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Gene Regulatory Networks Controlling Body Plan Development

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Gene Regulatory Networks Controlling Body Plan Development 

http://reasonandscience.heavenforum.org/t2318-gene-regulatory-networks-controlling-body-plan-development

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). 

[b]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.
[/b]
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. 
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.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.

Darwins doubt, page 202:
Davidson notes that, once established, the complexity of the dGRNs as integrated circuits makes them stubbornly resistant to mutational change—a point he has stressed in nearly every publication on the topic over the past fifteen years. “In the sea urchin embryo,” he points out, “disarming any one of these subcircuits produces some abnormality in expression.”

In each generation of each animal species, the body plan is formed by the execution of an inherited genomic regulatory program for embryonic development. The basic control task is to determine transcriptional activity throughout embryonic time and space, and here ultimately lies causality in the developmental process. The genomic control apparatus for any given developmental episode consists of the specifically expressed genes that encode the transcription factors required to direct the events of that episode, most importantly including the cis-regulatory control regions of these genes. The cis-regulatory sequences combinatorially determine which regulatory inputs will affect the expression of each gene and what other genes it will affect; that is, they hard-wire the functional linkages among the regulatory genes, forming network subcircuits. The subcircuits perform biologically meaningful jobs, for example, acting as logic gates, interpreting signals, stabilizing given regulatory states, or establishing specific regulatory states in given cell lineages; here the term “regulatory state” means the total of active transcription factors in any given cell at any given time. In turn the subcircuits are “wired” together to constitute the gene regulatory network (GRN), the genomically encoded developmental control system.

GRNs are inherently hierarchical: the networks controlling each phase of development are assemblages of subcircuits, the subcircuits are assemblages of specific regulatory linkages among specific genes, and the linkages are individually determined by assemblages of cis-regulatory transcription factor target sites. But at the highest level of its organization, the developmental GRN is hierarchical in an additional and, as we discuss below, very important sense. Development progresses from phase to phase, and this fundamental phenomenon reflects the underlying sequential hierarchy of the GRN control system. In the earliest embryonic phases, the function of the developmental GRN is establishment of specific regulatory states in the spatial domains of the developing organism. In this way the design of the future body plan is mapped out in regional regulatory landscapes, which differentially endow the potentialities of the future parts. Lower down in the hierarchy, GRN apparatus continues regional regulatory specification on finer scales. Ultimately, precisely confined regulatory states determine how the differentiation and morphogenetic gene batteries at the terminal periphery of the GRN will be deployed.

The result of relevant change in GRN structure is derived change in GRN operation, compared to the immediately ancestral GRNs. This will cause changes in developmental process, and ultimately in the product of that process, the body plan (Britten and Davidson, 1971, Davidson and Erwin, 2006, Erwin and Davidson, 2009).

 the evolution of the body plan is fundamentally a system-level problem to which GRN structure/function provides the most compelling direct access.

Evolution at cis-Regulatory Nodes

Because GRN topology is encoded directly in cis-regulatory sequences at its nodes, evolutionary changes in these sequences have great potency to alter developmental GRN structure and function. However, there are many kinds ofcis-regulatory changes that affect function in different ways, ranging from loss of function, to quantitative change in function, to qualitative gain of function resulting in redeployment of gene expression.

The Hierarchical Organization of Developmental GRNs

Knowing that the basic events causing GRN evolution are cis-regulatory alterations, particularly those resulting in qualitative additions to or subtractions from the developmental regulatory state, we can sharpen the question we are asking: how do the structural properties of GRNs affect the developmental consequences of such cis-regulatory alterations?

The Consequences of Hierarchical GRN Structure

As discussed above, the GRNs controlling embryonic development of the body plan are intrinsically hierarchical, essentially because of the number of successive spatial regulatory states that must be installed in the course of pattern formation, cell-type specification, and differentiation.

Question:  has the development stage and sequence not have to be precisely and correctly programmed right from the beginning ? What, if there is a evolutionary mutation in the genome, and a new gene product is the result, but there is no regulation when to express it, and how to locate and to transport it to the right place ? Furthermore, it must be interface compatible , that is well match and function,  and being able to interact properly with the neighbouring parts or  cells. 

 The consequences of any given cis-regulatory mutation will depend entirely on where in the GRN hierarchy the affected cis-regulatory node lies. As Figure 2 shows, changes that occur in the cis-regulatory control apparatus of a given differentiation gene could cause redeployment of that gene; changes in the cis-regulatory system determining expression of a controller of the battery could cause redeployment of the whole battery; changes upstream of that could affect redeployment of whole regulatory states, or of many other features. The circuitry drawn in Figure 2 is of course arbitrary but its import is general.



Figure 1

Regulatory Gene Co-option and Possible Consequences
The diagram shows cis-regulatory mutations that could result in co-optive change in the domain of expression of a regulatory gene and consequences at the level of gene regulatory networks (GRNs).
(A) Co-option event: The gene regulatory networks operating in spatial Domains 1 and 2 produce different regulatory states (colored balls, representing diverse transcription factors). A cis-regulatory module of Gene A, a regulatory gene, has target sites for factors present in the Domain 1 regulatory state and so Gene A and its downstream targets are expressed in Domain 1, but not in Domain 2 where only one of the three sites can be occupied. Two alternative types of cis-regulatory mutations are portrayed: appearance of new sites within the module by internal nucleotide sequence change; and transposition into the DNA near the gene of a module from elsewhere in the genome bearing new sites. Although these gain-of-function changes do not affect the occupancy of the cis-regulatory sites of Gene A in Domain 1, the new sites allow Gene A to respond to the regulatory state of Domain 2, resulting in a co-optive change in expression so that Gene A is now active in Domain 2 (modified from Davidson and Erwin, 2010).
(B) Gain-of-function changes in Domain 2 GRN architecture caused by co-option of Gene A: Gene A might control expression of an inductive signaling ligand, which could alter the fate/function of adjacent cells now receiving the signal from Domain 2 (left); Gene A might control expression of Gene B, another regulatory gene, and together with it cause expression of a differentiation (D) gene battery, which in consequence of the co-option is now expressed in Domain 2 (right).




