterning are based on the positional information and reaction-diffusion models. In brief, positional information involves the graded distribution of a morphogen across the tissue, from which the cells interpret their position to make fate choices. The morphogen concentration, therefore, provides positional coordinates along the embryonic axis. The reaction-diffusion model involves a self-amplifying local signal and a long-range inhibitor that is stimulated by the former. Interactions between these two components lead to the generation of periodic patterns of diffusible morphogens and consequently cell fates. While the significance of both models has long been recognized in developmental biology, these models are so far based purely on biochemical signaling. Here we discuss the new possibility that cytoskeletal forces, cell adhesion and polarity, and tissue-scale mechanical signals can confer positional information and control gene expression and fate at the cellular level, thereby allowing cells to coordinate embryonic patterning.

Cytoskeletal Forces Drive Tissue Patterning
Forces generated by motor proteins can couple with biochemical signals to generate morphogen gradients and patterns In Caenorhabditis elegans zygotes, actomyosin contraction induces large-scale, anterior-directed cortical flows, which in turn transport polarity and cell-fate-determining proteins to the anterior half of the cell. Importantly, if contraction itself causes flows that carry regulators of contraction, such as partitioning defective (PAR) proteins, this will lead to positive feedback and subcellular patterning (Figure A)


Cytoskeletal forces and geometric cues can mechanically feedback to morphogen signaling.
(A) Mechanochemical patterning in C. elegans zygote is established through the advection ( advection is the transport of a substance or quantity by bulk motion ) of anterior PAR proteins (red) by actomyosin-driven cytoplasmic flow. The binding of posterior PARs (blue) to the posterior membrane results in the amplification and stabilization of polarity by a reaction-diffusion mechanism. 
(B) In 8-cell early mouse embryos, contact asymmetry induces apical domain formation at the contact-free cell surface. Upon division, the daughter cells that inherit the apical domain adopt the trophectoderm ( The first epithelium to appear during mammalian embryogenesis is the trophectoderm)  fate while the apolar daughter cells may or may not become trophectoderm, depending on their eventual position.
(C) Luminal signaling can impact cell-fate acquisition in a multicellular context. In migrating zebrafish lateral line primordium, concerted apical constriction leads to the formation of a microlumen. Local trapping of FGF in these microlumens leads to enhanced signaling and restricted cellular differentiation in the neighboring cells. 
(D) Left: In the mouse gut, mechanical buckling of the epithelium due to growth leads to a local build-up of Shh signals in the villi, which then restricts progenitor cells to the base of these structures. Right: Geometric confinement of human embryonic stem cells leads to the spatial ordering of germ layers recapitulating human gastrulation. In the presence of BMP4, differential localization of TGF-b receptors at the colony edge (apical) and center (basolateral)
triggers differential signaling and generates a reaction-diffusion mechanism that involves the BMP inhibitor Noggin.


Cellular mechanisms of morphogenesis 2018 May 29
Biomechanics, tissue homeostasis, development, mechanotransduction, mechanosensing, cell contractility, tissue tension, intracellular tension, mechanical force, matrix stiffness, mechanical memory, actin-myosin contractility, cellular activities such as growth, proliferation, survival, and motility are all terms relevant to cell and tissue morphogenesis and homeostasis, and as such, relevant to explain organismal form and architecture.

In addition to generating forces, cells in tissues also respond to forces generated by the cells around them, and these mechanical signals can be translated into biochemical changes that influence tissue structure. 2 Epithelial a tissues utilize a combination of chemical and mechanical signals that allow them to extrude dying cells before they disrupt the mechanical integrity of the tissue, and to extrude live cells in overcrowded regions to restore homeostasis. Force-induced signaling mechanisms are important for development and influence tissue aging and disease in the adult.

Cellular adaptation to biomechanical stress across length scales in tissue homeostasis and disease 4 2018 Jul 1.
Human tissues are remarkably adaptable and robust, harboring the collective ability to detect and respond to external stresses while maintaining tissue integrity. Following injury, many tissues have the capacity to repair the damage - and restore form and function - by deploying cellular and molecular mechanisms reminiscent of developmental programs.  Cancer develops with deregulating a selection of developmental programs.

Cell and tissue shape is defined by Type II myosin, which establishes inter- and intracellular tension through motor contractility along the actin cytoskeleton. Actin filaments are anchored to cell-cell and cell-extracellular matrix (ECM) attachment points, and via cell surface receptors (e.g. integrins, cadherins), and the actin-myosin system is responsive to counter forces transferred from the ECM and other cells. Iterative interactions between cells and the surrounding environment modifies tissue tension and relays cell-cell and cell-ECM forces across a tissue, resulting in adaptations in the size, shape, and position of cells during development and tissue regeneration. Biomolecules that can respond to changes in mechanical forces are called mechanosensors. As an example, integrin receptors can respond to extra- or intracellular forces with changes in conformation. This then drives recruitment of “inside-out” or “outside-in signal” transduction complexes, in addition to altering cytoskeletal dynamics, which then modify protein activity and gene expression. In this way, cells possess an elaborate mechanism to integrate external biochemical cues together with physical interactions with neighboring cells and changes in the ECM to control tissue growth and morphology and maintain tissue homeostasis. When this biochemical–biomechanical balance is disrupted, chronic disease and cancer often follows.


Actin-Nucleating Factors Accelerate Polymerization and Generate Branched or Straight Filaments
In addition to the availability of active actin subunits, a second prerequisite for cellular actin polymerization is filament nucleation. Proteins that contain actin monomer binding motifs linked in tandem mediate the simplest mechanism of filament nucleation. These actin-nucleating proteins bring several actin subunits together to form a seed. In most cases, actin nucleation is catalyzed by one of two different types of factors: the Arp 2/3 complex or the formins. The first of these is a complex of proteins that includes two actin-related proteins, or ARPs, each of which is about 45% identical to actin. The Arp 2/3 complex nucleates actin filament growth from the minus end, allowing rapid elongation at the plus end ( A and B )


Nucleation and actin web formation by the Arp 2/3 complex. 
(A) The structures of Arp2 and Arp3 compared to the structure of actin. Although the face of the molecule equivalent to the plus end (top) in both Arp2 and Arp3 is very similar to the plus end of actin itself, differences on the sides and minus-end prevent these actin-related proteins from forming filaments on their own or coassembling into filaments with actin. 
(B) A model for actin filament nucleation by the Arp 2/3 complex. In the absence of an activating factor, Arp2 and Arp3 are held by their accessory proteins in an orientation that prevents them from nucleating a new actin filament. When an activating factor (indicated by the blue triangle) binds the complex, Arp2 and Arp3 are brought together into a new configuration that resembles the plus end of an actin filament. Actin subunits can then assemble onto this structure, bypassing the rate-limiting step of filament nucleation. 
(C) The Arp 2/3 complex nucleates filaments most efficiently when it is bound to the side of a preexisting actin filament. The result is a filament branch that grows at a 70° angle relative to the original filament. Repeated rounds of branching nucleation result in a treelike web of actin filaments. 
(D) Top, electron micrographs of branched actin filaments formed by mixing purified actin subunits with purified Arp 2/3 complexes. Bottom, reconstructed image of a branch where the crystal structures of actin (pink) and the Arp 2/3 complex have been fitted to the electron density. The mother filament runs from top to bottom, and the daughter filament branches off to the right where the Arp 2/3 complex binds to three actin subunits in the mother filament. 

The complex can attach to the side of another actin filament while remaining bound to the minus end of the filament that it has nucleated, thereby building individual filaments into a treelike web (Fig C and D).
Formins are dimeric proteins that nucleate the growth of straight, unbranched filaments that can be cross-linked by other proteins to form parallel bundles. Each formin subunit has a binding site for monomeric actin, and the formin dimer appears to nucleate actin filament polymerization by capturing two monomers. As the newly nucleated filament grows, the formin dimer remains associated with the rapidly growing plus end while still allowing the addition of new subunits at that end.  As the newly nucleated filament grows, the formin dimer remains associated with the rapidly growing plus end while still allowing the addition of new subunits at that end


Actin elongation mediated by formins
Formin proteins (green) form a dimeric complex that can nucleate the formation of a new actin filament (red) and remain associated with the rapidly growing plus end as it elongates. The formin protein maintains its binding to one of the two actin subunits exposed at the plus end as it allows each new subunit to assemble. Only part of the large dimeric formin molecule is shown here. Other regions regulate their activity and link it to particular structures
in the cell. Many formins are indirectly connected to the cell plasma membrane and aid the insertional polymerization of the actin filament directly beneath the membrane surface.



Profilin and formins. 
Some members of the formin protein family have unstructured domains or “whiskers” that contain several binding sites for profilin or the profilin–actin complex. These flexible domains serve as a staging area for addition of actin to the growing plus end of the actin filament when formin is bound. Under some conditions, this can enhance the rate of actin filament elongation so that filament growth is faster than that expected for a diffusion-controlled reaction, and faster in the presence of formin and profilin than the rate for pure actin alone

https://www.youtube.com/watch?v=d_uWtjyNb-Y


The actin cytoskeleton drives many essential biological processes, from cell morphogenesis to motility. Eukaryotic cells move, change their shape, and organize their interior through dynamic actin networks. Actin assembly requires nucleation of filaments, which elongate by the addition of subunits to filament ends. To move and quickly adapt their shape, most eukaryotic cells sustain vast amounts (>50 µM) of polymerizable subunits, which requires the monomer-binding protein profilin.

Assembly of functional actin networks requires control over the speed at which actin filaments grow.  Synergy between profilin and formins generates robust filament growth rates 7

Actin-Filament-Binding Proteins Alter Filament Dynamics
Actin filament behavior is regulated by two major classes of binding proteins: those that bind along the side of a filament and those that bind to the ends. Side-binding proteins include tropomyosin, an elongated protein that binds simultaneously to six or seven adjacent actin subunits along each of the two grooves of the helical actin filament. In addition to stabilizing and stiffening the filament, the binding of tropomyosin can prevent the actin filament from
interacting with other proteins; this aspect of tropomyosin function is important in the control of muscle contraction. An actin filament that stops growing and is not specifically stabilized in the cell will depolymerize rapidly, particularly at its plus end, once the actin molecules have hydrolyzed their ATP. The binding of plus-end capping protein (also called CapZ for its location in the muscle Z band) stabilizes an actin filament at its plus
end by rendering it inactive, greatly reducing the rates of filament growth and depolymerization


Filament capping and its effects on filament dynamics. 
A population of uncapped filaments adds and loses subunits at both the plus and minus ends, resulting in rapid growth or shrinkage, depending on the concentration of available free monomers (green line). In the presence of a protein that caps the plus end (red line), only the minus end is able to add or lose subunits; consequently, filament growth will be slower at all monomer concentrations above the critical concentration, and filament shrinkage will be slower at all monomer concentrations below the critical concentration. In addition, the critical concentration for the population shifts to that of the filament minus end.

At the minus end, an actin filament may be capped by the Arp 2/3 complex that was responsible for its nucleation, although many minus ends in a typical cell are released from the Arp 2/3 complex and are uncapped. Tropomodulin, best known for its function in the capping of exceptionally long-lived actin filaments in muscle, binds tightly to the minus ends of actin filaments that have been coated and thereby stabilized by tropomyosin. It can also transiently cap pure actin filaments and significantly reduce their elongation and depolymerization rates. A large family of tropomodulin proteins regulates actin filament length and stability in many cell types. For maximum effect, proteins that bind the side of actin filaments coat the filament completely, and must, therefore, be present in high amounts. In contrast, end-binding proteins can affect filament dynamics even when they are present at very low levels. Since subunit addition and loss occur primarily at filament ends, one molecule of an end-binding protein per actin filament (roughly one molecule per 200–500 actin subunits) can be enough to transform the architecture of an actin filament network.

Severing Proteins Regulate Actin Filament Depolymerization
Another important mechanism of actin filament regulation depends on proteins that break an actin filament into many smaller filaments, thereby generating a large number of new filament ends. The fate of these new ends depends on the presence of other accessory proteins. Under some conditions, newly formed ends nucleate filament elongation, thereby accelerating the assembly of new filament structures. Under other conditions, severing promotes the depolymerization of old filaments, speeding up the depolymerization rate by tenfold or more. In addition, severing changes the physical and mechanical properties of the cytoplasm: stiff, large bundles and gels become more fluid. One class of actin-severing proteins is the gelsolin superfamily. These proteins are activated by high levels of cytosolic Ca2+. Gelsolin interacts with the side of the actin filament and contains subdomains that bind to two different sites: one that is exposed on the surface of the filament and one that is hidden between adjacent subunits. According to one model, gelsolin binds the side of an actin filament until a thermal fluctuation creates a small gap between neighboring subunits, at which point gelsolin inserts itself into the gap to break the filament. After the severing event, gelsolin remains attached to the actin filament and caps the new plus end. Another important actin-filament destabilizing protein, found in all eukaryotic cells, is cofilin. Also called an actin-depolymerizing factor, cofilin binds along the length of the actin filament, forcing the filament to twist a little more tightly


Twisting of an actin filament induced by cofilin. 
(A) Three-dimensional reconstruction from cryoelectron micrographs of filaments made of pure actin. The bracket shows the span of two twists of the actin helix. 
(B) Reconstruction of an actin filament coated with cofilin, which binds in a 1:1 stoichiometry to actin subunits all along the filament. Cofilin is a small protein (14 kD) compared to actin (43 kD), and so the filament appears only slightly thicker. The energy of cofilin binding serves to deform the actin filament, twisting it more tightly and reducing the distance spanned by each twist of the helix.

This mechanical stress weakens the contacts between actin subunits in the filament, making the filament brittle and more easily severed by thermal motions, generating filament ends that undergo rapid disassembly. As a result, most of the actin filaments inside cells are shorter-lived than are filaments formed from pure actin in a test tube. Cofilin binds preferentially to ADP-containing actin filaments rather than to ATP-containing filaments. Since ATP hydrolysis is usually slower than filament assembly, the newest actin filaments in the cell still contain mostly ATP and are resistant to depolymerization by cofilin. Cofilin, therefore, tends to dismantle the older filaments in the cell.  The cofilin-mediated disassembly of old but not new actin filaments is critical for the polarized, directed growth of the actin network that is responsible for unidirectional cell crawling and the intracellular motility of pathogens. Actin filaments can be protected from cofilin by tropomyosin binding. Thus, the dynamics of actin in different subcellular locations depends on the balance of stabilizing and destabilizing accessory proteins.

Higher-Order Actin Filament Arrays Influence Cellular Mechanical Properties and Signaling
Actin filaments in animal cells are organized into several types of arrays: dendritic networks, bundles, and weblike (gel-like) networks


Actin arrays in a cell.
A fibroblast crawling in a tissue-culture dish is shown with four areas enlarged to show the arrangement of actin filaments. The actin filaments are shown in red, with arrowheads pointing toward the minus end. Stress fibers are contractile and exert tension. The actin cortex underlies the plasma membrane and consists of gel-like networks of dendritic actin networks that enable membrane protrusion at lamellopodia. Filopodia are spike-like projections of the plasma membrane that allow a cell to explore its environment.

Different structures are initiated by the action of distinct nucleating proteins: the actin filaments of dendritic networks are nucleated by the Arp 2/3 complex, while bundles are made of the long, straight filaments produced by formins. The proteins nucleating the filaments in the gel-like networks are not yet well defined. The structural organization of different actin networks depends on specialized accessory proteins. Arp 2/3 organizes filaments into dendritic networks by attaching filament minus ends to the side of other filaments. Other actin filament structures are assembled and maintained by two classes of proteins: bundling proteins, which cross-link actin filaments into a parallel array, and gel-forming proteins, which hold two actin filaments together at a large angle to each other, thereby creating a looser meshwork. Both bundling and gel-forming proteins generally have two similar actin-filament-binding sites, which can either be part of a single polypeptide chain or contributed by each of two polypeptide chains held together in a dimer


The modular structures of four actin-cross-linking proteins. 
Each of the proteins shown has two actin-binding sites (red) that are related in sequence. Fimbrin has two directly adjacent actin-binding sites, so that it holds its two actin filaments very close together (14 nm apart), aligned with the same polarity. The two actin-binding sites in α-actinin are separated by a spacer around 30 nm long, so that it forms more loosely packed actin bundles. Filamin has two actin-binding sites with a V-shaped linkage between them, so that it cross-links actin filaments into a network with the filaments oriented almost at right angles to one another. Spectrin is a tetramer of two α and two β subunits, and the tetramer has two actinbinding sites spaced about 200 nm apart

The spacing and arrangement of these two filament-binding domains determine the type of actin structure that a given cross-linking protein forms. Each type of bundling protein also determines which other molecules can interact with the cross-linked actin filaments. Myosin II is the motor protein that enables stress fibers and other contractile arrays to contract. The very close packing of actin filaments caused by the small monomeric bundling protein fimbrin apparently excludes myosin, and thus the parallel actin filaments held together by fimbrin are not contractile. On the other hand, α-actinin cross-links oppositely polarized actin filaments into loose bundles, allowing the binding of myosin and formation of contractile actin bundles


The formation of two types of actin filament bundles.
(A) Fimbrin cross-links actin filaments into tight bundles, which exclude the motor protein myosin II from participating in the
assembly. In contrast, α-actinin, which is a homodimer, cross-links actin filaments into loose bundles, which allow myosin (not shown) to incorporate into the bundle. Fimbrin and α-actinin tend to exclude one another because of the very different spacing of the actin filament bundles that they form. 
(B) Electron micrograph of purified α-actinin molecules. 

Because of the very different spacing and orientation of the actin filaments, bundling by fimbrin automatically discourages bundling by α-actinin, and vice versa, so that the two types of bundling protein are mutually exclusive. The bundling proteins that we have discussed so far have straight, stiff connections between their two actin-filament-binding domains. Other actin cross-linking proteins have either a flexible or a stiff, bent connection between their two
binding domains, allowing them to form actin filament webs or gels, rather than actin bundles. Filamin promotes the formation of a loose and highly viscous gel by clamping together two actin filaments roughly at right angles (Figure A)


(A) Each filamin homodimer is about 160 nm long when fully extended and forms a flexible, highangle link between two adjacent actin filaments. A set of actin filaments crosslinked by filamin forms a mechanically strong web or gel. 
(B) Magnetic resonance imaging of a normal human brain (left) and of a patient with periventricular heterotopia (right) caused by mutation in the filamin A gene. In contrast to the smooth ventricular surface in the normal brain, a rough zone of cortical neurons (arrowheads) is seen along the lateral walls of the ventricles, representing neurons that have failed to migrate to the cortex during brain development. Remarkably, although many neurons are not in the right place, the intelligence of affected individuals is frequently normal or only mildly compromised, and the major clinical syndrome is epilepsy that often starts in the second decade of life.

Cells require the actin gels formed by filamin to extend the thin, sheetlike membrane projections called lamellipodia that help them to crawl across solid surfaces. In humans, mutations in the filamin A gene cause defects in nerve-cell migration during early embryonic development. Cells in the periventricular region of the brain fail to migrate to the cortex and instead form nodules, causing a syndrome called periventricular heterotopia (Figure B above). Interestingly, in addition to binding actin, filamins have been reported to interact with a large number of cellular proteins of great functional diversity, including membrane receptors for signaling molecules, and filamin mutations can also lead to defects in development of bone, the cardiovascular system, and other organs. Thus, filamins may also function as signaling scaffolds by connecting and coordinating a wide variety of cellular processes with the actin cytoskeleton. A very different, well-studied web-forming protein is spectrin, which was first identified in red blood cells. Spectrin is a long, flexible protein made out of four elongated polypeptide chains (two α subunits and two β subunits), arranged so that the two actin-filament-binding sites are about 200 nm apart (compared with 14 nm for fimbrin and about 30 nm for α-actinin ). In the red blood cell, spectrin is concentrated just beneath the plasma membrane, where it forms a two-dimensional weblike network held together by short actin filaments whose precise lengths are tightly regulated by capping proteins at each end; spectrin links this web to the plasma membrane because it has separate binding sites for peripheral membrane proteins, which are themselves positioned near the lipid bilayer by integral membrane proteins. The resulting network creates a strong, yet flexible cell cortex that provides mechanical support for the overlying plasma membrane, allowing the red blood cell to spring back to its original shape after squeezing through a capillary. Close relatives of spectrin are found in the cortex of most other vertebrate cell types, where they also help to shape and stiffen the surface membrane. A particularly striking example of spectrin’s role in promoting mechanical stability is the long, thin axon of neurons in the nematode worm Caenorhabditis elegans, where spectrin is required to keep them from breaking during the twisting motions the worms make during crawling. Members of the ERM family (named for its first three members, ezrin, radixin, and moesin), help organize membrane domains through their ability to interact with transmembrane proteins and the underlying cytoskeleton. In so doing, they not only provide structural links to strengthen the cell cortex, but also regulate the activities of signal transduction pathways. Moesin also increases cortical stiffness to promote cell rounding during mitosis. Measurements by atomic force microscopy indicate that the cell cortex remains soft during mitosis when moesin is depleted. ERM proteins are thought to bind to and organize the cortical actin cytoskeleton in a variety of contexts, thereby affecting the shape and stiffness of the membrane as well as the localization and activity of signaling molecules.

The Lamellipodium
Electron tomography reveals unbranched networks of actin filaments in lamellipodia 25 April 2010
During development and in the adult eukaryotic organism, migratory cells are required to crawl between other cells and penetrate connective tissue lattices in their mission to create or repair organs. They achieve this using lamellipodia and filopodia, which are thin (0.1–0.2 μm) sheets and rods, respectively, that protrude from the cell, leading migration. The major structural elements are actin filaments, which in lamellipodia form networks and in filopodia are arranged in bundles. Actin filaments in lamellipodia and filopodia are polarized, with their fast-growing (plus) ends oriented towards the membrane, and the protrusion is driven by the nucleation and polymerization of actin at the membrane interface.

Arp 2/3 complex remains stably bound to the filament minus end, preventing subunit addition or loss at that end. Formin-dependent actin filament growth is strongly enhanced by the association of actin monomers with profilin. Like profilin activation, actin filament nucleation by Arp 2/3 complexes and formins occurs primarily at the plasma membrane and the highest density of actin filaments in most cells is at the cell periphery. The layer just beneath the plasma membrane is called the cell cortex, and the actin filaments in this region determine the shape and movement of the cell surface, allowing the cell to change its shape and stiffness rapidly in response to changes in its external environment.

A widely accepted scheme of how lamellipodia protrude is the dendritic nucleation model of actin polymerization. An integral component of this pathway is the Arp2/3 complex an actin-nucleator that localizes to lamellipodia and whose depletion, or lack of activation through missing cofactors, suppresses lamellipodia formation. Arp2/3 complex is an essential organizer of treadmilling actin filament arrays.   Arp2/3 complex serves as key upstream factor for the recruitment of modulators of lamellipodia formation such as capping protein or cofilin. Arp2/3 complex is thus decisive for filament organization and geometry within the network not only by generating branches and novel filament ends, but also by directing capping or severing activities to the lamellipodium. Arp2/3 complex is also crucial to lamellipodia-based migration of keratocytes. The actin cytoskeleton is fundamental for establishment and maintenance of forces in both individual cells and cell sheets or tissues and organizes into various structural arrays optimized for exerting specific functions. Migration is commonly initiated by the protrusion of sheets of cytoplasm, so-called lamellipodia, which are filled with networks of actin filaments, the structure, dynamics, and turnover. Lamellipodia and the structurally related membrane ruffles are common to a variety of migrating cell types, ranging from epithelial cells to neurons. Arp2/3 complex activation in lamellipodia is believed to be mediated by pentameric WASP-family verprolin homologous (WAVE) complex, harboring interaction surfaces for both ras-related C3 botulinum toxin substrate (Rac; Sra-1/PIR121) and Arp2/3 complex.

Lamellipodia protrusion is regulated, derived from the precise determination of biochemical activities of key regulators, including the Arp2/3 complex and its nucleation and branching activity, recognition of Scar/WAVE proteins as its activators at the lamellipodium tip, and conformational changes accompanying WAVE complex activation through coincident signals including phospholipids and Rac. 8

Evidence that Arp2/3 regulates protrusion was provided by the reconstitution of actin-driven motion in vitro, in a protein cocktail containing, amongst other components, Arp2/3, actin and beads carrying the carboxy-terminal WA domain of N‑WASP (a Wiskott-Aldrich Syndrome protein). In lamellipodia, the Arp2/3 complex is activated by the WAVE (WASP-family verprolin-homologous protein) complex downstream of Rac, and cycles with actin in a treadmilling mode10, with the WAVE complex concentrated at the lamellipodium tip. When the WA domain of WAVE or N‑WASP was mixed with actin and the Arp2/3 complex in solution, branched filaments were formed with the Arp2/3 complex localized at the branch points. 

https://www.youtube.com/watch?v=jonQiEtTHwY


Concomitant with these in vitro findings, electron microscope images of lamellipodia (in detergent extracted keratocytes and fibroblasts) showed actin filaments in the anterior region that appeared to be branched. Together, these results provided compelling support for the dendritic nucleation model.


6


Hypothetical scheme of actin network generation in lamellipodia.
In response to signaling events (including those downstream of Rac), nucleation-promoting factors (the WAVE complex), elongation complexes (Ena/VASP proteins) and nucleator/elongators (formins) are recruited to the membrane. We suggest that WAVE, VASP and formins associate in different combinations in multimolecular complexes to regulate the balance between network and bundle formation. Possible schemes are indicated. 
(a) Single actin filaments are nucleated by the docking of the Arp2/3 complex onto the WAVE complex. 
(b) Filaments elongate, initially tethered via the WAVE WH2 domain, with the Arp2/3 complex on the filament minus end. 
(c) VASP molecules associated with the WAVE complex take over the role of filament elongation (as oligomers) through common binding partners, releasing WAVE for further nucleation events. 
(d) Some growing actin plus ends tethered by VASP oligomers and associated proteins come together by lateral flow in the membrane and 
(e) initiate the formation of a filament pair through recruitment of an actin-bundling protein (X-linker). 
(f) Filament pairs could also be nucleated and elongated directly by formins (a doublet making up one ring) in combination with a bundling protein. Finally, long cross-linking proteins (such as filamin and α‑actinin) stabilize the network by forming filament interconnections (not shown). For clarity, the filament density in the scheme is lower than in the real cell.

https://www.youtube.com/watch?v=VVgXDW_8O4U



a Epithelium] is one of the four basic types of animal tissue, along with connective tissue, muscle tissue and nervous tissue. Epithelial tissues line the outer surfaces of organs and blood vessels throughout the body, as well as the inner surfaces of cavities in many internal organs. An example is the epidermis, the outermost layer of the skin. https://en.wikipedia.org/wiki/Epithelium

b A fibroblast is a type of biological cell that synthesizes the extracellular matrix and collagen, produces the structural framework (stroma) for animal tissues, and plays a critical role in wound healing. Fibroblasts are the most common cells of connective tissue in animals.


1. https://www.cell.com/current-biology/pdf/S0960-9822(17)30868-0.pdf
2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5973821/
3. https://en.wikipedia.org/wiki/Epithelium
4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5352546/
5. https://reasonandscience.catsboard.com/t2389-actin-filament-assembly-and-how-its-complex-engineering-process-points-to-intelligent-design
6. Published: 25 April 2010 Electron tomography reveals unbranched networks of actin filaments in lamellipodia
7. https://elifesciences.org/articles/50963
8. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3771948/

MYOSIN AND ACTIN

A crucial feature of the actin cytoskeleton is that it can form contractile structures that cross-link and slide actin filaments relative to one another through the action of myosin motor proteins. In addition to driving muscle contraction, actin-myosin assemblies perform important functions in non-muscle cells.

Actin-Based Motor Proteins Are Members of the Myosin Superfamily
The first motor protein to be identified was skeletal muscle myosin, which generates the force for muscle contraction. This protein, now called myosin II, is an elongated protein formed from two heavy chains and two copies of each of two light chains. Each heavy chain has a globular head domain at its N-terminus that contains the force-generating machinery, followed by a very long amino acid sequence that forms an extended coiled-coil that mediates heavy-chain dimerization


Myosin II.
(A) The two globular heads and long tail of a myosin II molecule shadowed with platinum can be seen in this electron micrograph.
(B) A myosin II molecule is composed of two heavy chains (each about 2000 amino acids long; green) and four light chains (blue). The light chains are of two distinct types, and one copy of each type is present on each myosin head. Dimerization occurs when the two α helices of the heavy chains wrap around each other to form a coiled-coil, driven by the association of regularly spaced hydrophobic amino acids (see Figure 3–9). The coiled-coil arrangement makes an extended rod in solution, and this part of the molecule forms the tail.

The two light chains bind close to the N-terminal head domain, while the long coiled-coil tail bundles itself with the tails of other myosin molecules. These tail–tail interactions form large, bipolar “thick filaments” that have several hundred myosin heads, oriented in opposite directions at the two ends of the thick filament


The myosin II bipolar thick filament in muscle.
(A) Electron micrograph of a myosin II thick filament isolated from frog muscle. Note
the central bare zone, which is free of head domains.
(B) Schematic diagram, not drawn to scale. The myosin II molecules aggregate by means of their tail regions, with their heads projecting to the outside of the filament. The bare zone in the center of the filament consists entirely of myosin II tails.
(C) A small section of a myosin II filament as reconstructed from electron micrographs. An individual myosin molecule is highlighted in green. The cytoplasmic myosin II filaments in non-muscle cells are much smaller, although similarly organized

Myosin Generates Force by Coupling ATP Hydrolysis to Conformational Changes
Motor proteins use structural changes in their ATP-binding sites to produce cyclic interactions with a cytoskeletal filament. Each cycle of ATP binding, hydrolysis, and release propels them forward in a single direction to a new binding site along the filament. For myosin II, each step of the movement along actin is generated by the swinging of an 8.5-nm-long α helix, or lever arm, which is structurally stabilized by the binding of light chains. At the base of this lever arm next to the head, there is a pistonlike helix that connects movements at the ATP-binding cleft in the head to small rotations of the so-called converter domain. A small change at this point can swing the helix-like a long lever, causing the far end of the helix to move by about 5.0 nm. These changes in the conformation of the myosin are coupled to changes in its binding affinity for actin, allowing the myosin head to release its grip on the actin filament at one point and snatch hold of it again at another. The full mechanochemical cycle of nucleotide-binding, nucleotide hydrolysis, and phosphate release (which causes the “power stroke”) produces a single step of movement


The cycle of structural changes used by myosin II to walk along an actin filament.
In the myosin II cycle, the head remains bound to the actin filament for only about 5% of the entire cycle time, allowing many myosins to work together to move a single actin filament


The cycle of structural changes used by myosin II to walk along an actin filament.
In the myosin II cycle, the head remains bound to the actin filament for only about 5% of the entire cycle time, allowing many myosins to work together to move a single actin filament

Actin and Myosin Perform a Variety of Functions in Non-Muscle Cells
Most non-muscle cells contain small amounts of contractile actin-myosin II bundles that form transiently under specific conditions and are much less well organized than muscle fibers. Non-muscle contractile bundles are regulated by myosin phosphorylation.


Light-chain phosphorylation and the regulation of the assembly of myosin II into thick filaments.
(A) The controlled phosphorylation by the enzyme myosin light-chain kinase (MLCK) of one of the two light chains (the so-called regulatory light chain, shown in light blue) on non-muscle myosin II in a test tube has at least two effects: it causes a change in the conformation of the myosin head, exposing its actinbinding site, and it releases the myosin tail from a “sticky patch” on the myosin head, thereby allowing the myosin molecules to assemble into short, bipolar, thick filaments. Smooth muscle is regulated by the same mechanism
(B) Electron micrograph of negatively stained short filaments of myosin II that have been induced to assemble in a test tube by phosphorylation of their light chains. These myosin II filaments are much smaller than those found in skeletal muscle cells

These contractile bundles function to provide mechanical support to cells, for example, by assembling into cortical stress fibers that connect the cell to the extracellular matrix through focal adhesions or by forming a circumferential belt in an epithelial cell, connecting it to adjacent cells through adherens junctions. Actin and myosin II in the contractile ring generate the force for cytokinesis, the final stage in cell division. 

Cell division and generating the contractile ring is of course absolutely life-essential for cell division and as such, life essential. As such, both are absolutely indispensable. 

