Gene Regulatory Networks Controlling Body Plan Development

Gene Regulatory Networks Controlling Body Plan Development 1 

https://reasonandscience.catsboard.com/t2318-gene-regulatory-networks-controlling-body-plan-development

Animal evolution has been permitted or driven by gene regulatory evolution. 1

EVOLUTIONARY BIOSCIENCE AS REGULATORY SYSTEMS BIOLOGY 1

Never in the modern history of evolutionary bioscience have such essentially different ideas about how to understand evolution of the animal body plan been simultaneously current. The first is the classic neo-Darwinian concept that evolution of animal morphology occurs by means of small continuous changes in primary protein sequence which in general require homozygosity to effect phenotype. The second paradigm holds that evolution at all levels can be illuminated by detailed analysis of cis-regulatory changes in genes that are direct targets of sequence level selection, in that they control variation of immediate adaptive significance. An entirely different way of thinking is that the evolution of animal body plans is a system level property of the developmental gene regulatory networks (dGRNs) which control ontogeny of the body plan.

Just as development is a system property of the regulatory genome, causal explanation of evolutionary change in developmental process must be considered at a system level.
Never in the modern history of evolutionary bioscience have such essentially different ideas about how to understand evolution of the animal body plan been simultaneously current.
The first is the classic neo-Darwinian concept that evolution of animal morphology occurs by means of small continuous changes in primary protein sequence which in general require homozygosity to effect phenotype.

The second paradigm holds that evolution at all levels can be illuminated by detailed analysis of cis-regulatory changes in genes that are direct targets of sequence level selection, in that they control variation of immediate adaptive significance.

Both approaches often focus on changes at single gene loci, and both are framed within the concepts of population genetics.

An entirely different way of thinking is that the evolution of animal body plans is a system level property of the developmental gene regulatory networks (dGRNs) which control ontogeny of the body plan. It follows that gross morphological novelty required dramatic alterations in dGRN architecture, always involving multiple regulatory genes, and typically affecting the deployment of whole network subcircuits.
Because dGRNs are deeply hierarchical, and it is the upper levels of these GRNs that control major morphological features in development, a question dealt with below in this essay arises: how can we think about selection in respect to dGRN organization?  The answers lie in the architecture of dGRNs and the developmental logic they generate at the system level, far from micro-evolutionary mechanism. While adaptive evolutionary variation occurs constantly in modern animals at the periphery of dGRNs, the stability over geological epochs of the developmental properties that define the major attributes of their body plans requires special explanations rooted deep in the structure/function relations of dGRNs.

Neo-Darwinian evolution is uniformitarian in that it assumes that all process works the same way, so that evolution of enzymes or flower colors can be used as current proxies for study of evolution of the body plan. It erroneously assumes that change in protein coding sequence is the basic cause of change in developmental program; and it erroneously assumes that evolutionary change in body plan morphology occurs by a continuous process. All of these assumptions are basically counterfactual. This cannot be surprising, since the neo-Darwinian synthesis from which these ideas stem was a pre-molecular biology concoction focused on population genetics and adaptation natural history, neither of which have any direct mechanistic import for the genomic regulatory systems that drive embryonic development of the body plan.

Sequence level changes in cis-regulatory modules controlling expression of these genes are demonstrated to be the cause of these variations, and in general they operate by altering the response of the cis-regulatory module to the pleisiomorphic spatial landscape of regulatory states. Evolutionary change in a cis-regulatory module controlling downstream gene expression is of course far less pleiotropically dangerous to the whole system than if either the coding region of the gene had been mutated or if the upstream regulatory landscape had been altered (Prud'homme et al., 2007).

The arguments are that essentially all evolutionary changes in morphology are at root cis-regulatory, which is indeed basically true; and that intra-modular mechanisms of cis-regulatory evolution will operate on similar principles wherever it occurs, also true. But these assumptions do not suffice to support the uniformitarian conclusion about body plan evolution: when the properties of the gene regulatory networks that actually generate body plans and body parts are taken into account, it can be seen that many entirely new and different mechanistic factors come into play. The result is that just as the paleontological record of evolutionary change in animal morphology is the opposite of uniformitarian (see the paper of D. Erwin in this collection), so, for very good reasons that are embedded in their structure/function relations, are the mechanisms of dGRN evolution.

This rather obvious argument gives rise to additional specific consequences, which taken together provide a new set of principles that apply to the mechanisms of body plan evolution (Britten and Davidson, 1971,Davidson and Erwin, 2006 and Peter and Davidson, in press). They are new in that none are specifically predicted by classical evolutionary theory.

No observations on single genes can ever illuminate the overall mechanisms of the development of the body plan or of body parts except at the minute and always partial, if not wholly illusory, level of the worm's eye view.

A distinguishing feature of dGRNs is their deep hierarchy, which essentially stems from the long sequence of successive spatial regulatory states required to be installed in building first the axial embryonic/larval body plan, and then constructing individual body parts

the universe of possible responses is vastly constrained by dGRN hierarchy at each level transition, inevitably resulting in what was classically termed “canalization” of the developmental process

For example, a frequently encountered type of subcircuit in upstream regions of dGRNs consists of two or three genes locked together by feedback inputs (Davidson, 2010). These feedback structures act to stabilize regulatory states, and there is a high penalty to change, in that interference with the dynamic expression of any one of the genes causes the collapse of expression of all, and the total loss from the system of their contributions to the regulatory state.

the development of an embryo is extremely canonical even though, as in sea urchins, the exact size of the egg, the temperature, or the amounts of many regulatory gene transcripts ( Materna et al., 2010) may vary considerably.