Figure 2

Evolutionary Consequences of cis-Regulatory Mutations
Functional evolutionary consequences of cis-regulatory mutations depend on their location in gene regulatory network (GRN) architecture. A GRN circuit encoding the control system of a differentiation gene battery (bottom tiers) activated in response to a signal from adjacent cells (top tier); linkages are in blue, red, and green. The double arrow indicates signal reception and transduction causing gene expression in the recipient cells. Note that the middle tier of circuitry consists of a dynamic feedback stabilization subcircuit. The numbered red “x” symbols denote mutational changes in the cis-regulatory modules controlling expression of these genes, keyed by number to the functional consequences listed in the box below. Loss-of-function mutations (1 and 2) are indicated in green, and co-optive gain-of-function mutations (3 and 4) resulting in expression of the affected gene in a new domain, as in Figure 1 A, are indicated in blue (modified from Erwin and Davidson, 2009).

 So in order to understand predictively the effect of a given cis-regulatory change, the GRN architecture and the position of the mutation therein must be known. This may seem a demanding requirement, but from the point of view of understanding evolution mechanistically, it places a powerful lever in our hands. First, it should enable a rational interpretation of evolutionary differences in development between related animals in terms of GRN structure (we consider examples below); second, in principle it could enable predicted effects to be tested experimentally by inserting the cis-regulatory change into a related form expressing the pleisiomorphic GRN, termed “synthetic exzperimental evolution” (Erwin and Davidson, 2009).

Another direct evolutionary consequence of GRN hierarchy has also been discussed (Davidson and Erwin, 2006, Davidson and Erwin, 2009), and this is the phenomenon of canalization. In developmental terms the establishment of a spatial regulatory state constrains subsequent processes: like a decrease in entropy, the number of possible regulatory states downstream is now decreased. If the regulatory state defines a progenitor field for a given organ, then all the subsequent stages in the development of that organ must take place within that domain. As in development so in evolution, and thus a co-optive mutation leading to qualitative evolutionary reorganization at cis-regulatory nodes of an upper-level GRN subcircuit is much more likely to entail numerous deleterious problems downstream than if the change were to occur further down in the hierarchy. Therefore upper levels of GRN hierarchy are much less likely to change  than are more peripheral levels, and this is the empirical mark of the classical canalization phenomenon.

Currently, no GRN is analyzed to a degree that we know its linkages and functions from its upstream to downstream peripheries, that is, from the beginning of the developmental process to the terminal differentiated state. We do know, however, that the GRN output is observable as individual gene expression patterns and, ultimately, as the developmental process. We can use these outputs to infer a framework within which to position individual regulatory subcircuits or evolutionary changes within the hierarchical GRN. To facilitate the discussion on GRN evolution we now define GRN parts according to the developmental functions they control and then go on to consider abstractly the impact of evolutionary changes occurring in each of these parts.



Figure 3

Hierarchy in Developmental Gene Regulatory Networks
The diagram shows a symbolic representation of hierarchy in developmental gene regulatory networks. The developmental process begins with the onset of embryogenesis at top. The outputs of the initial (i.e., pregastrular) embryonic gene regulatory networks (GRNs) are used after gastrulation to set up the GRNs, which establish regulatory states throughout the embryo, organized spatially with respect to the embryonic axes (axial organization and spatial subdivision are symbolized by orthogonal arrows and colored patterns). These spatial domains divide the embryonic space into broad domains occupied by pluripotent cell populations already specified as mesoderm, endoderm, future brain, future axial neuroectoderm, non-neural ectoderm, etc. The GRNs establishing this initial mosaic of postgastrular regulatory states, including the signaling interactions that help to establish domain boundaries, are symbolized as Box I.

The information of the GRN's to establish this initial mosaic of postgastrular regulatory states, and the signalling proteins and codification of the respective proteins has to be already setup and in place in order for the differentiation and development to be able to take place. Both are essential, and so irreducible. 

Within Box I domains the progenitor fields for the future adult body parts are later demarcated by signals plus local regulatory spatial information formulated in Box I, and given regulatory states are established in each such field by the earliest body-part-specific GRNs. Many such progenitor fields are thus set up during postgastrular embryogenesis, and a GRN defining one of these is here symbolized as Box II. Each progenitor field is then divided up into the subparts that will together constitute the body part, where the subdivisions are initially defined by installation of unique GRNs producing unique regulatory states. These “sub-body part” GRNs are symbolized by the oriented patterns of Box III. Because some body parts are ultimately of great complexity, the process of patterned subdivision and installation of successively more confined GRNs may be iterated, like a “do-loop,” symbolized here by the upwards arrow from Box III to Box II, labeled n ≥ 1. Toward the termination of the developmental process in each region of the late embryo, the GRNs specifying the several individual cell types and deployed in each subpart of each body part, are symbolized here as Box IV. Postembryonic generation of specific cell types (from stem cells) is a Box IV process as well. At the bottom of the diagram are indicated several differentiation gene batteries (“DGB1, 2, 3”), the final outputs of each cell type. Morphogenetic functions are also programmed in each cell type (not shown). For discussion and background, see text and Davidson, 2001,Davidson, 2006.

As shown in Figure 3, we can distinguish four causally connected developmental functions that are encoded by sections of the GRN represented by Boxes I–IV. 

So what must be explained, is the origin of correct connection sequence that permits the correct temporal gene expression. Doest that sequence not constiture a irreducible complex system ? If the information of one step is faulty or inexistent, the whole cascade collapes.

The most upstream part of the GRN indicated in Box I controls postgastrular pattern formation. It is animated by pregastrular spatial and signaling inputs (maternal anisotropies, maternal factors, early interblastomere signals, all used as directional cues, and then by the outputs of the initial zygotic GRNs). The functions of the GRNs set up in this phase of development, including their signaling interactions, are to establish broad domains that section the organism with respect to the major body axes. The immediate output of the GRNs of Box I is to set upregulatory state domains within spatially defined areas of the organism. These domains, such as the neuraxis or mesodermal layers, constrain the position of future body parts and also now provide initial regulatory inputs that will be utilized in subsequent derivatives of their territories. The fate patterns they produce are often broadly conserved within clades (the early postembryonic “phylotype”).