Contractile bundles contribute to the adhesion and forward motion of migrating cells. Non-muscle cells also express a large family of other myosin proteins, which have diverse structures and functions in the cell. Following the discovery of conventional muscle myosin, a second member of the family was found in the freshwater amoeba Acanthamoeba castellanii. This protein had a different tail structure and seemed to function as a monomer, and so it was named myosin I (for oneheaded). Conventional muscle myosin was renamed myosin II (for two-headed). Subsequently, many other myosin types were discovered. The heavy chains generally start with a recognizable myosin motor domain at the N-terminus and then diverge widely with a variety of C-terminal tail domains


Myosin superfamily
members. Comparison of the domain structure of the heavy chains of some myosin types. All myosins share similar motor domains (shown in dark green), but their C-terminal tails (light green) and N-terminal extensions (light blue) are very diverse. On the right are depictions of the molecular structure for these family members. Many myosins form dimers, with two motor domains per molecule, but a few (such as I, III, and XIV) seem to function as monomers, with just one motor domain. Myosin VI, despite its overall structural similarity to other family members, is unique in moving toward the minus end (instead of the plus end) of an actin filament. The small
insertion within its motorhead domain, not found in other myosins, is probably responsible for this change in direction.

The myosin family includes a number of one-headed and two-headed varieties that are about equally related to myosin I and myosin II, and the nomenclature now reflects their approximate order of discovery (myosin III through at least myosin XVIII). Sequence comparisons among diverse eukaryotes indicate that there are at least 37 distinct myosin families in the superfamily. All of the myosins except one move toward the plus end of an actin filament, although they do so at different speeds. The exception is myosin VI, which moves toward the minus end. The myosin tails (and the tails of motor proteins generally) have apparently diversified during evolution to permit the proteins to bind other subunits and to interact with different cargoes.

Some myosins are found only in plants, and some are found only in vertebrates. Most, however, are found in all eukaryotes. The human genome includes about 40 myosin genes. Nine of the human myosins are expressed primarily or exclusively in the hair cells of the inner ear, and mutations in five of them are known to cause hereditary deafness. These extremely specialized myosins are important for the construction and function of the complex and beautiful bundles of actin found in stereocilia that project from the apical surface of these cells; these cellular protrusions tilt in response to sound and convert sound waves into electrical signals. The functions of most of the myosins remain to be determined, but several are well characterized. The myosin I proteins often contain either a second actin-binding site or a membrane-binding site in their tails, and they are generally involved in intracellular organization—including the protrusion of actin-rich structures at the cell surface, such as microvilli, and endocytosis. Myosin V is a two-headed myosin with a large step size and is involved in organelle transport along actin filaments. In contrast to myosin II motors, which work in ensembles and are attached only transiently to actin filaments so as not to interfere with one another, myosin V moves continuously, or processively, along actin filaments without letting go. 


Myosin V carries cargo along actin filaments. 
(A) The lever arm of myosin V is long, allowing it to take a bigger step along an actin filament than myosin II 
(B) Myosin V transports cargo and organelles along actin cables, in this example moving a mitochondrion into the growing bud of a yeast cell.

Myosin V functions are well studied in the yeast Saccharomyces cerevisiae, which undergoes a stereotypical pattern of growth and division called budding. Actin cables in the mother cell point toward the bud, where actin is found in patches that concentrate where cell wall growth is taking place. Myosin V motors carry a wide range of cargoes— including mRNA, endoplasmic reticulum, and secretory vesicles—along the actin cables and into the bud. In addition, myosin V mediates the correct partitioning of organelles such as peroxisomes and mitochondria between mother and daughter cells (see Figure B above).

Non-muscle myosin II takes center stage in cell adhesion and migration


Non-muscle myosin II (NM II) 1 is an actin-binding protein that has actin 5 cross-linking and contractile properties and is regulated by the phosphorylation of its light and heavy chains. The three mammalian NM II isoforms have both overlapping and unique properties. Owing to its position downstream of convergent signaling pathways, NM II is central in the control of cell adhesion, cell migration, and tissue architecture. Myosins are motor proteins that play important roles in several cellular processes that require force and translocation. Myosin molecules can walk along, propel the sliding of or produce tension on actin filaments. This requires energy, which is provided by the hydrolysis of ATP, and requires myosins to have catalytic sites with ATPase activity. Myosin catalytic sites are usually found in the amino-terminal (head) region of the molecule, and they are often activated when myosin binds to actin. The carboxy-terminal region of some myosins binds to and moves cargo in a cell, whereas the C-terminal domains of other myosins self-associate into filaments, which allows their heads to tether actin filaments and exert tension. Myosins can also act indirectly through actin to bring adhesion-related proteins, such as integrins, or signal transduction molecules into close proximity. Most myosins belong to class II and, together with actin, make up the major contractile proteins of cardiac, skeletal and smooth muscle, in which the sliding cross-bridges that connect thick myosin filaments with thin actin filaments provide the force to, for example, pump blood, lift objects and expel babies. Importantly, myosin II molecules that resemble their muscle counterparts, with respect to both structure and function, are also present in all non-muscle eukaryotic cells. Like muscle myosin II, non-muscle myosin II (NM II) molecules are comprised of three pairs of peptides: two heavy chains of 230 kDa, two 20 kDa regulatory light chains (RLCs) that regulate NM II activity and two 17 kDa essential light chains (ELCs) that stabilize the heavy chain structure (FIG. a). 


Domain structure of NM II. 
a The subunit and domain structure of non-muscle myosin II (NM II), which forms a dimer through interactions between the α-helical coiled-coil rod domains. The globular head domain contains the
actin-binding regions and the enzymatic Mg2+-ATPase motor domains. The essential light chains (ELCs) and the regulatory light chains (RLCs) bind to the heavy chains at the lever arms that link the head and rod domains. In the absence of RLC phosphorylation, NM II forms a compact molecule through a head to tail interaction. This results in an assemblyincompetent form (10S; left) that is unable to associate with other NM II dimers. On RLC phosphorylation, the 10S structure unfolds and becomes an assembly-competent form (6S). S-1 is a fragment of NM II that contains the motor domain and neck but lacks the rod domain and is unable to dimerize. Heavy meromyosin (HMM) is a fragment that contains the motor domain, neck and enough of the rod to effect dimerization. 
b NM II molecules assemble into bipolar filaments through interactions between their rod domains. These filaments bind to actin through their head domains and the ATPase activity of the head enables a conformational change that moves actin filaments in an anti-parallel manner. Bipolar myosin filaments link actin filaments together in thick bundles that form cellular structures such as stress fibres.

Although these myosins are referred to as ‘non-muscle’ myosin IIs to distinguish them from their muscle counterparts, they are also present in muscle cells, where they have distinct functions during skeletal muscle development and differentiation, as well as in the maintenance of tension in smooth muscle. NM II has a fundamental role in processes that require cellular reshaping and movement, such as cell adhesion, cell migration, and cell division. NM II can use its actin cross-linking and contractile functions, which are regulated by phosphorylation and the ability of NM II to form filaments, to regulate the actin cytoskeleton. 

NM II in cell migration, adhesion, and polarity
NM II is an important regulator of adhesion and polarity in cell migration. These processes involve the dynamic remodeling of the actin cytoskeleton and the interaction of the cell with its environment. Each of the NM II isoforms affects these processes differently. NM II regulates protrusion and cell migration. In migrating cells, actin organizes into several distinct structures and its polymerization in cellular protrusions drives cell migration. Protrusions generally contain two actin-based structures: the lamellipodium and the lamellum. Different classes of regulatory molecules organize actin in these two structures. The actin nucleator Arp2/3 generates the lamellipodium.



Multiple roles of NM II in cell migration. 
a | A polarized, migrating fibroblast.b Areas of the cell in which non-muscle myosin II (NM II) has an active role are boxed and expanded in parts b–d. 
b | NM II regulates retrograde flow in the lamellum and promotes adhesion maturation, thereby limiting protrusion. Nascent adhesions form in the lamellipodium, in which dendritic actin branching mediated by the Arp2/3 complex also occurs. At the lamellipodium– lamellum interface, actin is depolymerized or bundled and adhesions disassemble or mature. A schematic of adhesions maturing in the lamellum is also shown. NM II localizes to actin bundles contacting growing adhesions, forming a striated pattern with α-actinin. In other cells, such as in neuronal growth cones, NM II may have a more direct role controlling retrograde flow in the peripheral zone. 
c | NM II participates in adhesion disassembly at the rear of the cell. NM IIA-mediated contraction, calpain-dependent cleavage of adhesion components and microtubule targeting coordinately induce adhesion disassembly. 
d | NM II has a role in nuclear positioning and orienting the microtubule-organizing centre (MTOC) and Golgi, which are important for cell polarization. NM II is thought to act in concert with the CDC42–partitioning defective 3 (PAR3) or PAR6–protein kinase Cζ (PKCζ) –glycogen synthase kinase 3 (GSK3) pathway to polarize the cell. Myotonic dystrophy kinase-related CDC42-binding kinase (MRCK; also known as CDC42BP) activates NM II and regulates its effect on nucleus repositioning. APC, adenomatous polyposis coli; DIAPH1, diaphanous 1; EB1, end binding protein 1 (also known as MAPRE1).


The lamellipodium and the lamellum are kinetically different: the lamelli podium is distinguished by a fast retrograde flow of actin, whereas the lamellum exhibits slower retrograde flow. The convergent zone between the two is characterized by active depolymeriz ation of the dendritic network and the reorganization of actin (FIG. b).

Adhesive signaling in NM II activation. 
NM II influences adhesive signaling through clustering and/or conformational changes, but adhesive signaling also controls NM II activation


NM II in integrin-mediated adhesion. 
Integrins that are bound to the extracellular matrix (ECM) are linked to the actin cytoskeleton through an actin linkage that is formed by multiple molecules, including talin, vinculin and α-actinin. Kinases such as focal adhesion kinase (FAK) and Src, and adaptors such as paxillin, are also recruited and trigger the downstream activation of Rho GTPases such as Rac through adaptor and activating proteins. Representative pathways and associations are shown, including the activation of Rac through paxillin by the CRK-associated substrate (p130CAS; also known as BCAR1)– CRK–dedicator of cytokinesis 1 (DOCK1; also known as DOCK180) and G protein-coupled receptor kinase interacting ArfGAP (GIT)–β−Pix (also known as ARHGEF7) pathways. Activated Rac induces actin polymerization through the Arp2/3 complex, which can also interact with some of the molecules of the actin linkage, such as
vinculin and FAK. Rac is also thought to locally inhibit NM II activation. The activation of RhoGEFs by integrins, and the subsequent activation of RHOA and Rho-associated, coiled coil-containing kinase (ROCK), activates NM II. ROCK activates NM II directly by phosphorylating the regulatory light chains (RLCs) or by inactivating myosin phosphatase, which in turn promotes RLC dephosphorylation. The pathways are spatially and temporally regulated. Additionally, the activation and inactivation of NM II itself affects adhesive signaling by triggering conformational changes in the mechanoresponsive molecules shown (pink boxes), which induces the clustering of the indicated adhesion proteins (blue boxes) by reinforcing or weakening the linkage of the adhesion and the actin cytoskeleton. AM, adaptor module; MYPT1, myosin phosphatase-targeting subunit 1 (also known as PPP1R12A); PP1, protein phosphatase 1.

Other signalling pathways activated by adhesion have the opposite effect and promote NM II activation through RHoA. Rac is activated by signals generated in small adhesions close to the leading edge that actively undergo turnover and reassemble. RHoA activation mediates actin filament formation and adhesion maturation as the protrusion moves away and the adhesions become more central. This induces further activation of NM II and the coalescence of thick actomyosin bundles. During adhesion maturation, Rac signaling decreases, and RHoA-mediated activation of NM II increases, which results in enhanced actomyosin bundling. NM II is a dual regulator of protrusion, through its effects on actin retrograde flow and adhesion-generated signaling. Increased activation of NM II results in large actin bundles and stable adhesions, decreased signaling to Rac, and decreased protrusion. Lower levels of active NM II result in less actin bundling and increased protrusion. This can explain, in part, the underlying differences in migration among different cell types. specifically, highly migratory cells such as leukocytes do not exhibit large adhesions, probably reflecting low levels of NM II activation or an intrinsic inability to rearrange their actin cytoskeleton into large bundles, whereas slow-moving cells such as fibroblasts have adhesions that tend to mature into large, elongated structures as a result of NM II activation and robust actin bundling. NM II activation promotes adhesion maturation through its actin-bundling and contractile activities. Cycles of Rac and Rho activation inactivate and activate NM II, resulting in protrusion and actin-bundling, respectively. The role of NM II in adhesion depends on its ability to exert force on adhesions, even though it is not physically present in the adhesions but attaches to the actin bundles with which adhesions are associated22. Thus, NM II influences adhesion from a distance.

NM II in cell–cell adhesion and morphogenesis. NM II is not only pivotal in controlling integrin-mediated adhesion and migration, it also regulates epithelial cell adhesion, polarization and morphogenesis. Well-defined cell–cell junctions are a key feature of epithelial sheets and represent a different type of adhesive structure that is controlled by NM II. Although these cell–cell junctions use cadherins as the main adhesion receptors, they contain scaffolds and signalling intermediates analogous to those found in integrin-mediated complexes. In aggregates, epithelial cells have apical and basolateral regions as well as integrin-based adhesions to the extracellular matrix (ECM) at the basal surface (FIG. below).


Roles of NM II in epithelial cell polarization. 
The different roles of non-muscle myosin II (NM II) in epithelial cell polarization. NM II is involved in apical constriction (step 1), which leads to important morphogenic movements such as dorsal closure (closure of the epidermis over the amnioserosa during embryogenesis) in Drosophila melanogaster. In addition, NM II regulates nuclear positioning (step 2), in a similar manner to how it does this in fibroblasts. NM II and RHOA signaling also stabilize cell–cell contacts by reinforcing them through actin cross-linking (known as contact compaction; step 3). The initial contacts are formed as a result of Rac-driven actin polymerization, but NM IIA is required for contact formation and reinforcement and cadherin clustering. NM II also mediates crosstalk between homophilic cadherin contact-initiated signaling and extracellular matrix (ECM) remodeling triggered by integrin activation and clustering (step 4).

Epithelial cell sheets can move as multicellular cohorts, with the leading cells showing protrusions and the trailing cells retracting, or they can detach from the sheet and move away as single cells. In the free edges of the sheet, the migrating cells undergo a polarity switch from apicobasal to front–back. NM II controls the formation and stability of the cell-cell junctions. NM IIA is required for cadherin clustering

Signaling in Cell Differentiation and Morphogenesis [url=2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3367549/]2[/url]
Cells in the developing embryo are in constant communication with their neighbors, and the molecules they use to send and receive signals are essential for normal embryogenesis. Several intracellular signaling pathways have been identified, some of which are activated in response to secreted growth factors. In cases where the secreted factors form a concentration gradient and cell fate is specified as a function of growth factor concentration, these molecules are referred to as morphogens. Examples include the

sonic hedgehog (SHH),
wingless (WNT),
retinoic acid (RA),
bone morphogenetic protein (BMP),
fibroblast growth factor (FGF) 1




1. https://sci-hub.ren/https://www.nature.com/articles/nrm2786
2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3367549/

2.Cell fate determination and differentiation ( phenotype, or what cell type each one will become )

1. Cell fate determination depends on various kinds of codified information, communication and feedback systems, signaling, and bar-code marking.  These information systems prescribe, drive, direct, operate, control, and induce reacting to stimuli, provide patterning cues, regulation, determine and permit the making of decisions based on memory, control transcription, remodel chromatin structure and state, control cell-cell interactions, differential gene expression, regulate which genes are transcribed in a cell ( which genes are turned on and off ), influence the arrangement of different cell types during embryological development, give cues to cleavage patterns, create asymmetry from homogeneity, induce concentration gradients, cell positioning, and many more. 
2. These are not simply chemical reactions, but actions directed by prescribed, instructional complex information input.  This orchestration depends on a network of logic interactions programmed into the DNA sequence, epigenetic information, and signaling networks that amount essentially to a hardwired biological computational device. Animal forms depend on tightly integrated networks of genes, proteins, and other molecules to regulate their development.
3. Neither proponents of "evo-devo," nor proponents of other proposed materialistic theories of evolution, have identified a mutational mechanism capable of generating anything even remotely resembling a complex integrated circuit. In our experience, the functional integration of parts in complex systems generally—are known to be produced by intelligent agents—specifically, by engineers. Intelligence is the only known cause of such effects. Developing animals employ a form of integrated circuitry, and certainly one manifesting a tightly and functionally integrated system of parts and subsystems, and intelligence is the only known cause of these features. Once established, the complexity of integrated circuits and signaling networks makes them stubbornly resistant to mutational change. Disarming any one of these hierarchically structured systems produces some abnormality in expression.

Answering the questions about how cells, tissues, and organisms form, precedes the question of how they can eventually diversify, evolve, change and morph from one species to another through a macroevolutionary primary speciation transition zone, where novel organismal features arise, like wings, eyes, ears, legs, arms, and so forth. The fact is, that science is still FAR from being able to answer that question in an exhaustive manner. One of the crucial questions is cell fate determination and differentiation, tissue formation, and what, what cell phenotype type each cell will become. Many questions have been provided, but many open questions remain. 

Within an embryo, several processes play out at the cellular and tissue level to create an organism. These processes include cell proliferation, differentiation, and cellular movement 7

Cell determination starts early and progressively narrows the options as the cell steps through a programmed series of intermediate states—guided at each step by its genome, its history, and its interactions with neighbors. The process reaches its limit when a cell undergoes terminal differentiation to form one of the highly specialized cell types of the adult body. Although there are cell types in the adult that retain some degree of pluripotency, their range of options is generally narrow.

Cell determination depends on: 
1. Cell-Cell communication and transcriptional control through up to eleven different signaling pathways
2. Cell-cell adhesion and cell signaling; hundreds of human genes encode signal proteins, cell-surface receptors, cell adhesion proteins, or ion channels that are either not present in yeast or present in much smaller numbers.
3. Transcription regulation and chromatin structure: more than 1000 human genes encode transcription regulators, but only about 250 yeast genes do so. The development of animals is dominated by cell-cell interactions and by differential gene expression.
4. Noncoding microRNAs (miRNAs); there are at least 500 of these in humans. Along with the regulatory proteins, they play a significant part in controlling gene expression during animal development, but the full extent of their importance is still unclear.
5. Transcription factors — proteins that regulate which genes are transcribed in a cell — appear to be essential to determining the pathway particular stem cells take as they differentiate.
6. Cell decision-making depending on cell memory Underlying the richness and astonishingly complex outcomes of development is cell memory. Both the genes a cell expresses and the way it behaves depend on the cell’s past, as well as on its present circumstances.
7. The chromatin state — the packaging of DNA with both histone and non-histone proteins — has marked effects on gene expression and is believed to contribute to the establishment and the maintenance of cell identities. Many histone modifiers and chromatin remodelers have been implicated in stem cell pluripotency, cellular differentiation and development.
8. The DNA methylation code is like a barcode or marker, the methyl group indicates, for instance, which genes in the DNA are to be turned on.
9. The Sugar Code forms information-rich structures that influence the arrangement of different cell types during embryological development.
10. Factors secreted into its cytoplasm during cleavage.  (proteins, small regulatory RNAs and mRNA)  Early cleavage patterns appear to bias blastomeres to a particular fate
11. Positive feedback can create asymmetry from homogeneity. In cases where the external or stimuli that would cause asymmetry are very weak or disorganized, through positive feedback the system can spontaneously pattern itself.
12. Concentration-gradients of morphogens: Cell-extrinsic process that relies on cues and interactions between cells or from concentration-gradients of morphogens.
13. Positional value: cell specification occurs based on where within the embryo the cell is positioned. Position within the morula ( early-stage embryo consisting of 16 cells) being the most significant contributor to eventual cell fate decisions.
14. The primitive endoderm (PE)  is an extra-embryonic cell type whose descendants provide patterning cues and nutrient supplies to the developing embryo. 
15. Lateral inhibition. Very often mediated by exchange of signals at cell– cell contacts via the Notch signaling pathway, driving cell diversification by enabling individual cells that express one set of genes to direct their immediate neighbors to express a different set.
16. Reaction-diffusion systems. A substance A (a shortrange activator) may stimulate its own production in the cells that contain it and in their immediate neighbors, while also causing these cells to produce a signal I (a long-range inhibitor) that diffuses widely and inhibits the production of A in cells farther away. If the cells all start the same, but one group gains a slight advantage by making a little more A than the rest, the asymmetry can be self-amplifying.
17. Asymmetric cell division, in which some important molecule or molecules are distributed unequally between the two daughters. This asymmetric inheritance ensures that the two daughter cells develop differently. 
18. Sequential induction. It is chiefly through sequential inductions that the body plan of a developing animal, after being first roughed out in miniature, becomes elaborated with finer and finer details as development proceeds.
19. Master transcription regulators exert their powerful differentiation-inducing activity by binding to many different regulatory sites in the genome and thereby controlling the expression of large numbers of downstream target genes.
20. A mechanism based on the size of embryonic cells helps to determine the type of mature tissues they will eventually produce. 8 

Modes of specification

There are three general ways a cell can become specified for a particular fate; they are autonomous specification, conditional specification and syncytial specification.^[16]

Autonomous specification

This type of specification results from cell-intrinsic properties; it gives rise to mosaic development. The cell-intrinsic properties arise from a cleavage of a cell with asymmetrically expressed maternal cytoplasmic determinants (proteins, small regulatory RNAs and mRNA). Thus, the fate of the cell depends on factors secreted into its cytoplasm during cleavage. 

Positive feedback can create asymmetry from homogeneity. In cases where the external or stimuli that would cause asymmetry are very weak or disorganized, through positive feedback the system can spontaneously pattern itself. Once the feedback has begun, any small initial signaling is magnified and thus produces an effective patterning mechanism.^[19] This is normally what occurs in the case of lateral inhibition in which neighboring cells induce specification via inhibitory or inducing signals (see Notch signaling). This kind of positive feedback at the single cell level and tissue level is responsible for symmetry breaking, which is an all-or-none process whereas once the symmetry is broken, the cells involved become very different. Symmetry breaking leads to a bistable or multistable system where the cell or cells involved are determined for different cell fates. The determined cells continue on their particular fate even after the initial stimulatory/inhibitory signal is gone, giving the cells a memory of the signal.^[19]

Conditional specification

In contrast to the autonomous specification, this type of specification is a cell-extrinsic process that relies on cues and interactions between cells or from concentration-gradients of morphogens. Inductive interactions between neighboring cells is the most common mode of tissue patterning. In this mechanism, one or two cells from a group of cells with the same developmental potential are exposed to a signal (morphogen) from outside the group. Only the cells exposed to the signal are induced to follow a different developmental pathway, leaving the rest of the equivalence group unchanged. Another mechanism that determines the cell fate is regional determination (see Regional specification). As implied by the name, this specification occurs based on where within the embryo the cell is positioned, it is also known as positional value.^[20] This was first observed when mesoderm was taken from the prospective thigh region of a chick embryo, was grafted onto the wing region and did not transform to wing tissue, but instead into toe tissue.^[21]

Syncytial specification

This type of a specification is a hybrid of the autonomous and conditional that occurs in insects. This method involves the action of morphogen gradients within the syncytium. As there are no cell boundaries in the syncytium, these morphogens can influence nuclei in a concentration-dependent manner.

At first glance, one would no more expect the worm, the flea, the eagle, and the giant squid all to be generated by the same developmental mechanisms than one would suppose that the same methods were used to make a shoe and an airplane. Remarkably, however, research in the past 30 years has revealed that much of the basic machinery of development is essentially the same in all animals—not just in all vertebrates, but in all the major phyla of invertebrates too. Recognizably similar, related molecules define the specialized animal cell types, mark the differences between body regions, and help create the animal body pattern. Homologous proteins are often functionally interchangeable between very different species. Thus, a human protein produced artificially in a fly, for example, can perform the same function as the fly’s own version of that protein.

The shared anatomical features of animals develop through conserved mechanisms. After fertilization, the zygote usually divides rapidly, or cleaves, to form many smaller cells; during this cleavage, the embryo, which cannot yet feed, does not grow. This phase of development is initially driven and controlled entirely by the material deposited in the egg by the mother. The embryonic genome remains inactive until a point is reached when maternal mRNAs and proteins rather abruptly begin to be degraded. The embryo’s genome is activated, and the cells cohere to form a blastula—typically a solid or a hollow fluid-filled ball of cells. Complex cell rearrangements called gastrulation (from the Greek “gaster,” meaning “belly”) then transform the blastula into a multilayered structure containing a rudimentary internal gut


The early stages of development, as exemplified by a frog.
(A) A fertilized egg divides to produce a blastula—a sheet of epithelial cells often surrounding a cavity. During gastrulation, some of the cells tuck into the interior to form the mesoderm (green) and endoderm (yellow). Ectodermal cells (blue) remain on the outside. 
(B) A cross-section through the trunk of an amphibian embryo shows the basic animal body plan, with a sheet of ectoderm on the outside, a tube of endoderm on the inside, and mesoderm sandwiched between them. The endoderm forms the epithelial lining of the gut, from the mouth to the anus. It gives rise not only to the pharynx, esophagus, stomach, and intestines, but also to many associated structures. The salivary glands, liver, pancreas, trachea, and lungs, for example, all develop from the wall of the digestive tract and grow to become systems of branching tubes that open into the gut or pharynx. The endoderm forms only the epithelial components of these structures— the lining of the gut and the secretory cells of the pancreas, for example. The supporting muscular and fibrous elements arise from the mesoderm. The mesoderm gives rise to the connective tissues—at first, to the loose mesh of cells in the embryo known as mesenchyme, and ultimately to cartilage, bone, and fibrous tissue, including the dermis (the inner layer of the skin). The mesoderm also forms the muscles, the entire vascular system—including the heart, blood vessels, and blood cells—and the tubules, ducts, and supporting tissues of the kidneys and gonads. The notochord forms from the mesoderm and serves as the core of the future backbone and the source of signals that coordinate the development of surrounding tissues. The ectoderm will form the epidermis(the outer, epithelial layer of the skin) and epidermal appendages such as hair, sweat glands, and mammary glands. It will also give rise to the whole of the nervous system, central and peripheral, including not only neurons and glia but also the sensory cells of the nose, the ear, the eye, and other sense organs.

Some cells of the blastula remain external, constituting the ectoderm, which will give rise to the epidermis and the nervous system; other cells invaginate, forming the endoderm, which will give rise to the gut tube and its appendages, such as lung, pancreas, and liver. Another group of cells moves into the space between ectoderm and endoderm and forms the mesoderm, which will give rise to muscles, connective tissues, blood, kidney, and various other components. Further cell movements and accompanying cell differentiations create and refine the embryo’s architecture. The ectoderm, mesoderm, and endoderm formed during gastrulation constitute the three germ layers of the early embryo. Many later developmental transformations will produce the elaborately structured organs. But the basic body plan and axes set up in miniature during gastrulation are preserved into adult life, when the organism may be billions of times larger.

Concomitant with the refinement of the body plan, the individual cells become more and more restricted in their developmental potential. During the blastula stages, cells are often totipotent or pluripotent—they have the potential to give rise to all or almost all of the cell types of the adult body. The pluripotency is lost as gastrulation proceeds: a cell located in the endodermal germ layer, for example, can give rise to the cell types that will line the gut or form gut-derived organs such as the liver or pancreas, but it no longer has the potential to form mesoderm-derived structures such as skeleton, heart, or kidney. Such a cell is said to be determined for an endodermal fate. Thus, cell determination starts early and progressively narrows the options as the cell steps through a programmed series of intermediate states—guided at each step by its genome, its history, and its interactions with neighbors. The process reaches its limit when a cell undergoes terminal differentiation to form one of the highly specialized cell types of the adult body. Although there are cell types in the adult that retain some degree of pluripotency, their range of options is generally narrow.



The lineage from blastomere to differentiated cell type.
As development proceeds, cells become more and more specialized. Blastomeres have the potential to give rise to most or all cell types. Under the influence of signalling molecules and gene regulatory factors, cells acquire more restricted fates until they differentiate into highly specialized cell types, such as the pancreatic β-islet cells that secrete the hormone insulin.

Genes involved in Cell-Cell communication and transcriptional control are especially important for animal development
What are the genes that animals share with one another but not with other kingdoms of life? These would be expected to include genes required specifically for animal development but not needed for unicellular existence. Comparison of animal genomes with the genome of budding yeast—a unicellular eukaryote— suggests that three classes of genes are especially important for multicellular organization.

The first class includes genes that encode proteins used for cell-cell adhesion and cell signaling; hundreds of human genes encode signal proteins, cell-surface receptors, cell adhesion proteins, or ion channels that are either not present in yeast or present in much smaller numbers.

The second class includes genes encoding proteins that regulate transcription and chromatin structure: more than 1000 human genes encode transcription regulators, but only about 250 yeast genes do so. The development of animals is dominated by cell–cell interactions and by differential gene expression.

The third class of noncoding RNAs has a more uncertain status: it includes genes that encode microRNAs (miRNAs); there are at least 500 of these in humans. Along with the regulatory proteins, they play a significant part in controlling gene expression during animal development, but the full extent of their importance is still unclear.

The loss of individual miRNA genes in C. elegans, where their functions have been well studied, rarely leads to obvious phenotypes, suggesting that the roles of miRNAs during animal development are often subtle, serving to fine-tune the developmental machinery rather than to form its core structures.

Each gene in a multicellular organism is associated with many thousands of nucleotides of noncoding DNA that contains regulatory elements. These regulatory elements determine when, where, and how strongly the gene is to be expressed, according to the transcription regulators and chromatin structures that are present in the particular cell.

Genetically identical human cells are classified by their distinct behaviors into cell types, implying that nongenetic factors—including chromatin organization—contribute to their distinctive gene expression patterns. 1  Being stably heritable through cell division, both chromatin organization and the unique pattern of gene expression are therefore epigenetic. In higher organisms, all cells share the same genome, but every cell expresses only a limited and specific set of genes that defines the cell type. During cell division, not only the genome but also the cell type is inherited by the daughter cells. This intriguing phenomenon is achieved by a variety of processes that have been collectively termed epigenetics. governed by extremely rich and exquisitely multiscale physical mechanisms. 2  These include chemical modifications of DNA and histone proteins, and the physics that govern the three-dimensional organization of the genome in cell nuclei. Cells use many different physical principles, electrostatic and mechanical properties related to chemical modifications of DNA and histones. A limited set of physical rules ( instructional information ) plays a key role in cell differentiation.

During development, the determination of the cell type (cell fate) depends on DNA methylation which is a key control parameter of this process: genes that are specific for the desired tissue are kept unmethylated, whereas the others are methylated. Moreover, patterns of DNA methylation are faithfully propagated throughout successive cell divisions. The gene body DNA methylation codes are universal similar to the universality of the genetic code and 

should consequently be considered as part of the inheritance system. 3  Living organisms incorporate complex interaction between genes and epigenetic factors and the environment which shapes the organismal form that develops and adapts over time.

For a complete understanding of biological processes such as development and adaptation, it is necessary to understand as many integrative elements of biological systems as possible. Epigenetic information can be stored in a multitude of bearers such as histone modifications, non-coding RNA, the topology of the nucleus, and methylation of DNA.