Whatever continuous variation occurs at individual cis-regulatory sequences, the dGRN circuit output preserves its Boolean morphogenetic character.

Therefore the action of selection differs across dGRN structure. Selection does not operate to produce continuous adaptive change except at the dGRN periphery.

the system level output is very impervious to change, except for catastrophic loss of the body part or loss of viability altogether. As long realized and much discussed in a non-mechanistic way in advance of actual knowledge of dGRN structure and function (for review see Gibson and Wagner, 2000), this imperviousness has something to do with whatever processes generate canalization and/or “buffering” of the genetic control system. We can now begin to understand canalization mechanistically in terms of dGRN hierarchy and subcircuit structure, as above, but in so far as “buffering” is taken to mean protection against “environmental fluctuations” as in many evolutionary mathematical models, it is irrelevant to animal embryonic processes, since in the main these depend not at all upon environmental inputs.

the fundamental role of upper level dGRNs is to set up in embryonic space a progressive series of regulatory states, which functionally define first the regions of the body with respect to its axes; then the location of the progenitor fields of the body parts; then the subparts of each body part.

In embryonic development the transcriptional processes mediated by dGRNs are intrinsically insensitive to varying cis-regulatory input levels.

1. https://www.pnas.org/content/115/38/E8909

Some principles that emerge from the precept that evolution of the animal body plan occurs by alteration of genomic developmental GRNs

At the outset, the main point of difference between this and all other approaches to understanding the origin of the body plan is that this is a system approach to developmental evolution, in which answers derive from the topologies of regulatory gene interaction circuitry. No observations on single genes can ever illuminate the overall mechanisms of the development of the body plan or of body parts. The same must be true as well for major  change in the body plan or in body parts.

The mechanism underlying structural change in dGRNs depends on position change of cis-regulatory modules  to a new spatial and/or temporal domain of the developing animal. This tells us where to look in the regulatory system for differences in developmental patterning.  An important point is that while these mechanisms include gradual, continuous, and reversible SNP mutations, they also (and perhaps more importantly) encompass irreversible and discontinuous mutational events such as transposon-mediated sequence insertion and other mechanisms of sequence change that cannot be accommodated in neo-Darwinian algorithms.


The subcircuits at each level provide feeds to the next level in the same or, via signaling, in other specified spatial domains. But each subcircuit produces a finite set of inputs for the next level, and only recipient nodes that contain target site combinations can respond to those particular inputs. Thus, the universe of possible responses is vastly constrained by dGRN hierarchy at each level transition, inevitably resulting in what was classically termed “canalization” of the developmental process. A few years ago remarkably conserved subcircuits, termed network “kernels” that operate high in the dGRN hierarchy were discovered. These produce regulatory states in the fields of cells that will later in development give rise to specific body parts (e.g., a pan-bilaterian heart progenitor field kernel; . A testable theory to explain the hierarchical shape of Linnean bilaterian phylogeny (superphylum, phylum, class, etc), or  “clumpiness” of the phylogenetic distribution of animal morphologies, is based on kernels . The conservation of developmental process within each animal clade generates the phylogenetic distribution of the morphologies these processes generate. The prediction follows that the underlying cause is the phylogenetic distribution of dGRN kernels conserved within all members of a superphylum or phylum or class; that is, these shared kernels would account for the shared morphogenetic characters of each clade. This theory requires that the kernels similarly canalize downstream developmental process in each member of each given clade. 

On purely internal considerations, some aspects of dGRN structure appear much more impervious to change than others. For example, a frequently encountered type of subcircuit in upstream regions of dGRNs consists of two or three genes locked together by feedback inputs. These feedback structures act to stabilize regulatory states, and there is a high penalty to change, in that interference with the dynamic expression of any one of the genes causes the collapse of expression of all, and the total loss from the system of their contributions to the regulatory state. On the other hand, peripheral far downstream subcircuits such as differentiation gene batteries can change freely without affecting major patterning functions or causing network collapse. Generalizing, if we knew enough about the structure and functions of the constituent subcircuits, and their contextual upstream and downstream linkages, the architecture of the dGRN should predict its flexible and its less flexible linkages. Other features often thought of as properties of single genes, such as pleiotropy or epistasis, are likewise due to the positions genes occupy in network topology.  since no one gene produces body parts or executes a whole element of the developmental process, while on the other hand such functions are executed by dGRN subcircuits, the most powerful form of change in dGRN structure should be those alterations that result in redeployment of whole subcircuits.  An important place in dGRN structure to look for change is in linkages that control subcircuit deployment: such linkages include those that determine where signal ligands will be expressed; those that link one subcircuit to another; and those that serve as switches on the outside of morphogenetic subcircuits, so to speak, allowing or prohibiting their expression. much evidence indicates that hox gene functions often fall into this latter class. rather, they are often wired as one way connections, and are likely to be intrinsically less resistant to change without catastrophe.

dGRN hierarchy and selection

In dGRNs the effector genes ( differentiation genes and morphogenesis genes ). that constitute terminal differentiation and morphogenetic gene batteries, and their immediate controllers, lie at the network periphery. Their functions are terminal from the genetic control point of view, in that they lie at the ends of upstream cascades of regulatory steps, and they lack direct transcriptional feedbacks directed upstream. The cis-regulatory modules for which functionally adaptive sequence variation has been demonstrated all lie at such peripheral positions in the respective dGRNs. Yet the outputs of the upper-level pattern formation circuits of dGRNs which specify the overall body plan, and the clade-specific organization of individual body parts, do not display continuous variation in the types of forms they generate. Thus, the disposition and morphologies of the major components of the body plan are invariant at the levels which define unequivocally the phylum, class, and order, to which an animal belongs; and thus, the development of an embryo is extremely canonical. Whatever continuous variation occurs at individual cis-regulatory sequences, the dGRN circuit output preserves its Boolean morphogenetic character.