In Box II, progenitor fields for specific body parts (for example, the heart progenitor field or the limb bud) are defined within these early domains. These are sets of cells each expressing the specific GRNs indicated at the level of Box II. The progenitor field then must be subdivided into regions that give rise to the future constituent pieces of the body part, each of which is foreshadowed by a new GRN (for example, the aorta or ventricle of the heart or the autopod of the limb). Within Box III thus lie the GRNs that control both the identity and the spatial boundaries of these subparts. This patterning GRN thus implements a coordinate system within the progenitor domain that is crucial for morphology and function of the body part. Both patterning GRNs (e.g., Box I and Box III) are oriented along the same axes, and the downstream body-part-specific patterning GRN therefore depends at least indirectly on the upper-level postgastrular patterning GRNs. Depending on the complexity of the body part, multiple rounds of spatial regulatory state subdivision and installation of further regional GRNs may be required. Thus, the progression from Box II- and Box III-type GRNs may be reiterated (backward arrow in Figure 3). Only following these patterning processes, the terminal cell-fate specification GRNs (Box IV) become activated in spatially restricted domains within the body part progenitor field. At the lower periphery of developmental GRNs are the differentiation gene batteries, that is, the protein-coding effector genes plus their immediate transcriptional regulatory drivers.

What kinds of subcircuit topologies are found at these different levels of GRN hierarchy? So far, a number of GRNs have been elaborated that indicate the recurrent use of subcircuits in given developmental contexts (Peter and Davidson, 2009). One such subcircuit, the positive feedback subcircuit, links two or more regulatory genes by multiple activating regulatory interactions and acts to stabilize regulatory states. This is necessary in body-part-specific GRNs (Box II) or cell-fate GRNs (Box IV), given that pattern formation processes usually occur only in a limited temporal window. Recurrent activating linkages keep the genes expressed even when the initial activating regulatory input fades. A positive intercellular feedback subcircuit can result in a “community effect” (Bolouri and Davidson, 2010), the stabilizing activation of similar regulatory states within a field of cells. Here a gene encoding an intercellular signaling ligand is expressed under the control of the same signal transduction system it activates. The pattern-forming GRNs of Box I and Box III in Figure 3, in contrast, operate largely by means of transient signal inputs as well as repressive exclusion functions that control spatial subdivision. Patterning processes are not concerned with stabilization or homogenization of regulatory states, and they contain few positive feedback loops. The biological function of individual subcircuit topologies predicts the probability of its occurrence at specific positions within the GRN hierarchy.

If one had to predict the GRN parts most likely modified in the evolution of body plans, a place to begin would be to define where in the developmental process and therefore in the GRN hierarchy differences occur. Morphological differences between species of different phyla affect the basic body plan, the overall organization of the organism. During development, the body plan is established mainly by the upstream embryonic patterning mechanisms and the individual body-part specification programs that they activate in given positions. Phylum-level morphological differences are therefore expected to occur in the GRNs underlying Boxes I and II. Among classes within the same phylum, the position with respect to the body axes or the internal structures of individual body parts may differ. Differences in the positions of body parts relative to each other could occur even when embryonic patterning GRNs and body-part specification GRNs are conserved, simply by rewiring the connections between these functions (such as the linkages connecting Box I and Box II; see also the discussion of hox gene functions below). This could result in alterations in the positions of given body parts. Morphological differences within body parts are more likely to be caused by differences in the spatial assignment of cell-fate domains determined by the body-part patterning GRNs of Box III. Based on these arguments one would expect that mutations in regulatory linkages within the patterning functions are more likely to be the cause of morphological changes, whereas specification GRNs active within given cell types or body-part progenitor fields are more likely to be conserved.

Given the predicted prevalence of specific network topologies for given biological functions, there might be a direct correlation between regional network topology and rate of evolutionary change. Regulatory linkages used for patterning embryos or body parts frequently rely on inductive signals that connect GRNs underlying specification in different domains and ensure orchestrated progression of development. In organisms of different spatial geometry, inductive signaling relationships will differ, and thus, inductive signaling interactions are likely to show a higher rate of evolutionary change. Indeed they do, as discussed elsewhere (Davidson and Erwin, 2006, Erwin and Davidson, 2009). The high level of conservation of positive feedback subcircuits has been previously proposed in the Kernel theory of Davidson and Erwin (2006)). These Kernels consist of a few regulatory genes linked by recursive positive regulatory interactions, and they are usually used upstream in GRNs that control the specification of progenitor fields for particular body parts, and they are conserved at large evolutionary distances.
In summary, evolution of GRNs to produce new developmental outcomes must involve new subcircuit deployments. This places a premium on co-optive change at the switches, signals, and inter-subcircuit inputs that encode subcircuit deployment. Evolution of new developmental GRN features must thus proceed to some extent as a process in which diverse subcircuits are combined, recombined, activated, and inactivated in given spatial domains of the embryo.

Evolution by Regulatory Changes in Single Genes

Though the jobs of development require the outputs of multigene subcircuits of given topologies, we see from the above that there are points of “flexibility” in developmental GRNs, where co-optive gain-of-function, or loss-of-function, regulatory changes may have large effects. By focusing on naturally occurring variations between closely related animals where visible evolutionary change has occurred recently, the most evolutionarily flexible aspects of the regulatory system are revealed. In the examples that follow, in which single genes are responsible for the changes observed, it has furthermore been possible to obtain experimental evidence for the evolutionary mechanism underlying the phenotypic variation in form.