Transcription factors — proteins that regulate which genes are transcribed in a cell — appear to be essential to determining the pathway particular stem cells take as they differentiate. 4

Some Transcription Regulators Can Activate a Program That Defines a Cell Type or Creates an Entire Organ
There are genes whose products act as triggers for the development of a specific cell type or even a specific organ, initiating, and coordinating the whole complex program of gene expression that is required. An example is the MyoD/myogenin family of transcription regulators. These proteins drive cells to differentiate into muscle, expressing muscle-specific actins and myosins and all the other specialized cytoskeletal, metabolic, and membrane proteins that a muscle cell needs. Analogously, members of the Achaete/Scute family of transcription regulators drive cells to become neural progenitors. In both these examples, the proteins belong to the basic helix–loop–helix (bHLH) class of transcription regulators, and the same is true for many of the other proteins that induce the differentiation of particular cell types. These master transcription regulators exert their powerful differentiation-inducing activity by binding to many different regulatory sites in the genome and thereby controlling the expression of large numbers of downstream target genes. In one well-studied case, that of an Achaete/Scute family member called Atonal homolog 1 (Atoh1), the number of direct target genes in the mouse genome is more than 600. It is important to note, however, that even such powerful drivers of cell differentiation can have radically different effects according to the context and history of the cells in which they act: Atoh1, for example, drives the differentiation of certain classes of neurons in the brain, of sensory hair cells in the inner ear, and of secretory cells in the lining of the gut. Other genes encoding transcription regulators can drive the formation and assembly of the multiple cell types that constitute an entire organ. A famous example is the transcription regulator Eyeless. When it is artificially expressed in a patch of cells in the leg precursors of Drosophila, a well-organized eye-like organ develops on the leg, with the various eye cell types correctly arranged; conversely, loss of the Eyeless gene results in flies that lack eyes. Moreover, loss of the Eyeless homolog Pax6 in vertebrates likewise leads to loss of eye structures. Similar organ-selector proteins are known for foregut, heart, pancreas, and other organs. They are all master transcription regulators that directly regulate hundreds of target genes, the products of which then specify and construct the different elements of the appropriate organ. However, as in the example of Atoh1, they usually exert their specific effect only in combination with the right partners, which are only expressed in cells that were appropriately primed during their earlier development.

Cell Memory Underlies Cell Decision-Making
Underlying the richness and astonishingly complex outcomes of development is cell memory. Both the genes a cell expresses and the way it behaves depend on the cell’s past, as well as on its present circumstances. The cells of our
body—the muscle cells, the neurons, the skin cells, the gut cells, and so on—maintain their specialized characters largely because they retain a record of the extracellular signals their ancestors received during development, rather than because they continually receive such instructions from their surroundings. Despite their radically different phenotypes, they retain the same complete genome that was present in the zygote; their differences arise instead from differential gene expression. 

Several Model Organisms Have Been Crucial for Understanding Development
The differences between species are usually more striking to our human eye than the similarities. But at the level of the underlying molecular mechanisms and the macromolecules that mediate them, the reverse is true: the similarities among all animals are profound and extensive. All animals have retained unmistakably similar sets of genes and proteins that are responsible for generating their body plans and for forming their specialized cells and organs. This astonishing degree of evolutionary conservation ( non-evolution) was discovered not by broad surveys of animal diversity, but through intensive study of a small number of representative species. For animal developmental biology, the most important have been the fly Drosophila melanogaster, the frog Xenopus laevis, the roundworm Caenorhabditis elegans, the mouse Mus musculus, and the zebrafish Danio rerio. 

Chromatin modifiers and remodelers: regulators of cellular differentiation
Nearly all cells of an organism share the same genome but show different phenotypes and carry out diverse functions. Individual cell types, which are characterized by distinct gene expression patterns, are generated during development and are then stably maintained. The chromatin state — the packaging of DNA with both histone and non-histone proteins — has marked effects on gene expression and is believed to contribute to the establishment and the maintenance of cell identities. Indeed, developmental transitions are accompanied by dynamic changes in chromatin states. The assembly and the compaction of chromatin are regulated by multiple mechanisms, including DNA modifications (for example, cytosine methylation and cytosine hydroxymethylation), post-translational modifications (PTMs) of histones (for example, phosphorylation, acetylation, methylation, and ubiquitylation), the incorporation of histone variants (for example, H2A.Z and H3.3), ATP-dependent chromatin remodeling and non-coding RNA (ncRNA)-mediated pathways. PTMs of histones may either directly affect chromatin compaction and assembly or serve as binding sites for effector proteins, including other chromatin-modifying or chromatin-remodeling complexes, and ultimately influence transcription initiation and/or elongation.

Many histone modifiers and chromatin remodelers have been implicated in stem cell pluripotency, cellular differentiation and development. 5

The make and maintenance of specialized Cell types
The organization of DNA in an intricate, dynamic nucleoprotein assembly termed chromatin is accomplished by a remarkable feat of biological engineering. Although all cells must be able to switch genes on and off in response to changes in their environments, the cells of multicellular organisms have this capacity to an extreme degree. Transcription factors are positioned at multiple sites along long stretches of DNA and that these proteins bring into play coactivators and co-repressors. The Drosophila Even-skipped (Eve) gene expression plays an important part in the development of the Drosophila embryo. If this gene is inactivated by mutation, many parts of the embryo fail to form, and the embryo dies early in development. This cytoplasm contains a mixture of transcription factors that are distributed unevenly along the length of the embryo, thus providing positional information that distinguishes one part of the embryo from another. Although the nuclei are initially identical, they rapidly begin to express different genes because they are exposed to different transcription regulators.

Molecular genetic mechanisms that create and maintain specialized cell types
Although all cells must be able to switch genes on and off in response to changes in their environments, the cells of multicellular organisms have this capacity to an extreme degree. In particular, once a cell in a multicellular organism becomes committed to differentiate into a specific cell type, the cell maintains this choice through many subsequent cell generations, which means that it remembers the changes in gene expression involved in the choice. This phenomenon of cell memory is a prerequisite for the creation of organized tissues and for the maintenance of stably differentiated cell types.

Complex genetic switches that regulate Drosophila development are built up from smaller molecules
Drosophila Even-skipped (Eve) gene expression plays an important part in the development of the Drosophila embryo. If this gene is inactivated by mutation, many parts of the embryo fail to form, and the embryo dies
early in development. At the stage of development when Eve begins to be expressed, the embryo is a single giant cell containing multiple nuclei in a common cytoplasm. This cytoplasm contains a mixture of transcription
factors that are distributed unevenly along the length of the embryo, thus providing positional information that distinguishes one part of the embryo from another.



The nonuniform distribution of transcription regulators in an early Drosophila embryo. 
At this stage, the embryo is a syncytium; that is, multiple nuclei are contained in a common cytoplasm. Although not shown in these drawings, all of these proteins are concentrated in the nuclei.

Although the nuclei are initially identical, they rapidly begin to express different genes because they are exposed to different transcription factors. For example, the nuclei near the anterior end of the developing embryo are exposed to a set of transcription factors that is distinct from the set that influences nuclei at the middle or at the posterior end of the embryo. The regulatory DNA sequences that control the Eve gene “read” the concentrations of transcription factors at each position along the length of the embryo, and they cause the Eve gene to be expressed in seven precisely positioned stripes, each initially five to six nuclei wide. How is this remarkable feat of information processing carried out? Although there is still much to learn, several general principles have emerged from studies of Eve and other genes that are similarly regulated.


The blue stripes (top and bottom panels) are eve expression, the red in the center (bottom panel) is Kruppel expression, and each green dot represents a single nucleus. 1 The seven stripes of the protein encoded by the Evenskipped (Eve) gene in a developing Drosophila embryo. At this stage in development, the egg contains approximately 4000 nuclei. The Eve and Giant proteins are both located in the nuclei, and the Eve stripes are about four nuclei wide.

The regulatory region of the Eve gene is very large (approximately 20,000 nucleotide pairs). It is formed from a series of relatively simple regulatory modules, each of which contains multiple cis-regulatory sequences and is responsible for specifying a particular stripe of Eve expression along the embryo. 6


Lateral Inhibition Can Generate Patterns of Different Cell Types
Morphogen gradients, and other kinds of inductive signal, exploit an existing asymmetry in the embryo to create further asymmetries and differences between cells: already, at the outset, some cells are specialized to produce the morphogen and thereby impose a pattern on another class of cells that are sensitive to it. But what if there is no clear initial asymmetry? Can a regular pattern arise spontaneously within a set of cells that are initially all alike?
The answer is yes. The fundamental principle underlying such de novo pattern formation is positive feedback: cells can exchange signals in such a way that any small initial discrepancy between cells at different sites becomes self-amplifying, driving the cells toward different fates. This is most clearly illustrated in the phenomenon of lateral inhibition, a form of cell–cell interaction that forces close neighbors to become different and thereby generates fine-grained patterns of different cell types. Consider a pair of adjacent cells that start off in a similar state. Each of these cells can both produce and respond to a certain signal molecule X, with the added rule that the stronger the signal a cell receives, the weaker the signal it generates (Figure below).


Genesis of asymmetry through lateral inhibition and cell 1 cell 2 positive feedback. 
In this example, two cells interact, each producing a substance X that acts on the other cell to inhibit its production of X, an effect known as lateral inhibition. An increase of X in one of the cells leads to a positive feedback that tends to increase X in that cell still further, while decreasing X in its neighbor. This can create a runaway instability, making the two cells become radically different. Ultimately, the system comes to rest in one or the other of two opposite stable states. The final choice of state represents a form of memory: the small influence that initially directed the choice is no longer required to maintain it.

If one cell produces more X, the other is forced to produce less. This gives rise to a positive feedback loop that tends to amplify any initial difference between the two adjacent cells. Such a difference may arise from a bias imposed by some present or past external factor, or it may simply originate from spontaneous random fluctuations, or “noise”—an inevitable feature of the genetic control circuitry in cells (discussed in Chapter 7). In either case, lateral inhibition means that if cell 1 makes a little more of X, it will thereby cause cell 2 to make less; and because cell 2 makes less X, it delivers less inhibition to cell 1 and so allows the production of X in cell 1 to rise higher still; and so on, until a steady state is reached where cell 1 produces a lot of X and cell 2 produces very little. In the standard case, the signal molecule X acts in the receiving cell by regulating gene transcription, and the result is that the two cells are driven along different pathways of differentiation. In almost all tissues, a balanced mixture of different cell types is required. Lateral inhibition provides a common way to generate the mixture. Lateral inhibition is very often mediated by exchange of signals at cell– cell contacts via the Notch signaling pathway, driving cell diversification by enabling individual cells that express one set of genes to direct their immediate neighbors to express a different set.

Short-Range Activation and Long-Range Inhibition Can Generate Complex Cellular Patterns
Lateral inhibition mediated by the Notch pathway is not the only example of pattern generation through positive feedback: there are other ways in which, through the same basic principle, a system that starts off homogeneous and symmetrical can pattern itself spontaneously, even in the absence of an external morphogen. Positive feedback processes mediated by diffusible signal molecules can operate over broad arrays of cells to create many types of spatial patterns. Mechanisms of this sort are called reaction-diffusion systems. For example, a substance A (a shortrange activator) may stimulate its own production in the cells that contain it and in their immediate neighbors, while also causing these cells to produce a signal I (a long-range inhibitor) that diffuses widely and inhibits the production of A in cells farther away. If the cells all start the same, but one group gains a slight advantage by making a little more A than the rest, the asymmetry can be self-amplifying.


Pattern generation by a reaction-diffusion system. 
From (A) a uniform field of cells, (B) local positive feedback and (C) long-range inhibition can (D) generate patterns within the initially uniform field. The patterns can be complex, resembling the spots of a leopard (as shown) or the stripes of a zebra; or they can be simple, with creation of a single cluster of specialized cells that can, for example, go on to serve as the source of a morphogen gradient.

Such short-range activation combined with long-range inhibition can account for the formation of clusters of cells within an initially homogeneous tissue that become specialized as localized signaling centers.

Asymmetric Cell Division Can Also Generate Diversity
Cell diversification does not always depend on extracellular signals: in some cases, daughter cells are born different as a result of an asymmetric cell division, in which some important molecule or molecules are distributed unequally between the two daughters. This asymmetric inheritance ensures that the two daughter cells develop differently. 


Two ways of making sister cells different.

Asymmetric division is a common feature of early development, where the fertilized egg already has an internal pattern and cleavage of this large cell segregates different determinants into separate blastomeres. We shall see that asymmetric division also plays a part in some later developmental processes.

Initial Patterns Are Established in Small Fields of Cells and Refined by Sequential Induction as the Embryo Grows
The signals that organize the spatial pattern of cells in an embryo generally act over short distances and govern relatively simple choices. A morphogen, for example, typically acts over a distance of less than 1 mm—an effective range for diffusion —and directs choices between several developmental options for the cells on which it acts. Yet the organs that eventually develop are much larger and more complex than this. The cell proliferation that follows the initial specification accounts for the size increase, while the refinement of the initial pattern is explained by a series of local inductions plus other interactions that add successive levels of detail on an initially simple sketch. For example, as soon as two types of cells are present in a developing tissue, one of them can produce a signal that induces a subset of the neighboring cells to specialize in a third way. The third cell type can in turn signal back to the other two cell types nearby, generating a fourth and a fifth cell type, and so on.



Patterning by sequential induction. 
A series of inductive interactions can generate many types of cells, starting from only a few.

This strategy for generating a progressively more complicated pattern is called sequential induction. It is chiefly through sequential inductions that the body plan of a developing animal, after being first roughed out in miniature, becomes elaborated with finer and finer details as development proceeds.

Asymmetric Cell Divisions Make Sister Cells Different
Cell diversification does not always have to depend on extracellular signals: in some cases, sister cells are born different as a result of an asymmetric cell division, during which some significant set of molecules is divided unequally between them. This asymmetrically segregated molecule (or set of molecules) then acts as a determinant for one of the cell fates by directly or indirectly altering the pattern of gene expression within the daughter cell that receives it. We have already encountered the asymmetric segregation of molecules in the context of the early frog embryo: VegT RNA is localized in the vegetal region of the fertilized egg. Following cell division, only vegetal daughter cells will inherit VegT RNA. Asymmetric divisions often occur at the beginning of development, but they are also encountered at some later stages. As mentioned for the sensory bristle, they can set the scene for an exchange of Notch signals between the daughter cells, with the signaling occurring after the cells have become separate and reinforcing the differences between them. In the central nervous system, asymmetric divisions have a key role in generating the very large numbers of neurons and glial cells that are needed. A special class of cells becomes committed as neural precursors, but instead of differentiating directly as neurons or glial cells, these undergo a long series of asymmetric divisions through which a succession of additional neurons and glial cells are added to the population. The process is best understood in Drosophila, although there are many hints that something similar
occurs also in vertebrate neurogenesis. In the embryonic central nervous system of Drosophila, the nerve-cell precursors, or neuroblasts, are initially singled out from the neurogenic ectoderm by a typical lateral-inhibition mechanism that depends on Notch. Each neuroblast then divides repeatedly in an asymmetric fashion


Neuroblasts and asymmetric cell division in the central nervous system of a fly embryo.
The neuroblast originates as a specialized ectodermal cell. It is singled out by lateral inhibition and emerges from the basal (internal) face of the ectoderm. It then goes through repeated division cycles, dividing asymmetrically to generate a series of ganglion mother cells. Each ganglion mother cell divides just once to give a pair of differentiated daughters (typically a neuron plus a glial cell).

At each division, one daughter remains as a neuroblast, while the other, which is much smaller, become specialized as a ganglion mother cell. Each ganglion mother cell will divide only once, giving a pair of neurons, or a neuron plus a glial cell, or a pair of glial cells, with Notch-mediated interactions helping to drive the daughters along different paths. The neuroblast itself becomes smaller at each division, as it parcels out its substance into one ganglion mother cell after another. Eventually, typically after about 12 cycles, the process halts, presumably because the neuroblast becomes too small to pass the cell-size checkpoint in the cell-division cycle. Later, in the larva, neuroblast divisions resume, but now they are accompanied by cell growth, permitting the process to continue indefinitely and to generate the much larger numbers of neurons and glial cells required in the adult fly.


1. https://advances.sciencemag.org/content/6/12/eaax7798
2. https://sci-hub.ren/https://journals.aps.org/rmp/abstract/10.1103/RevModPhys.88.025002
3. https://www.nature.com/articles/s41598-018-37407-8
4. https://www.nature.com/scitable/topicpage/cell-differentiation-and-tissue-14046412/
5. https://www.nature.com/articles/nrg3607
6. https://ittakes30.wordpress.com/2010/06/30/eve-and-the-tree-of-knowledge/
7. https://en.wikipedia.org/wiki/Cell_fate_determination
8. https://www.quantamagazine.org/for-embryos-cells-size-can-determine-fate-20190812/

3.Cell size

Cell size is fundamental to cell physiology because it sets the scale of intracellular geometry, organelles, and biosynthetic processes.  Size is one of the most fundamental characteristics of life and has a large impact on animal and cell physiology. Thus, cell size is tightly associated with cell function and metabolism so that different types of cells vary enormously in size. Animal cells maintain size homeostasis through two distinct classes of mechanisms. The first mechanism class is size-dependent cell cycle progression so that larger cells transition through the cell cycle to divide earlier than smaller cells. The second class of mechanism is size-dependent adjustment of growth rate, in which cells closer to the target size grow more rapidly than smaller or larger cells. Here, growth rate refers to the mass accumulation rate per unit mass. We note that this form of homeostatic cell-level size control is distinct from the size ‘control’ phenomena where cells of different cell types or in different environmental conditions modulate their average size. Cell size homeostasis in proliferating animal cells is achieved through the coupling between cell size, growth rate, and cell cycle progression. Animal cells exhibit a variety of cell line-dependent behaviors (sizers, timers, and adders) and researchers have begun to unravel their molecular mechanisms. 3

Cells of a given type maintain a characteristic cell size to function efficiently in their ecological or organismal context. They achieve this through the regulation of growth rates or by actively sensing the size through geometric, external cue, and titration mechanisms ( cells can perform quantitative chemical analysis to determine the concentration of intracellular substances, like proteins etc). 1 Cells size varies greatly depending on cell type and species. Among eukaryotic cells, ∼1-mm frog oocytes are 1000 times larger in diameter than ∼1-μm phytoplankton, a billion-fold difference in volume. Even within an organism, cells of different types may be of very different sizes: human blood cells are tiny (<10 μm) compared with greater than1-m-long neurons. For each type of cell, efficient function depends on appropriate size. The function of cells in multicellular organisms strongly depends on their size.

Active regulation of cell-cycle progression in response to cell size requires that cells have a method to accurately measure their size. Cell size is not rigidly fixed and responds to external factors, particularly nutrient levels. This plasticity appears important for cell physiology and function under changing environments 2 Mechanisms of cell size control are complex, condition-specific, often redundant, and differ between organisms and cell types. Membrane potential resulting from gradients of monovalent inorganic ions such as Na+, K+, and Cl− together with concentrations of impermeant intracellular molecules are critical determinants of cell size for all cell types.  Na+K+2Cl− (NKCC) co-transporters have a role in hyper-osmotic cell volume regulation, they show that NKCC isoforms have functional selectivity, with only NKCC1 functioning in cell volume recovery.

Many influences determine cell sizes, such as input from the environment and signals from other cells. Cell sizes can be altered by signals from food particles or from molecules that are part of ordinary metabolic cycles. 4 Organs, too, have multiple techniques to maintain exact cell sizes, but these are not yet well understood. Even with differing environmental factors, they know what size the new cells should be. Cells produced from stem cells are not the size of the stem cell itself and can be ten times larger. The number of human cells, not the average cell size, makes one person larger than another. Even during rapid growth, organs maintain cell sizes.

For an individual cell, size is determined by activity in phases of the multi-staged reproduction cycle for new cells. When new cells emerge, only certain-sized cells can proceed to the next stage in the process. For example, cells measure protein production as they go through different phases of the cycle. These levels of proteins become signals to tell a too small cell that it has to stay in a particular phase longer to catch up in size. Cellular size is often increased between stages of DNA copying and the separation of the two DNA strands, for instance. Sending secreted signal molecules from one cell to another is another technique used to alter cell size. These signals activate receptors on the second cell that signal internally to the nucleus to adjust the cell’s size. Signals include immune cytokine signals and growth factors (often proteins or hormones) that trigger cells to divide in order to produce new, smaller cells. Some signals increase growth and others decrease it. These factors have varied effects in different organs and are not yet well understood.

1. https://cshperspectives.cshlp.org/content/8/4/a019083.full
2. https://www.frontiersin.org/articles/10.3389/fcell.2017.00115/full
3. https://sci-hub.tw/https://www.sciencedirect.com/science/article/abs/pii/S0168952520300275
4. The secret language of Cells, M.D.Jon Lieff, page 27

4. Development timing

Developmental events unfold over minutes, hours, days, weeks, months, or even years, with each organism following its own strict timetable. The cascades of inductive interactions and transcriptional regulatory events take time, as signals are transmitted and transcription regulators are synthesized and then bind to DNA to activate or repress their target genes. Development can be compared with an orchestral performance. There are many players, and each must do the right thing at the right time; yet there is no leader or conductor to set the tempo and coordinate the timing of all the different events. Each developmental process must thus occur at an appropriate rate, tuned by evolution by an intelligent designer to fit with the timing of other processes in the embryo or in the environment. The control of timing is one of the most important problems in developmental biology, but also one of the least understood.

Molecular Lifetimes Play a Critical Part in Developmental Timing
Developmental processes are complex, but they are built up from simple steps. A first challenge is to understand the timing of these steps. How long does it take, for example, to switch the expression of a gene on or off? This is not like throwing a light switch: it involves delays. First, it takes time to make an mRNA molecule: the RNA polymerase must travel the length of the gene, the primary RNA transcript must be spliced and otherwise processed, and the resulting mRNA must be exported from the nucleus and delivered to the site where it will be translated. This adds up to what one might call the gestation time of the individual molecule. Second, it takes time for the individual mRNA molecules to accumulate to their fully effective concentration, this accumulation time is dictated by the average lifetime of the molecules—the longer they last, the higher their ultimate concentration, and the longer the time taken to attain it. Similar delays occur at the next step, where the mRNA is translated into protein: synthesis of each individual protein molecule involves a gestation delay, and attainment of an effective concentration of protein molecules involves an accumulation delay that depends on the protein’s lifetime. The time for the whole gene switching process is just the sum of the gestation delays and the accumulation delays (basically, the molecular lifetimes) for both the mRNA and the protein molecules. Somewhat counterintuitively, it is the combined length of these delays, rather than the rate of molecular synthesis (the number of molecules synthesized per second), that chiefly determines the switching time. The same additive principle applies to long cascades of gene switching, where gene A activates gene B, and gene B activates gene C, and so on. It also applies in other circumstances, such as in signaling pathways where one protein directly regulates the activation of the next. In all these cases, molecular lifetimes, along with gestation delays, play a key part in determining the pace of development. The lifetimes of mRNA and protein molecules are enormously variable, from a few minutes or hours to days or more, explaining much of the variation we see in the tempo of developmental events. Gene switching delays, however, are not the be-all and end-all of developmental timing. Development involves many other kinds of delay that contribute to timing. Chromatin structure takes time to remodel. Inductive signals take time to diffuse across a field of cells. Cells take time to move and rearrange themselves in space. Nevertheless, the timing of gene switching plays a fundamental part in developmental timing, as illustrated in an especially clear and striking way by a gene-expression oscillator that controls the segmentation of the vertebrate body axis.


Somite formation in the chick embryo. 
(A) A chick embryo at 40 hours of incubation. (B) How the temporal oscillation of gene expression in the presomitic mesoderm becomes converted into a spatial alternating pattern of gene expression in the formed somites. In the
posterior part of the presomitic mesoderm, each cell oscillates with a cycle time of 90 minutes. As cells mature and emerge from the presomitic region, their oscillation is gradually slowed down and finally brought to a halt, leaving them in a state that depends on the phase of the cycle they happen to be in at the critical moment. In this way, a temporal oscillation of gene expression traces out an alternating spatial pattern.

The somites form (as bilateral pairs) one after another, in a regular rhythm, starting in the region of the head and ending in the tail. Depending on the species, the final number of somites ranges from less than 40 (in a frog or a zebrafish) to more than 300 (in a snake). The posterior, most immature part of the mesodermal slab, called the presomitic mesoderm, supplies the required cells: as the cells proliferate, this mesoderm retreats tailward, extending the embryo (Figure B above). In the process, it deposits a trail of somites formed from cells that group together into blocks as they emerge from the anterior end of the presomitic region. The special character of the presomitic mesoderm is maintained by a combination of fibroblast growth factor (FGF) and Wnt signals, produced by a signaling center at the tail end of the embryo, and the range of these signals seems to define the length of the presomitic mesoderm. The somites emerge with clocklike timing, but what determines the rhythm of the process? In the posterior part of the presomitic mesoderm, the expression of certain genes oscillates in time. Snapshots of gene expression taken by fixing embryos for analysis at different times in the oscillation cycle reveal what is happening, and the oscillations can now also be observed in time-lapse movies of embryos containing fluorescent reporters of individual oscillating genes. One new somite pair is formed in each oscillation cycle, and, in mutants where the oscillations fail to occur, somite segmentation is disrupted: the cells may still break up, belatedly, into separate clusters, but they do so in a haphazard, irregular way. The gene-expression oscillator controlling regular segmentation is called the segmentation clock. The length of one complete oscillation cycle depends on the species: it is 30 minutes in a zebrafish, 90 minutes in a chick, 120 minutes in a mouse. As cells emerge from the presomitic mesoderm to form somites—in other words, as they escape from the influence of the FGF and Wnt signals—their oscillation stops. Some become arrested in one state, some in another, according to the phase of the oscillation cycle at the time they leave the presomitic region. In this way, the temporal oscillation of gene expression in the presomitic mesoderm leaves its trace in a spatially periodic pattern of gene expression in the maturing mesoderm; this in turn dictates how the tissue will break up into physically separate blocks, through effects on the pattern of cell–cell adhesion (see Figure B). How does the segmentation clock work? The first somite oscillator genes to be discovered were Hes genes, which are key components of the Notch signaling pathway. They are directly regulated by the activated form of Notch, and they code for inhibitory transcription regulators that inhibit the expression of other genes, including Delta. As well as regulating other genes, the products of Hes genes can directly regulate their own expression, creating a remarkably simple negative feedback loop. Autoregulation of certain specific Hes genes (depending on species) is thought to be the basic generator of the oscillations of the somite clock. Although the machinery has been modified in various ways in different species, the underlying principle seems to be conserved. When the key Hes gene is transcribed, the amount of Hes protein product builds up until it is sufficient to block Hes gene transcription; synthesis of the protein ceases; the protein then decays, permitting transcription to begin again; and so on, cyclically.


Delayed negative feedback giving rise to oscillating gene expression.
(A) A single gene, coding for a transcription regulator that inhibits its own expression, can behave as an oscillator. For oscillation to occur, there must be a delay (or several delays) in the feedback circuit, and the lifetimes of the mRNA and protein (which contribute to the delay) must be short compared with the total delay. The total delay determines the period of oscillation. It is thought that a feedback circuit like this, based on a pair of redundantly acting genes called Her1 and Her7 in the zebrafish—or their counterpart, Hes7, in the mouse—is the pacemaker of the segmentation clock governing somite formation. 
(B) The predicted oscillation of Her1 and Her7 mRNA and protein, computed using rough estimates of the feedback circuit parameters appropriate to this gene in the zebrafish. Concentrations are measured as numbers of molecules per cell. The predicted period is close to the observed period, which is 30 minutes per somite in the zebrafish (depending on temperature).

The period of oscillation, which determines the size of each somite, depends on the delay in the feedback loop. This equals the sum of the gestation delays and accumulation delays (that is, the molecular lifetimes) of the Hes mRNA and protein molecules, according to the additive principle. Mathematical modeling allows us to relate these basic molecular parameters to the cycle time of the segmentation clock: to a first approximation, the cycle period is simply equal to twice the total delay in the negative feedback loop, and thus twice the sum of the delays occurring at each step of the loop. The feedback loop is intracellular, and each cell in the presomitic mesoderm can generate oscillations on its own. But these oscillations at the single-cell level are somewhat erratic and imprecise, reflecting the fundamentally noisy, stochastic nature of the control of gene expression. A mechanism is needed to keep all the cells in the presomitic mesoderm that will form a particular somite oscillating in synchrony. This is achieved through cell–cell communication via the Notch signaling pathway, to which the Hes genes are coupled. The gene regulatory circuitry is such that in this context Notch signaling does not drive neighboring cells to be different, as in lateral inhibition, but does just the opposite: it keeps them in unison. In mutants where Notch signaling fails, including mutants defective in Delta or Notch itself, the cells drift out of synchrony and somite segmentation is again disrupted. This leads to gross deformity of the vertebral column—an extraordinary display of the consequences of the
noisy temporal control of gene expression at the single-cell level, writ large in the structure of the vertebrate body as a whole.

Intracellular Developmental Programs Can Help Determine the Time-Course of a Cell’s Development
Although signaling between cells plays an essential part in driving the progress of development, this does not mean that cells always need signals from other cells to prod them into changing their character as development proceeds. Some of these changes are intrinsic to the cell (like the ticking of the segmentation clock) and depend on intracellular developmental programs that can operate even when the cell is removed from its normal environment. The best-understood example is in the development of neural precursor cells, or neuroblasts, in the embryonic Drosophila central nervous system. These cells are initially singled out from the neurogenic ectoderm of the embryo by a typical lateral-inhibition mechanism that depends on Notch, and they then proceed through an entirely predictable series of asymmetric cell divisions to generate ganglion mother cells that divide to form neurons and glial cells. The neuroblast changes its internal state as it goes through its set program of divisions, generating different cell types with a reproducible sequence and timing. These successive changes in neuroblast specification occur through the sequential expression of specific transcription regulators. For example, most embryonic neuroblasts sequentially express the transcription regulators Hunchback, Krüppel, Pdm, and Cas in a fixed order

A Gene-Expression Oscillator Acts as a Clock to Control Vertebrate Segmentation
The main body axis of all vertebrates has a repetitive, periodic structure, seen in the series of vertebrae, ribs, and segmental muscles of the neck, trunk, and tail. These segmental structures originate from the mesoderm that lies as a long slab on either side of the embryonic midline. This slab becomes broken up into a regular repetitive series of separate blocks, or somites—cohesive groups of cells, separated by clefts

Cell division counting: How do cells know when to stop self-replicating?
Why is body growth in animals rapid in early life but then progressively slows, thus imposing a limit on adult body size? This growth deceleration in mammals is caused by potent suppression of cell proliferation in multiple tissues and is driven primarily by local, rather than systemic, mechanisms. This progressive decline in proliferation results from a genetic program that occurs in multiple organs and involves the down-regulation of a large set of growth-promoting genes. This program does not appear to be driven simply by time but rather depends on growth itself, suggesting that the limit on adult body size is imposed by a negative feedback loop. Different organs appear to use different types of information to precisely target their adult size. For example, skeletal and cardiac muscle growth is negatively regulated by myostatin, the concentration of which depends on muscle mass itself. Liver growth appears to be modulated by bile acid flux, a parameter that reflects organ function. In the pancreas, organ size appears to be limited by the initial number of progenitor cells, suggesting a mechanism based on cell-cycle counting. 

During the early development of Xenopus laevis, a counting mechanism is in place to ensure that the zygote divides exactly 12 times before cell division slows. 2

Cell size homeostasis: Metabolic control of growth and cell division March 2019
Joint regulation of growth rate and cell division rate determines cell size.  Animal cells achieve cell size homeostasis involving multiple signaling pathways converging at metabolic regulation of growth rate and cell cycle progression. While several models have been developed to explain cell size control, a comparison of the two predominant models shows that size homeostasis is dependent on the ability to adjust cellular growth rate based on cell size.3

Single-cell studies have highlighted the fact that despite substantial variability in growth rate at a single-cell level, this translates into stable cell size distribution and a more predictable average growth rate at the population level. Somewhat counterintuitively, comparison of the ‘adder’ and ‘sizer’ models suggests that proper maintenance of size homeostasis requires that larger cells grow slower than small cells in relative terms. How do cells sense their size to modulate their growth rate accordingly? The molecular mechanisms by which cells regulate their size remain poorly understood and are likely to be more complex than anticipated. We need to clarify how different signaling pathways contribute to cell size regulation, particularly through regulation of metabolism. For example, while manipulation of mTOR activity alters cell size, this kinase is not a key contributor to cell-size sensing in animal cells [14]. Instead, the p38 MAPK pathway reduces cell size variability while coordinating cell size and cell cycle progression [33]. Neither is mTOR activity involved in setting the cell size dependent mitochondrial activity, whereas the mevalonate/cholesterol pathway is involved [5]. How can these results be reconciled? Does the whole question of cell size control need to be subdivided into two or more processes? The role of CDK4 in regulating cell's target size also warrants further investigation 

Many Human Cells Have a Built-In Limitation on the Number of Times They Can Divide
Many human cells divide a limited number of times before they stop and undergo a permanent cell-cycle arrest. Fibroblasts taken from normal human tissue, for example, go through only about 25–50 population doublings when cultured in a standard mitogenic medium. Toward the end of this time, proliferation slows down and finally halts, and the cells enter a nondividing state from which they never recover. This phenomenon is called replicative cell senescence. Replicative cell senescence in human fibroblasts seems to be caused by changes in the structure of the telomeres, the repetitive DNA sequences and associated proteins at the ends of chromosomes. When a cell divides, telomeric DNA sequences are not replicated in the same manner as the rest of the genome but instead are synthesized by the enzyme telomerase. Telomerase also promotes the formation of protein cap structures that protect the chromosome ends. Because human fibroblasts, and many other human somatic cells, do not produce telomerase, their telomeres become shorter with every cell division, and their protective protein caps progressively deteriorate. Eventually, the exposed chromosome ends are sensed as DNA damage, which activates a p53-dependent cell-cycle arrest. 