Selection cannot operate to produce continuous adaptive change except at the dGRN periphery. The lack of continuous variation in morphogenetic traits defining class and phylum level clades is obvious in the striking stasis revealed by the fossil record. In other words, while cis-regulatory sequence variation may have continuing adaptive significance at the dGRN periphery at upper levels of the dGRN hierarchy it does not have the same significance because the system level output is very impervious to change, except for catastrophic loss of the body part or loss of viability altogether. As long realized and much discussed in a non-mechanistic way in advance of actual knowledge of dGRN structure and function, this imperviousness has something to do with whatever processes generate canalization and/or “buffering” of the genetic control system. We can now begin to understand canalization mechanistically in terms of dGRN hierarchy and subcircuit structure,but in so far as “buffering” is taken to mean protection against “environmental fluctuations” as in many evolutionary mathematical models, it is irrelevant to animal embryonic processes, since in the main these depend not at all upon environmental inputs.

Then what structural features of dGRN design do account for the imperviousness of upper level system output to continuous cis-regulatory variation and to continuous selective functional change? Or, a very closely related question, what accounts for the evolutionary stasis over geologic time of body plan phylogeny in the ? here we see the real-time distribution of fossil variants of echinoderm body plans.

The definitive properties of the five surviving echinoderm classes have remained stable essentially since the Cambrian and Ordovician. The answer to the questions posed is that there are multiple intrinsic design features of modern dGRN structure that all contribute at the system level to imperviousness to continuous variation and to evolutionary morphogenetic stasis. 

To consider this question we must first remind ourselves what is the main function of upper level dGRNs for body plan formation. The fundamental role of upper level dGRNs is to set up in embryonic space a progressive series of regulatory states, which functionally define first the regions of the body with respect to its axes; then the location of the progenitor fields of the body parts; then the subparts of each body part. At each stage the output is a mosaic of sharply bounded regional regulatory states. This constitutes a Boolean checkerboard of diverse dGRN subcircuit expressions. Our problem thus resolves into understanding the system properties that “booleanize” dGRN subcircuit output, thus converting quantitatively and qualitatively varying sets of inputs into the same spatial regulatory state checkerboards for each member of the species at each stage. There are at least six different aspects to the solution to this problem.

Transcriptional dynamics of developmental gene regulatory cascades
In embryonic development the transcriptional processes mediated by dGRNs are intrinsically insensitive to varying cis-regulatory input levels. First, from the basic physical chemistry of target site occupancy, we know that modest changes in transcription factor concentration have little effect on target site occupancy; and second, in a dynamic analysis, in a typical embryonic gene cascade target genes are activated long before input factors approach steady state. This means that these “forward drive” systems operate over a great range of input concentrations, in contrast to typical physiological or biochemical macromolecular pathways in which quantitative output is usually mediated by exact control of steady-state input levels.

dGRN subcircuits controlling spatial regulatory state in development which execute Boolean logic transactions
Such subcircuits include the “X, 1-X” processors of ; these set up given regulatory states in a domain “X” and completely prohibit the expression of the given regulatory state everywhere else. For example, Tcf/β-catenin-mediated Wnt signaling operates to permit expression of target genes in cells receiving the signal but in all other cells, the dominant repressor Groucho replaces the Tcf cofactor β-catenin and transcriptionally represses the same target genes . Other subcircuits set sharp boundaries of expression by a variety of design devices; others mutually exclude regulatory states; etc. Boolean truth tables can be used to represent the function of each such subcircuit.

Transcriptional repression, utilized in most spatial control dGRN subcircuits
While some mechanisms of repression merely result in decreasing rate of output, others dominantly silence gene expression in a given cell. There are many and diverse biochemical mechanisms of transcriptional repression but a prominent feature of dominant developmental repression is that it is a multistep, non-equilibrium, one-way process which, following the initial appearance of the sequence-specific transcriptional repressor, alters the configuration of the transcription complex so it can no longer function even after the transcriptional repressor has disappeared. Thus, inclusion of repression in subcircuit topology increases all-or-nothing behavior.

Specific feedback state lockdowns
Noticed when dGRN circuitry first began to be revealed experimentally, it is an almost invariant observation that after a transient specification function first installs a spatial regulatory state, a feedback circuit is soon set up such that genes of the regulatory state are locked into a dynamic positive mutual embrace and the state is now stabilized. This general design feature clearly contributes to imperviousness to input variation since once these “stabilization motors” are activated they enable the system to forget upstream events so long as they worked at all, and the feedback circuitry has the capacity to strongly amplify the dGRN output. New levels of expression are established irrespective of the initial inputs. As development proceeds, such “reloading” and “restabilizing” devices are brought into play in each region of the organism, often at each stage.

Evolutionary inflexibility due to highly conserved canalizing dGRN kernels
These subcircuits operate at upper levels of dGRN hierarchy so as to affect characters of the body plan that are definitive for upper level taxa, i.e., they control the early stages of just the types of developmental process of which the invariance per taxon constitutes our problem. Since they preclude developmental alternatives, they may act to “booleanize” the evolutionary selective process: either body part specification works the way it is supposed to or the animal fails to generate the body part and does not exist.