Genomic Basis of Rapid Evolutionary Trait Loss

A canonical example, recently elaborated at the sequence level, and causally confirmed by experiment, is reduction of pelvic spines in stickleback fish. Following the end of the last Ice Age, marine stickleback fish were marooned in multiple lakes formed as the glaciers melted, and during the last 10,000–20,000 years independent populations of two different genera of these fish have repeatedly lost external pelvic spines. The exact selective advantages of pelvic reduction and spine loss are not defined, but as it has happened many times independently, there clearly are some (Shapiro et al., 2006 and references therein). Genetic complementation tests show that diverse isolates bear the same or overlapping genetic lesions, and this is so even in crosses of species from different genera displaying the same spine reduction phenotype. The underlying genomic event turns out to be deletion of a cis-regulatory module that controls expression of the pitx1 regulatory gene in the pelvic buds during larval development (Chan et al., 2010). Most significantly, when this cis-regulatory module was cloned upstream of a sequence encoding the Pitx1 protein and introduced into reduced spine fish, it rescued the spineless phenotype. The cis-regulatory module lies in an unstable, repetitive sequence-filled genomic region, possibly accounting for its repeated deletion (Chan et al., 2010). The pitx1 gene is clearly involved in pattern formation functions upstream of pelvic girdle specification, and in spineless fish there is no pitx1 expression in the pelvic buds even though the coding region of the gene is intact (Cole et al., 2003, Shapiro et al., 2006). In amniotes pitx1 operates in the patterning system that organizes the subparts of the appendages developing from the hindlimb buds, and forced expression in forelimb buds transforms them into hindlimbs (Logan and Tabin, 1999, Szeto et al., 1999). Thus this gene operates upstream in a portion of the GRN, the function of which is to generate the spatial regulatory states that presage the parts of the amniote hindlimb, and also of the pelvis, which is rudimentary in mice deficient in pitx1 (Szeto et al., 1999). Though pitx1 could execute more downstream roles in pelvic skeletal formation as well, its expression prior to the terminal phases of pelvic skeletogenesis indicates that it also functions in a Box III body-part-specific patterning GRN in stickleback fish.

However, rapidly evolving, reduced, or regressive phenotypes can be due to gain-of-function as well as loss-of-function mutations. The Mexican cave fish Astyanax exists both in riverine surface waters and in various cave populations that became isolated , and the regressively evolved traits of the cave populations have been studied for over a half century. A recurrent change in cave Astyanax is degeneration of eyes during larval development. During embryogenesis of cavefish, the eyes initially develop similarly to those of surface conspecifics, including expression of many regulatory genes (Jeffery, 2005, Jeffery, 2009). But then many things go wrong in eye development including apoptotic degeneration of lens and retina. A cause is ectopic spatial expression of sonic hedgehog (shh) from the normal medial interocular region across the top of the ocular fields in cave fish. As shown experimentally by introduction of shh mRNA in surface Astyanax, excess Shh causes expression of transcription repressors (vax1 and pax2a), which interfere with pax6 expression and thus the downstream pax6 ocular patterning subcircuit (Jeffery, 2009,Yamamoto et al., 2004, Baumer et al., 2002). Also, excess Shh indirectly promotes apoptosis in lens and retina. Though yet undefined at the sequence level, in cave Astyanax, regulatory changes have evidently caused a spatial gain of function in shh transcription resulting in regression of the eyes.

The simplest cases of evolutionary trait loss are deleterious mutations in far downstream differentiation genes. Pigmentation is among the regressive traits in cave Astyanax. Two pigmentation phenotypes have been shown to be due to mutations in the protein-coding sequences of receptors directly involved in pigmentation, oca2 (Protas et al., 2006) and mc1r (Gross et al., 2009). However, in stickleback fishes where there is also loss of pigmentation in lacustrine forms,cis-regulatory changes rather than coding region mutations are responsible (Miller et al., 2007). Here the gene responsible encodes kit ligand (Steele factor) and this gene has pleiotropic effects, so that total loss of function would be severely deleterious. Loss of function in a single cis-regulatory module, on the other hand, has specific effects that under certain conditions are adaptive. Because this is a general feature of cis-regulatory versus coding sequence mutations, it predicts that evolutionary changes in any pleiotropically active gene, as are most regulatory genes, will generally target specific cis-regulatory modules (as discussed, for example, by Chan et al., 2010, Miller et al., 2007, Prud'homme et al., 2006). Inverting this argument, we see a powerful evolutionary explanation for the modularity generally typical of the cis-regulatory systems controlling expression of regulatory and signaling genes in animal genomes (Davidson, 2001, Davidson, 2006). GRN evolution by regulatory gain and loss of function of expression of these genes would be utterly impossible were these control systems not in general modular, given that almost all such genes function in multiple time-space compartments, and in multiple GRNs during development. Physical and functional modularity in the control systems of regulatory genes is thus among the fundamental characteristics of animal genomes that permit and, indeed, that produce evolution of development by GRN reorganization.


Morphological Variation due to Single-Gene Regulatory Changes

Whereas the foregoing concerns rapidly occurring evolutionary changes in single-gene functions that are of adaptive significance, we now face a conundrum. How do we extrapolate from recent evolutionary events to the much more ancient processes by which order- and class-level differences in body plan arose, let alone phylum-level differences?
Recent studies focusing on the adaptive evolution of external traits in and among Drosophila species have revealed processes of cis-regulatory sequence microevolution. Such processes account for variation in pigmentation patterns due to regulatory changes affecting expression of the yellow differentiation gene (Gompel et al., 2005, Rokas and Carroll, 2006) and the ebony differentiation gene (Rebeiz et al., 2009). Similarly, cis-regulatory evolution in the shavenbaby (ovo) regulatory gene, which controls the differentiation and morphogenesis of trichomes (short hair-like surface appendages), determines where this gene is expressed, and thereby the minute pattern differences in trichome distribution distinguishing Drosophila species (McGregor et al., 2007). These studies afford multiple real examples of cis-regulatory site addition, and quantitative as well as qualitative cis-regulatory gain and loss of function due to internal DNA sequence change (see Table 1). They provide general and specific indication of the flexibility and changeability of cis-regulatory modules in local evolution, at the level of function and deployment of differentiation gene batteries, the lowest level in the hierarchy of Figure 3.