How DNA damage arrests the cell cycle in G1. 
When DNA is damaged, various protein kinases are recruited to the site of damage and initiate a signaling pathway that causes cell-cycle arrest. The first kinase at the damage site is either ATM or ATR, depending on the type of damage. Additional protein kinases, called Chk1 and Chk2, are then recruited and activated, resulting in the phosphorylation of the transcription regulatory protein p53. Mdm2 normally binds to p53 and promotes its ubiquitylation and destruction in proteasomes. Phosphorylation of p53 blocks its binding to Mdm2; as a result, p53 accumulates to high levels and stimulates transcription of numerous genes, including the gene that encodes the CKI protein p21. The p21 binds and inactivates G1/S-Cdk and S-Cdk complexes, arresting the cell in G1. In some cases, DNA damage also induces either the phosphorylation of Mdm2 or a decrease in Mdm2 production, which causes a further increase in p53.

Rodent cells, by contrast, maintain telomerase activity when they proliferate in culture and therefore do not have such a telomere-dependent mechanism for limiting proliferation. The forced expression of telomerase in normal human fibroblasts, using genetic engineering techniques, blocks this form of senescence. Unfortunately, most cancer cells have regained the ability to produce telomerase and therefore maintain telomere function as they proliferate; as a result, they do not undergo replicative cell senescence.

Abnormal Proliferation Signals Cause Cell-Cycle Arrest or Apoptosis, Except in Cancer Cells
Many of the components of mitogenic signaling pathways are encoded by genes that were originally identified as cancer-promoting genes, because mutations in them contribute to the development of cancer. The mutation of a single amino acid in the small GTPase Ras, for example, causes the protein to become permanently overactive, leading to constant stimulation of Ras-dependent signaling pathways, even in the absence of mitogenic stimulation. Similarly, mutations that cause an overexpression of Myc stimulate excessive cell growth and proliferation and thereby promote the development of cancer. Surprisingly, however, when a hyperactivated form of Ras or Myc is experimentally overproduced in most normal cells, the result is not excessive proliferation but the opposite: the cells undergo either permanent cell-cycle arrest or apoptosis. The normal cell seems able to detect abnormal mitogenic stimulation, and it responds by preventing further division. Such responses help prevent the survival and proliferation of cells with various cancer-promoting mutations. Although it is not known how a cell detects excessive mitogenic stimulation, such stimulation often leads to the production of a cell-cycle inhibitor protein called Arf, which binds and inhibits Mdm2. As discussed earlier, Mdm2 normally promotes p53 degradation. Activation of Arf therefore causes p53 levels to increase, inducing either cell-cycle arrest or apoptosis . 

Cell-cycle arrest or apoptosis induced by excessive stimulation of mitogenic pathways.
Abnormally high levels of Myc cause the activation of Arf, which binds and inhibits Mdm2 and thereby increases p53 levels (see previous picture). Depending on the cell type and extracellular conditions, p53 then causes either cell-cycle arrest or apoptosis.

How do cancer cells ever arise if these mechanisms block the division or survival of mutant cells with overactive proliferation signals? The answer is that the protective system is often inactivated in cancer cells by mutations in the genes that encode essential components of the blocking mechanisms, such as Arf or p53 or the proteins that help activate them.

Cell-intrinsic timing, biological clocks, cell division counting, and other mechanisms control the growth and division of cells.  Evolution, or design? 
If each cell in our face were to undergo just one more cell division, we would be considered horribly malformed. If each cell in our arms underwent just one more round of cell division, we could tie our shoelaces without bending over. How do our cells know when to stop dividing? Our arms are generally the same size on both sides of the body. How is cell division so tightly regulated? 

“A person's right and left legs almost always end up the same length, and the hearts of mice and elephants each fit the proper rib cage. How genes set limits on cell size and number continues to mystify.” 4 One of the remaining fundamental mysteries in biology is how organs and organisms were programmed to stop growing at the right time. This cessation or near cessation of growth does not occur abruptly, but rather is the end result of a progressive decline in growth rate.

It is difficult to examine conception, the division of cells, and the transition from zygote to the fetus and not see a rigorous and meticulous series of patterns begin to emerge. If random occurrence and simple chaotic incidence had been the cause for human life, one could expect a far greater number of genetic mutations, anomalies and aberrations. The symmetry of the human body from conception to birth is overwhelming, and it is extremely unlikely that random occurrence is responsible.

A variety of genes are involved in the control of cell growth and division. The cell cycle is the cell’s way of replicating itself in an organized, step-by-step fashion. This cycle of duplication and division, known as the cell cycle, is the essential mechanism by which all living things reproduce. The duplication of eukaryotic cells is an all fine-tuned biochemical processes that depend on the precise structural arrangement of the cellular components. The only way to make a new cell is to duplicate a cell that already exists. Tight regulation of this process ensures that a dividing cell’s DNA is copied properly, any errors in the DNA are repaired, and each daughter cell receives a full set of chromosomes. The cycle has checkpoints (also called restriction points), which allow certain genes to check for problems and halt the cycle for repairs if something goes wrong. A minimal number of Cell-cycle regulators are required, which makes them irreducibly complex. Why would a prebiotic soup, or, let's suppose, life began with simple cells which divide by fission, produce any of these regulators without the others, if by themselves, without the others, there is no function for them?

The Cell cycle
https://reasonandscience.catsboard.com/t2109-the-cell-cycle

If a cell has an error in its DNA that cannot be repaired, it may undergo programmed cell death (apoptosis). Apoptosis is a common process throughout life that helps the body get rid of cells it doesn’t need. Cells that undergo apoptosis break apart. Apoptosis is a tightly regulated process – controlled by the integration of multiple pro-and anti-apoptotic signals. Ultimately the induction of apoptosis occurs through the activation of the caspase proteases that are responsible for coordinating the hallmarks of an apoptotic death:  cell shrinkage, chromatin condensation, membrane blebbing and DNA fragmentation. Bad things happen when apoptotic pathways are disrupted. A shut down of the pathway through mutation allows for growth of cancer and neurological disorders

Apoptosis, or Programmed Cell Death, is key to multicellular life and multicellular computing. Orchestrated apoptosis helps the growing embryo to sculpt many aspects of its final form. It is also a part of normal "maintenance." Every year the average human loses perhaps half of his/her body weight in cells via apoptosis! Apoptosis also protects the organism from "rogue" cells because such cells self-destruct when their internal mechanisms go wrong unless the apoptosis mechanism itself is compromised, as happens in the development of cancer. 1 Because apoptosis is so crucial to the growth and survival of multicellular organisms, it is carefully intertwined with other three multicellular principles. In other words, computing will adopt eventually in the future architectures similar to multicellular
biological organisms.

The four biological principles - specialization, messaging, stigmergy and apoptosis - had to emerge together since they depend upon each other.  Interdependence is a hallmark of intelligent design. 2

Multicellular computing adopts these four major organizing principles of multicellular biological systems because they help tame the spiralling problems of complexity and out-of-control interactions in the Internet.
Human-made computer networks are far behind multicellular biological networks - a computer virus is able to affect millions of computers, an attack a few years ago is an example, where millions of computers were blocked, and the owners had to pay to get their operational system back. In life, when a cell drives havoc, it is isolated and self-destructs. The whole organism is not affected.  Multicellular computing is biomimetics at its best, and the WWW is moving forward to get close to what High-tech computer networks in cells and brain neuronal networks do. Apoptosis-like mechanisms will eventually be developed, to shut down or disconnect computers infected by viruses. Computer science can learn a lot from computing and signalling networks in multicellular organisms.

Each cell participates simultaneously in all four principles
Specialization - All healthy Metazoan cells are specialized. Even adult stem cells are somewhat specialized. What is perhaps the most specialized aspect of the cells, other than their unique shape and function, is their unique repertoire of message receptors that determine the set of message molecules to which they can respond. They all share common behaviours too. Included in the common behaviour are participation in the cues and signals of their stigmergy relationship with the rest of the body, and obedience to apoptosis messages. In other words, multicellular organisms are characterized by specialized behaviours,  appropriate messaging, stigmergy and apoptosis behaviours.

Polymorphic Messaging - Complex messenger proteins often act as "bundles" of messages. That is, one messenger protein may have separate domains, each with a different messaging function. And often, the different message domains address each of the other three architectural principles. For example, one domain initiates signal cascades specific to the unique specialized function of that type of cell, another domain on the same complex molecular messenger facilitates or verifies physical attachment to the extracellular matrix (i.e., deals explicitly with the stigmergy structure), and yet another provides signals that either suppress or encourage apoptosis! The existence of these multi-part messages shows how fundamental these principles are. A single multi-part message speaks to the functional relationship of the cell to the whole organism/tissue/organ rather than to just a single cell function.

Question: Does that indicate its origin in a stepwise, evolutionary fashion, or intelligent setup and implementation?

Stigmergy - Virtually all cells other than simple red blood cells that lack a nucleus and most organelles are affected by stigmergy cues and/or signals. Even unattached cells such as other blood and lymph born cells are affected by and affect blood borne stigmergy signals, e.g. hormones. Cells that are attached to the Extracellular Matrix (ECM), i.e., the stigmergy structure, leave long-lasting cues (persistent messages) in those structures that affect other cells. In turn, the cells respond to such cues in ways that may cause them to modify the physical structures; that's how the structures are built in the first place. Cells that are normally attached or in direct contact with the ECM require constant feedback from the ECM. Absent the appropriate attachment cues, they suicide (undergo apoptosis).

Apoptosis - Almost all cells except cancerous cells participate in apoptosis signalling all the time. Even very simple cells such as red blood cells that lack a nucleus can undergo apoptosis.

Body growth in animals is rapid in early life but then progressively slows, thus imposing a limit on adult body size. This growth deceleration in mammals is caused by potent suppression of cell proliferation in multiple tissues and is driven primarily by local, rather than systemic, mechanisms. 4  This progressive decline in proliferation results from a GENETIC PROGRAM that occurs in multiple organs and involves the down-regulation of a large set of growth-promoting genes. The limit on adult body size is imposed by a negative feedback loop. Different organs appear to use different types of information to precisely target their adult size. Organ size appears to be limited by the initial number of progenitor cells, suggesting a mechanism based on cell-cycle counting. Growth disorders result in the unrestrained growth of cancer. Growth of different organs and structures is coordinated temporally and conditionally to maintain proportionality of these body parts. Coordinated growth of different organs is that body growth is orchestrated by a hormonal or other systemic mechanisms. Biological clocks allow the body to grow for a defined period of time before slowing, thus achieving a certain size. Such timing mechanisms may be employed during embryonic development in certain cell types. Time, rather than the number of cell divisions, signals the start of differentiation, indicating the presence of a cell-intrinsic timing mechanism. Furthermore, other, even more complex regulatory mechanisms that consist of multiple components with overlapping functions guarantee that defects in one component do not totally abolish the timing function. In some developmental systems, growth appears to be limited by a cell-division counter. During the early development of Xenopus laevis, a counting mechanism is in place to ensure that the zygote divides exactly 12 times before cell division slows.

Genetic programming, regulation, set up negative feedback loops, precise targeting, cell-cycle counting, orchestration, biological clocks, cell-intrinsic timing mechanism, cell-division counting.  Design and programming by intelligence, or set up by unguided evolutionary development ? You decide. I certainly go with the first option.

Cell division and counting, an amazing orchestrated process
The time required for human cell division is 20 hours for the first two days, and in subsequent divisions, curiously extends to 31 hours perhaps because of the extra processing required to generate specialization instructions. Researchers found that after a certain number of cycles the number of cells never correlated to the number expected, presumably because not all cells duplicate during all division cycles, or some duplicate at a much slower speed, or perhaps need to wait until other cells have divided before receiving the instruction to divide further. if the duplication speed remains at about 30 hours, no cell in the finished product, the newborn baby, could be older than 224 cell divisions, more or less, which indicates a fantastic direction of branching and timing, all of which must be encoded somehow within the pluripotent single, original stemcell.  Interestingly there are also about 210 different kinds of cells in the human body, so if we allow for a little error either way in both these measurements we could arrive at a very similar figure for each.

If a newborn baby has five trillion cells, then by purely doubling cells every 30 hours at maximum speed this number could be achieved in 42 divisions or about 7.5 weeks.  Even if we reduce the percent of cells constantly dividing to a more reasonable 50% (instead of 100%) at each level, it still works out at about 72 stages of duplication, or less than 13 weeks.

Clearly, then, the overwhelming majority of nine months is not spent in comparatively routine, automated duplication but in more complicated knowledge-based processes, such as monitoring and error checking, perhaps testing various organs and priming them for activity with the required enzymes and proteins before handing off development to the next stage.  This implies a specific store of rules and knowledge far greater than that currently possessed by all of mankind’s specialists combined, is maintained somewhere and referred to continually to detail the assembly of each specialized part and measure its function against set targets.  Otherwise, the odds are against a single pregnancy ending up with anything remotely resembling a living, fully functioning human being.

But this is a very important point suggesting that the creation of a baby is not due to duplication mechanisms – amazing as they are – but to intelligence of some sort, to an information and reference system somewhere.  After all, it is mechanically possible to build a human being within only two or three months, and evolution in an exposed habitat would surely favor the quickest possible pregnancy: a dog’s gestation is around 9 weeks – about four times quicker than ours -  while a dolphins is around 52 weeks, a third longer.

If we take into account the coiling time required for the new DNA and all the processes involved with the separation of the actual cell into two (where, in at least 210 stages, the new one must vary significantly from the original, since there are at least 210 different kinds of cells) the DNA duplication time is going to be very much faster than this since it is only one stage in the process.  Nevertheless, some cells present in the newborn infant may have been the result of this staggering rate of base pair copying -280 per second- carried out continuously for a period of more than 24 million seconds.

The total number of DNA base pairs duplicated to achieve the construction of the last few cells would, by that time, be 672 billion: as such, fetal development does indeed seem a fair analogy to evolution, but especially so in its overlooked aspects of synchronized timing and complexity.  Even twins raised completely separately emerge in much the same condition, showing that internal preferences remain very similar, even those presumed to be a random affair.

The finished product, as can be observed in twins, proves the fidelity to the original design.  But that quality after all seems the most prominent feature of all biological life and suggests that it arises not from mechanical duplications which are generally understood but from complex laws and sources of data which are not yet known.  There can be no suggestion that the process tends to take random turns at any point: if this were so, identical twins could never be identical

Where functional changes are observed over time, this can only be a result of a very consistent process, fully accounted for somewhere within a database governing duplication and cell specialization directives.

The whole idea of random mutations hinges on the persistent and rapid appearance of significant errors, but within each of us, we have a completed experiment in which given the same starting point, the end result is identical. After all, nobody is saying that identical twins have a completely different process of birth than anyone else: they simply share the same starting point. 4

1. https://bitesizebio.com/20969/apoptosis-gone-wrong-cell-deaths-role-in-disease/
2. http://evolutionofcomputing.org/Multicellular/IntertwinedPrinciples.html
3. Development biology, Gilbert / Barresi, page 3
4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3365796/


1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3365796/
2. https://royalsocietypublishing.org/doi/10.1098/rsob.170190
3. https://www.sciencedirect.com/science/article/pii/S0167488918302544
4. M. Herbert & colleagues, Journal of Reproduction and Fertility, 1995: 103, pg 209-214

5. Mechanisms of pattern formation

https://reasonandscience.catsboard.com/t2990-how-does-biological-multicellular-complexity-and-a-spatially-organized-body-plan-emerge#7863

Surprisingly, the earliest steps of animal development are among the most variable, even within a phylum. A frog, a chicken, and a mammal, for example, even though they develop in similar ways later, make eggs that differ radically in size and structure, and they begin their development with different sequences of cell divisions and cell specializations. Gastrulation f occurs in all animal embryos, but the details of its timing, of the associated pattern of cell movements, and of the shape and size of the embryo as gastrulation proceeds are highly variable. Likewise, there is great variation in the time and manner in which the primary axes of the body become marked out. However, this polarization of the embryo usually becomes discernible very early, before gastrulation begins: it is the first step of spatial patterning. Three axes generally have to be established. The animal-vegetal (A-V) axis, in most species, defines which parts are to become internal (through the movements of gastrulation) and which are to remain external. (The bizarre name dates from a century ago and has nothing to do with vegetables.) The anteroposterior (A-P) axis specifies the locations of future head and tail. The dorsoventral (D-V) d  axis specifies the future back and belly.

The anterior-posterior (AP or anteroposterior) axis is the line extending from head to tail (or mouth to anus in those organisms that lack a head and tail). The dorsal-ventral (DV or dorsoventral) axis is the line extending from back (dorsum) to belly (ventrum). The right-left axis separates the two lateral sides of the body. Although humans (for example) may look symmetrical, recall that in most of us, the heart is in the left half of the body, while the
liver is on the right. Somehow, the embryo knows that some organs belong on one side and other organs go on the other.


The embryo does not know, but the intelligent designer with foresight knows it and programmed the development program correctly. 

At one extreme, the egg is spherically symmetrical, and the axes only become defined during embryogenesis. The mouse comes close to being an example, with little obvious sign of polarity in the egg. Correspondingly, the blastomeres a produced by the first few cell divisions seem to be all alike and are remarkably adaptable. If the early mouse embryo is split in two, a pair of identical twins can be produced—two complete, normal individuals from a single cell. Similarly, if one of the cells in a two-cell mouse embryo is destroyed by pricking it with a needle and the resulting “half-embryo” is placed in the uterus of a foster mother to develop, in many cases a perfectly normal mouse will emerge.

At the opposite extreme, the structure of the egg defines the future axes of the body. This is the case for most species, including insects such as Drosophila. Many other organisms lie between the two extremes. The egg of the frog Xenopus, for example, has a clearly defined A-V axis even before fertilization: the nucleus near the top defines the animal pole, while the mass of yolk (the embryo’s food supply, destined to be incorporated in the gut) toward the bottom defines the vegetal pole. Several types of mRNA molecules are already localized in the vegetal cytoplasm of the egg, where they produce their protein products. After fertilization, these mRNAs and proteins act in and on the cells in the lower and middle part of the embryo, giving the cells their specialized characters, both by direct effects and by stimulating the production of secreted signal proteins. 

For example, mRNA encoding the transcription regulator VegT is deposited at the vegetal pole during oogenesis b . After fertilization, this mRNA is translated, and the resulting VegT protein activates a set of genes that code for signal proteins that induce mesoderm c and endoderm. The dorsoventral  D-V axis of the Xenopus embryo, by contrast, is defined through the act of fertilization. Following entry of the sperm, the outer cortex of the egg cytoplasm rotates relative to the central core of the egg, so that the animal pole of the cortex becomes slightly shifted to one side. 


The frog egg and its asymmetries. 
(A) Side view of a Xenopus egg photographed just before fertilization.
(B) The asymmetric distribution of molecules inside the egg, and how this changes following fertilization so as to define a dorsoventral as well as an animal-vegetal asymmetry. Fertilization, through a reorganization of the microtubule cytoskeleton, triggers a rotation of the egg cortex (a layer a few μm deep) through about 30° relative to the core of the egg; the direction of rotation determined by the site of sperm entry. Some components are carried still further to the future dorsal side by active transport along microtubules. The resulting dorsal concentration of Wnt11 mRNA leads to dorsal production of the Wnt11 signal protein and defines the dorsoventral polarity of the future embryo. Vegetally localized VegT defines the vegetal source of signals that will induce endoderm and mesoderm.

Treatments that block the rotation allow cleavage to occur normally but produce an embryo with a central gut and no dorsal structures or D-V asymmetry. Thus, this cortical rotation is required to define the D-V axis of the future body by creating the D-V axis of the egg. 

The site of sperm entry that biases the direction of the cortical rotation in Xenopus, perhaps through the centrosome that the sperm brings into the egg— inasmuch as the rotation is associated with a reorganization of the microtubules nucleated from the centrosome in the egg cytoplasm. The reorganization leads to a microtubule-based transport of several cytoplasmic components, including the mRNA coding for Wnt11, a member of the Wnt family of signal proteins, moving it toward the future dorsal side (see Figure above). This mRNA is soon translated and the Wnt11 protein secreted from cells that form in that region of the embryo activates the Wnt signaling pathway. This activation is crucial for triggering the cascade of subsequent events that will organize the dorsoventral axis of the body. (The A-P axis of the embryo will only become clear later, in the process of gastrulation.) Although different animal species use a variety of different mechanisms to specify their axes, the outcome has been relatively well conserved in evolution: head is distinguished from tail, back from belly, and gut from skin. It seems that it does not much matter what tricks the embryo uses to break the initial symmetry and set up this basic body plan.

Studies in Drosophila have revealed the genetic control mechanisms underlying development
It is the fly Drosophila, more than any other organism, that has provided the key to our present understanding of how genes govern development. Decades of genetic study culminated in a large-scale genetic screen, focusing especially on the early embryo and searching for mutations that disrupt its pattern. This revealed that the key developmental genes fall into a relatively small set of functional classes. The discovery of these genes and the subsequent analysis of their functions was a famous tour de force and had a revolutionary impact on all of developmental biology, earning its discoverers a Nobel Prize. Some parts of the developmental machinery revealed in this way are conserved between flies and vertebrates, some parts not. But the logic of the experimental approach and the general strategies of genetic control that it revealed have transformed our understanding of multicellular development in general. To understand how the early developmental machinery operates in Drosophila, it is important to note a peculiarity of fly development. Like the eggs of other insects, but unlike most vertebrates, the Drosophila egg—shaped like a cucumber— begins its development with an extraordinarily rapid series of nuclear divisions without cell division, producing multiple nuclei in a common cytoplasm—a syncytium. The nuclei then migrate to the cell cortex, forming a structure called the syncytial blastoderm. After about 6000 nuclei have been produced, the plasma membrane folds inward between them and partitions them into separate cells, converting the syncytial blastoderm into the cellular blastoderm. 



Development of the  Drosophila egg from fertilization to the cellular blastoderm stage

The initial patterning of the Drosophila embryo depends on signals that diffuse through the cytoplasm at the syncytial stage and exert their actions on genes in the rapidly dividing nuclei, before the partitioning of the egg into separate cells. Here, there is no need for the usual forms of cell-cell signalling; neighbouring regions of the syncytial blastoderm can communicate by means of transcription regulatory proteins that move through the cytoplasm of the giant multinuclear cell.

Egg-polarity genes encode macromolecules deposited in the egg to organize the axes of the early Drosophila embryo
As in most insects, the main axes of the future body of Drosophila are defined before fertilization by a complex exchange of signals between the developing egg, or oocyte, and the follicle cells e that surround it in the ovary. In the stages before fertilization, the anteroposterior and dorsoventral axes of the future embryo become defined by four systems of egg-polarity genes that create landmarks—either mRNA or protein—in the developing oocyte. Following fertilization, each landmark serves as a beacon, providing a signal that organizes the developmental process in its neighbourhood. The nature of the genes emerged from studies of mutants in which the patterning of the embryo was altered. One class of mutations gave embryos with disrupted polarity—for example, tail-end structures at both ends of the body, with no head-end structures. This class of mutations identified the set of egg-polarity genes. The egg-polarity gene responsible for the signal that organizes the anterior end of the embryo is called Bicoid. A deposit of Bicoid mRNA molecules is localized, before fertilization, at the anterior end of the egg. Upon fertilization, the mRNA is translated to produce Bicoid protein. This protein is an intracellular morphogen and transcription regulator that diffuses away from its source to form a concentration gradient within the syncytial cytoplasm, with its maximum at the head end of the embryo 


The Bicoid protein gradient. 
(A) Bicoid mRNA is deposited at the anterior pole during oogenesis.
(B) Local translation followed by diffusion generates the Bicoid protein gradient.
(C) Absence of the Bicoid protein gradient in embryos from Bicoid homozygous mutant mothers. 

The different concentrations of Bicoid along the A-P axis help determine different cell fates by regulating the transcription of genes in the nuclei of the syncytial blastoderm. Of the three other egg-polarity gene systems, two contribute to patterning the syncytial nuclei along the A-P axis and one to patterning them along the D-V axis. Together with the Bicoid group of genes, and acting in a broadly similar way, their gene products mark out three fundamental partitions of body regions—head versus rear, dorsal versus ventral, and endoderm versus mesoderm and ectoderm— as well as a fourth partition, no less fundamental to the body plan of animals: the distinction between germ cells and somatic cells. 




The organization of the four egg-polarity gradient systems in Drosophila. 
Nanos is a translational repressor that governs the formation of the abdomen. Localized Nanos mRNA is also incorporated into the germ cells as they form at the posterior of the embryo, and Nanos protein is necessary for germ-line development. Bicoid protein is a transcriptional activator that determines the head and thoracic regions. Toll and Torso are receptor proteins that are distributed all over the membrane but are activated only at the sites indicated by the coloring, through localized exposure to the extracellular ligands Spaetzle (the ligand for Toll) and Trunk (the ligand for Torso). Toll activity determines the mesoderm and Torso activity determines the formation of terminal structures.

The egg-polarity genes have a further special feature: they are all maternal-effect genes, in that it is the mother’s genome rather than the zygote’s genome that is critical. For example, a fly whose chromosomes are mutant in both copies of the Bicoid gene but who is born from a mother carrying one normal copy of Bicoid develops perfectly normally, without any defects in the head pattern. However, if that offspring is a female, she cannot deposit any functional Bicoid mRNA into her own eggs, which will therefore develop into headless embryos, regardless of the father’s genotype. The egg-polarity genes act first in a hierarchy of gene systems that define a progressively more detailed pattern of body parts. 

Three Groups of Genes Control Drosophila Segmentation Along the A-P Axis
The body of an insect is divided along its A-P axis into a series of segments. The segments are repetitions of a theme with variations: each segment forms highly specialized structures, but all built according to a similar fundamental plan 


The origins of the Drosophila body segments. 
(A) At 3 hours, the embryo (shown in side view) is at the blastoderm stage and no segmentation is visible, although a fate map can be drawn showing the future segmented regions (color). 
(B) At 10 hours, all the segments are clearly defined (T1: first thoracic segment; A1: first abdominal segment). 
(C) The segments of the Drosophila larva and their correspondence with regions in the embryo. 
(D) The segments of the Drosophila adult and their correspondence with regions in the embryo.

The gradients of transcription regulators set up along the A-P axis in the early embryo by the egg-polarity genes are the prelude to creation of the segments. These regulators initiate the orderly transcription of segmentation genes, which refine the pattern of gene expression to define the boundaries and ground plan of the individual segments. Segmentation genes are expressed by subsets of cells in the embryo, and their products are the first components that the embryo’s own genome contributes to embryonic development; they are therefore called zygotic-effect genes, to distinguish them from the earlier-acting maternal-effect genes. Mutations in segmentation genes can alter either the number of segments or their basic internal organization. The segmentation genes fall into three groups according to their mutant phenotypes 


Examples of the phenotypes of mutations affecting egg-polarity genes and the three types of segmentation genes. 
In each case, the areas shaded in green on the normal larva (left) are deleted in the mutant or are replaced by mirror-image duplicates of the unaffected regions

It is convenient to think of the three groups as acting in sequence, although in reality their functions overlap in time. First to be expressed is a set of at least six gap genes, whose products mark out coarse A-P subdivisions of the embryo. Mutations in a gap gene eliminate one or more groups of adjacent segments: in the mutant Krüppel, for example, the larva lacks eight segments. Next comes a set of eight pair-rule genes. Mutations in these genes cause a series of deletions affecting alternate segments, leaving the embryo with only half as many segments as usual; although all the mutants display this two-segment periodicity, they differ in the precise pattern. Finally, there are at least 10 segment-polarity genes, in which mutations produce a normal number of segments but with a part of each segment deleted and replaced by a mirror-image duplicate of all or part of the rest of the segment. In parallel with the segmentation process, a further set of genes—the homeotic selector, or Hox, genes—serves to define and preserve the differences between one segment and the next, as we describe shortly. The phenotypes of the various segmentation mutants suggest that the segmentation genes form a coordinated system that subdivides the embryo progressively into smaller and smaller domains along the A-P axis, each distinguished by a different pattern of gene expression. Molecular genetics has helped to reveal how this system works.

A Hierarchy of Gene Regulatory Interactions Subdivides the Drosophila Embryo
Like Bicoid, most of the segmentation genes encode transcription regulator proteins. Their control by the egg-polarity genes and their actions on one another and on still other genes can be deciphered by comparing gene expression in normal and mutant embryos. By using appropriate probes to detect RNA transcripts or their protein products, one can observe genes switch on and off in changing patterns. By comparing these patterns in different mutants, one can begin to discern the logic of the entire gene control system. The products of the egg-polarity genes provide the global positional signals in the early embryo. The Bicoid protein acts as a morphogen and activates different sets of genes at different positions along the A-P axis: some gap genes are only activated in regions with high levels of Bicoid, others only where levels of Bicoid are lower. After the gap gene products refine their positions by mutual repression, they provide a second tier of positional signals that act more locally to regulate finer details of patterning. Gap genes act by controlling the expression of yet other genes, including the pair-rule genes. The pair-rule genes, in turn, collaborate with one another and with the gap genes to set up a regular, periodic pattern of expression of the segment polarity genes, which collaborate with one another to define the internal pattern of each individual segment. 


An example of the regulatory hierarchy of egg-polarity, segmentation, and Hox genes. 
As discussed in the text, there are three groups of segmentation genes. The photographs show mRNA expression patterns of representative examples of genes of each type.

The initial steps in the creation of the segmental pattern occur before cellularization of the syncytial blastoderm and are governed by the combinatorial effects of transcription regulators, for the regulation of the expression of the pair-rule gene Even-skipped. After cellularization, the segment-polarity genes further subdivide each segment into smaller domains. A large subset of the segment-polarity genes codes for components of two signaling pathways—the Wnt pathway and the Hedgehog pathway, including the secreted signal proteins Wingless (the first-named member of the Wnt family) and Hedgehog. (The Hedgehog pathway was first discovered through study of Drosophila segmentation, and it takes its name from the prickly appearance of the surface of the Hedgehog mutant embryo.) Wingless and Hedgehog are synthesized in different bands of cells that serve as signaling centers within each segment. The two proteins mutually maintain each other’s expression, while regulating the expression of genes such as Engrailed in neighboring cells. In such a manner, a series of sequential inductions creates a fine-grained pattern
of gene expression within each segment.


Mutual maintenance of Hedgehog and Wingless expression.
Engrailed is a transcription regulator (blue) that drives the expression of Hedgehog. Hedgehog encodes a secreted protein (red) that activates its signaling pathway in neighboring cells and thereby drives them to express the Wingless gene. In turn, Wingless encodes a secreted protein (green) that acts back on neighbors of the Wingless-expressing cell to maintain their expression of Engrailed and Hedgehog. As indicated, the same control loop repeats along the A-P axis of the fly.