The significance of crown group dGRN design
At first glance subcircuit deployment in dGRNs can appear “overwired” or even redundant. Typically a regulatory state is installed in a given domain by a signal, or a gate of one sort or another; and the same state is not just activated exclusively in the right place but also specifically repressed everywhere else; dynamic feedback loops stabilize and enforce regulatory states; and not uncommonly many of the above devices are all deployed together in the same dGRN.  interference with expression of any of the key genes of these subcircuits always causes an immediate loss of function phenotype, such as ectopic expression if a spatial repression function is interrupted in cis(by mutation of repressor target sites) or trans (by application of a morpholino). For instance in the sea urchin embryo the regulatory genes of the initial endoderm specific dGRN are all activated by means of a Wnt signaling gate mediated by β-catenin/Tcf because their cis-regulatory modules include essential Tcf target sites . The requisite Wnt signal and its biochemical response in recipient cells, nuclearized β-catenin, are present only in the appropriate vegetal cell lineages of the embryo, and this might be thought quite sufficient to ensure expression of the endoderm genes only in those cells. However, in all other cells, as noted above, in the absence of nuclearized β-catenin the same endoderm specific genes are actively repressed outside the prospective endoderm by the alternative Tcf co-factor Groucho. Logically this could be regarded as a redundant spatial control, but it is clearly not, since if the Tcf sites of the cis-regulatory modules governing expression of endoderm genes are mutated, wild ectopic expression results. This result is instructive: we see that the wiring enables these genes to utilize powerful ubiquitous activators in addition to their spatial control gates, though eventually control is handed off to the spatially confined cross-regulatory endoderm specific dGRN. As a second example, in the skeletogenic micromere lineage the gcm gene is inactive while gcm is directly turned on as a result of Notch signaling in the adjacent mesoderm cells in response to Delta expression in the skeletogenic cells . On top of this, an additional element of circuitry ensures independently that gcm is not expressed in the skeletogenic cells, a negative consequence of skeletogenic alx1 expres sion. But nor is this a redundant spatial control: if alx1 expression is prevented, gcmis indeed transcribed in skeletogenic cells, and so we learn that Delta signals among the micromeres would trigger gcm expression if not prevented from doing so. Examples could easily be multiplied, but without doing so their import can be generally summarized. Each apparently redundant spatial control mechanism turns out to have a special function, often not evident a priori. The overall control principle is that the embryonic process is finely divided into precise little “jobs” to be done, and each is assigned to a specific subcircuit or wiring feature in the upper level dGRN. No subcircuit functions are redundant with another, and that is why there is always an observable consequence if a dGRN subcircuit is interrupted. Since these consequences are always catastrophically bad, flexibility is minimal, and since the subcircuits are all interconnected, the whole network partakes of the quality that there is only one way for things to work. And indeed the embryos of each species develop in only one way.


https://evolutionnews.org/2013/10/when_theory_tru/
To claim, that dGRNs might have been more elastic in the past contradicts what developmental biologists have learned over several decades investigating how these networks actually function from mutagenesis studies of many different biological "model systems," including Drosophila (fruit flies), Caenorhabditis (nematodes), Strongylocentrotus (sea urchins), Danio (zebrafish) and other animals.6 Moreover, as Marshall himself has noted elsewhere, there is a good reason for this inflexibility. As Marshall explains, “many of the characters that evolved during the origin of the phyla are no longer able to change. The reason for this is that selectable variation is absent: either the characters are invariant or mutants that carry this variation are sterile or lethal.”

Thus, we can think of a crown group dGRN as an evolutionarily terminal, finely divided, extremely elegant control system that allows continuing alteration, variation, and evolutionary experimentation only after the body plan per se has formed, i.e., in structural terms, at the dGRN periphery, and in developmental terms, late in the process. It is no surprise, from this point of view, that cell type re-specification by insertion of alternative differentiation drivers is changed only at the dGRN periphery, quite a different matter from altering body plan. In terms of their general hierarchical depth, the dGRNs of all living (non-degenerate) bilaterians are probably approximately similar , though the number of subcircuits required at each given developmental stage or dGRN level to complete the body plan is likely much greater for some forms than others. Deconstructing the evolutionary process by which stem group body plans were stepwise formulated will require us to traverse the conceptual pathway to dGRN elegance, beginning where no modern dGRN provides a model. The basic control features of the initial dGRNs of the Precambrian and early Cambrian must have differed in fundamental respects from those now being unraveled in our laboratories. The earliest ones were likely hierarchically shallow rather than deep, so that in the beginning adaptive selection could operate on a larger portion of their linkages. Furthermore, we can deduce that the outputs of their subcircuits must have been polyfunctional rather than finely divided and functionally dedicated, as in modern crown group dGRNs. A general result of these arguments is that considerations of evolutionary change in dGRN structure may at last provide a unified conceptual framework for understanding the stages of crown group evolution, and in the same breath the sequential history of change that has produced the different hierarchical levels of animal dGRNs.

But some things never change, and a principle that must have been obtained from early in metazoan evolution is that developmental jobs are controlled through the logic outputs of genetic subcircuits. Thus, how evolution of the animal body plan has occurred is a question that in the end can only be addressed in the terms of transcriptional regulatory systems biology.