Mechanistic studies of intra- and interspecific evolutionary variation illuminate the next level up as well, that is, evolutionary changes (other than simple loss of function) in the Box III-type pattern formation GRNs that determine the morphological characteristics of given body parts. The results have thus far often resolved into demonstration of alterations in the deployment of signal systems in the development of these parts; that is, the underlying evolutionary change is in the cis-regulatory apparatus controlling time and place of inductive signaling, just as predicted earlier. The causal developmental mechanism underlying the adaptively diverse beak morphologies of Darwin's classic series of Galapagos finch species was solved in these terms by Abzhanov et al., 2006, Abzhanov et al., 2004). Species with heavy beaks displayed earlier and higher expression of bone morphogenetic protein 4 (BMP4) in pre-beak neural crest mesenchyme, and species with elongated, pointed beaks expressed Ca2+/calmodulin at higher levels, indicating that beak length depends on extent of Ca2+ signaling. Remarkably, experimental overexpression of BMP4 by retroviral gene transfer into developing frontonasal tissues of chicken embryos produces robust beaks, and experimental overexpression of the downstream mediator of Ca2+ signaling, CaMKII, produced elongated beaks, confirming the causality. To take another example, a recent study shows that short legs in dog breeds such as dachshunds and basset hounds is due to a retrogene encoding fibroblast growth factor 4 (FGF4), inserted and evidently controlled by cis-regulatory elements carried in non-LTR transposons (Parker et al., 2009).

Changes in upstream patterning apparatus can account for differences in body plan at inter-ordinal to inter-class levels, and such changes are not found in comparing organisms that diverged only a few million or a few thousand years ago or less. For example, one of the characters distinguishing bats and rodents, which are of different mammalian orders and in fact belong to different super-orders, is the much longer relative length of the forearm skeleton in bats. A candidate regulatory gene known to affect limb skeletal elongation is prx1 (mhox), and in bats this gene is upregulated after the early limb bud stage compared to mice (Cretekos et al., 2008). The (indirect) causality of this change was then demonstrated by inserting the bat prx1 limb enhancer into the mouse gene, with the result that the forelimbs of the recipient mouse now develop with relatively longer dimensions. In an essentially similar case, the tbx5 gene, deeply embedded in the vertebrate heart formation GRN (for review, Davidson, 2006), turns out to be regulated differently during heart formation in reptiles than in birds and mammals, a class-level difference. Expression of this gene is confined to the left ventricle in the developing amniote heart but is expressed across the common ventricle in the three-chambered reptile heart (Koshiba-Takeuchi et al., 2009). When uniform tbx5 expression is forced in the mouse heart, or left ventricle tbx5expression is prevented, that is, if a reptilian tbx5 spatial regulatory expression is imposed, the mouse develops a three-chambered heart lacking an interventricular septum. Understanding of developmental GRN structure tells us that these examples differ from the foregoing in that they imply the existence of Box III GRN subcircuits in which the targeted genes participate. In contrast, in the peripheral gene examples above, the phenotype is wholly encompassed by changes in a single cis-regulatory system.

Hox Gene Functions in Upper-Level GRN Patterning Systems

Genes of the trans-bilaterian hox complexes have been the subject of a vast amount of phenomenological research, which has revealed the many and various effects on developmental morphology of hox gene knockouts or ectopic hox gene expression. The variety of effects precludes any simple interpretation of the functions of these genes in terms of developmental GRN structure, for the simple reason that they work at diverse levels. Studies of direct hox gene targets reveal both other regulatory genes and far downstream genes encoding proteins active in apoptosis, cell-cycle control, cell adhesion, cell polarity, noncanonical signaling, and cytoskeletal functions (Cobb and Duboule, 2005, Hueber and Lohmann, 2008, Pearson et al., 2005). However, Hox genes are most famous for their developmental effects on the placement and the internal organization of body parts. The most important evolutionary and developmental attributes of hoxgene complex function can be reduced to two statements: first, in organisms in which coherent hox complexes exist they are expressed in a vectorial or sequential fashion with respect to the coordinates of the body plan or the body part; and second, they can act as switches that allow (or activate) GRN patterning subcircuits in given locations of the body plan or body part, or alternately they prohibit (or repress) these subcircuits in given locations.

The genomic organization of hox gene clusters indicates that distinct mechanisms account for the locations in the body plan where individual hox genes are expressed in development. In Drosophila a plethora of cis-regulatory modules control each aspect of expression of each gene. Particularly well-known at the cis-regulatory level is the bithorax region (Ho et al., 2009, Maeda and Karch, 2009, Simon et al., 1990). Each specific hox gene enhancer responds to local upstream regulatory states that are the product of earlier developmental GRNs, just as in any other developmental process. Similarly, many very well-characterized cis-regulatory modules that control very specific spatial and temporal aspects of anterior hox gene expression are known in mammals, and often conserved to fish (Tumpel et al., 2009). The prevalence of local cis-regulatory hox gene control modules explains how these genes can function in animals that lack large hox gene clusters. It is interesting that hox genes are not required for embryonic development of organisms that utilize fixed cell lineages for specification (Davidson, 1990), for instance in C. elegans, which lacks both a coherent hox complex and many hox genes (Aboobaker and Blaxter, 2010); in sea urchins (Martinez et al., 1999); or in Ciona, which also lacks a coherent hoxcomplex (Ikuta et al., 2010). However, in addition to control by local enhancers, another entirely different mechanism that speaks directly to both the evolutionary maintenance of the hox gene cluster(s) and the vectorial expression of hox genes relative to one another has come to light in mammals and other tetrapods.

Over the last decade, transcriptional control of the mouse hoxd complex has been extensively examined by deletions, rearrangements, and insertions of reporter transgenes, including ectopically positioned hox genes at various locations in the complex . To summarize very briefly, early expression in the tetrapod limb bud is controlled not only by local enhancers but also by distant regulatory regions located outside the hox gene clusters. One of these operates from the 3′ (anterior) end of the cluster and causes the progressive expression of first anterior and then middle hox genes in the limb bud region that will give rise to the forearm. Meanwhile the posterior hox genes are repressed by a counteracting locus control region operating from beyond the 5′ end of the complex in the anterior cells of the early limb bud, allowing expression of these genes only in the posterior limb bud cells. A second phase of hoxdexpression is controlled by other complex distant enhancers located 200 kb away from the 5′ end of the cluster, which are required to pattern the autopod region of the tetrapod limb where the digits form (Tarchini and Duboule, 2006). This “global control region” (GCR) is responsible for a graded expression of the five posteriorhox genes across the anterior/posterior (A/P) dimension of the autopod.