Egg-Polarity, Gap, and Pair-Rule Genes Create a Transient Pattern That Is Remembered by Segment-Polarity and Hox Genes
The gap genes and pair-rule genes are activated within the first few hours after fertilization. Their mRNA products initially appear in patterns that only approximate the final picture; then, within a short time, this fuzzy initial pattern resolves itself into a regular, crisply defined system of stripes. But this pattern itself is unstable and transient: as the embryo proceeds through gastrulation and beyond, the pattern disintegrates. The genes’ actions, however, have passed on an enduring memory of their patterns of expression by inducing the expression of certain segment polarity genes along with Hox genes. After a period of pattern refinement mediated by cell-cell interactions, the expression patterns of these new groups of patterning genes is stabilized to provide positional labels that serve to maintain the segmental organization of the larva and adult fly. The segment-polarity gene Engrailed provides a good example. Its RNA transcripts form a series of 14 bands in the cellular blastoderm, each approximately one-cell wide. These stripes lie immediately anterior to similar stripes of expression of another segment polarity gene, Wingless. As the cells in the developing embryo continue to grow, divide, and move, a mutually reinforcing signal between the Wingless expressing cells and the Engrailed expressing cells maintains narrow stripes of their expres​sion(see Figure above). After three cell cycles, newly expressed regulators stabilize an Engrailed expression pattern that will last throughout the life of the fly, long after the signals that induced and refined it have disappeared. The segment borders will form at the posterior edge of each such Engrailed stripe


The pattern of expression of Engrailed, a segment-polarity gene.
The Engrailed pattern is shown in a 10-hour embryo and an adult (whose wings have been removed in this preparation). The pattern is revealed by constructing a strain of Drosophila containing the control sequences of the Engrailed gene coupled to the coding sequence of the reporter LacZ, whose product is detected histochemically through the brown product generated by immunohistochemistry against LacZ (10-hour embryo) or through the blue product generated by a reaction that LacZ catalyzes (adult). Note that the Engrailed pattern, once established, is preserved throughout the animal’s life.

In addition to regulating the segment-polarity genes, the products of pair-rule genes collaborate with those of gap genes to induce the precisely localized activation of a further set of genes—originally called homeotic selector genes and now often called Hox genes. It is the Hox genes that permanently distinguish one segment from another.  This role is critical in a wide range of animals, including ourselves.

The Genetics of Axis Specification in Drosophila

Early Drosophila Development
In Drosophila development, cell membranes do not form until after the thirteenth nuclear division. Prior to this time, the dividing nuclei all share a common cytoplasm and material can diffuse throughout the whole embryo. The specification of cell types along the anterior-posterior and dorsal-ventral axes is accomplished by the interactions of components within the single multinucleated cell. Moreover, these axial differences are initiated at an earlier developmental stage by the position of the egg within the mother’s egg chamber.

Gene segmentation
Cell fate commitment in Drosophila appears to have two steps: specification and determination. Early in fly development, the fate of a cell depends on cues provided by protein gradients. This specification of cell fate is flexible and can still be altered in response to signals from other cells. Eventually, however, the cells undergo a transition from this loose type of commitment to an irreversible determination. At this point, the fate of a cell becomes cell-intrinsic. The transition from specification to determination in Drosophila is mediated by segmentation genes that divide the early embryo into a repeating series of segmental primordia along the anterior-posterior axis. Segmentation genes were originally defined by zygotic mutations that disrupted the body plan, and these genes were divided into three groups based on their mutant phenotypes



• Gap mutants lack large regions of the body (several contiguous segments; Figure A).
• Pair-rule mutants lack portions of every other segment (Figure B).
• Segment polarity mutants show defects (deletions, duplications, polarity reversals) in every segment (Figure C)



Three types of segmentation gene mutations. 
The left side shows the early-cleavage embryo (yellow), with the region where the particular gene is normally transcribed in wild-type embryos shown in blue. These areas are deleted as the mutants develop into late-stage embryos.


a In humans, blastomere formation begins immediately following fertilization and continues through the first week of embryonic development. About 90 minutes after fertilization, the zygote divides into two cells. These mitotic divisions continue and result in a grouping of cells called blastomeres. During this process, the total size of the embryo does not increase, so each division results in smaller and smaller cells. When the zygote contains 16 to 32 blastomeres it is referred to as a "morula.

b 

c In all bilaterian animals, the mesoderm is one of the three primary germ layers in the very early embryo. The other two layers are the ectoderm (outside layer) and endoderm (inside layer), with the mesoderm as the middle layer between them

d




Axes of a bilaterally symmetrical animal. 
(A) A single plane, the midsagittal plane, divides the animal into left and right halves. (B) Cross sections bisecting the anterior-posterior axis.




e The epithelium of follicle cells encases germline cells to create an egg. 1 Maintain the epithelium or permit migrations essential for oogenesis. Cell-cell communication is important, but the same signals are used repeatedly to control distinct events. Understanding intrinsic mechanisms that alter responses to developmental signals will be important to understand the regulation of cell shape and organization.


f Gastrulation is a phase early in the embryonic development of most animals, during which the single-layered blastula is reorganized into a multilayered structure known as the gastrula. Before gastrulation, the embryo is a continuous epithelial sheet of cells; by the end of gastrulation, the embryo has begun differentiation to establish distinct cell lineages, set up the basic axes of the body (e.g. dorsal-ventral, anterior-posterior), and internalized one or more cell types including the prospective gut. 2


Gastrulation in Drosophila. 
In this cross section, the mesodermal cells at the ventral portion of the embryo buckle inward, forming the ventral furrow. This furrow becomes a tube that invaginates into the embryo and then flattens
and generates the mesodermal organs.


Gastrulation in Drosophila. 
The anterior of each gastrulating embryo points upward in this series of scanning electron micrographs. 
(A) Ventral furrow beginning to form as cells flanking the ventral midline invaginate. 
(B) Closing of ventral furrow, with mesodermal cells placed internally and surface ectoderm flanking the ventral midline. 
(C) Dorsal view of a slightly older embryo, showing the pole cells and posterior endoderm sinking into the embryo. 
(D) Schematic representation showing dorsolateral view of an embryo at fullest germ band extension, just prior to segmentation. The cephalic furrow separates the future head region (procephalon) from the germ band,
which will form the thorax and abdomen. 
(E) Lateral view, showing fullest extension of the germ band and the beginnings of segmentation. Subtle indentations mark the incipient segments along the germ band. Ma, Mx, and Lb correspond to the mandibular,
maxillary, and labial head segments; T1–T3 are the thoracic segments; and A1–A8 are the abdominal segments. 
(F) Germ band reversing direction. The true segments are now visible, as well as the other territories of the dorsal head, such as the clypeolabrum, procephalic region, optic ridge, and dorsal ridge.


Newly hatched first instar larva.


1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2430523/
2. https://en.wikipedia.org/wiki/Gastrulation

6. HOX genes

1. Cells store codified information in DNA, and at least 24 epigenetic codes, which are complex instructional informational blueprints, essential for cells to make copies of themselves, animal development, adaptation, and body architecture
2. All Codes, and blueprints we know the origin of come from an intelligent mind. Evolution is a non-directed, non-intelligent process and does not suffice to explain the origin of biodiversity and body architecture.
3. Therefore we have 100% inference that DNA comes from an intelligent mind and 0% inference that it is not.

In order to know what mechanisms eventually provoke change and if unguided evolution of an organism is a viable explanation, it must be known what mechanisms do form phenotype,  body architecture, organs, various cell types, cell migration etc. Development biology ( Evo-devo ) is a rather new branch of biology. Development biology might be the most complex branch of biology, and many open questions remain.

Homeoboxes have been found in fungi, plants and animals. In each "kingdom" homeobox genes occupy a key position in the genetic control of either cell differentiation, morphogenesis and or body plan specification.

All Hox genes and many other developmental transcription factors contain the homeobox: conserved arrangement on chromosomes that is the same as their order of activation along the body axis. The regulation is very precise. The degree of sequence conservation of the homeodomain is extremely high indicating strong functional constraints leading to a high pressure to retain the homeobox sequences constant.

What the Hox code represents is a somewhat digital mechanism for regulating axial patterning. 

It is obvious that not any arrangement of Homeobox genes will give rise to functional body architecture. The precise arrangement is key to have functional body plans.  Mutations in hox genes do produce aberrant body plans..like legs where there should be antennae in fruit flies. But it is a mistake to assume that this is evidence that hox genes causally lay out the body plan, just like it would be a mistake to assume that a fault in a TV, causing a disruption in the reception of the signal, shows that the TV box produces the signal itself. Linear DNA cannot produce 3D form. There is a higher orchestration, which directs the correct linear arrangement of Homeobox genes in the genome.

Hox Genes in Development: The Hox Code
https://www.nature.com/scitable/topicpage/hox-genes-in-development-the-hox-code-41402

This colinearity, arrangement, order of activation and precise regulation of Hox gene clusters indicates there is a HOX Code, which sets the right pattern of Hox gene cluster arrangement for correct sequential expression of segments  in the embryo.  

There is uncertainty in our understanding of homeobox gene cluster evolution at present. This relates to our still rudimentary understanding of the dynamics of genome rearrangements and evolution over the evolutionary timescales being considered when we compare lineages from across the animal kingdom. The mechanisms responsible for the synchronous regulation of Hox genes and the molecular function of their colinearity remain unknown. Despite 35 years of active research, the mechanisms of Hox gene regulation have remained elusive. It has been argued that chromatin structure and histone demethylation play important roles in activation of Hox genes, but the mechanism precisely directing chromatin modifications to specific loci at the right time remains mysterious.

What does this elucidate? Life is not only composed of organic carbon-based matter but essentially, instructional information. Not any kind of information, but complex, specifying information, blueprints, which precisely orchestrates and directs how to develop, build, adopt animals, plants, fungi, bacterias, and perpetuate life in all its various forms.

The Hox Code, Code biology, Barbieri, page 107
In 1979, David Elder proposed a model that was capable of accounting for the regularities that exist in the bodies of many segmented worms (annelids). The segments of these animals are often subdivided into annuli whose number varies according to a simple rule: if a segment contains n annuli, the following segment contains either the same number n (repetition) or n plus or minus 1 (digital modification). This type of rules is known to the designers of electronic circuits as a Gray code, a code that is binary (because it employs circuits that have only one of two states), combinatorial (because its outcomes are obtained by combinations of circuits) and progressive (because consecutive outcomes must be coded by combinations that differ in the state of one circuit only). The results obtained with these rules describe with great accuracy what is observed in segmented worms. The body plan of these animals is based on a combinatorial code. The coding principle cannot be the classical “one gene one pattern”, but “one combination of genes-one pattern” and for this reason it is called it epigenetic code (Elder 1979). After the discovery of the Hox genes, it became increasingly clear that they are used in many different permutations, according to a combinatorial set of rules that became known as Hox code. The term Hox code was introduced to account for the finding that the individual characteristics of the vertebrae are determined by different combinations of Hox genes. This is true in most other organs and it became standard practice to refer to any combination of Hox genes as a Hox code. The epigenetic code, in particular, is a Hox code because it is Hox genes that are responsible for the body plan of the segmented worms. It must be underlined that the Hox genes can be used in different combinations not only in various parts of a body, but also in different stages of embryonic development. At the phylotypic stage, for example, the Hox genes specify characteristics of the phylum, whereas in later stages they determine characteristics at lower levels of organization. There is, in short, a hierarchy of Hox gene expressions, and therefore a hierarchy of Hox codes. At this point, however, we have to face a key definition problem: is it legitimate to say that the Hox codes are true organic codes? More precisely, that they have the basic features that we find, for example, in the genetic code? An organic code is a mapping between two independent worlds and cannot exist without a set of adaptors that physically realize the mapping. The Hox codes have been defined instead as patterns of combinatorial gene expression and do not require adaptors because a molecular pattern in one world is not a mapping between two independent worlds. We have therefore two different definitions of code, one based on mapping and the other on patterns, or sequences, and it is important to keep them separate because they have different biological implications.

Hox Genes Permanently Pattern the A-P Axis
As animal development proceeds, the body becomes more and more complex. But again and again, in every species and at every level of organization, we find that complex structures are made by repeating a few basic themes, with variations. Thus, a limited number of basic differentiated cell types, such as muscle cells or fibroblasts, recur with subtle individual variations in different sites. These cell types are organized into a limited variety of tissue types, such as muscle or tendon, which again are repeated with subtle variations in different regions of the body. From the various tissues, organs such as teeth or digits are built— molars and incisors, fingers and thumbs and toes—a few basic kinds of structure, repeated with variations. Wherever we find this phenomenon of modulated repetition, we can break down the developmental biologist’s problem into two kinds of questions:

what is the basic construction mechanism common to all the objects of the given class, and how is this mechanism modified to give the observed variations in different animals? 

The segments of the insect body provide a good example. We have thus far sketched the way in which the rudiment of a single body segment is constructed and how cells within each segment become different from one another. We now consider how one segment becomes determined or specified, to be different from another. The first glimpse of the answer to this problem came over 80 years ago, with the discovery of a set of mutations in Drosophila that cause bizarre disturbances in the organization of the adult fly. In the Antennapedia mutant, for example, legs sprout from the head in place of antennae, whereas in the Bithorax mutant, portions of an extra pair of wings appear where normally there should be the much smaller appendages called halteres


Ultrabithorax, or Ubx, is one of three genes in the Bithorax gene complex (a Hox gene cluster). 
Ubx is responsible for all of the differences between the second and third thoracic segments. (A, B) Ubx loss-of-function mutations transform the haltere-bearing segment (A) into a wingbearing segment, resulting in four-winged flies (B). (C) Ubx gain-of-function in the second thoracic segment transforms this wing-bearing segment into a halterebearing segment, resulting in wingless flies.

These mutations transform parts of the body into structures appropriate to other positions, and they are called homeotic mutations (from the Greek “homoios,” meaning similar) because the transformation is between structures of a recognizably similar general type, changing one kind of limb, or one kind of segment, into another. It was eventually discovered that a whole set of genes, the homeotic selector genes, or Hox
genes, serve to permanently specify the A-P characters of the whole set of animal segments. These genes are all related to one another as members of a multigene family. There are eight Hox genes in the fly, and they all lie in one or the other of two gene clusters known as the Bithorax complex and the Antennapedia complex.

The genes in the Bithorax complex control the differences among the abdominal and thoracic segments of the body, while those in the Antennapedia complex control the differences among thoracic and head segments. Comparisons with other species show that the same genes are present in essentially all animals, including humans. These comparisons also reveal that the Antennapedia and Bithorax complexes are the two halves of a single entity, called the Hox complex, that is split, and whose members operate in a coordinated way to exert their control over the head-to-tail pattern of the body. The products of the Hox genes, the Hox proteins, are transcription regulators, all of which possess a highly conserved, 60-amino-acid-long DNA-binding homeodomain. The corresponding motif in the DNA sequence is called a “homeobox,” from which, by abbreviation, the Hox complex takes its name. There are many homeobox-containing genes, but only those located in a Hox complex are Hox genes.

Hox Proteins Give Each Segment Its Individuality
The Hox proteins can be viewed as molecular address labels possessed by the cells of each segment: these labels give the cells in each region a positional value—that is, an intrinsic character that differs according to a cell’s location. If the address labels in a developing Drosophila segment are changed, the segment behaves as though it were located somewhere else; if all the Hox genes in an embryo are deleted, the body segments in the larva will all be alike. To a first approximation, each Hox gene is normally expressed in those regions that develop abnormally when that gene is mutated or absent. How does each Hox protein give a segment its permanent identity? All the Hox proteins are similar in their DNA-binding regions, but they are very different in the regions that interact with the other proteins with which the Hox proteins form transcriptional regulatory complexes. The different protein partners act together with the Hox proteins to dictate which DNA binding sites will be recognized, as well as whether the effect on transcription at those sites will be activation or repression. Acting in this way, the Hox proteins modulate the actions of many other transcription regulators. Hundreds of genes are under this type of Hox-modulated control, including genes for cell–cell signaling, transcriptional regulation, cell polarity, cell adhesion, cytoskeletal function, cell growth, and cell death, all conspiring (in ways that are not yet understood) to give each segment its distinctive Hox-dependent character.

Hox Genes Are Expressed According to Their Order in the Hox Complex
How, then, is the expression of the Hox genes themselves regulated? The coding sequences of the eight Hox genes in the Antennapedia and Bithorax complexes in Drosophila are interspersed amid a much larger quantity of regulatory DNA. This DNA includes binding sites for the products of the egg-polarity and segmentation genes, thereby serving as an interpreter of the multiple items of spatial information supplied to it by all these transcription regulators. The net result is that the particular set of Hox genes transcribed is appropriate for each location along the A-P body axis. The pattern of Hox gene expression exhibits a remarkable regularity that suggests an additional form of control. The sequence in which the genes are ordered along the chromosome, in both the Antennapedia and the Bithorax complexes, corresponds almost exactly to the order in which they are expressed along the A-P axis of the body


The patterns of expression compared to the chromosomal locations of the genes of the Hox complex. 
The diagram shows the sequence of genes in each of the two subdivisions of the chromosomal complex. This corresponds, with minor deviations, to the spatial sequence in which the genes are expressed, shown in the photograph of a Drosophila embryo at the so-called germ band retraction stage, about 10 hours after fertilization. The embryo has been stained by in situ hybridization with differently labeled probes to detect the mRNA products of different Hox
genes in different colors.

This hints at some process of gene activation, perhaps dependent on chromatin structures that propagate along the Hox complexes, switching on one Hox gene after another according to their order along the chromosome. The most “posterior” of the Hox genes that are expressed in a cell generally dominates, driving down expression and activity of the “anterior” genes and dictating the character of the segment. The gene regulatory mechanisms underlying these phenomena are still not well understood, but their consequences are profound. We shall see that the serial organization of gene expression in the Hox complex is a fundamental feature that has been highly conserved.

Trithorax and Polycomb Group Proteins Enable the Hox Complexes to Maintain a Permanent Record of Positional Information
The spatial pattern of expression of the genes in the Hox complex is set up by signals acting early in development, but the consequences are long-lasting. Although the pattern of expression undergoes complex adjustments as development proceeds, the Hox complexes serve to stamp each cell and its progeny with a permanent record of the A-P position that the cell occupied in the early embryo. In this way, the cells of each segment are equipped with a long-term memory of their location along the A-P axis of the body. This memory trace is somehow imprinted on the Hox complexes, and it governs the segment-specific identity not only of the larval segments, but also of the structures of the adult fly. The molecular mechanism of this memory of positional information relies on two types of regulation. One is from the Hox genes themselves: many of the Hox proteins autoactivate the transcription of their own genes, thereby helping to keep the genes on indefinitely. Another crucial input is from two large, complementary sets of proteins, called the Trithorax group and the Polycomb group, which stamp the chromatin of the Hox complex with a heritable record of its embryonic state of activation or repression. These are key general regulators of chromatin structure that can be shown to be critical for cell memory: if genes of the Trithorax or Polycomb group are defective, the pattern of expression of the Hox genes is set up correctly at first, but it is not correctly maintained as the embryo grows older. The two sets of regulators act in opposite ways. Trithorax group proteins are needed to maintain the transcription of Hox genes in cells where transcription has already been switched on. In contrast, Polycomb group proteins form stable complexes that bind to the chromatin of the Hox complex and maintain the repressed state in cells where Hox genes have not been activated at the critical time


The role of genes of the Polycomb group
(A) Photograph of a wild-type Drosophila embryo. 
(B) Photograph of a mutant embryo defective for the gene Extra sex combs (Esc) and derived from a mother also lacking this gene. The gene belongs to the Polycomb group. Essentially all segments have been transformed to resemble the most posterior abdominal segment. In the mutant, the pattern of expression of the homeotic selector genes, which is roughly normal initially, is unstable in such a way that all these genes soon become switched on all along the body axis.

The D-V Signaling Genes Create a Gradient of the Transcription Regulator Dorsal
The patterning along the dorsoventral (D-V) axis begins with maternal gene products that define this axis in the egg, and it then progresses through zygotic gene products that further subdivide the D-V axis in the embryo. Initially, a protein that is produced by follicle cells underneath the future ventral region of the embryo leads to the localized activation of a transmembrane receptor, called Toll, on the ventral side of the egg membrane. The various maternal
genes required for this process are called D-V egg-polarity genes. (Curiously, Drosophila Toll and vertebrate Toll-like proteins also operate in innate immune responses). The localized activation of Toll controls the distribution of Dorsal, a transcription regulator of the NFκB family. The Toll-regulated activity of Dorsal, like that of NFκB, depends on the translocation of Dorsal from the cytosol, where it is held in an inactive form, to the nucleus, where it regulates gene expression. In the newly laid egg, both Dorsal mRNA and protein are distributed uniformly in the cytosol. After the nuclei in the syncytial blastoderm have migrated to the surface of the embryo, but before cellularization, Toll receptor activation on the ventral side induces a remarkable redistribution of the Dorsal protein. On the dorsal side, the protein remains in the cytosol, but ventrally it becomes concentrated in the nuclei, with a smooth gradient of nuclear localization between these two extremes


The concentration gradient of Dorsal protein in the nuclei of the blastoderm. 
In wild-type Drosophila embryos, the protein is present in the dorsal cytoplasm and absent from the dorsal nuclei; ventrally, it is depleted in the cytoplasm and concentrated in the nuclei. In a mutant in which the Toll pathway is
activated everywhere and not just ventrally, Dorsal protein is everywhere concentrated in the nuclei; the result is a ventralized embryo. Conversely, in a mutant in which the Toll signaling pathway is inactivated, Dorsal protein everywhere remains in the cytoplasm and is absent from the nuclei; the result is a dorsalized embryo

Once inside the nucleus, the Dorsal protein acts as a morphogen and turns on or off the expression of different sets of genes depending on Dorsal’s concentration. The expression of each responding gene depends on its regulatory DNA—specifically, on the number and affinity of the binding sites that this DNA contains for Dorsal and other transcription regulators. In this way, the regulatory DNA interprets the positional signal provided by the nuclear Dorsal protein gradient, so as to define a D-V series of territories—distinctive bands of cells that run the length of the embryo. Most ventrally—where the nuclear concentration of Dorsal protein is highest—it switches on, for example, the expression of a gene called Twist, which is specific for mesoderm. Most dorsally, where the nuclear concentration of Dorsal protein is lowest, the cells switch on a gene called Decapentaplegic (Dpp). And in an intermediate region, where the nuclear concentration of Dorsal protein is high enough to repress Dpp but too low to activate Twist, the cells switch on another set of genes, including one called Short gastrulation (Sog)


How morphogen gradients guide a patterning process along the dorsoventral axis of the Drosophila embryo. 
(A) Initially, a gradient of Dorsal protein defines three broad territories of gene expression, marked here by the expression of three representative genes—Dpp, Sog, and Twist. 
(B) Slightly later, the cells expressing Dpp and Sog
secrete, respectively, the signal proteins Dpp (a TGFβ family member) and Sog (an antagonist of Dpp). These two proteins then diffuse and interact with one another (and with certain other factors) to create the dorsoventral (D–V) territories shown.

Products of the genes directly regulated by the Dorsal protein generate in turn more local signals, which define finer subdivisions along the D-V axis. These signals act during cellularization and take the form of conventional extracellular signal proteins. In particular, Dpp codes for a secreted TGFβ-family protein, which forms a local morphogen gradient in the dorsal part of the embryo. Sog encodes another secreted protein that is produced by the neurogenic ectoderm (which gives rise to the nervous system) and acts as an antagonist of Dpp protein. The opposing diffusion gradients of these two signal proteins create a steep gradient of Dpp activity: the highest Dpp activity levels, in combination with certain other factors, cause development of the most dorsal tissue of all—an extraembryonic membrane. Intermediate levels cause development of dorsal epidermis; and the absence of Dpp activity allows the development of neurogenic ectoderm (Figure B above). 

A Hierarchy of Inductive Interactions Subdivides the Vertebrate Embryo
The molecular genetic analysis of Drosophila development has uncovered how a cascade of transcription regulators and signaling pathways subdivides the embryo. The same principle of progressive pattern refinement is used during the development of all animal embryos, including vertebrates. Remarkably, conservation is not restricted to the general strategy of pattern formation, but also extends to many of the molecules involved.  The earliest phases of vertebrate development are surprisingly variable, even between closely related species, and it is even hard to say precisely how the axes of an early fly embryo correspond to those of an early frog or mouse embryo. Nevertheless, we shall see that amid this display of plasticity, some features of early development turn out to be highly conserved. The same is true of later developmental stages also, often to an astonishing degree. Vertebrate embryos are patterned by the interplay of signaling molecules and transcription regulators. The origins of the embryonic axes and the three germ layers in the frog can be traced back to the blastula. By labeling individual blastomeres, we can track cells through all their divisions, transformations, and migrations and see what they become and where they come from. The precursors of ectoderm, mesoderm, and endoderm are arranged in order along the animal-vegetal axis of the blastula: the endoderm derives from the most vegetal blastomeres, the ectoderm from the most animal, and the mesoderm from a middle set. Within each of these territories, the cells have diverse fates according to their positions along the D-V axis of the later embryo. For ectoderm, epidermal precursors are located ventrally, and future neurons are found dorsally; for mesoderm, precursors for notochord, muscle, kidney, and blood are arranged from dorsal to ventral. All this can be represented by a fate map that shows which cell types derive from which regions of the blastula


Blastula fate map in a frog embryo. 
The endoderm derives from the most vegetal blastomeres (yellow), the ectoderm from the most animal (blue), and the mesoderm from a middle set (green) that contributes also to endoderm and ectoderm. Different cell types
derive from different positions along the dorsoventral axis.

The fate map confronts us with the central question: how are the cells in different positions driven toward their different fates? We have already explained how maternal factors deposited in the developing frog egg define its animal-vegetal axis, and how cortical rotation triggered by fertilization defines the orientation of the dorsoventral axis. But how does the establishment of axes lead on to the subdivision of the embryo into the future body parts? The maternal gene products lead to the formation of signaling centers on the vegetal and dorsal sides of the embryo. The dorsal signaling center in particular has a special place in the history of developmental biology. Experiments in the
early twentieth century identified it as a small cluster of cells, located on the dorsal side of the amphibian embryo, with an extraordinary property: when the cells were transplanted to an opposite site, they could trigger a radical reorganization of the neighboring tissue, causing it to form a second whole-body axis


Induction of a secondary axis by the Organizer. 
An amphibian embryo receives a graft of a small cluster of cells taken from a specific site, called the Organizer region, on the dorsal side of another embryo at the same stage. Signals from the graft organize the behavior of neighboring cells of the host embryo, causing development of a pair of conjoined (Siamese) twins.

The discovery of this signaling center, called the Organizer, led the way to a pioneering analysis of the chain of inductive interactions that establish the framework of the vertebrate body. In contrast to the Drosophila syncytial embryo, the fertilized frog egg undergoes rapid cleavage divisions that result in an embryo consisting of thousands of cells. Patterning must therefore be mediated by extracellular signal molecules that diffuse through the embryo from cell to cell, not by transcription regulators that move through the cytoplasm of a syncytium. Not surprisingly, the Organizer is now known to be a major source of secreted protein signals.

A Competition Between Secreted Signaling Proteins Patterns the Vertebrate Embryo
The signal molecules that pattern the frog embryo along the animal-vegetal (A-V) axis belong to the TGFβ family: they are secreted by a signaling center at the vegetal pole and form concentration gradients along the A-V axis. The Nodal protein acts over a relatively short range: cells near the vegetal pole are exposed to high levels of it and respond by switching on genes that promote the development of endoderm; cells further away are exposed to lower levels and activate genes that promote the formation of mesoderm. The cells at the vegetal pole that produce Nodal also produce a more rapidly diffusing TGFβ-like protein called Lefty, which antagonizes Nodal. The result is a high ratio of Lefty to Nodal at the animal pole, where Lefty predominates and Nodal signaling is blocked; this causes the cells there to develop as ectoderm


How Nodal and bone morphogenic protein (BMP) signaling pattern the embryonic axes. 
Nodal and its antagonist Lefty pattern the animal-vegetal axis, while BMP and its antagonists Chordin and Noggin pattern the dorsoventral axis. (A) In the animal pole region, where Nodal levels are low relative to Lefty, Lefty blocks
Nodal from binding to its receptors. In the vegetal region, there is an excess of Nodal, resulting in Nodal pathway activation. (B) Along the dorsoventral axis, BMP is widely present but Chordin and Noggin are concentrated at the dorsal side: there, they bind to BMP and block its binding to receptors. The resulting patterns of Nodal and BMP activity are illustrated at the bottom of the figure.

Thus, a mid-range activation by Nodal, combined with a long-range inhibition by Lefty, sets up the pattern of progenitors along the A-V axis for the three germ layers—endoderm, mesoderm, and ectoderm. The frog’s dorsal signaling system uses a different set of secreted signals from that of the vegetal signaling system to subdivide the germ-layer territories according to location along the D-V axis of the embryo. It exerts its influence by secreting two inhibitory signal proteins, called Chordin and Noggin. These antagonize the action of bone morphogenetic proteins (BMPs; members of yet another subclass of the TGFβ family), which themselves are secreted throughout the embryo. In this way, Chordin and Noggin form a dorsal-to-ventral gradient that blocks BMP signaling on the dorsal side but allows it to remain high on the ventral side (Figure B above). Ectodermal cells that experience high levels of BMP signaling are driven to epidermal fates, whereas cells that experience little or no BMP signaling remain neural. Knowing the signals that specify the three germ layers and various tissue types of the vertebrate body, one can reproduce this specification in a culture dish. Frog cells taken from the animal-pole region of the embryo, for example, will differentiate into blood (a ventral mesodermal tissue) when diverted from their original fate by exposure to intermediate concentrations of Nodal and high concentrations of BMP. Similarly, mouse or human embryonic stem cells can be coaxed to generate specific cell types by exposing them in culture to appropriate combinations of signal molecules. In this way, the insights gained through studies of animal development can be used to generate the cell types needed for regenerative medicine, as we discuss in the next chapter.

Comment: Signaling molecules specify the patterning of the developing embryo. 

The Insect Dorsoventral Axis Corresponds to the Vertebrate Ventral-Dorsal Axis
The signaling systems that pattern the D-V axis in Drosophila and in vertebrates are similar. In Drosophila, as we saw, Dpp and its inhibitor Sog are responsible, whereas in vertebrates, BMP and its inhibitors Chordin and Noggin do the job. Dpp is a member of the BMP family, while Sog is a homolog of Chordin. Both in flies and frogs, high levels of the inhibitors define the region that is neurogenic, and high levels of BMP/Dpp activity define the region that is not. These and other molecular parallels strongly suggest that this aspect of body patterning has been conserved in evolution from insects to vertebrates. Curiously, however, the axis is inverted: dorsal in the fly corresponds to ventral in the vertebrate. At some point in evolution, it seems that the ancestor of one of these classes of animals took to living life upside-down.


The vertebrate body plan as a dorsoventral inversion of the insect body plan. 
Note the correspondence with regard to the circulatory system as well as the gut and nervous system. In insects, the circulatory system is represented by a tubular heart and a main dorsal blood vessel, which pumps blood out into the tissue spaces through one set of apertures and receives blood back from the tissues through another set. Unlike vertebrates, insects have no system of capillary vessels to contain the blood as it percolates through the tissues. Nevertheless, heart development depends on homologous genes in vertebrates and insects, reinforcing the relationship between the two body plans.

Hox Genes Control the Vertebrate A-P Axis
The conservation of developmental mechanisms between Drosophila and vertebrates extends beyond the D-V signaling system. Hox genes are found in almost every animal species studied, where they are often grouped in complexes similar to the insect Hox complex. In mice and humans, for example, there are four such complexes—called the HoxA, HoxB, HoxC, and HoxD complexes—each on a different chromosome. Individual genes in each complex can be recognized by their sequences as counterparts of specific members of the Drosophila set. Indeed, mammalian Hox genes can function in Drosophila as partial replacements for the corresponding Drosophila Hox genes. It appears that each of the four mammalian Hox complexes is, roughly speaking, the equivalent of one complete insect Hox complex (that is, an Antennapedia complex plus a Bithorax complex)


The Hox complexes of an insect and a mammal, compared and related to body regions.
The genes of the Antennapedia and Bithorax complexes of Drosophila are shown in their chromosomal order in the top line. The corresponding genes of the four mammalian Hox complexes are shown below, also in chromosomal order. The gene expression domains in fly and mammal are indicated in a simplified form by color in the cartoons of animals above and below. There is a remarkable parallelism. However, the details of the patterns depend on developmental stage and vary somewhat from one mammalian Hox complex to another. Also, in many cases, genes shown here as expressed in an anterior domain are also expressed more posteriorly, overlapping the domains of more posterior Hox genes. 