Accurate models of the cross-talk between signaling pathways and transcriptional regulatory networks within cells are essential to understand complex response programs.2 Setting up the body plan during embryonic development requires the coordinated action of many signals and transcriptional regulators in a precise temporal sequence and spatial pattern. 3 During vertebrate embryonic development the body plan is laid down from a single cell, the fertilised egg. This involves the allocation of multipotent cells to the three germ layers, subdivision of the germ layers into organ primordia, spatial patterning and finally differentiation into special cell types. Thus, multipotent progenitor cells undergo a series of cell fate decisions during which their developmental potential becomes gradually restricted. Ultimately, the instructions for developmental programmes are encoded in the genome with non-coding regulatory regions and their interacting factors controlling temporal and spatial deployment of cell fate determinants and differentiation genes. Formation of the body plan requires coordinated and sequential action of many such factors controlling spatiotemporal distribution of cell fate specific proteins and differentiation factors.  As cells become specified each population is characterised by a specific set of transcription factors defining its regulatory state. GRNs establish functional linkages between the signalling inputs, transcription factors and their targets, thus providing a view of cell fate decisions at the molecular level .  In short, GRNs are “wiring diagrams” that explain how cells or organs develop. GRNs have a hierarchical structure with a clear beginning and terminal states, and therefore have directionality: each state depends on the previous. They define genetic circuits or modules, each with a specific task. It is thus easy to decipher how individual sub-circuits are used repeatedly in different contexts and how the assembly of new modules has allowed cell diversification.  Importantly however, GRNs not only provide information about the genetic hierarchy of network components, but must also identify the cis-regulatory elements that integrate this information.

1) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3135751/
2. https://genome.cshlp.org/content/23/2/365.full
3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3626748/

Eric Davidson (1937-2015) on Gene Regulatory Networks

With the passing of paleontologist David Raup and biologist and science historian Will Provine in recent months, we've been given the opportunity to reflect on two famous evolutionary scientists who were brave enough to critique neo-Darwinism. But there's another one we've missed -- Eric Davidson, the developmental biologist from Caltech who, sadly, passed away this past August.
Davidson is famous for formulating the concept of developmental gene regulatory networks (dGRNs), a description of how genes interact with one another to regulate their expression in the early stages of development. The activity of a dGRN is very influential in determining the body plan of an animal.
Recently, an email correspondent contacted me to ask about dGRNs. This person had been in communication with an evolutionary biologist who claimed that dGRNs are very flexible and could show how new animals evolved. I didn't know Eric Davidson so I can't share any personal anecdotes, but it seems like an appropriate time to review what the great dGRN expert Eric Davidson said on this point.
As Stephen Meyer explains in Darwin's Doubt, Davidson believed, based upon his experimental work, that dGRNs aren't very flexible at all. Davidson observed that mutations affecting the dGRNs that regulate body-plan development lead to "catastrophic loss of the body part or loss of viability altogether."1 He explained:

There is always an observable consequence if a dGRN subcircuit is interrupted. Since these consequences are always catastrophically bad, flexibility is minimal, and since the subcircuits are all interconnected, the whole network partakes of the quality that there is only one way for things to work. And indeed the embryos of each species develop in only one way.

He further wrote:

Interference with expression of any [multiply linked dGRNs] by mutation or experimental manipulation has severe effects on the phase of development that they initiate. This accentuates the selective conservation of the whole subcircuit, on pain of developmental catastrophe..."2

But perhaps most strikingly, Davidson, in discussing hypothetical "flexible" dGRNs, acknowledged that we are speculating "where no modern dGRN provides a model" since they "must have differed in fundamental respects from those now being unraveled in our laboratories."1
Meyer cited much of this evidence in Darwin's Doubt. Now consider how UC Berkeley paleontologist Charles Marshall responded to Stephen Meyer when Marshall reviewed Meyer's book in the journal Science. Marshall wrote: "Today's GRNs have been overlain with half a billion years of evolutionary innovation (which accounts for their resistance to modification), whereas GRNs at the time of the emergence of the phyla were not so encumbered."3
The only reason Marshall would have said this is if modern dGRNs are in fact "so encumbered" that they could not provide a model for evolution. Marshall was forced to ignore modern experimental data and speculate that perhaps in the past dGRNs were different.
Thus, while Marshall's title, "When Prior Belief Trumps Scholarship," was an accusation aimed against Meyer's work, I think that it's far more apt to turn that right around at Marshall's own arguments, as Meyer explained in his response to Marshall.
As a result of all of this, Davidson concluded that, "contrary to classical evolution theory, the processes that drive the small changes observed as species diverge cannot be taken as models for the evolution of the body plans of animals."4 He elaborated:

Neo-Darwinian evolution ... assumes that all process works the same way, so that evolution of enzymes or flower colors can be used as current proxies for study of evolution of the body plan. It erroneously assumes that change in protein- coding sequence is the basic cause of change in developmental program; and it erroneously assumes that evolutionary change in body- plan morphology occurs by a continuous process. All of these assumptions are basically counterfactual. This cannot be surprising, since the neo-Darwinian synthesis from which these ideas stem was a premolecular biology concoction focused on population genetics and . . . natural history, neither of which have any direct mechanistic import for the genomic regulatory systems that drive embryonic development of the body plan.1

The bottom line is that experimental research on dGRNs in modern animals shows that they do NOT appear flexible. Experts acknowledge this. They even acknowledge that it poses a challenge to neo-Darwinism. Those who claim otherwise are simply mistaken.


References:
(1) Eric Davidson, "Evolutionary Bioscience as Regulatory Systems Biology."Developmental Biology, 357:35-40 (2011).
(2) Eric H. Davidson and Douglas Erwin. "An Integrated View of Precambrian Eumetazoan Evolution." Cold Spring Harbor Symposia on Quantitative Biology, 74: 1-16 (2010).

(3) Charles R. Marshall, "When Prior Belief Trumps Scholarship," Science, 341 (September 20, 2013): 1344.