Global Control Regions and Regulatory Landscapes in Vertebrate Development and Evolution  
The proper implementation of the genetic program controlling cell differentiation and, ultimately, metazoan development requires the highly coordinated actions of multiple genes. Consequently, these genes need to be tightly regulated in both time and space, and with respect to their quantitative outputs. Small changes in a single gene expression pattern can lead to severe morphological alterations, as exemplified by haplo-insufficiencies in many human syndromes , ectopic gene expression in cancers , or even slight heterochronic variations (e.g., Juan and Ruddle, 2003; Zakany et al., 1997). Yet gene products rarely act alone, but usually interact with partners either to form large multiproteins complexes (e.g., Polycomb-group complexes, muscle contractile apparatus) or to be part of a sequential metabolic pathway (e.g., retinoic acid). Therefore, it is important that genes whose products are part of the same functional pathway are expressed at the same time and in the same cells.

The GCR probably had an ancient role in controlling colinear expression in the central nervous system, a basal axial organization function that in terms of our Figure 3 would reside somewhere in Box I; part of the active GCR elements are conserved from fish to mammals. However some limb-specific elements of the GCR likely evolved in tetrapods, particularly the autopod control device and its patterning GRN, which would make the autopod a novel evolutionary invention with respect to the fish antecedents (Gonzalez et al., 2007, Woltering and Duboule, 2010). More generally, it is an interesting speculation that distant hox complex control regions were superimposed during chordate evolution (they are absent fromDrosophila), and control by local hox gene enhancers was the primal regulatory mode (Spitz et al., 2001). However, because the regulatory landscape to which the local enhancers must respond can be very different in different organisms, they themselves must have evolved in clade-specific ways.

Given these systems, deeply conserved and otherwise, by which hox gene expression is regionally controlled, we come to their mode of interaction with the GRNs that control development of specific body parts. Sometimes individual hoxgenes act by participating, like any other regulatory genes in patterning GRNs, for example in early hindbrain specification, a Box I function. Together with other important regulatory genes such as krox and Kreisler, the anterior group hoxa and hoxb genes establish recursively wired, extremely conserved, rhombomere-specific GRNs (Tumpel et al., 2007, Tumpel et al., 2009). But more often they operate in another,  flexible way, such that change in their functions has been directly correlated, in many comparative observations, with  change in both the positioning and organization of body parts.

In many more cases than those mentioned here the function of these regionally expressed genes is rather to provide a one-way switch that provides “go” or “no go” instruction to body-part-specific GRN patterning circuitry. In evolution the deployment of these switches, and the linkages between them and the body-part-specific subcircuits, are far more flexible than is the internal structure of these subcircuits. 

Unless all forms were “sprung forth fully blown” like Athena from the head of Zeus, the evolution of the diverse body plans of animals requires large-scale processes of change in ancestral developmental GRN architecture. 

Conservation

The hierarchical Linnean classification system we use, including modern corrections based on molecular phylogenetics, essentially arranges animal body plans on the basis of their evolutionarily shared and derived characters (avoiding convergent associations). Shared body plan characters of given clades ultimately imply conserved developmental regulatory circuitry (Davidson and Erwin, 2009). But other apparently older characters are shared over huge phylogenetic distances across cladistic boundaries, being represented in multiple bilaterian phyla and in diverse body plans. These are particular body parts, such as hearts, and the major domains of brains, and particular cell types, such as muscle and neurons.
Because of their very widespread distribution, some differentiation gene batteries are probably among the oldest features of modern developmental GRNs (Davidson, 2006, Davidson and Erwin, 2009). But just as a cell type is not the same thing as a body part, so a differentiation gene battery is not the same thing as a cell type. During evolution the identity of the effector genes can change radically, whereas the biological function of the cell type remains the same; and in addition, the cell type often has cell-biological or -morphological characteristics that are not encoded the same way as is activation of sets of effector genes. So we have to consider what GRN structures actually lie at the root of trans-phyletic cell-type conservation. A few examples may clarify this issue.

Comparative GRN analysis is beginning to reveal “kernels” (Davidson and Erwin, 2006), in which regulatory genes wired together in certain conserved linkages execute upstream regulatory functions in development of given body parts. These circuits are characterized by extensive feedback wiring, and where tested, interference with expression of any of their genes results in developmental catastrophe. 

 These features, and developmental canalization due to the upstream position of such kernels in the body-part GRN, explain their exceptional evolutionary conservation. Examples include what may be a pan-bilaterian (i.e., from flies to mice) kernel for heart specification (Davidson, 2006) and an (at least) pan-echinoderm kernel underlying mesoderm specification in both sea urchin and sea star development (McCauley et al., 2010) (these lineages have not shared a common ancestor since the end of the Cambrian). Similarly, a fundamental Box II subcircuit may underlie mesoderm specification in vertebrate embryogenesis (Swiers et al., 2010). A recursively wired triple feedback circuit has been proposed as a kernel underlying the pluripotent state of endothelial/hematopoietic precursors that arise in vertebrate development (Pimanda et al., 2007). There are also many less coherent observations, not yet at the level of an explicit GRN, in which detailed patterning similarities plus some gene interaction data strongly suggest the existence of GRN kernels that yet await elucidation. One convincing example is the brain, where a large amount of work has illuminated striking similarities in both A/P and mediolateral patterns of regulatory gene expression as well as homologous gene interactions between Drosophila and mouse (Davidson, 2006,Denes et al., 2007, Lowe et al., 2003, Seibert and Urbach, 2010, Tessmar-Raible et al., 2007).


1) http://www.cell.com/cell/fulltext/S0092-8674(11)00131-0
2) http://bejerano.stanford.edu/readings/public/80_Function_GCRs.pdf



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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.


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.

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Views of body plan evolution



At present several entirely different explanatory approaches compete to illuminate the mechanisms by which animal body plans have evolved. Their respective relevance is briefly considered here in the light of modern knowledge of genomes and the regulatory processes by which development is controlled. 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. Of the many different aspects of evolution, we are here to be concerned with how the developmental mechanisms generating the body plan architectures recognized in Linnaen systematics at the level of phylum and class evolve, and how these mechanisms have been maintained, often since the Cambrian or Ordovician. Ideas about the nature of the underlying evolutionary mechanisms, and what to do to study them, generally associate with one of several paradigmatic views. Two of these views, though mutually incompatible, share the conviction that evolution of the body plan can be illuminated by study of adaptive evolution of detailed properties of modern organisms that are generated by far downstream developmental processes such as terminal differentiation.