Hox gene expression and function are similar in different animals
Left: A cladogram indicating major bilaterian taxa with diagrams showing the basic body plans of different species. 
Right: Complexes of Hox genes from the indicated taxa. The Drosophila Hox complex (HOM-C) boxed includes eight homeotic Hox genes and three that have taken on novel functions (bcd, zen, and ftz). Orthology to the Drosophila Hox genes is indicated by the color code. The homeotic Hox genes are expressed collinearly with their positions in the complex: lab is expressed more anteriorly than pb, which is expressed more anteriorly than Dfd, and so on with Abd-B expressed most posteriorly. This colinearity, as well as position of individual Hox orthologs within Hox complexes, is similar amongst various animal species.

And here the evolutionary storytelling goes: The complexes are thought to have evolved as follows: first, in some common the ancestor of worms, flies, and vertebrates, a single primordial homeotic selector gene underwent repeated duplication to form a series of such genes in tandem—the ancestral Hox complex. In the Drosophila sublineage, this single complex became split into separate Antennapedia and Bithorax complexes. Meanwhile, in the lineage leading to the mammals, the whole complex was repeatedly duplicated to give four Hox complexes. The parallelism is not perfect because apparently some individual genes have been duplicated and others
lost. Still, others have been co-opted for different purposes (genes in parentheses in the top line) over the time that has elapsed since the complexes diverged. The ordering of the genes within each vertebrate Hox complex is essentially the same as in the insect Hox complex, suggesting that all four vertebrate complexes originated by duplications of a single primordial complex and have preserved its basic organization. Most tellingly, when the expression patterns of the Hox genes are examined in the vertebrate embryo, it turns out that the members of each complex are expressed in a head-to-tail series along the axis of the body, just as they are in Drosophila. As in Drosophila, vertebrate Hox gene expression patterns are often aligned with vertebrate segments.

Comment: The authors conveniently neglect another possible ( and in my view more plausible) explanation: Namely that the creator, both of flies, and mammals, created both using the same design principles! The authors also do not mention, where the order of the first supposed common ancestor came from. 

This alignment is especially clear in the hindbrain, where the segments are called rhombomeres. The products of the vertebrate Hox genes, the Hox proteins, specify positional values that control the A-P pattern of parts in the hindbrain, neck, and trunk (as well as some other parts of the body). As in Drosophila, when a posterior Hox gene is artificially expressed in an anterior region, it can convert the anterior tissue to a posterior character. Conversely, loss of posterior Hox genes allows the posterior tissue where they are normally expressed to adopt an anterior. Because of redundancy between genes in the four Hox gene clusters, the transformations observed in mouse Hox mutants are not always so straightforward as those in the fly, and they are often incomplete. Nonetheless, it seems clear that the fly and the mouse use essentially the same molecular machinery to impart individual characteristics to successive regions along at least a part of the A-P axis.

Notch-Mediated Lateral Inhibition Refines Cellular Spacing Patterns
After the establishment of the basic body plan and the generation of organ precursors, many further steps of pattern refinement are required to achieve the adult pattern of terminally differentiated cells in tissues and organs. Lateral inhibition mediated by Notch signaling is crucial for both cell diversification and fine-grained patterning in an enormous variety of tissues in all animals. One example is the development of sensory bristles in Drosophila, most easily seen on the fly’s back, but also present on most of its other exposed surfaces. Each of these is a miniature sense organ, consisting of a sensory neuron and a small set of supporting cells. Some bristles respond to chemical stimuli, others to mechanical stimuli, but they are all constructed in a similar way


The basic structure of a mechanosensory bristle. 
The lineage of the four cells of the bristle—all descendants of a single sensory mother cell—is shown on the left. The sensory mother cell, once it is specified, generates this set of cells through a short program of division cycles.
In each generation of the progeny, lateral inhibition operates again to drive the newborn cells toward different fates: one of the ultimate progeny will become the neuron; another, the shaft of the bristle; others, supporting cells of various sorts. As the sensory mother cell and its progeny divide, certain proteins are allocated preferentially to one of each pair of newborn sister cells, biasing the outcome of the lateral-inhibition competition mediated by Notch signaling.

The proneural genes Achaete and Scute mark the patches of epidermis within which bristles will form. Mutations that eliminate the expression of these genes at some of their usual sites block the development of bristles at just those sites, and mutations that cause expression in abnormal sites cause bristles to develop there. The initial cells expressing the proneural genes are called proneural cells, and they are primed to take the neurosensory pathway of differentiation, but which of the cells will actually do so depends on competitive interactions among them. In the first round of these interactions, a single cell within each small group of proneural cells is picked to serve as the progenitor of the bristle. This single cell is called the sensory mother cell. It becomes distinct from the other cells of the cluster through lateral inhibition mediated by the Notch signaling pathway. The cells in the proneural cluster initially all express both the transmembrane receptor Notch and its transmembrane ligand Delta, along with proteins that regulate the signaling activity of Delta. Wherever Delta activates Notch, an inhibitory signal is transmitted that diminishes the tendency of the Notch-activated cell to specialize as a sensory mother cell. At first, all the cells in the cluster inhibit one another. However, receipt of the signal in a given cell diminishes that cell’s ability to fight back by delivering the inhibitory Delta signal in return. This creates a competitive situation, from which a single cell in each cluster—the future sensory mother cell—eventually emerges as winner, sending a strong inhibitory signal to its immediate neighbors but receiving no such signal in return


Lateral inhibition.
(A) The basic mechanism of Notch-mediated competitive lateral inhibition, illustrated for just two interacting cells. In this diagram, the absence of color on proteins or effector lines indicates inactivity. 
(B) The outcome of the same process operating in a larger patch of cells. At first, all cells in the patch are equivalent, expressing both the transmembrane receptor Notch and its transmembrane ligand Delta. Each cell has a tendency to specialize (as a sensory mother cell), and each sends an inhibitory signal to its neighbors to discourage them from also specializing in that way. This creates a competitive situation. As soon as an individual cell gains any advantage in the competition, that advantage becomes magnified. The winning cell, as it becomes more strongly committed to differentiating as a sensory mother cell, also inhibits its neighbors more strongly. Conversely, as these neighbors lose their capacity to differentiate as sensory mothers, they also lose their capacity to inhibit other cells from doing so. Lateral inhibition thus makes adjacent cells follow different fates. Although the interaction is thought to be normally dependent on cell-cell contacts, the future sensory mother cell may be able to deliver an inhibitory signal to cells that are more than one cell diameter away—for example, by sending out long protrusions to touch them.

If a cell that would normally become a sensory mother cell is genetically disabled from doing so, a neighboring proneural cell, freed from lateral inhibition, will become a sensory mother cell instead. The sensory mother cell goes through a short program of further divisions to generate the set of cells that form the final bristle. Notch signaling acts repeatedly at successive stages in this program to drive the descendants of the sensory mother cell along different pathways and assign them to their various specialized fates. However, it does so in conjunction with additional mechanisms that bias the outcome of the competition mediated by lateral inhibition. Determinants that are asymmetrically localized inside the dividing cells have this role in sensory bristle development.

Homeotic genes
It is a fascinating thought that the single cell zygote contains all the information required for the development of the adult organism. Understanding how this information is encoded and deciphered is a major uncompleted scientific challenge. 10

Homeotic genes act within cells to select their developmental fate. Homeotic genes, and other genes with analogous functions in controlling cell fate are therefore known as selector genes. They determine segmental identity. Systematic screening for homeotic genes led to the identification of eight linked genes, collectively referred to as Hox genes, that affect the specification of particular segment identities. The complete loss of any Hox gene function causes transformations of segmental identity and is lethal in early development.

One of the most intriguing features of these Hox genes is that they are linked in two gene complexes in Drosophila, the Bithorax and Antennapedia Complexes; each complex contains several distinct homeotic genes. Furthermore, the order of the genes on the chromosome and within the two complexes corresponds to the rostral (head) to caudal (rear) order of the segments that they influence, a relationship described as colinearity



The Hox genes of Drosophila
Eight Hox genes regulate the identity of regions within the adult and embryo. The color coding represents the segments and structures that are affected by mutations in the various Hox genes.

The ability to visualize Hox and other gene expression patterns during development was crucial to understanding the correlation between gene function and phenotypes. All Hox genes are expressed in spatially restricted, sometimes overlapping domains within the embryo. These genes are also expressed in subsets of the developing larval imaginal discs, which proliferate during larval development and differentiate during the pupal stages to give rise to the adult fly. Homeotic gene products exert their effects by controlling gene expression during development and that the homeodomain binds to DNA in a sequence-specific manner. The homeobox gene family is large and diverse. In fact, the homeodomain motif is found in approximately 20 other distinct families of homeobox-containing genes, all of which encode DNA-binding proteins.

Sections of genes codify Transcription factors, which are used by the cell to turn other genes on or off. There is a class of proteins, containing a region of about sixty amino acids called “homeobox.” This class of proteins is called Hox proteins. In subsequent years homeotic proteins and other classes of control proteins have proven to be master regulators of developmental programs in animals. In animals, a master switch sets in train a whole cascade of lesser switches, where the initial regulatory protein turns on the genes for other regulatory proteins, which turn on other regulatory proteins, and so on. Eventually, after a pyramid of control switches, a regulatory protein activates a gene that actually does some of the construction work to build an animal’s body. But there’s another complication. A gene in an animal cell might be regulated not by just one or a few proteins, but by more than ten. What’s more, there may be dozens of sites near the gene at which the regulatory proteins might bind, with multiple separate sites for some regulatory proteins. 1

Animal bodies contain many different kinds of cells that have to be positioned in definite relationships to other cells, in order to be formed into organs and to connect to other parts of the body.

Cal Tech biologist Eric Davidson emphasizes what the task of building an animal demands:
The most cursory consideration of the developmental process produces the realization that the program must have remarkable capacities, for development imposes extreme regulatory demands…Metaphors often have undesirable lives of their own, but a useful one here is to consider the regulatory demands of building a large and complex edifice, the way this is done by modern construction firms. All of the structural characters of the edifice, from its overall form to minute aspects that determine its local functionalities such as placement of wiring and windows, must be specified in the architect’s blueprints. The blueprints determine the activities of the construction crews from beginning to end.

Homeobox Genes and the Vertebrate Body Plan
This family of related genes determines the shape of the body. It subdivides the embryo along the head-to-tail axis into fields of cells that eventually become limbs and other structures.  Starting as a fertilized egg with a homogeneous appearance, an embryo made of skin, muscles, nerves and other tissues gradually arises through the division of cells. Long before most cells in the emerging body begin to specialize, however, a plan that designates major regions of the body-the head, the trunk, the tail and so on is established. This plan helps seemingly identical combinations of tissues arrange themselves into distinctly different anatomical structures, such as arms and legs. Individual genes mediate some of the developmental decisions involved in establishing the embryonic body plan.

Comment: The make of plans and blueprints prior to something is made, and making decisions, is ALWAYS the result of intelligence. 

Key is a family of genes, known as homeobox genes, that subdivides the early embryo into fields of cells with the potential to become specific tissues and organs. 

Hox genes encode a group of transcription factors, responsible for developmental processes and the establishment of the body plan. All Hox genes and many other developmental transcription factors contain the homeobox, a DNA sequence encoding the functional DNA-binding domain. Hox genes are known for their colinearity: conserved arrangement on chromosomes that is the same as their order of activation along the body axis. The regulation is very precise, for example, the regions of activity of Hox genes are tightly confined to specific rhombomeres ( see picture below ) or to segments of the vertebrate anteroposterior body axis



The vertebrate Hox genes are synchronized: the expression domains of paralogs ( either of a pair of genes that derive from the same ancestral gene )  from the A, B, C and D clusters are virtually identical

The mechanisms responsible for the synchronous regulation of Hox genes and the molecular function of their colinearity remain unknown. Despite 35 years of active research, the mechanisms of Hox gene regulation have remained elusive. It has been argued that chromatin structure and histone demethylation play important roles in activation of Hox genes, but the mechanism precisely directing chromatin modifications to specific loci at the right time remains mysterious. Ultraconserved regions and regulatory elements have been found within the coding sequences of Hox genes, but the key questions remain unanswered. It is unknown what mechanism could be responsible for the exceptional synchronous colinearity of Hox gene clusters and the conserved synteny of other pairs of groups of homeobox-containing genes, however, the topology of chromatin has been proposed to play a role in the regulation of these genes. 2

1. Behe, Edge of evolution, page 116
2. https://www.nature.com/articles/srep35415#ref32
3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1891803/

7. Position and place in the body. This is crucial. Limbs like legs, fins, eyes etc. must all be placed at the right place.

The development of an organism from an undifferentiated single cell into a spatially complex structure requires spatial patterning of cell fates across tissues. Several different signaling pathways are essential for the formation of organized tissues, organismal form, and architecture. Spatial patterning of a developing animal requires that cells become different according to their positions in the embryo, which means that cells must respond to extracellular signals produced by other cells, especially their neighbors. In what is probably the most common mode of spatial patterning, a group of pluripotent cells start out with the same developmental potential, and a signal from cells outside the group then induces one or more members of the group to change their character. Some inductive signals depend on cell–cell contact; others act over a longer range and are mediated by molecules that diffuse through the extracellular medium or are transported in the bloodstream. Most of the known events in animal development are governed by a small number of signaling pathways.

A core set of key morphogens regulates development. Morphogens are signaling molecules forming long-range concentration gradients that pattern a field of cells. After morphogens are secreted from a group of cells, they are transported across tissues to establish a gradient. The transport of morphogens is mediated by kinesin and dynein motor proteins along the microtubule networks of axons. Morphogens near the local source get attached to the shuttle and the morphogen-shuttle complex is transported across tissues. Subsequently, the morphogen-shuttle complex is degraded, resulting in the morphogen being locally immobilized, and start forming a local gradient. Bioelectric signals and resting membrane potentials across tissues are also and additionally crucial for proper patterning in multiple organisms. Furthermore, there are also neurotransmitters ( chemical messengers that transmit a message from a nerve cell across the synapse to a target cell) establish left-right patterning in embryos through regulation of ion fluxes. Calcium (Ca2+) signaling also plays a key role in mediating cellular responses to morphogens. In addition to mediating cellular differentiation and proliferation, Ca2+ signals also influence cellular migration in response to morphogen gradients.

Robustness is a ubiquitous feature of biological systems that ensures specific functions of the system are maintained despite external and internal perturbations. Furthermore, directional transport of substances through the nervous system is necessary to achieve scale-free morphogen patterning and body axis polarity determination.

Cells sense and interpret their position as a function of the amount of signal they receive, thus obtaining ‘‘positional information’’.  Within larger tissues, neural networks provide directed information, via physiological signaling, that supplements positional information through diffusion. This positional information specifies gene expression and subsequent fates of cells in tissues.

A few key-sentences: Cells operate upon biological design rules - Developing cells have a vocabulary - There is intercellular communication - Cells communicate with each other through cell-cell gap junctions, which exercise modulation, that is control, direct, induce, and regulate pattern formation - There is so-called physiological signaling, and there are positive diffusion regulators -  There are as well signals that propagate modulation of ion fluxes through voltage-gated ion channels -  There are signaling molecules that provide information for direction cues - There are as well bioelectrical signals -  Cells know how to interpret their position by obtaining ‘‘positional information’ through a coordinate system, which is modulated and mediated through morphogens that permit the formation of specific patterns through gradients. These regulate development; those also specify cell fates.

There is a general importance of mechanical forces in regulating growth and morphogenesis. Mechanical forces, cellular mechanics, and tissue mechanics are key components of morphogenesis and can influence pattern formation.

My comment: As a general scheme, it can be observed, that there are not simply chemical reactions, but molecules and cells operate, behave, migrate, position themselves, organize, form tissues, specialize based on instructions through various signaling networks and mechanisms, which have as source various epigenetic signaling pathways. Integrated, network-based information systems permit little if no errors, which undoubtedly lead to disease. This is prevented by robustness which has to be set up from the beginning. This leads to my understanding to intelligent design as the best explanation.    




Morphogen Gradients and Pattern Formation 1

Small Numbers of Conserved Cell–Cell Signaling Pathways Coordinate Spatial Patterning
Spatial patterning of a developing animal requires that cells become different according to their positions in the embryo, which means that cells must respond to extracellular signals produced by other cells, especially their neighbors. In what is probably the commonest mode of spatial patterning, a group of cells starts out with the same developmental potential, and a signal from cells outside the group then induces one or more members of the group to change their character. This process is called inductive signaling. Generally, the inductive signal is limited in time and space so that only a subset of the cells capable of responding—the cells close to the source of the signal—take on the induced character 


Inductive signaling.

Some inductive signals depend on cell–cell contact; others act over a longer range and are mediated by molecules that diffuse through the extracellular medium or are transported in the bloodstream. Most of the known inductive events in animal development are governed by a small number of highly conserved signaling pathways, including 

transforming growth factor-β (TGFβ), 
Wnt, 
Hedgehog, 
Notch, 
receptor tyrosine kinase (RTK) pathways

The discovery of the limited vocabulary that developing cells use for intercellular communication has emerged over the past 25 years as one of the great simplifying features of developmental biology.

Compartment boundaries are the sources of morphogens. Morphogens are signaling molecules that are produced from a localized source forming long-range concentration gradients that pattern a field of cells (Figure 7a). Cells interpret their position as a function of the amount of signal they receive, thus obtaining ‘‘positional information’’ (Wolpert, 1989, 1996). Signaling molecules have to fulfill two stringent criteria to qualify as morphogens: (1) their effect must be exerted in a concentration-dependent manner and (2) they must act directly on target cells at a distance from the source (i.e., not through a secondary relay mechanism). When these criteria are met, the local concentration can be interpreted as a measure of distance from the source of the signal (Figure 7a). The three signaling proteins that qualify as morphogens in Drosophila wing development are Hedgehog (Hh), Decapentaplegic (Dpp), and Wingless (Wg) (e.g., Wg is shown in Figure 7).


Figure 7 Example of a morphogen gradient: Wingless (Wg).
(a) Drawing of a section of the wing pouch epithelium (upper panel) and of the wing pouch in an apical view (lower panel). The cells in the center express Wg which generates a gradient across the adjacent tissue. Cells at different distances receive different levels of Wg. 
(b) Schematic drawing of the Wg gradient and target gene activation in the epithelium in response to Wg (upper panel). The Wg gradient activates the target genes hindsight (cyan) and distalless (blue) at different threshold levels. The expression domains of Hindsight and Distalless are depicted in the same colors in the lower panel. (c) Wg downregulates its own receptor Drosophila Frizzled 2 (DFz2; blue), shaping its gradient and rendering the
cells at a distance from the source of Wg more sensitive.

It is generally accepted that cells interpret the gradient by eliciting differential transcriptiona responses depending on the concentration of morphogen they are exposed to. This requires cells to make decisions depending on different threshold levels of signaling pathway activity. The concept implies that a single event – namely the production of a secreted molecule at a localized source – can lead to the formation of several different cell
types in a correct spatial relationship to each other (Figure 7). This represents a highly efficient way of generating complex patterns in previously uncommitted cells (review: Gurdon and Bourillot, 2001).

Different modes of morphogen movement/transport have been invoked to explain long-range gradient formation: extracellular diffusion of the secreted molecule, cycles of receptor- mediated endocytosis and resecretion (planar transcytosis) , membranous exosomes (argosomes), and cytoplasmic extensions (cytonemes). Although there is no compelling evidence to date that mechanisms other than diffusion contribute productively to gradient formation, it is certainly plausible that these mechanisms could do so. Cytonemes and argosomes exist, but have not yet been shown to be required to mediate ligand movement or to transduce signals. Evidence presented in favor of endocytosis and resecretion as a mechanism of ligand transport has been questioned. Further experimentalevidence is needed to support or refute these possibilities. Several factors have been implicated in shaping morphogen gradients: posttranslational modification of the ligand can influence diffusibility (e.g., acylation of Hh and Wg (Ingham, 2000, 2001)).
Regulation of receptor levels can influence ligand movement  (Figure 7c). Cell surface heparan-sulfate proteoglycans, and secreted enzymes that modify them, can modulate ligand
movement . Secreted ligand binding proteins can influence movement or stability . In addition, cellular responses to these ligands can also be modulated, contributing to shaping the activity gradients rather than the ligand gradients per se.

Interplay between morphogen‐directed positional information systems and physiological signaling 03 December 2019 4
The development of an organism from an undifferentiated single cell into a spatially complex structure requires spatial patterning of cell fates across tissues. Positional information, proposed by Lewis Wolpert in 1969, has led to the characterization of many components involved in regulating morphogen signaling activity. Quantitative and systems-based approaches are increasingly needed to define general biological design rules that govern positional information systems in developing organisms. There are various roles of physiological signaling in modulating and mediating morphogen-based pattern formation. Similarities between neural transmission and morphogen-based pattern formation mechanisms suggest underlying shared principles of active cell-based communication. Within larger tissues, neural networks provide directed information, via physiological signaling, that supplements positional information through diffusion. Further, mounting evidence demonstrates that physiological signaling plays a role in ensuring robustness of morphogen-based signaling. We conclude by highlighting several outstanding questions regarding the role of physiological signaling in morphogen-based pattern formation. Elucidating how physiological signaling impacts positional information is critical for understanding the close coupling of developmental and cellular processes in the context of development, disease, and regeneration. 2 

Lewis Wolpert proposed positional information as a mechanism whereby differential gene expression of cells results in spatial patterns of cellular differentiation. Within the concept of positional information, a coordinate system defines the magnitude and directionality of the positional information sensed by cells. This positional information specifies gene expression and subsequent fates of cells in tissues (Figure below)


A, classical view of positional information and morphogenesis. 
The classical view of positional information stems from the French Flag Model in which source cells secrete morphogens that are transported through a tissue due to diffusion. A, Morphogen molecules (green) are secreted from source cells where morphogen concentration will be the highest. Morphogens travel to neighboring cells to establish a concentration gradient. Cells will have differential biological responses to the morphogen gradient dependent upon multiple threshold levels. Blue cells are located where morphogen concentrations are above the higher threshold. White cells sense morphogen concentrations that are above the lower threshold. Red cells detect low to no morphogens. 
B, Cells respond to morphogens to determine cell fates and cell morphology in a dose-dependent response.
C, The ability of progenitor cells to create morphogen gradient-based patterns is dependent upon tissue geometry, size, temporal signaling of morphogens. 
D, Governing diffusion equation of the morphogen concentration in accordance with Fick's second law of diffusion with a nonlinear degradation profile (k1), a source term dependent upon location (k2), and effective diffusion of the molecule (Deff). This results in a powerlaw relationship in which the gradient is position-dependent and reflects the local steepness of the gradient

Following Wolpert, Gierer and Meinhardt proposed models of morphogen distribution to demonstrate that relatively simple molecular mechanisms can explain the formation of a spatially patterned tissue. Subsequent experimental work has demonstrated that a core set of morphogens generate and relay positional information to cells to directly induce cellular responses based on the cells' location. Because morphogens play crucial roles during the specification of cell fates, they contribute to many aspects of development. A core set of key morphogens regulates development. Examples include members of the Hedgehog (Hh) family that are involved in Drosophila appendage formation and chick neural tube development. As a second example, Wingless (Wg)/Int-1 (Wnt) proteins contribute to Drosophila appendage development and human degenerative diseases. Bone morphogenetic proteins (BMPs), such as Drosophila Decapentaplegic (Dpp), are utilized in dorsal-ventral patterning of the Drosophila embryo and pattern formation and growth control of limb primordia. BMPs also regulate the formation of early germ layers of mammals. Morphogen signaling includes conveying positional information, and their ability to induce pattern formation through gradients. One morphogen can control the expression of another during morphogenetic processes, such as Hh-induced Dpp activity in developing Drosophila and Hh participating in crosstalk with Wnt in cancer. On the other hand, computational modeling has proven critical for explaining increasingly complex datasets and counter-intuitive results from genetic perturbations to morphogenetic patterning systems. Additional computational efforts have uncovered the role of the nervous system in facilitating regeneration in Planaria.  This among many other studies support a close analogy between embryonic patterning and brain-like signal processing. However, in many cases the underlying kinetics and dynamics in gradient formation and maintenance remain poorly understood.

Morphogen-based signaling during development requires active cellular and physiological processes, as has been noted through analysis of the importance of lipid metabolism in morphogen transport. Here, we denote the term physiological signaling to represent cell regulatory mechanisms at the ionic and molecular level that control key cellular functions such as trafficking through exo- and endocytosis, metabolic processes of cellular growth and division, or the regulation of cell mechanics. Increasingly, evidence demonstrates the central role of physiological signaling events in mediating morphogen activity and emerging parallels between neurotransmission and morphogen transport across non-neural tissues. In particular, we highlight the functional roles of secondary messengers, such as calcium (Ca2+), in mediating morphogen secretion, transport, downstream information processing, and providing robustness of positional information. 

CELLULAR MECHANICS DIRECTLY INFLUENCE PATTERN FORMATION AND MORPHOGEN GRADIENTS 
Early studies of morphogenesis posited the notion that chemical-based morphogen gradients are the primary signals to pattern cell differentiation. However, there is emerging evidence demonstrating that mechanical events can also be a primary trigger in pattern formation. Mechanical forces drive cellular self-organization and structural rearrangements of a feather follicle's shape and gene expression in chicken embryos. In particular, mechanical activation of β-catenin initiated downstream follicle gene expression of bmp2, a key component of the TGF-β signaling pathway. This study thus serves as a striking example of the general importance of mechanical forces in regulating growth and morphogenesis.  Mechanical forces, cellular mechanics, and tissue mechanics are key components of morphogenesis and can influence pattern formation. The underlying mechanisms in which mechanics provide positional information to cells require further elucidation.

MORPHOGEN SOURCES ARE DYNAMIC AND CONTROLLED BY PHYSIOLOGICAL SIGNALING 
The conceptual French Flag model, an iconic description of positional information proposed by Wolpert, specifies that morphogens are secreted from a cluster of cells and form a graded distribution throughout the tissue to specify multiple cell types dependent on the concentration sensed by cells (Figure A above). Within this model, the secretion of morphogens from source cells is the initial step in the formation of positional information (Figure B above). This initial framework has since expanded from a static viewpoint to include dynamic changes to the size, geometry, location, mechanics, and temporal signaling of morphogen secreting cells (Figure C above). An example of a dynamic morphogen source occurs during the morphogenesis of dorsal appendages of the Drosophila melanogaster eggshell. The morphological boundaries of these structures depend on the spatial patterning transcription factor Broad r, which is regulated by the epidermal growth factor receptor (EGFR) signaling pathway through a feedback regulatory network. The patterning of Broad is established when Gurken, an EGFR ligand, is secreted from the underlying oocyte forming a posterior-to-anterior gradient. Later, a dorsoventral gradient forms after translocation of the oocyte nucleus to the dorsal anterior cortex.41-43 This suggests that morphogen sources are spatiotemporally dynamic and do not always adhere to the framework of passive diffusion-based transport from a stationary source (Figure D). This computational-based analysis of how subsequent rounds of EGFR activation refine spatial patterns was experimentally confirmed. Given the potential for spatiotemporally dynamic morphogen sources, secretion mechanisms of morphogens may provide insight into how this is possible.

Source cells secrete morphogens through exocytosis, an active transport mechanism of molecules to the cell membrane through vesicles. Fusion of vesicles to the cell membrane releases the morphogen. Therefore, morphogen secretion is influenced by physiological regulation of exocytosis. For example, inwardly rectifying potassium (Irk) channels regulate the release of Dpp and cytosolic Ca2+ in the developing Drosophila wing imaginal disc45 (Figure below).


Irk channels regulate Dpp secretion and cytosolic Ca2+. 
In the Drosophila wing disc, inwardly rectifying potassium channels (Irk) regulate both the secretion of bone morphogenic protein Dpp and cytosolic Ca2+ levels. Expression of Dpp along the dorsal-ventral (D-V) axis in the wing disc is indicated in red. Dpp expression is not present along the anterior-posterior (A-P) axis of the wing disc. Inhibition of Irk channels using dominant negative Irk2 mutants (Irk2DN) decreased the duration and amplitude of Ca2+ oscillations in Dpp-producing cells (yellow square indicates region of interest). Further, loss of Irk2 channel function increased the baseline concentration level of Dpp-GFP while simultaneously decreasing discrete Dpp release events. Thus, Irk2 inhibition in wing discs simultaneously alters intracellular Ca2+ activity and Dpp release 

Irk channels facilitate flow of potassium (K+) into the cell and have a fundamental role in restoring the membrane potential to its resting potential. Inhibition of Irk channels in Dpp producing cells, reduced Dpp secretion independent of Dpp expression.  Irk channels regulate vesicle release by changing membrane potential while also altering intracellular Ca2+ dynamics. A potential explanation for similar outcomes in Dpp and Ca2+ after Irk channel inhibition is that intracellular Ca2+ dynamics regulate Dpp release, although whether or not Ca2+ has a causative effect requires further study.  Ca2+ is crucial to regulation of epithelial physiology during development. However, whether Ca2+ directly regulates Dpp secretion is currently unknown. Given that misregulation of Ca2+ is frequently associated with developmental genetic disorders resulting in neoplasms, it is necessary to further characterize the roles of Ca2+ and other second messengers in morphogen transport and morphogenesis.

PARALLELS BETWEEN NEUROTRANSMISSION AND MORPHOGEN-BASED TRANSPORT SYSTEMS 
After morphogens are secreted from a group of cells, they are transported across tissues to establish a gradient. How morphogens disperse and form gradients is still under debate despite progress in understanding the molecular mechanisms of morphogen transport through imaging studies and biophysical measurements. Several morphogen transport models have been proposed in the literature. These range from passive mechanisms, such as free or hindered diffusion (first Figure D), to cell-based dispersal by transcytosis or cytonemes. Multiple transport mechanisms may be involved, and this likely varies across developmental contexts. The simplest mechanism of morphogen transport is passive diffusion where molecules disperse by random motion. However, this model does not fully capture the complexity of morphogen transport dynamics due to evidence demonstrating that a single source of morphogen is not always sufficient to establish a gradient. For example, during Drosophila wing disc development, DWnt6 is expressed in an identical pattern to Wg,57 while both BMP ligands Gbb and Dpp are necessary to establish proper morphogen gradients. In facilitated diffusion, morphogens are largely immobile until they bind to a positive diffusion regulator that enhances motility. Shuttling is a special case of facilitated diffusion in which molecular shuttles, not morphogens, are generated from a localized source. Morphogens near the local source get attached to the shuttle and the morphogen-shuttle complex is transported across tissues. Subsequently, the morphogen-shuttle complex is degraded, resulting in the morphogen being immobilized to stationary negative diffusion regulators and the formation of a gradient.

Beyond extracellular-diffusion-based morphogen transport, morphogens also can be transported through cell-based mechanisms via transcytosis or along cellular extensions known as cytonemes. In the case of transcytosis, signaling molecules are taken up by cells through endocytosis and subsequently released through exocytosis into the extracellular space. Endocytosis is the process in which cargo molecules, including morphogens, are absorbed and distributed into a series of endosomes with distinct physical and biochemical properties. An endosome's dynamics, such as fission, fusion, and maturation, influences its ability to sort and concentrate cargo molecules. For the case of morphogens, endosome dynamics are thus able to establish concentration gradients and signaling activities across tissues. Significant questions remain regarding which is the dominant mode of morphogen transport in a given developmental context. In the case of the Drosophila wing disc, Dpp can move through regions that are mutant for the type I transforming growth factor beta receptor thickveins (tkv) and downstream transcriptional repressor (Brinker). This is significant given that support for transcytosis stems from experiments utilizing the Dpp receptor Tkv. Due to the apparent conflicts between experimental studies, much work remains to clarify the roles of the physiological processes governing morphogen transport. An example of morphogen signaling being affected by exo- and endocytosis is seen in development of the Drosophila air sac primordia (ASP), which depends on Dpp signaling. Cytoneme-based signaling utilizes many of the same components found in neural synapses. In the Drosophila ASP (“receiving cells”), specialized filopodia-like cytonemes endocytose Dpp from the adjacent Drosophila wing disc cells (“sending cells”; Figure 3A). 