(4) Eric Davidson, The Regulatory Genome: Gene Regulatory Networks in Development and Evolution. Burlington: Elsevier, 2006, p. 195.


http://www.evolutionnews.org/2015/10/eric_davidson_1100141.html

Molecular Genetics and the evolution of animal design,

Sean B.Carroll, From DNA to Diversity: Molecular Genetics and the Evolution of Animal Design, page 28:

how do different cells acquire the unique morphologies and functional properties required in the diverse organs and tissues of the body? We now understand that this process occurs through the selective expression of distinct subsets of the many thousands of genes in any animal’s genome in different cells. How genes are turned on and off in different cells over the course of animal development is an exquisitely orchestrated regulatory program whose features are only now coming into detailed view.

If morphological diversity is all about development, and development results from genetic regulatory programs, then is the evolution of diversity directly related to the evolution of genetic regulatory programs? Simply put, yes. But to understand how diversity evolves, we must first understand the genetic regulatory mechanisms that operate in development. In other words, what is the genetic toolkit of development and how does it operate to build animals?

The foremost challenge for embryology has been to identify the genes and proteins that control the development of animals from an egg into an adult. Early embryologists discovered that localized regions of embryos and tissues possess properties that have long-range effects on the formation and patterning of the primary body axes and appendages. Based on these discoveries, they postulated the existence of substances responsible for these activities. However, the search for such molecules proved fruitless until the relatively recent advent of genetic and molecular biological technologies. The most successful approach to understanding normal development has involved the isolation of single gene mutations that have discrete and often large-scale effects on body pattern. Lets  take an inventory of the essential genetic toolkit for animal development. We concentrate on genes first discovered in insects, where systematic screens for developmental genes were pioneered. Importantly, however, it turns out that related genes are present in many other animals. We describe how members of the genetic toolkit were identified and what kinds of gene products they encode. In addition, we illustrate the general correlation between these genes’ patterns of expression with the development of the morphological features they affect. Finally, we briefly survey their distribution and function in other animals. Only a small fraction of all genes in any given animal constitute the toolkit that is devoted to the formation and patterning of the body plan and body parts. Two classes of gene products with the most global effects on development are of special interest: families of proteins called transcription factors that regulate the expression of many other genes during development, and members of signaling pathways that mediate short- and long-range interactions between cells. The expression of specific transcription factors and signaling proteins marks the location of many classically defined regions within the embryo. These proteins control the formation, identity, and patterning of most major features of animal design and diversity.

BEFORE THE TOOLKIT—ORGANIZERS, FIELDS, AND MORPHOGENS

Long before any genes or proteins affecting animal development were characterized, embryologists sought to identify the basic principles governing animal design. In their search, they focused on the large-scale organization of the primary body axes, the differentiation of various germ layers (ectoderm, mesoderm, and endoderm), and the polarity of structures such as appendages and insect segments. By manipulating embryos and embryonic tissues, primarily by transplantation and ablation, researchers discovered many important properties of developing embryos and tissues. Much of the fascination of embryology stems from the remarkable activities of discrete regions within developing embryos in organizing the formation of body axes and body parts. Furthermore, these classical concepts of embryonic organization present a very useful framework for considering how that organization can change during evolution. We will briefly review some of these experiments and ideas before addressing their genetic and molecular manifestations. The first demonstration of organizersaregions of embryos or tissues that have long range effects on the fate of surrounding tissues was achieved by Mangold and Spemann in 1924. They transplanted the lip of the blastopore, the invagination where mesoderm and endoderm move inside the amphibian embryo, of a newt gastrula into another newt embryo and found that the transplanted tissue could induce a second complete body axis (Fig. 2.1a). The additional embryo induced was partly derived from the transplanted graft and partly derived from the host. The equivalent of the “Spemann organizer” in amphibians has been found in chick and mouse embryos, and it is now recognized to be a structure characteristic of all chordate embryos.

Other organizers with long-range effects on surrounding tissues have been identified in the developing vertebrate limb bud. Transplantation of a discrete patch of posterior tissue to an ectopic anterior site induces the formation of limb structures (digits, tendons, muscles) with mirror-image polarity to the normal anteroposterior order (Fig. 2.1b). By contrast, transplantation or removal of anterior tissue has no effect on limb development, suggesting that this posterior region of the limb bud, dubbed the zone of polarizing activity (ZPA), organizes anteroposterior (that is, the thumb-to-pinkie axis) polarity and limb formation. Another organizer operates from the most distal tip of the limb bud, the apical ectodermal ridge (AER). Removal of this region truncates the limb and deletes distal elements (digits), whereas transplantation of the AER to an early limb bud can induce outgrowth of a duplicate limb (Fig. 2.1b). One explanation for the long-range polarizing and inductive effects of the Spemann organizer, ZPA, and AER is that these tissues are sources of inducer molecules, or morphogens athat is, substances whose concentrations vary within a tissue and to which surrounding cells and tissues respond in a concentration-dependent manner. The response to a morphogen depends, then, on the distance of the responding tissue from the source. For example, if the ZPA is a source of a morphogen, then diffusion of this substance can establish a gradient of inducer concentration. Induction of different digit types depends on the morphogen concentration,
with low levels of morphogen inducing anterior digits (thumb) and high levels inducing posterior digits (pinkie) (Fig. 2.1b).