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.

Views of body plan evolution

Of the first of these approaches (e.g., Hoekstra and Coyne, 2007), I shall have nothing to say, as mechanistic developmental biology has shown that its fundamental concepts are largely irrelevant to the process by which the body plan is formed in ontogeny. In addition it gives rise to lethal errors in respect to evolutionary process. 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.
The second paradigm holds that general evolutionary process will be revealed by studies of continuous variation in cis-regulatory modules affecting expression of adaptively meaningful genes. Its experimental application has indeed been enormously revealing in respect to the sequence level cis-regulatory mechanisms by which much natural variation arises. For example, very clear examples of functional evolutionary changes in cis-regulatory modules have come from recent studies on regulation of pigmentation genes between and within various Drosophila species. Among these are adaptively significant variations in regulation of the yellow gene, which accounts for a variety of spatial pigmentation patterns in higher Dipteran wings and body surfaces including sexually dimorphic markings ( Gompel et al., 2005Prud'homme et al., 2006Prud'homme et al., 2007 and Jeong et al., 2006); and of the ebony gene, which controls the degree of melanization in differently pigmented populations living in diverse Ugandan environments ( Rebeiz et al., 2009). 


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). 


Another, essentially similar, recent demonstration of cis-regulatory evolutionary change in an adult trait concerned some detailed pattern differences in trichome distribution that distinguish Drosophila species ( McGregor et al., 2007). Trichome assembly is controlled by genes requiring expression of the regulatory gene shavenbaby ( Chanut-Delalande et al., 2006), and several cis-regulatory modules determine the exact spatial expression of this gene in response to the upstream regulatory landscape. Evolutionarily arising differences in the DNA sequences of these modules collectively determine differences in spatial expression of this gene, and thereby generate the species' specific differences in trichome pattern. Prud'homme et al. (2007;opcit.) explicitly proposed that experimental analysis of functional cis-regulatory differences affecting adaptive traits among related species will illuminate larger evolutionary changes in development of the body plan, just as such analysis illuminates selective changes in intra- or inter- specific color patterns or surface morphology (another uniformitarian view).


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.

Suppose that we begin with the following syllogism, which to a systems developmental biologist seems inescapable: since dGRNs control ontogeny of the body plan, and since evolution of the body plan requires genomic alteration of the developmental program, then relevant explanations must be couched in terms of those genomic alterations that change the structure and function of dGRNs. 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. In the interest of conciseness these principles are summarized in Table 1 and briefly discussed in the following.





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

Many of the arguments referred to in Table 1 have been presented earlier, as indicated. At the outset, the main point of difference between this and all other approaches to understanding evolution 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 except at the minute and always partial, if not wholly illusory, level of the worm's eye view. The same must be true as well for major evolutionary change in the body plan or in body parts.
The purpose of Table 1 is to indicate the specific consequences for considerations of evolutionary process that derive from dGRN structure/function relationships (cf.Davidson, 2010 for review of this subject). The first principle in Table 1 is that the mechanism underlying structural change in dGRNs is redeployment of cis-regulatory modules due to sequence changes that result in co-option of regulatory gene expression 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. Co-option can occur by various mechanisms at the genomic sequence level. 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 (for current review, Peter and Davidson, in press).
Principle 2 follows from the point that such co-optive changes in general belong to the cis-regulatory gain of function class. As initially pointed out by Ruvkun et al. (1991), laboratory experiments show that where the genes affected are regulatory genes operating in embryonic development, these are almost always haplosufficient mutations; one copy expressed in a new location does the job (otherwise, of course, none of the regulatory genes isolated by haploid recessive screens would have been found!). The fundamental importance of haplosufficiency is that in evolution an individual bearing such a mutation will become a clonal founder of a novel population expressing a new developmental regulatory state (Davidson and Erwin, 2010), unless it is developmentally deleterious. To make a long story short, it follows that change in dGRN structure does not require the population genetics functions that result in homozygosity; that such co-optive dGRN change is likely to happen, and that it could happen at a relatively high rate were there not stabilizing circuitry in dGRNs that precludes alternative outcomes and locks down regulatory states once they are established ( Erwin and Davidson, 2009Davidson and Erwin, 2010 and Peter and Davidson, in press). In addition, as discussed below, dGRNs are insensitive to quantitative regulatory state changes.
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 (Peter and Davidson, in press and Davidson, 2010). Principles 3–5 derive from the hierarchical characteristic of dGRNs. Principle 3 is to the effect that the significance or functionality of any given cis-regulatory mutation affecting expression of a regulatory gene will depend entirely on where in the dGRN the affected cis-regulatory node is located ( Erwin and Davidson, 2009). The effects of given cis-regulatory DNA sequence changes on GRN function cannot be inferred simply from results obtained in the “flat” regulatory landscape where the phenomenon studied is the effects of SNPs or small indels on either protein coding sequence, or on cis-regulatory function in the control of expression of peripheral effector genes.

Implicit in the hierarchical structure of GRNs is the mechanism of evolutionary canalization, as indicated in Table 1 at principle 4. 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 (Waddington, 1957 and Gibson and Wagner, 2000). A few years ago remarkably conserved subcircuits, termed network “kernels” that operate high in the dGRN hierarchy were discovered (Davidson and Erwin, 2006). 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; Davidson, 2006). A testable theory to explain the hierarchical shape of Linnean bilaterian phylogeny (superphylum, phylum, class, etc), or what Erwin (this collection) terms the “clumpiness” of the phylogenetic distribution of animal morphologies, is based on kernels (Davidson and Erwin, 2006 and Erwin and Davidson, 2009). 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. The argument is commutative. This theory requires that the kernels similarly canalize downstream developmental process in each member of each given clade. But since on first principles hierarchical dGRNs must produce canalization (principle 4), then in order to account for the phylogenetic distribution of shared morphological characters, the existence of kernels could have been predicted, as stated in principle 5 of Table 1.