Morphogen transport mechanisms. 
A, Extended filopodia called cytonemes are present in the Drosophila air sac primordium (ASP). Cytonemes projecting from the ASP take up Dpp from the adjacent wing imaginal disc. Correlated transients of Ca2+ concentrations are observed in cytonemes. Cytoneme mediated transport requires Synaptotagmin-4 (Syt4), which helps in vesicle fusion and receptor internalization, and glutamate receptor GluRII in the ASP. Wing disc cells secreting Dpp require Synaptobrevin, Synaptotagmin-1, the glutamate transporter, and voltage-gated calcium channels. Blue circles represent morphogens, red circles represent pMad molecules in cells, and orange circles represent dpERK molecules in cells. B, In Planaria, morphogens are transported on microtubule arrays along the axons from the nervous system to the wound edge during regeneration. Vector transport is identified as a fundamental requirement for scale-free self-assembly of morphogens during Planaria homeostasis and regeneration. Figure is adapted with permission from Reference 

Dpp signaling was compromised when Synaptotagmin-1, Synaptobrevin, the glutamate receptor, or voltage-gated Ca2+ channels were inhibited in the secreting disc cells, resulting in a reduction in the size of the ASP. This result parallels neurotransmission as Synaptobrevin is an intrinsic membrane protein that regulates neurotransmitter release through Ca2 +-dependent exocytosis. The receiving cells of the ASP require Synaptotagmin-4 and the glutamate receptor GluRII. This is noteworthy given that Ca2+ is a crucial regulator of endocytosis 3 and neurotransmitter regulation. Huang et al further demonstrated that Ca2+ transients observed in cytonemes correlate with Dpp uptake. This suggests that signal uptake and transport within cytonemes depends on local Ca2+ concentrations. This study, thus, underscores the role of physiological signals, like Ca2+, in morphogen mediated transport and internalization that have not yet been fully explored. Further evidence for second messenger signaling and morphogen transport lies in the interaction between Ca2+ and endocytic and exocytic regulators. Knockdown of Ca2+ signaling-dependent exocytic components in the prothoracic gland in Drosophila brain, resulted in the accumulation of unreleased steroid hormone ecdysone. Whether the same exocytic machinery controls secretion of key morphogens is currently unknown. Reverse genetic RNAi screening could be employed to map out the components regulating morphogen secretion and transport through exocytosis. Additionally, synthesis of new alleles with mutations in Ca2+ binding domains of key morphogen regulatory proteins will further enable confirmation of the role of physiological signals in regulating morphogen secretion.

A key player in the endocytic process is the regulatory guanosine triphosphatase (GTP) protein Rab5. Rab5 proteins aid in the formation of transport vesicles and regulate molecular cargo degradation and recycling. Rab5 is required for endosome integrity in the presynaptic terminal in Drosophila neuromuscular synapses. Impaired Rab5 function affects both Exo- and endocytosis rates and decreases the ability of neurotransmitter release, while overexpression of Rab5 increases the release efficacy of neurotransmitter. This is particularly interesting because Ca2+ is an important regulator of neurotransmitter release. Recent work has shown that Ca2+ channels regulate bulk endocytosis, a form of endocytosis of synaptic vesicles at nerve terminals, in addition to coupling exo- and endocytosis. Further, Rab5-dependent endocytosis requires Ca2+ signaling to increase the rate of membrane capacitance, which determines how quickly the membrane potential can respond to a change in current and is linearly related to changes in membrane surface area. The converse was also shown in which low Ca2+ concentrations decreased membrane capacitance. Thus, an increase in surface area reflects increased exocytosis and decreased surface area reflects an increase in endocytosis. Additionally, discovery of a feedback loop showed BMP and Scrib, a scaffold protein associated with cellular proliferation, promotes Rab5 endosome-dependent BMP/Dpp signaling during morphogenesis in Drosophila.  Given that Ca2+ affects Rab5-related endocytosis and Rab5 affects Dpp signaling, it can be inferred that Ca2+ concentrations may serve as a potential modulator of morphogen transport through coupled exo- and endocytosis. However, this inference remains to be tested directly. Another possible mechanism through which physiological signals such as Ca2+ affect transport of morphogens is through Ca2+ binding domains of transport proteins. Evidence for this lies in the presence of Ca2+ binding EF-domains in proteins involved in the formation of BMP and Dpp gradients in the Drosophila wing disc. For example, Drosophila Pentagone (Pent) directly interacts with Dally to provide long range distribution of the Dpp ligand. Structurally Pent has a similar domain composition to that of human SMOC protein, and both Pent and SMOC proteins contain Ca2+ binding EF domains. Xenopus SMOC-1 (XSMOC-1) protein acts as a BMP antagonist in Xenopus embryos even in the presence of constitutively active BMP receptor.84 Further analysis suggests that SMOC-1 antagonizes BMP signaling downstream of receptor binding through activation of MAPK signaling. Later studies demonstrated the ability of Drosophila-specific Pent to similarly inhibit BMP signaling in Xenopus downstream of the BMP receptor after injection of synthetic pent mRNA into Xenopus embryos. 

Following this, Thomas et al utilized the SMOC deletion mutant constructs XSMOC-1ΔEC (lacking the extracellular Ca2+ binding domain) and XSMOC-1EC (containing the extracellular Ca2+ binding domain only) to demonstrate that normal XSMOC-1 and XSMOC1ΔEC, but not XSMOC-1EC convert the fate of naïve Xenopus ectoderm explants to anterior neural tissue. Thus, embryonic cell fate decisions to become neural tissue via SMOC, and BMP inhibition does not require the extracellular Ca2+ binding domain. However, the potentiation of BMP signaling by the extracellular Ca2+ binding domain was shown in further studies by observing the affinities of XSMOC-1EC and BMP2 for each other and other heparan sulfate proteoglycans (HSPGs). Experiments utilizing in vitro diffusion assays on agarose gels embedded with or without HSPGs demonstrated that the binding of BMPs to HSPGs restricts their range of effect and that BMP diffusion can be enhanced by the binding of SMOC to HSPGs to expand the range of effect. The SMOC extracellular Ca2+ binding domain expands the range of BMP signaling through competitive binding to HSPGs. These studies demonstrate the importance of the SMOC extracellular Ca2+ binding domain in the context of development. Further investigations are needed into the role of physiological signaling within the context of the extracellular environment.

 The coupling between neuronal and non-neural tissues in defining positional information extends beyond similarities in signaling mechanisms. A recent study provided further insight into the role of the nervous system in morphogen-based pattern formation and regeneration in Planaria.  A remarkable feature of regeneration in Planaria is the reformation of key morphogen gradients such as Hh and Notum Regulating Factor (NRF) after fragmentation or wounding of the organism. Pietak et al developed a quantitative model of regenerating Planaria to elucidate the mechanism of morphogen gradient activity that ensures robust body-plan regulation. The model predicts the fraction of heteromorphoses in regenerating Planaria fragments. Through an iterative combination of computational simulations and experiments, they found that vector transport of morphogens was required to explain regeneration of pattern formation. Morphogen vector-based transport is defined as the directional transport of morphogens by a vector field. The vector transport field coincided with the nerve polarity throughout regenerating planarian tissue. In their Markov chain model, the transport of morphogens, such as Hh and NRF, is mediated by kinesin and dynein motor proteins along the microtubule networks of axons (Figure B above)

They further demonstrated that the head-tail axis is controlled by the net polarity of neurons. In contrast, a purely diffusion-based model of patterning could not explain the scaling of steady-state concentrations of morphogens of fragments of various sizes. Further, diffusion would be too slow to regenerate the pattern (requiring more than a week on a 1 cm scale organism), while Planaria can reform within 72 hours or less. Thus, the nervous system plays an instructive role in regulating long-distance axial patterning repair during regeneration.

Bioelectric signals and resting membrane potentials across tissues are crucial for proper patterning in multiple organisms. Changes in membrane potential in regenerating Planaria, induced by ionophore treatment, permanently impacted gene expression, patterning, and polarity. After ionophore washout from the treated tissue, the induced changes in resting membrane potential persisted. This mechanism parallels synaptic plasticity in the brain where action potentials, modulated by voltage-gated ion channels, propagate signals. Work done in Xenopus and chick provides insight to this occurrence where serotonin (5-HT), an endogenous neurotransmitter, establishes left-right patterning in embryos through regulation of ion fluxes. A follow-up study showed that extracellular 5-HT availability drives innervation through tissues via gap junctional communication modulation. Combining their findings, Levin and colleagues propose that extracellular 5-HT, a positively charged molecule, navigates through gap junctions to accumulate in hyperpolarized cells, mimicking the reuptake function of 5-HT transporters (SERTs). These 5-HT sequestering, hyperpolarized cells can depolarize in response to loss of chloride ions via glycine-gated chloride channel activation. Without normal hyperpolarization, 5-HT is exported to the extracellular space by SERTs, which can induce growth and hyperinnervation of tissue. The importance of ion channels in facilitating communication in bacterial biofilm communities shows the generality of physiological signaling for mediating cell-to cell communication mechanisms. For example, bacterial biofilm communities utilize synchronized oscillations of short-range connectivity among a few cells and community-wide signaling, to minimize competition for resources. In sum, further quantitative investigations are needed to explore the role of neurotransmission, gap junction communication, and the nervous system in morphogen based transport systems. The establishment of morphogen gradients through neural signaling still needs to be integrated with known physiological regulators of patterning. This will be critical for considering how patterns form across large spatial domains where diffusion-based mechanisms become ineffective.

PHYSIOLOGICAL SIGNALS MODULATE MORPHOGEN-BASED POSITIONAL INFORMATION SYSTEMS 
Ca2+ signaling also plays a key role in mediating cellular responses to morphogens. Several studies in various model systems have shown the interplay between Hh signaling and Ca2+ dynamics. For example, Shaw et al showed that intracellular Ca2+ mobilization regulates the level of Sonic hedgehog (Shh)-dependent expression domains of nkx2.2b, isl1, nkx6.1, and pax3 genes in the developing nervous system of zebrafish embryos during the 18-somite stage. Furthermore, they showed that reduced expression of ryanodine receptor (RyR), an intracellular Ca2+-release channel, shifted the allocation of Shh-dependent cell fates in the somitic muscle and neural tubes in a manner that resembled the effects of reduced Shh signaling. These findings were discovered by utilizing loss-of-function mutations, antisense morpholino knockdowns, and pharmacological treatments to perturb RyR activity.

In another study, Belgacem and colleagues demonstrated that Shh acutely increases Ca2+ through the activation of the transducer Smoothened (Smo), which then recruits heterotrimeric GTP-binding protein-dependent pathways. They demonstrated that Shh increases Ca2+ spike activity of developing spinal neurons and propose that Ca2+ spike frequency encodes Shh concentration and is required for proper neuronal differentiation (Figure A). 


Physiological roles of Ca2+ in mediating morphogen response. 
A, A gradient of the morphogen Sonic Hedgehog (Shh) directs the patterning of neuronal differentiation in the developing Xenopus spinal cord. Shh increases Ca2+ spike activity in developing spinal neurons. A current model suggests that Ca2+ spikes convert noisy Hh signaling into binary outputs to specify cell fates. This was demonstrated by a positive relationship between activation of Hedgehog signaling through activation of the Shh transducer Smoothened (Smo) and stimulating Ca2+ spike activity. Loss of the Ca2+ spikes resulted in decreased GABAergic neuronal cell fates. This suggests Ca2+ spike frequency encodes Shh concentration and is required for proper neuronal differentiation . 
B, Tissue-wide, long-range Ca2+ oscillations have been observed in mesenchymal cells. Synergistic actions of Shh and Wnt signaling allowed synchronized Ca2+ oscillations to coordinate cell movements during chicken feather elongation

In addition to mediating cellular differentiation and proliferation, Ca2+ signals also influence cellular migration in response to morphogen gradients. Coordinated cell migration during chicken feather elongation is accompanied by dynamic changes of bioelectric currents and Ca2+ signaling. Specifically, Shh-responsive cells contained synchronized Ca2+ oscillations in which Shh plays a key role in mediating interactions between the epithelium and mesenchyme during feather morphogenesis. Voltage-gated Ca2+ channels and Connexin43-based gap junctions regulate Ca2+ dynamics during feather elongation.  Shh signaling and β-Catenin signaling activates Connexin-43 expression transcriptionally to establish the gap junctional network (Figure B). The establishment of gap junctional networks in growing tissues thus alters the spatial range of Ca2+ signaling

Further, a review of the role of gap junctions in regulation of pattern formation details that left-right asymmetry in C. elegans neurons involves Ca2+ signaling and communication through gap junctions.94,95 These studies further demonstrate the role of second messenger systems in mediating morphogen-induced responses of cells and the role of physiological signaling in morphogen-based pattern formation. Quantitative experiments investigating the interplay between second messengers and gap junctions during pattern formation are greatly needed. 

FEEDBACK FROM PHYSIOLOGICAL SIGNALING ENSURES ROBUSTNESS OF MORPHOGEN-BASED SIGNALING 
Important aspects of morphogen gradients as a source of positional information are robustness in the presence of genetic or environmental noise and the proper scaling of morphological patterns with respect to size. Robustness is an ubiquitous feature of biological systems that ensures specific functions of the system are maintained despite external and internal perturbations. Robustness of positional information can influence development significantly. Several recent studies have begun to elucidate the mechanisms governing pattern robustness. For instance, in the context of Planarian regeneration, it was shown that pure reaction-diffusion mechanism of morphogens fails to provide scale-free morphogen gradients.  The authors hypothesized that in addition to the classical mechanism involving diffusion, directional transport of substances through the nervous system is necessary to achieve scale-free morphogen patterning and body axis polarity determination. 

Question: How was that directing achieved? What if the robustness was not there right from the beginning?  

Overall, this study supports the idea of scale invariance in developing systems in which morphogen gradients are scaled properly despite external and internal perturbations. However, despite extensive work into the scaling mechanisms that regulate scaling of patterns at the tissue, organ, and organism level, biochemical mechanisms underlying patterning robustness remain to be discovered Regulation of downstream responses by physiological signals also suggest that physiological signals alter the robustness of morphogenetic processes. Ca2+ gradients are generated along the dorsal-ventral axis of the Drosophila embryo. These concentration gradients are formed during embryonic stage 5 with higher Ca2+ levels on the dorsal side. They also showed that manipulation of the Ca2+ gradient affects the specification of amnioserosa, located dorsally in Drosophila. This study underscores the importance of physiological signaling pathways that utilize Ca2+ in contributing to the specification of the dorsal embryonic region.  Ca2+ gradients affect robustness of morphogenetic processes. An outstanding question is whether this mechanism of specification of positional information is influenced by Ca2+ gradients in other model systems.  

Increasingly, evidence is accumulating for significant crosstalk of Ca2+ and ROS between endoplasmic reticulum, an established site of Ca2+ storage, and mitochondria, a generation site of ROS.102 In particular, Ca2+ diminished ROS from ROS generation sites within the mitochondria under normal conditions and enhanced ROS generation when generation sites were inhibited by pharmacological agents.103 Further, quantitative in vivo microscopy in Drosophila and zebrafish embryos identified ROS as crucial signals that regulate cell polarity after wounding.104 Hunter et al utilized fluorescent imaging after wounding Drosophila embryos and demonstrated Ca2+-dependent mitochondrial ROS production correlates with the site of actomyosin cable assembly. This suggests that wound-induced ROS production promotes healing in Drosophila and zebrafish embryos. Taken together, these studies demonstrate that physiological signaling, in the form of ROS and Ca2+, act to regulate robustness of morphogenesis. Further experiments are needed to elucidate how ROS and Ca2+ affect robustness of positional information in the context of development, regeneration, and disease.

Analysis on gene modular network reveals morphogen-directed development robustness in Drosophila30 June 2020 4
Genetic robustness is an important characteristic to tolerate genetic or nongenetic perturbations and ensure phenotypic stability. Morphogens, a type of evolutionarily conserved ( no evolutionary change) diffusible molecules, govern tissue patterns in a direction-dependent or concentration-dependent manner by differentially regulating downstream gene expression. However, whether the morphogen-directed gene regulatory network possesses genetic robustness remains elusive. In the present study, we collected 4217 morphogen-responsive genes along A-P axis of Drosophila wing discs from the RNA-seq data, and clustered them into 12 modules. By applying mathematical model to the measured data, we constructed a gene modular network (GMN) to decipher the module regulatory interactions and robustness in morphogen-directed development. The computational analyses on asymptotical dynamics of this GMN demonstrated that this morphogen-directed gene modular network (GMN) is robust to tolerate a majority of genetic perturbations, which has been further validated by biological experiments. Furthermore, besides the genetic alterations, we further demonstrated that this morphogen-directed GMN can well tolerate nongenetic perturbations (Hh production changes) via computational analyses and experimental validation. Therefore, these findings clearly indicate that the morphogen-directed GMN is robust in response to perturbations and is important for Drosophila to ensure the proper tissue patterning in wing disc.

Multimodal transcriptional control of pattern formation in embryonic development December 27, 2019 5
Predicting how the gene expression patterns that specify animal body plans arise from interactions between transcription factor proteins and regulatory DNA remains a major challenge in physical biology. While the modulation of transcriptional bursting has been implicated as the primary lever for controlling gene expression, we find that this alone cannot quantitatively recapitulate pattern formation. Instead, we find that the pattern arises through the joint action of 2 regulatory strategies—control of bursting and control of the total duration of transcription—that originate from distinct underlying molecular mechanisms. During embryonic development, tightly choreographed patterns of gene expression—shallow gradients, sharp steps, narrow stripes—specify cell fates. The correct positioning, sharpness, and amplitude of these patterns of cytoplasmic mRNA and protein ensure the reliable determination of animal body plans. Predicting developmental outcomes demands a quantitative understanding of the flow of information along the central dogma: how input transcription factors dictate the output rate of mRNA production, how this rate of mRNA production dictates cytoplasmic patterns of mRNA, and how these mRNA patterns lead to protein patterns that feed back into the gene regulatory network. While the connection between transcription factor concentration and output mRNA production rate has been the subject of active research over the last 3 decades, the connection between this output rate and the resulting cytoplasmic patterns of mRNA has remained largely unexplored. For example, a graded stripe of cytoplasmic mRNA within an embryo could arise as a result of radically different transcriptional dynamics at the single-nucleus level


Multiple modes of pattern formation by single-cell transcriptional activity.
(A–D) Cytoplasmic mRNA patterns (A) could arise from transcription factors exerting control over the mean transcription rate (B), the transcriptional time window dictating when a nucleus is transcriptionally active or quiescent (C), or the fraction of active nuclei (D) or some combination thereof.

The detailed cytoplasmic distribution of mRNA that makes these stripes is key to the transmission of spatial information along the gene regulatory network that drives Drosophila development and reinforcing the need to develop models of gene regulation capable of connecting quantitative variations in input transcription factor patterns to graded output rates of transcription. We found that all 3 regulatory strategies outlined in Fig. above quantitatively contribute to the formation of eve stripe 2.

First, a smaller fraction of nuclei become active and engage in transcription in the periphery of the stripe than in the center, although this regulation of the fraction of active nuclei makes only a minor contribution to stripe formation. Second,  the rate of mRNA production is significantly elevated in the center of the stripe.  This analog control of the transcription rate is insufficient to quantitatively recapitulate the cytoplasmic mRNA stripe pattern. In addition to the control of the rate of mRNA production among nuclei, there is a pronounced regulation of the window of time during which eve loci were engaged in transcription across the stripe, with those in the stripe center expressing for approximately 3 times longer than those in the flanks. While it is widely appreciated that genes are transcriptionally competent for limited windows of time during development, we found that—in the case of eve stripe 2—this binary transcriptionally engaged/disengaged logic is not merely a necessary precondition for pattern formation—it is the primary driver thereof. Thus, we conclude that the regulation of eve stripe 2 is multimodal in nature, with contributions from 3 distinct regulatory strategies (Fig.  B–D above). Nonetheless, stripe formation can be quantitatively explained almost entirely through the interplay between 2 distinct control strategies: binary control of the duration of transcriptional engagement.  (Fig. C) and control of the mean rate of transcription (Fig. B).

To describe eve stripe 2 transcriptional dynamics, we need to account for both the short, transient ON periods dictated by transcriptional bursts and a longer transcriptional time window that describes the period over which loci engage in this transcriptional bursting. One or more of these bursting parameters are subject to spatially controlled regulation.

In Drosophila development, information encoded in a handful of maternally deposited protein gradients propagates through increasingly complex layers of interacting genes, culminating in the specification of the adult body plan. A priori, there are several distinct regulatory strategies at the single-cell level capable of generating spatially differentiated patterns of cytoplasmic mRNA  each with distinct implications for the nature of the underlying molecular processes at play. The average rate of transcription is mainly modulated across the embryo by tuning the frequency of transcriptional bursting. It has remained unclear whether this modulation of the rate of transcription (and thereby mRNA production) is the dominant modality by which input concentrations of transcription factors drive the formation of patterns of gene expression or whether, instead, it is simply the most readily apparent mechanism among multiple distinct control strategies.

While it is widely appreciated that genes are expressed for discrete windows of time over the course of development, in the case of this eve stripe 2 reporter—this binary transcriptionally engaged/quiescent logic is actively regulated by transcription factors to drive pattern formation. Important is the temporal component of transcriptional regulation in specifying developmental outcomes.  The limited readout time imposed by short nuclear cycles in early Drosophila development places strict constraints on the kinds of regulatory architecture that could be responsible for driving observed patterns of hunchback gene expression. The pioneer factor Zelda plays a key role in regulating both the timing and probability of transcriptional activation following mitosis.  Our work complements these previous observations by exploring yet another aspect of the interplay between timing and transcriptional regulation.

Electromagnetism & Morphogenesis
1. One of the widely debated key questions in evolutionary biology is if traditional claims based on genetic mutations, natural selection, drift, and gene flow explain sufficiently the creation and maintenance of organismal form, complex biological systems,  from cellular to body, guiding morphogenesis.
2. Recent groundbreaking scientific discoveries are demonstrating that one key issue, cell migration (galvanotropism, galvanotaxis or electrotaxis) is not due to change in allele frequencies in genes, but endogenous electric currents and fields, generated by molecules, program the formation of extracellular molecular gradients which play ai instructive role, guiding and generating cues of the migratory trajectory of cells to their end destination in the body during development. Complex pattern formation requires mechanisms to coordinate individual cell behavior towards the anatomical needs of the organism. Alongside the well-studied biochemical and genetic signals functions an important and powerful system of bioelectrical communication. All cells, not just excitable nerve, and muscle utilize ion channels and pumps to drive standing gradients of ion content and transmembrane resting potential. Bioelectrical properties are key determinants of cell migration, differentiation, and proliferation. Spatio-temporal gradients of transmembrane voltage potentials are instructive cues that encode positional information and organ identity, and thus regulates the creation and maintenance of large-scale shape. In a variety of model systems, it is now clear that bioelectric pre patterns function during embryonic development, organ regeneration, and cancer suppression."
3. Furthermore, the collective oscillations of calcium Ca2+ ions on the surface of cell membranes also contribute to generating endogenous electromagnetic fields and there is information encoded both in the amplitude modulation and in the frequency modulation of Ca2+ oscillations. Calcium (Ca2+) oscillations are ubiquitous signals present in all cells providing efficient means to transmit intracellular biological information. They regulate a wide spectrum of cellular processes, including fertilization, proliferation, differentiation, muscle contraction, learning, and cell death. Information encoded in Calcium (Ca2+) oscillations generate a huge spatial and temporal diversity of signals since a Ca2+ response can exhibit infinite patterns.  Through an intricate concert of action between several Ca2+ transporters in the cell, the cytosolic Ca2+ concentration can start to oscillate, much like a radio signal. Specific information can thereby be efficiently encoded in the signal and transmitted through the cell without harming the cell itself. These endogenous electric fields generate three-dimensional coordination systems for embryo development. The genome is tightly linked to bioelectric signaling, via ion channel proteins that shape the gradients, downstream genes whose transcription is regulated by voltage, and transduction machinery that converts changes in bioelectric state to second-messenger cascades. The data clearly indicates that bioelectric signaling is an autonomous layer of control not reducible to a biochemical or genetic account of cell state.
4. Programming, the generation of instructions for complex pattern formation, coordination, communication, the encoding of information, orchestration of actions, the generation of informational radio signals, it's encoding, transmission, and decoding are all things exclusively done by intelligent minds with preset goals.
5. All those things are observed during morphogenesis and the development of complex organismal architecture. Therefore, the source of the biological development of complex multicellular organisms is best explained by intelligent design.

https://reasonandscience.catsboard.com/t2982-electromagnetism-morphogenesis

The recent groundbreaking scientific research which explains the real mechanisms of biodiversity
https://reasonandscience.catsboard.com/t2293-the-recent-groundbreaking-scientific-research-which-explains-the-real-mechanisms-of-biodiversity

Frequency decoding of calcium oscillations
https://sci-hub.ren/https://www.sciencedirect.com/science/article/pii/S0304416513005163

Establishing an animal body plan depends on many mechanisms that go beyond genetic information.
https://reasonandscience.catsboard.com/t2354-morphogen-gradients-and-pattern-formation

The recent groundbreaking scientific research which explains the real mechanisms of biodiversity ( The bioelectric code)
https://reasonandscience.catsboard.com/t2293-the-recent-groundbreaking-scientific-research-which-explains-the-real-mechanisms-of-biodiversity


1. INSECT DEVELOPMENT MORPHOGENESIS, MOLTING AND METAMORPHOSIS, page 70
2. https://anatomypubs.onlinelibrary.wiley.com/doi/pdf/10.1002/dvdy.140
3. https://en.wikipedia.org/wiki/Endocytosis
4. https://www.nature.com/articles/s41421-020-0173-z
5. https://www.pnas.org/content/117/2/836
6. https://sci-hub.tw/https://www.sciencedirect.com/science/article/pii/S0960982219303173

8. What communication it requires to communicate with other cells, and the setup of the communication channels

Signalling: Main topics on signalling
https://reasonandscience.catsboard.com/t2811-signalling-maintopics-on-signalling

Two major themes emerged from studies of these conserved genes: First, these genes are regulatory genes (sometimes called master regulatory genes) that are often members of large families of genes encoding proteins
with related functions. Second, these genes are utilized, albeit in somewhat different ways, in many, many animal species examined


The metazoan genetic toolkit for development. 
A large body of work demonstrates that the genetic toolkit governing development is highly conserved throughout the animal kingdom. Many of the constituent signaling molecules and transcription factor families are pan-metazoan. Examples of these ancient gene families are pictured in a toolkit (left). TF denotes “transcription factor” and SP denotes “signaling pathway.” It is claimed that as animal life became more complex, these toolkit genes were repurposed to allow for the development of new cell types, tissues, and structures, indicated by color changes shared between groups of toolkit genes and a corresponding embryo region (e.g., TF 2, TF 5, TF 6, and SP C families have been changed from ancestral green to derived magenta in Porifera lineage). Importantly, these toolkit genes are often used in more than one time or place in the embryo to help build unique gene networks and allow for greater cell diversity, which is indicated by genes boxed by an additional color representing a second embryo region (for example, TF 1 has cyan font boxed by red in the protostome lineage to indicate use in both the cyan- and red-colored embryo regions). In spite of the impressive reuse and repurposing of these toolkit components, there are also many examples of toolkit genes with deeply conserved developmental functions, indicated by a lack of color change (as examples, TF 3, TF 4, and SP B families are shown in green font in every depicted toolkit). The addition of completely new toolkit factors occurs but is rare (for example, TF 7 in the bilaterian lineage).

What has been more difficult to understand is how animal life can be so rich in diversity if essentially every animal is built by the same genetic toolkit.

Darwins Doubt, Stephen Meyer, page 235
Some evo-devo advocates such as Sean B. Carroll and Jeffrey Schwartz have pointed specifically to homeotic (or Hox) genes— master regulatory genes that affect the location, timing, and expression of other genes—as entities
capable of producing such large-scale change in animal form. These evo-devo advocates have broken with classical neo-Darwinism primarily in their understanding of the size or increment of mutational change.

MAJOR BUT NOT VIABLE, VIABLE BUT NOT MAJOR
Despite the enthusiasm surrounding the field, evo-devo fails, and for an obvious reason: its main proposal, that early-acting developmental mutations can cause stably heritable, large-scale changes in animal body plans, contradicts the results of one hundred years of mutagenesis experiments. The experiments of scientists such as Nüsslein-Volhard and Wieschaus have shown definitively that early-acting body-plan mutations invariably generate embryonic lethals— dead animals incapable of further evolution. The results of these experiments have generated the dilemma for evolutionary biologists that geneticist John McDonald aptly described as the “great Darwinian paradox.” Early-acting regulatory mutations do not produce viable alterations in form that will persist in populations, as evolution absolutely requires. Instead, these mutations are eliminated immediately by natural selection because of their invariably destructive consequences. On the other hand, later-acting mutations can generate viable changes in the features of animals, but these changes do not affect global animal architectures. This generates a dilemma: major changes are not viable; viable changes are not major. In neither case do the kinds of mutation that actually occur produce viable major changes of the kind necessary to build new body plans. In 2007, I coauthored a textbook with several colleagues titled Explore Evolution. In it, we explained this “either/or” (“major-not-viable, viable-not-major”) dilemma and suggested that it posed a challenge to theories that rely on the mutation and selection mechanism to explain the origin of major morphological changes. 

WHAT ABOUT HOX GENES?
Hox (or homeotic) genes regulate the expression of other protein-coding genes during the process of animal development. Some biologists have likened them to the conductor of an orchestra who plays the role of coordinating the contributions of the players. And because Hox genes affect so many other genes, many evo-devo advocates think that mutations in these genes can generate largescale changes in form. For example, Jeffrey Schwartz, at the University of Pittsburgh, invokes mutations in Hox genes to explain the sudden appearance of animal forms in the fossil record. In his book Sudden Origins, Schwartz acknowledges the discontinuities in the fossil record. As he notes, “We are still in the dark about the origin of most major groups of organisms. They appear in the fossil record as Athena did from the head of Zeus—full-blown and raring to go, in contradiction to Darwin’s depiction of evolution as resulting from the gradual accumulation of countless infinitesimally minute variations.” What resolves this mystery? Schwartz, an evo-devo advocate, reveals his answer: “A mutation affecting the activity of a homeobox [Hox] gene can have a profound effect—such as turning . . . larval tunicates into the first chordates. Clearly, the potential homeobox genes have for enacting what we call evolutionary change would seem to be almost unfathomable.” But can mutations in Hox genes transform one form of animal life—one body plan—into another? There are several reasons to doubt that they can. First, precisely because Hox genes coordinate the expression of so many other different genes, experimentally generated mutations in Hox genes have proven harmful. In fruit flies “most mutations in homeotic [Hox] genes cause fatal birth defects.” In other cases, the resulting Hox mutant phenotype, while viable in the short term, is nonetheless markedly less fit than the wild type. For example, by mutating a Hox gene in a fruit fly, biologists have produced the dramatic Antennapedia mutant, a hapless fly with legs growing out of its head where the antennae should be. Other Hox mutations have produced fruit flies in which the balancers (tiny structures behind wings that stabilize the insect in flight, called “halteres”) are transformed into an extra pair of wings. Such mutations alter the structure of the animal, but not in a beneficial or permanently heritable way. The Antennapedia mutant cannot survive in the wild; it has difficulty reproducing, and its offspring die easily. Similarly, fruit-fly mutants sporting an extra set of wings lack the musculature to make use of them and, absent their balancers, cannot fly. As Hungarian evolutionary biologist Eörs Szathmáry notes with cautious understatement in the journal Nature, “macromutations of this sort [i.e., in Hox genes] are probably frequently maladaptive.”