Transplantation and ablation experiments have been used to investigate the long-range organizing activities of embryonic tissues.
(a) The Spemann organizer. The dorsal blastopore lip of an early amphibian embryo can induce a second embryonic axis and embryo
when transplanted to the ventral region of a recipient embryo. 
(b) Limb organizers. The apical ectodermal ridge (AER) is required for formation of distal limb elements. Removal leads to loss of structures; transplantation to specific ectopic sites induces extra elements. The zone of polarizing activity (ZPA) organizes the anteroposterior pattern; transplantation to an ectopic site induces extra digits with reverse polarity.
(c) Insect egg organizer. Ligation of the insect Euscelis embryo (marked by the gray line) early in development deletes the thorax and abdomen; later ligations leave more segments intact. However, transplantation of the posterior pole cytoplasm (marked by the black dot) into the anterior of a ligated embryo induces the formation of a complete embryo. This result demonstrates that the posterior cytoplasm has organizer activity. 
(d) Within insect segments, epithelial polarity is organized by signaling sources. Ablation of a segment boundary (indicated by the interruption of the black line) reorganizes segment polarity (indicated by the orientation of small black hairs).

Organizers have been demonstrated and morphogens postulated in insects as well as vertebrates. Ligature and cytoplasmic transplantation experiments first suggested that the anteroposterior axis of certain insect embryos is influenced by two organizing centers, one at each pole of the egg (Fig. 2.1c), that behave as sources of morphogens. Similarly, the polarity of cells within insect segments appears to be organized by signals that produce a graded pattern (Fig. 2.1d). One difficulty with this picture of morphogen-producing organizers arises when we attempt to explain the boundaries of their range of influence. All of the cells in a growing embryo are in contact with other cells, so how is it that some parts respond and others do not? One explanation involves the concept of the morphogenetic field. Early embryologists demonstrated that some parts of developing animals, such as the forelimb field, could be transplanted to another site and still differentiate properlyathat is, into a forelimb. In addition, if undetermined cells were introduced into the field, they could become incorporated into the limb. These transplantable, self-regulating fields are discrete physical units or modules of embryoni development. They form bounded domains within which specific programs of morphogenesis occur. The term “primary field” applies to the entire embryo before the axes are determined; the limbs, eyes, and other organs are termed “secondary fields,” or organ primordia.

Secondary fields may be further subdivided into “tertiary fields,” defined by physical or developmental boundaries. Compartments are one special type of subdivision. First demonstrated within the wing imaginal disc of the fruit fly Drosophila melanogaster, compartments are composed of populations of cells that do not intermix with cells outside the compartment. Further progress in understanding the nature of organizers, morphogens, and fields stalled after their discovery and description in the first half of the 1900s. The impasse was ultimately broken by the discovery of genes whose products governed the activity of organizers, behaved as morphogens, and controlled the formation and identity of embryonic fields. These genes make up the “toolkit” for animal development.

Biological systems demonstrate engineering principles, which point to design

https://reasonandscience.catsboard.com/t2318-gene-regulatory-networks-controlling-body-plan-development#5746

The specific genetic changes that give rise to the evolutionary origins of novel protein-protein interactions have rarely been documented in detail 
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0001054

Although numerous investigators assume that the global features of genetic networks are moulded by natural selection, there has been no formal demonstration of the adaptive origin of any genetic network. The mechanisms by which genetic networks become established evolutionarily are far from clear.  Many physicists, engineers and computer scientists, and some cell and developmental biologists, are convinced that biological networks exhibit properties that could only be products of natural selection; however, the matter has rarely been examined in the context of well-established evolutionary principles.  Alon states that it is “…wondrous that the solutions found by evolution have much in common with good engineering,”There is no evidence that genetic pathways emerge de novo in response to a selective challenge.

https://www.nature.com/articles/nrg2192

François Jacob pictured evolution as a tinkerer, not an engineer. Engineers and tinkerers arrive at their solutions by very different routes. Rather than planning structures in advance and drawing up blueprints (as an engineer would), evolution as a tinkerer works with odds and ends, assembling interactions until they are good enough to work. It is therefore wondrous that the solutions found by evolution have much in common with good engineering design. 
http://science.sciencemag.org/content/301/5641/1866.full

Maybe it's not that wondrous if we consider that the solutions in questions might be explained by the conscious actions of a powerful engineer, namely creator God ?!!

The cell can be viewed as an overlay of at least three types of networks, which describes protein-protein, protein-DNA, and protein-metabolite interactions. Second, biological systems viewed as networks can readily be compared with engineering systems, which are traditionally described by networks such as flow charts and blueprints. Remarkably, when such a comparison is made, biological networks are seen to share structural principles with engineered networks. Here are three of the most important shared principles, modularity, robustness to component tolerances, and use of recurring circuit elements.
http://science.sciencemag.org/content/301/5641/1866.full

The first principle, modularity
is an oft-mentioned property of biological networks. For example, proteins are known to work in slightly overlapping, coregulated groups such as pathways and complexes. Engineered systems also use modules, such as subroutines in software (13) and replaceable parts in machines. The following working definition of a module is proposed based on comparison with engineering: A module in a network is a set of nodes that have strong interactions and a common function. A module has defined input nodes and output nodes that control the interactions with the rest of the network. A module also has internal nodes that do not significantly interact with nodes outside the module. Modules in engineering, and presumably also in biology, have special features that make them easily embedded in almost any system. For example, output nodes should have “low impedance,” so that adding on additional downstream clients should not drain the output to existing clients

The second common feature of engineering and biological networks is robustness to component tolerances.
In both engineering and biology, the design must work under all plausible insults and interferences that come with the inherent properties of the components and the environment. Thus, Escherichia coli needs to be robust with respect to temperature changes over a few tens of degrees, and no circuit in the cell should depend on having precisely 100 copies of protein X and not 103. This point has been made decades ago for developmental systems

The third feature common to engineering and biological networks is the use of recurring circuit elements. 
An electronic device, for example, can include thousands of occurrences of circuit elements such as operational amplifiers and memory registers. Biology displays the same principle, using key wiring patterns again and again throughout a network. Metabolic networks use regulatory circuits such as feedback inhibition in many different pathways

Non-Coding DNA “Extremely” Conserved, Essential for Regulation   11/26/2004

A paper in PLOS Biology¹ compared non-coding DNA from widely-separated vertebrates and found them not only “extremely conserved” in many cases, but essential for regulating the gene-coding regions.

Understanding the intricate and finely tuned process of gene regulation in vertebrate development remains a major challenge facing post-genomic research.  In order to begin to understand how genomic information can coordinate regulatory processes, we have adopted an approach integrating comparative genomics and a medium-throughput functional assay. Nearly 1,400 non-coding DNA sequence elements were identified that exhibitextreme conservation throughout the vertebrate lineage.... Most, if not all, of the CNE [conserved noncoding element] sequences appear to be associated with genes involved in the control of development, many of them transcription factors.  A significant proportion of genes identified in this study are homologous to those identified in the sea urchin and other invertebrates as master regulators of early development, leading us to believe that they interact in GRNs [gene regulatory networks].  Consequently, it is extremely likely that the CNEs identified compose at least part of the genomic component of GRNs in vertebrates, acting as critical regions of regulatory control for their associated genes.  Such regions would mediate up- or down-regulation of expression, effecting a cascade of downstream events.

They speculate that these sequences are not mere binding sites, because that would not explain the high degree of sequence conservation.  “Consequently,” they say, “we have not ruled out the possibility that the CNEs may have a completely different mode of action or act in numerous different ways.”  The team of 16 scientists from the UK were struck with the similarity of these noncoding sequences between human, rat, mouse and pufferfish.  They performed some limited functional analysis on the sequences and found that some affect genes that are physically distant, often megabases away.  Though apparently essential, “They are amongst the most highly conserved of all sequences in vertebrate genomes yet they are completely unrecognisable in invertebrates.”  It seems, however, that invertebrates have analogous sequences for gene regulation, as stated in their introduction:

Identification and characterisation of cis-regulatory regions [i.e., on the same strand of DNA] within the non-coding DNA of vertebrate genomes remain a challenge for the post-genomic era.  The idea that animal development is controlled by cis-regulatory DNA elements (such as enhancers and silencers) is well established and has been elegantly described in invertebrates such as Drosophila and the sea urchin.  These elements are thought to comprise clustered target sites for large numbers of transcription factors and collectively form the genomic instructions for developmental gene regulatory networks (GRNs).  However, relatively little is known about GRNs in vertebrates.

More work will need to be done to find out if this is true for vertebrates, as it appears from this study, and if so, how these vertebrate CNEs work.  Some could prevent gene expression, for example, as well as enhance it.  “Whatever their mode of action, the striking degree of conservation displayed by this set of CNEs suggests they play critically important functional roles,” they deduce.  In conclusion, they state, “Given their strong association with genes involved in developmental regulation, they are most likely to contain the essential heritable information for the coordination of vertebrate development.”

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¹Woolfe et al., “Highly Conserved Non-Coding Sequences Are Associated with Vertebrate Development,” PLOS Biology, Vol 3 Issue 1 (Jan 2005), published online 11/15/2004: DOI: 10.1371/journal.pbio.0030007.

The authors make only the meagerest references to evolution, none of it helpful to the Darwin Party.  They merely state as matters of belief the “evolutionary divergence” between humans and mice, and make other similar assumptions that said divergent animals evolved from a common ancestor.  They merely assume some genes evolve quickly and others slowly.  But when it comes to explaining how such extremely conserved sequences could survive the inexorable pressure of natural selection for oodles of aeons, they admit there is nothing but guesswork:

A number of other ideas on the evolutionary mechanisms responsible for “ultra-conservation” have been suggested, involving decreased mutation rate, increased DNA repair, and multiply-overlapping transcription factor binding sites, but without more functional studies such hypotheses remain speculative.

Elsewhere, they remark with astonishment about specific examples of CNEs in all four species (human, rat, mouse, pufferfish) that show 100% identity, “demonstrating an extraordinary level of conservation for genomes separated by 900 million years of divergent evolution.”  Maybe no divergent evolution.  Maybe no 900 million years.

http://creationsafaris.com/crev200411.htm

Gene regulatory elements during development

Regulatory DNA Seems Largely Responsible for the Differences Between Animal Species
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. 


Regulatory DNA defines the gene expression patterns in development. 
The genome is the same in a muscle cell as in a skin cell, but different genes are active because these cells express different transcription regulators that bind to gene regulatory elements. For example, transcription regulators in skin
cells recognize a regulatory element in gene 1, leading to its activation, whereas a different set of regulators is present in muscle cells, binding to and activating gene 3. Transcriptional regulators that activate the expression of gene 2 are present in both cell types.

Consequently, a change in the regulatory DNA, even without any change in the coding DNA, can alter the logic of the gene-regulatory network and change the outcome of development. It seems that changes in regulatory DNA are largely responsible for the dramatic differences between one class of animals and another. We can view the protein products of the coding sequences as a conserved kit of common molecular parts, and the regulatory DNA as instructions for assembly: with different instructions, the same kit of parts can be used to make a whole variety of different body structures.

The direct link between evolutionary changes in gene regulatory networks and selection pressures remains elusive.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8483737/

Gene regulatory networks coordinate the expression of genes across physiological states and ensure a synchronized expression of genes in cellular subsystems, critical for the coherent functioning of cells.
https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-021-04213-5