Second, Hox genes in all animal forms are expressed after the beginning of animal development, and well after the body plan has begun to be established. In fruit flies, by the time that Hox genes are expressed, roughly 6,000 cells have already formed, and the basic geometry of the fly—its anterior, posterior, dorsal, and ventral axes—is already well established. So Hox genes don’t determine body-plan formation. Eric Davidson and Douglas Erwin have pointed out that Hox gene expression, although necessary for correct regional or local differentiation within a body plan, occurs much later during embryogenesis than global body-plan specification itself, which is regulated by entirely different genes. Thus, the primary origin of animal body plans in the Cambrian explosion is not merely a question of Hox gene action, but of the appearance of much deeper control elements— Davidson’s “developmental gene regulatory networks” (dGRNs). Davidson argues that it is extremely difficult to alter dGRNs without damaging their ability to regulate animal development. Third, Hox genes only provide information for building proteins that function as switches that turn other genes on and off. The genes that they regulate contain information for building proteins that form the parts of other structures and organs. The Hox genes themselves, however, do not contain information for building these structural parts. In other words, mutations in Hox genes do not have all the genetic information necessary to generate new tissues, organs, or body plans. Nevertheless, Schwartz argues that biologists can explain complex structures such as the eye just by invoking Hox mutations alone. He asserts that “[t]here are homeobox genes for eye formation and that when one of them, the Rx gene in particular, is activated in the right place and at the right time, an individual has an eye.” He also thinks that mutations in Hox genes help arrange organs to form body plans. In a review of Schwartz’s book, Eörs Szathmáry finds Schwartz’s reasoning deficient. He too notes that Hox genes don’t code for the proteins out of which body parts are made. It follows, he insists, that mutations in Hox genes cannot by themselves build new body parts or body plans. As he explains, “Schwartz ignores the fact that homeobox genes are selector genes. They can do nothing if the genes regulated by them are not there.” Though Schwartz says he has “marveled” at “the importance of homeobox genes in helping us to understand the basics of evolutionary change,” Szathmáry doubts that mutations in these genes have much creative power. After asking whether Schwartz succeeds in explaining the origin of new forms of life by appealing to mutations in Hox genes, Szathmáry concludes, “I’m afraid that, in general, he does not.” Nor, of course, do Hox genes possess the epigenetic information necessary for body-plan formation. Indeed, even in the best of cases mutations in Hox genes still only alter genes. Mutations in Hox genes can only generate new genetic information in DNA. They do not, and cannot, generate epigenetic information. Instead, epigenetic information and structures actually determine the function of many Hox genes, and not the reverse. This can be seen when the same Hox gene (as determined by nucleotide sequence homology) regulates the development of different anatomical features found in different phyla. For instance, in arthropods the Hox gene Distal-less is required for the normal development of jointed arthropod legs. But in vertebrates a homologous gene (e.g., the Dlx gene in mice) builds a different kind of (nonhomologous) leg. Another homologue of the Distal-less gene in echinoderms regulates the development of tube feet and spines—anatomical features classically thought not to be homologous to arthropod limbs, nor to limbs of tetrapods. In each case, the Distal-less homologues play different roles determined by the higher-level organismal context. And since mutations in Hox genes do not alter higher-level epigenetic contexts, they cannot explain the origin of the novel epigenetic information and structure that establishes the context and that is necessary to building a new animal body plan.

Small numbers of Cell-Cell signaling pathways coordinate spatial patterning
Spatial patterning of a developing animal requires that cells become different according to their positions in the embryo, which means that cells must respond to extracellular signals produced by other cells, especially their neighbours. In what is probably the commonest mode of spatial patterning, a group of cells starts out with the same developmental potential, and a signal from cells outside the group then induces one or more members of the group to change their character. This process is called inductive signalling. Generally, the inductive signal is limited in time and space so that only a subset of the cells capable of responding—the cells close to the source of the signal—take on the induced character. Some inductive signals depend on cell–cell contact; others act over a longer range and are mediated by molecules that diffuse through the extracellular medium or are transported in the bloodstream. Most of the known inductive events in animal development are governed by a small number of highly conserved signaling pathways, including 

- transforming growth factor-β (TGFβ), 
- Wnt, 
- Hedgehog, 
- Notch, and 
- receptor tyrosine kinase (RTK) pathways. 

The discovery of the limited vocabulary that developing cells use for intercellular communication has emerged over the past 25 years as one of the great simplifying features of developmental biology. But how can this small number of signalling pathways generate the huge diversity of cells and patterns? Three kinds of mechanisms are responsible. First, through gene duplication, the basic components of a pathway often come to be encoded by small families of closely related homologous genes. This allows for diversity in the operation of the pathway, according to which family member is employed in a given situation. Notch signalling, for example, may be mediated by Notch1 in one tissue, but by its homolog Notch4 in another. Second, the response of a cell to a given signal protein depends on the other signals that the cell is receiving concurrently As a result, different combinations of signals can generate a large variety of different responses. Third, and most fundamental, the effect of activating a signalling pathway depends on the previous experiences of the responding cell: past influences leave a lasting mark, registered in the state of the cell’s chromatin and the selection of transcription regulatory proteins and RNA molecules that the cell contains. This cell memory enables cells with different histories to respond to the same signals differently. Thus, the same few signalling pathways can be used repeatedly at different times and places with different outcomes, so as to generate patterns of unlimited complexity.

Genes Involved in Cell-Cell Communication and Transcriptional Control Are Especially Important for Animal Development 
What are the genes that animals share with one another but not with other kingdoms of life? These would be expected to include genes required specifically for animal development but not needed for unicellular existence. A comparison of animal genomes with the genome of budding yeast—a unicellular eukaryote— suggests that three classes of genes are especially important for multicellular organization. The first class includes genes that encode proteins used for cell-cell adhesion and cell signaling; hundreds of human genes encode signal proteins, cell-surface receptors, cell adhesion proteins, or ion channels that are either not present in yeast or present in much smaller numbers. The second class includes genes encoding proteins that regulate transcription and chromatin structure: more than 1000 human genes encode transcription regulators, but only about 250 yeast genes do so. The development of animals is dominated by cell-cell interactions and by differential gene expression. The third class of noncoding RNAs has a more uncertain status: it includes genes that encode microRNAs (miRNAs); there are at least 500 of these in humans. Along with the regulatory proteins, they play a significant part in controlling gene expression during animal development, but the full extent of their importance is still unclear. The loss of individual miRNA genes in C. elegans, where their functions have been well studied, rarely leads to obvious phenotypes, suggesting that the roles of miRNAs during animal development are often subtle, serving to fine-tune the developmental machinery rather than to form its core structures.

9. Sensory and stimuli functions of cells
Animals possess sensory organs that collect information and convey this information to the central nervous system. 2
Sensory cells have extreme sensitivity for the adequate stimuli and efficient signal amplification. They use different solutions to the problem of detecting and encoding complex information, the sensory modalities of touch, vibration detection, hearing, vision, and olfaction. Pain perception is described as a sensory modality with very special features that differ fundamentally from those of other modalities. Polymodality of sensory cells, modulation by the immune system, and suppression by endorphins represent characteristic properties of the pain system, linked to its unique protective function. The process of sensory perception begins when sensory cells detect stimuli in the environment. Light acts on photoreceptors, sound on mechanoreceptors, and odorants on olfactory receptors - all highly specialized cells of eyes, ears, noses that are exposed to the outside world. Eyes, ears and noses are of almost unimaginable sensitivity, in fact, a sensitivity that has reached physical limits: A single photon, the movement of one atom, or a single odorant molecule can be detected by sensory cells of some animal species. This beautifully illustrates the power of the intelligent designer. The single-photon response of vertebrate photoreceptors indeed represents a sensory system driven to perfection. 

How sensory cells work
It requires a multitude of different sensory cells to carry out all our vital functions. The brain has to be informed on every relevant detail so as to be able to coordinate these functions in a sensible way.

Comment: That raises the question of how sensory cells could have emerged without the brain, if there is just a function for them, if duly interconnected with the brain, which processes the information.

The spectrum of information acquired by the brain is quite dazzling. The posture of the body, its supply with nutrients and oxygen, the state of the cardiovascular and digestive systems, as well as the body temperature and ion concentrations are constantly monitored by various types of sensory cells. Information about objects in the environment, their shape, color, chemical composition, their distance and movement are collected and conveyed to the brain. This steady and complex flow of information is then integrated and used to generate expedient behavior. However, the brain itself can only process information that is encoded in a language that consists of electrical discharges, termed action potentials. The task of a sensory cell is to convert the relevant stimuli into this language- a process that is called transduction. Transduction differs greatly between light-sensitive cells and cells that detect mechanical stimuli. But a few common principles can be outlined that illustrate the working of all sensory cells.


Functional components of a sensory cell.
An adequate stimulus acts on the cell's sensor, a structure specialized for the detection of this stimulus. The sensor triggers a transduction system which generates a chemical or electrical signal inside the cell. An amplification process increases the signal strength, using metabolic energy to boost the cellular response that is elicited by the stimulus. Finally, an output signal is generated in form of a series of electrical action potentials (spikes) that inform the brain about the detection of the stimulus

Comment: This is an irreducible, integrated, and interdependent system, where 1. sensor cells 2. a transduction system, 3. an amplification process, 4. metabolic energy, 5. the brain which interprets the information trough the language of the Neuronal spike-rate Code

The pivotal components of each sensory cell are specific sensory molecules that are contained in specialized cellular structures like cilia, microvilli or other membrane structures. Sensory molecules are highly specialized for their particular stimulus. If it is the right stimulus - the adequate stimulus - then the cell will respond even to very weak stimulation. The sensor may also respond to other stimuli, but not with high sensitivity. Thus, we may see stars with our photoreceptors at night in our bedroom in absolute darkness when we bump our eye against the edge of our wardrobe, thus receiving a strong mechanical stimulus. But the identity of a sensory cell and, indeed, of the entire sensory modality is defined by the adequate stimulus which elicits the sensory response; the ability of dim light to stimulate a sensory response defines a photoreceptor.

Following the uptake of the stimulus the next functional step is the transduction of the sensory signal - meaning the conversion of the extracellular stimulus into an intracellular signal. All cells operate with a certain repertoire of intracellular signals. These may be chemical or electrical signals which trigger the cell's internal responses to stimulation. There is only a limited number of such signals. Roughly ten different chemicals and basically four types of electrical signals carry such signals within all cell types of the body. In sensory cells, the sensor molecule must actuate at least one of them - for example a calcium signal or an electrical depolarization. In most cases, this task is fulfilled by ion channels residing in the plasma membrane of a sensory cell. Ion channels are proteins that can trigger both chemical and electrical signals, because ions - like the calcium ion

Ca2+ - can enter the cell through ion channels. These ion channels are termed transduction channels as their job is to start the transduction process. Once they are activated by the stimulus, the cell can start to process
the sensory signals. Sensory transduction virtually always includes a step of signal amplification. If the adequate stimulus consists of only a few photons or a few molecules of odorant, not much energy is fed into the sensory cell. However, this little energy must be converted into a robust output signal, usually a series of electrical potential changes that can be conveyed to the brain for analysis. The difference in energy between the input
and the output of a sensory cell is added to the sensory signal, thereby amplifying it. Different sensory cells have different amplification strategies. Not surprisingly, the most effective amplification strategies known are operating in sensory cells with high detection sensitivity.

Transduction of the stimulus energy to a first cellular response is followed by a process called encoding. Encoding leads to the generation of an electrical signal that contains the sensory information. Ideally, all aspects of the stimulus would be translated into the electrical code. Stimulus intensity, stimulus duration and other relevant parameters should be encoded in such a way that the brain is able to extract all this information by analysing the action-potential activity received from a sensory cell. The duration and shape of action potentials are uniform and therefore not useful for coding. The sensory information must, therefore, be encoded in the number of action potentials and in the time between them. This coding principle is called frequency modulation (FM). It is a very reliable method of information coding, as we know from the excellent quality of FM-coded music and speech in radio transmission. Thus, the final task of a sensory cell is to convert the amplified sensory signal into a message encoded in a frequency modulation that is then read out and deciphered in the brain, a process that leads in most cases to perception.

Touch, medium flow and mechanosensitive hairs
Touching things or being touched is arguably the most basic sensory experience. Even Paramecium, a unicellular ciliate, is able to register touch when it bumps into an obstacle. And, what is more important, it can properly respond to this experience. It stops, then swims backwards for a short distance, readjusts its heading, and continues to swim in the new direction - apparently to bypass the obstacle and to continue on its way. This is quite a remarkable accomplishment for a single-cell organism, and it illustrates that the processing of touch information is almost as old as life itself. Today's complex animals use all kinds of specialized structures to feel even the slightest touch. One of the most successful developments for this purpose was the combination of a hair-like structure and a sensory cell. Imagine a hair shaft, delicately suspended in a soft, elastic membrane, able to move into any direction upon the slightest touch, and connected at its base to the dendrite of a sensory cell and its sensor structure. If anything touches this hair, a force will act on the sensor and start the transduction process


Mechanosensory cells. 
A The combination of a hair and a sensory cell allows insects to detect medium flow signals like wind with extreme sensitivity. The deflection of a hair that is supported by a membrane is converted into a mechanical stimulus detected by a mechanosensory cell. 
B Model of the mechansosensory transduction channel that operates in the touch receptor of the nematode worm Caenorhabditis elegans. The channel consists of several proteins that are coassembled in the plasma membrane of the mechanosensory cell. It is tethered to the microtubule system inside the cell and to the cuticula outside of the cell. When the cuticula moves upon being touched, the transduction channel is pulled open and allows cation current to flow into the cell. This channel is an example of a multi-protein complex - in this case made of multiple MEC proteins. 

We find touch-sensitive hair-like structures on the surface of insects which are able to detect with these highly-sensitive mechanoreceptors air currents that may indicate an approaching mate or predator. The combination of hair-like structures and sensory cells  works perfectly well in higher animals. The motile whiskers of rats are a good example. The animals can feel their way in the dark by probing their environment with their whiskers. If anything touches these hairs, sensory cells at their base are activated. Physiologists think that the input from all whiskers is integrated by the animal's brain to form an image of objects that surround the rat's head - that the rat "sees" with its whiskers. While we do not sport whiskers ourselves, we have a less sophisticated form of the hair - sensory cell combination: the hair-follicle receptors. Each hair on our forearm is equipped with a sensory cell that picks up each movement of the hair and hence contributes essential information to the touch sensation of our skin. Although touch is such a basic, omnipresent sensory modality, we do not know much about the transduction mechanisms in mechanoreceptor cells. We know that our skin contains at least seven different types of mechanoreceptors apart from the hair-follicle receptors, and we have a good idea what they are there for. Some detect vibrations, others the touch intensity or the speed of an object moving along our skin. However, we do not know how the mechanical stimulus is converted into an output signal. The main reason for this ignorance is that our touch sensors are hidden in the skin and very difficult to study. Fortunately, one of biology's most popular model organisms, the nematode worm Caenorhabditis elegans, lends itself also to studies of touch reception. These animals respond to a gentle touch with an evasive movement that can be triggered by each of its six touch-sensitive cells. The underlying transduction mechanism was examined with immense effort by Martin Chalfie over a period of almost thirty years. It turned out that the transduction channel of the touch-sensitive cells is a protein complex in the sensor membrane, connected to the cuticula, the skin of the worm (Fig.B). When something touches the cuticula, the transduction channel is pulled open and causes an electrical signal. Thus, the worm operates its touch receptors by linking the transduction channel to the cuticula, just like the insect links its mechanoreceptor cell to a hairlike structure. Conceivably our touch receptors also work with such tethered transduction channels - but this is not yet known. Despite the manifold tasks that touch receptors perform in our lives, from explorative, tactile activity to social signalling, we know little about how they work. In fact, our sense of touch is the least understood of all our senses despite its fundamental importance. Maybe the worm can help us here.

From vibration detection to hearing
For many animals the perception of vibrations is even more important than touch sensation. Being touched by a predator marks the moment when it is often too late to escape. Vibrations, on the other hand, travel over some distance and can alert the animal well before the predator can strike. Vibrations in water or soil are caused byanimalsmoving around. They spread into the surroundings and warn every animal that is able to detect them. It is therefore not surprising that both fish and land animals have developed sensory organs for the detection of vibratory signals. Some of the vibration sensors found in animals are almost incredibly sensitive. Cockroaches possess inside their legs sensory organs that consist of a tiny horizontal membrane supported in air by a ring of sensory cells. Whenever the

How sensory cells work
It requires a multitude of different sensory cells to carry out all our vital functions. The brain has to be informed on every relevant detail so as to be able to coordinate these functions in a sensible way. The spectrum of
information acquired by the brain is quite dazzling. The posture of the body, its supply with nutrients and oxygen, the state of the cardiovascular and digestive systems, as well as the body temperature and ion concentrations
are constantly monitored by various types of sensory cells. Information about objects in the environment, their shape, colour, chemical composition, their distance and movement are collected and conveyed to the brain. This steady and complex flow of information is then integrated and used to generate expedient behaviour. However, the brain itself can only process information which is encoded in a language that consists of electrical discharges, termed action potentials.

The vertebrate head is equipped with complex paired sense organs that help in the active exploration of the environment.
The eyes comprise a lens for image formation and a retina with a large number of photoreceptors responding to light, several types of neurons involved in first steps of visual information processing (bipolar, amacrine, horizontal cells) and retinal ganglion cells, whose axons form the optic nerve connecting the retina to the forebrain.

The nose contains chemoreceptors allowing the detection of pheromones (vomeronasal cells) and other chemical stimuli (olfactory cells) which send their axons into the forebrain forming the vomeronasal and olfactory nerves. 

The inner ear has several sensory areas with mechanoreceptors (hair cells) employed for the perception of gravity and position and movement in space (vestibular areas: saccule, utricle, semicircular canals) as well as for sound (auditory area: cochlea) which transmit information via the vestibulocochlear nerve to the hindbrain. The lateral line system of fishes and amphibians uses a series of regularly spaced receptor organs on the body surface containing similar hair cells for the detection of water movements, while modified hair cells serve as electroreceptors in some lineages. 

Finally, there are additional small receptor organs (taste buds with chemosensory cells) mediating gustation and receptor cells or free nerve endings of sensory neurons mediating pain, temperature, or touch sensation.

The ability of any living organism to probe and sense stimuli emanating from the surrounding environment (exteroception), as well as to monitor bodily parameters (interoception), is of fundamental importance for its survival. Highly specialized primary sensory cells are tuned exquisitely to their respective sensory modality: photoreceptors can detect single photons, chemoreceptors respond to single molecules and mechanoreceptors sense mechanical deflections on the nanometer scale.

1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3077259/
2. https://sci-hub.st/https://link.springer.com/chapter/10.1007/978-3-211-99751-2_1

10.What specific new regulatory functions do cells have to acquire

Multicellular organisms are formed by specialized cells assembled in tissues. Individual cells contact and interact with other cells and with the extracellular matrix-a network of secreted proteins and carbohydrates that fills the intercellular spaces. The extracellular matrix helps cells to bind together and regulates a number of cellular functions, such as adhesion, migration, proliferation, and differentiation. It is formed by macromolecules, locally secreted by resident cells. The two main classes of macromolecules are polysaccharide glycosaminoglycans, usually covalently linked to proteins in the form of proteoglycans, and fibrous proteins of two functional types, structural (collagen, elastin) and adhesive (fibronectin, laminin, vitronectin, etc.). Receptors for extracellular matrix macromolecules are present in virtually all of the cells studied. They belong to the superfamily of integrins, afi heterodimers, which, in most cases, recognize the Arg-GlyAsp sequence of extracellular matrix proteins. On the exterior side of the cell, integrins link an extracellular matrix macromolecule, whereas in the cytosol, they bind the cytoskeleton, thereby forming a membrane bridge between extracellular and intracellular fibers. This structure enables the cell to adhere to the substratum. Similar to hormone- or growth factor-receptor binding, the interaction of the integrin with its specific ligand induces immediate signal transduction and influences cellular activities. 1

1. https://jasn.asnjournals.org/content/jnephrol/2/10/S83.full.pdf

11. When will the development program of the organism express the genes to grow the new cells during development?

Delphine Aymoz: Timing of gene expression in a cell‐fate decision system 2018 Apr 25 1
During development, morphogens provide extracellular cues allowing cells to select a specific fate by inducing complex transcriptional programs. The mating pathway in budding yeast offers simplified settings to understand this process. Pheromone secreted by the mating partner triggers the activity of a MAPK pathway, which results in the expression of hundreds of genes. The interplay between transcription factor binding and nucleosomes contributes to determining the kinetics of transcription in a simplified cell‐fate decision system. Cell‐fate decisions play a key role in crucial processes such as tissue repair, immune response, or embryonic development. In order to make choices, cells integrate cues from neighboring cells as well as from morphogens. Signal transduction cascades relay this information inside the cell to translate these extracellular signals into defined biological responses. The cellular output includes the induction of complex transcriptional programs where specific genes are expressed to different levels and at various times. Ultimately, these different expression programs will determine the fate of individual cells.

Temporal coordination of gene expression during development 31 May 2016 3
During development, cell specification and differentiation requires the spatial and temporal coordination of gene expression. This is facilitated through accurate interaction between intercellular signalling and gene regulatory networks. Several regulatory motifs driving gene expression have been identified at the cellular level, and we are beginning to understand how morphogens determine the spatial regulation of cells in a tissue.

Richard J White: A high-resolution mRNA expression time course of embryonic development in zebrafish Nov 16, 2017 3
Gene regulatory interactions are the fundamental basis of embryonic development and transcription is one of the major processes by which these interactions are mediated. A time-resolved comprehensive analysis of relative mRNA expression levels is an important step towards understanding the regulatory processes governing embryonic development.

The zebrafish is a unique vertebrate model system as it possesses high morphological and genomic conservation with humans, but also experimental tractability of embryogenesis otherwise only found in invertebrate model organisms such as Drosophila melanogaster or Caenorhabditis elegans. Features such as very large numbers of offspring, ex vivo development and embryonic translucency have enabled comprehensive forward and reverse genetic screens as well as high-throughput drug discovery approaches. Together with a high quality genome only comparable in vertebrates to mouse and human, this has led to many important discoveries in areas such as zygotic genome activation, blood stem cell biology, and findings directly affecting human health.

The morphological processes underlying the transformation of a fertilized egg into a free swimming fish have been studied extensively owing to the ease with which embryogenesis can be observed and manipulated. This has identified many genes that drive crucial steps of the differentiation process.

The first baseline expression study in zebrafish was conducted by Mathavan et al. (2005) using microarrays. This profiled the expression of 14,904 genes across 12 time points from unfertilised egg to 2 days post-fertilisation (dpf). Other baseline transcriptome work in zebrafish has focused on either certain aspects of development such as the maternal-zygotic transition, the identification of specific transcript types or promoters.

Jacqueline Deschamps: Embryonic timing, axial stem cells, chromatin dynamics, and the Hox clock May 1, 2021 4
Collinear (Three or more points , , , ..., are said to be collinear if they lie on a single straight line. ) regulation of Hox genes in space and time has been an outstanding question ever since the initial work of Ed Lewis in 1978. Here we discuss recent advances in our understanding of this phenomenon in relation to novel concepts associated with large-scale regulation and chromatin structure during the development of both axial and limb patterns. This sequential transcriptional activation marks embryonic stem cell-like axial progenitors in mammals and, consequently, how a temporal genetic system is further translated into spatial coordinates via the fate of these progenitors.

During the development of vertebrate animals, Hox genes provide positional information to the emerging embryonic axial tissues, thereby instructing them how to undergo appropriate morphogenesis. The underlying molecular mechanisms are diverse, depending on the ontogenetic and phylogenetic contexts. Various mechanisms seem to be in place in bilaterian animals, depending on their developmental strategies to secure an appropriate spatial coordination in Hox gene expression along the emerging embryonic axes. However, in many classes of bilateria, this coordination is associated with the process of “collinearity,” whereby clustered Hox genes are functional in a series of spatial domains that recapitulates the order of the respective genes in their genomic cluster. Nevertheless, this rule has many exceptions, and animals as diverse as the urochordate Oikopleura or the fruit fly Drosophila display collinear Hox gene expression either without any Hox clusters or with a split series of genes, respectively, thus illustrating that various mechanisms can eventually deliver similar information.

Vertebrates organize their bodies through progressive growth from anterior to posterior structures. Hox genes are activated in a timed sequence, which follows their 3′-to-5′ genomic order—a process referred to as “temporal collinearity”. This time-controlled transcriptional activation (the Hox clock) has been observed thus far only in animals containing an intact cluster of genes and occurs in a growth zone, a progenitor region located at the posterior aspect of the extending body axis. Therefore, temporal collinearity is a property displayed by animals with an anterior-to-posterior developmental progression in time, whereas strategies relying mostly on other developmental principles that do not involve a progressive anterior-to-posterior determination do not call for this process. As a consequence, they may not display an intact Hox cluster. In Drosophila, for instance, transcriptional activation of Hox genes is not sequential in time but instead depends on the regional activities of both gap and segmentation genes.

Mammals achieve the appropriate Hox-mediated spatial patterning through the initial timed-sequenced activation of their Hox clusters in response to early embryonic signals. During axial elongation, they use this early timing mechanism to synchronize Hox gene expression with the progressive generation of the trunk and tail from the posterior embryonic growth zone. This Hox clock is critical for the spatial distribution of the patterning information both in various axial tissues and along the appendicular axes (Fig. 1).


Figure 1. Collinear expression of Hox genes during the development of trunk axial tissues and limbs. (Left) Early: Schematic drawings of posteriorly overlapping transcript domains of HoxD genes in developing trunk axial tissues and early limb buds of an embryonic day 9.5 (E9.5) mouse embryo. Hox-expressing tissues are neural tube (midline); somites (blocks along the neural tube) labeled as cervical (C), thoracic (T) and lumbar (L); forelimb mesoderm (lateral bulges at the level of somites 7–12); and nascent mesoderm and neurectoderm in the tailbud. (t) Time of anterior to posterior development. Colinearity between the positions of the genes in the cluster and the anterior extension of their expression domains along the antero–posterior embryonic axis is illustrated both in the schematic embryo and by the bars under the cluster drawn below. Hox1 is expressed from an anterior limit that is the most rostral of all Hox genes in the embryo (expression not shown in the embryo; no color in the bar), and Hox8 is expressed from an anterior limit that is less rostral than that of Hox1 in the embryo. Posterior to this Hox8 anterior expression boundary, all genes between 1 and 8 are expressed (green color in the schematic embryo and in the bar corresponding to Hox8). Similarly, posterior to the Hox10 expression boundary in the embryo, all genes between Hox1 and Hox10 are expressed (lighter blue in the schematic embryo and in the bar corresponding to Hox10), and similar representations illustrate the expression of Hox11, Hox12, and Hox13. These domains thus tend to overlap posteriorly in the embryo, like Russian dolls. Expression of other Hox genes is not shown. (Right) Late: Hox transcript distribution in the E10.5 developing tailbud and forelimb bud. The two domains in late forelimb buds mark the future proximal (arm and forearm) and distal (digits) parts of the adult limbs, respectively. (t) Time of development of proximal to distal limb structures. Color codes indicate the cumulative amounts of combinations of Hox transcripts. Anterior is to the top in all schemes.

Initial Hox gene activation responds to time-dependent embryonic signals acting on enhancers located on the early side of the cluster, and collinear Hox gene expression is relayed to the regional tissue anlagen that generates axial structures. The cis-acting mechanisms underlying this time-dependent developmental sequence may constitute an important constraint in the organization of an evolutionarily conserved Bauplan in vertebrates.

Relay from early temporal to spatial Hox expression: a mechanism conserved in vertebrates
Recent findings in zebrafish in addition to previous literature suggest that the translation of an initial Hox expression timing into successive anterior-to-posterior instructions to axial tissues is a feature conserved throughout vertebrates despite the apparent differences in how these organisms gastrulate. In amphibian and murine embryos, for example, Wnt signals precede the onset of Hox gene activation in a ring around the marginal zone in Xenopus laevis or along the corresponding structure in mice—the primitive streak, respectively. In both species, sequential Hox gene transcription further expands into presumptive trunk tissues as gastrulation proceeds. However, slight differences exist; Hox transcripts are found in a large marginal zone in amphibians that converges toward the blastopore lip, whereas the tiny initial murine Hox domains expand anteriorly along the streak toward the regions containing MPs and NMPs. The situation in bird embryos is somewhat similar to that in mice, while the situation in fish is closer to that of amphibians. The embryonic domain fated to contribute to trunk tissues is thus larger in early fish and amphibian embryos than in mice, where a small population of axial progenitors ensures most of the axial growth in the trunk. In zebrafish, NMPs contribute significantly to axial growth only in the tailbud, and a low number of multipotent progenitors are found at relatively late stages in the Xenopus tailbud.

Despite these differences, the translation of sequential Hox gene activation into spatial cues in axial tissue anlagen occurs in all vertebrates thus far examined, including amphibians. Since this feature is crucial for patterning along the main axis and because it is dependent on a timing device that is itself intrinsically linked to the physical arrangement of genes along a genomic cluster, we conclude that the meta-cis arrangement of Hox genes is constrained by the necessity to properly and safely implement the Hox clock. The existence of this particular constraint in all vertebrates was proposed earlier to coincide with the narrow passage of the phylotypic hourglass; i.e., a short time window during which various developmental strategies converge toward completion of axial growth. Recent data indicate that the core of this obligatory process includes the relay of temporally acquired Hox codes to the precursors of anterior to posterior axial tissues.

A link between the Hox clock and the somitic clock?
Once the successive waves of Hox gene expression have progressively labeled the axial progenitors for trunk tissues in the node-abutting posterior growth zone, the progenitors transmit their Hox addresses to their daughter cells in both the mesoderm and neurectoderm of the emerging axial tissues. Early progenitors relay an anterior code, while later progenitors transmit a more posterior genetic address until the progenitor population is exhausted by the end of axial elongation. In nascent paraxial mesoderm, the proliferation of Hox-instructed cells takes place concomitantly with the segmentation of the PSM into somites, a series of discrete compartments that foreshadows the future vertebrae. The sequential production of somites results from an oscillatory mechanism (the “somitic clock”), and potential connections between the two clocks were documented in both mice  and chicks, where early expression of several anterior Hox genes was observed to cycle and follow a particular phase of the somitogenesis cycle.

Nevertheless, this connection between both clocks has not yet been functionally validated, and thus a mechanism synchronizing these clocks is still elusive. The necessary genetic approaches are complicated due to the high level of redundancy found in the developing PSM, where all four Hox gene clusters are at work. Alternatively, the somitic clock may operate independently from the Hox clock; i.e., by segmenting a tissue where the set of Hox addresses would already be properly distributed due to its earlier activation.

Hox genes and the control of body length
The length of the trunk depends on the activity of axial progenitors. In addition to niche factors, which are essential for maintaining these progenitors, the pluripotency factor Oct4 is a crucial player in determining progenitor activity. A sufficiently high level of Oct4 expression in the posterior aspect of the early mouse embryo is essential to maintain the pluripotency network active in the epiblast, and Oct4 levels normally decrease at the three- to five-somite stage. Experimental stimulation of early Oct4 expression produced a longer trunk in mice, as shown by using the Cdx2 promoter driving Oct4 in early mouse embryos. Aires et al. (2016) increased both the level and the time of expression of Oct4 in posterior epiblasts, including in the caudal lateral epiblast and the NMP region. This presumably overruled the reduction in Oct4 expression and associated decline in pluripotency, which normally occurs in posterior epiblasts after the three- to five-somite stage, and allowed for an extended activity of the pluripotency network, leading to a longer trunk.

Hox genes, together with the related Cdx genes, also play a role in trunk extension. Therefore, they do more than merely confer axial identity to the emerging tissues. Cdx genes are required in a dosage-dependent manner for the generation of post-occipital embryonic axial tissues (van Rooijen et al. 2012). Cdx2 transcriptionally activates Wnt and Fgf signaling pathway components in posterior embryonic tissues (Amin et al. 2016) and thus maintains the proficiency of the axial progenitor niche. Accordingly, experimental stimulation of both the Wnt and Fgf pathways rescues the axial truncation of Cdx mutants at least in part (Young et al. 2009; van Rooijen et al. 2012). Middle or trunk Hox genes can substitute for Cdx genes in axial extension, demonstrating that these Hox gene products stimulate trunk growth, presumably by maintaining a proficient niche of axial progenitors. The trunk growth-stimulating action of Cdx and Hox genes is amplified by a feed-forward activation of Hox genes by Cdx2 .

After the trunk-to-tail transition, posterior Hox genes become highly expressed in most caudal embryonic tissues. These genes—in particular Hox13—retroinhibit more anterior Hox genes. In addition, they antagonize Cdx2 and more centrally located Hox genes in their task of axial stimulation by directly interfering with activation of the Wnt and Fgf pathways. Posterior Hox genes thus intervene to slow down and interrupt the axial elongation process by preventing Cdx and middle Hox genes from further activating axial progenitors, MPs, and NMPs at caudal axial levels.

1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5916086/#:~:text=Some%20cells%20will%20induce%20the,first%20wave%20of%20gene%20expression.
2. https://cordis.europa.eu/article/id/191067-gene-expression-during-development-timing-matters
3. https://elifesciences.org/articles/30860
4. http://genesdev.cshlp.org/content/31/14/1406.full