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 (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. 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 (Davidson and Erwin, 2006 and Erwin and Davidson, 2009). 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 evolutionarily flexible and its evolutionarily less flexible linkages (Peter and Davidson, in press), leading to principle 6 in Table 1. 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. Principle 7 states the self-evident: 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 evolutionary change in dGRN structure should be those co-optive alterations that result in redeployment of whole subcircuits. A very good example is the evident redeployment of an adult skeletogenic GRN to an embryological address in sea urchin evolution, at least a large part of the mechanism by which the “modern” sea urchins acquired skeletogenic function in their embryonic micromere lineages (Gao and Davidson, 2008). Putting principle 6 together with principle 7 we see that an important place in dGRN structure to look for evolutionary change is in linkages that control subcircuit deployment: as principle 8 indicates, 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. As reviewed by Peter and Davidson (in press), much evidence indicates that hox gene functions often fall into this latter class. An ancillary point is that these kinds of linkage usually lack the feedback relations that act to stabilize developmental state (and evolutionary status); 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 that constitute terminal differentiation and morphogenetic gene batteries, and their immediate controllers, lie at the network periphery (Davidson, 2006 and Davidson, 2010). 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 same is true of many quantitative developmental traits which affect post-embryonic developmental process. The cis-regulatory modules for which functionally adaptive evolutionary sequence variation has been demonstrated, such as in the paradigmatic studies cited above on the yellow, theebony, and the shavenbaby genes of Drosophila, all lie at such peripheral positions in the respective dGRNs. Here we can readily perceive continuous Darwinian processes of sequence change, and selective adaptive variation in cis-regulatory output, as shown explicitly in the cited studies, among many other less well worked out examples. Throughout the dGRN, at every level of hierarchy, the processes of sequence change in cis-regulatory modules must be the same. 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 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. Or consider the particular example used by Prud'homme et al. (2007) to argue for the uniformity of evolutionary process at all levels of dGRN hierarchy,viz. the repression of wing patterning functions in the haltere imaginal disk by Ubx in Diptera ( Weatherbee et al., 1998 and Galant et al., 2002). In fact we do not see variation in the amount of “wingness” vs. “haltereness” displayed in the development of this imaginal disk; in bees, which have four wings, Ubx has different cis-regulatory targets than in flies ( Weatherbee et al., 1999), and there is either the one morphological output, four wings, or the other, two wings and two halteres, across this region of insect phylogeny. 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 lack of continuous variation in morphogenetic traits defining class and phylum level clades is obvious in the striking evolutionary stasis revealed by the fossil record (Davidson and Erwin, 2006Erwin and Davidson, 2009 and Erwin, in press). 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 (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.
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 bilateria ( Erwin and Davidson, 2009)? A dramatic illustration of such stasis is reproduced in Fig. 1 (Bottjer et al., 2006): here we see the real time distribution of fossil variants of echinoderm body plans.




Fig. 1. 
Evolutionary history of the major echinoderm groups. Cambrian echinoderms are recognized by the possession of stereom, but the phylogenetically most basal groups (such as stylophorans) lack the water vascular system, are highly asymmetrical, and possess gill slits. Pentameral symmetry is seen in two major Early Cambrian lineages, the edrioasteroids and eocrinoids. All stem-group echinoderm lineages became extinct by the Carboniferous (indicated with crosses). Crown-group echinoderms, indicated by the yellow circle, consist of the five major extant lineages in addition to numerous extinct lineages not shown. Most class-level crown groups first appear in the latest Paleozoic–early Mesozoic, including echinoids. The lineage leading to echinoids is indicated in purple. Known stratigraphic ranges are shown with thick lines, and inferred range extensions are shown with thin lines. Reproduced from Bottjer et al. (2006). Copyright (2006) AAAS.

The early Cambrian was a period of (relatively) rapid evolutionary exploration of diverse developmental pathways as the programs directing the formation of crown group echinoderm characters were stepwise added into the stem group dGRNs. But following the period of morphological change the definitive properties of the five surviving echinoderm classes have remained stable essentially since the Cambrian and Ordovician (cf. Erwin, in press). The answer to the questions posed at the beginning of this paragraph 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. A short discussion of such features follows, and in the final section of this paper are some further considerations of the meaning of the most interesting of these dGRN design properties.

To consider this question we must first remind ourselves what is the main function of upper level dGRNs for body plan formation. This has been discussed in detail in developmental (Peter and Davidson, 2009 and Davidson, 2010) and evolutionary (Peter and Davidson, in press) contexts; a very brief summary is that 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, as shown by Bolouri and Davidson (2003) 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 Peter and Davidson (2009[Febs Lett]); 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 (for multiple examples see Peter and Davidson, 2010). Other subcircuits set sharp boundaries of expression by a variety of design devices; others mutually exclude regulatory states; etc. As Peter and Davidson (2009) showed, 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 (Davidson et al., 2002), 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 (for review, Davidson, 2006). 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

As discussed above 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.


Multiplicity of dGRN subcircuits ensuring given developmental outcomes

The characteristic tempo of evolutionary change illustrated in Fig. 1, in which a period of intense morphogenetic novelty is succeeded by long epochs of body plan stasis, suggests that early in clade history dGRNs were in some way different from crown group dGRNs (Erwin and Davidson, 2009 and Erwin, in press). This is of course another prima facie contradiction of the uniformitarian assumption that current observations on adaptive evolutionary change in specific peripheral cis-regulatory systems can illuminate early animal evolution. One way of thinking about this is to imagine that the evolutionary stability of crown group dGRN structure is due to the addition of more and more circuitry to control developmental pathways and exclude alternatives. These changes would have affected control of those embryonic stages at which the body plan is being specified by regional installation of regulatory states ( Erwin and Davidson, 2009 and Peter and Davidson, in press). The implication is that stem group dGRNs, for example those of the early Cambrian echinoderms of Fig. 1, were structured differently from modern crown group dGRNs in respect to the multiplicity of the subcircuits brought to bear on each phase of the developmental process.

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; domains are set up by alternative regional activation and their boundaries are then enforced by cross boundary repressive signaling and/or specific repressive exclusion of possible alternative regulatory states; dynamic feedback loops stabilize and enforce regulatory states; and not uncommonly many of the above devices are all deployed together in the same dGRN (for examples,Oliveri et al., 2008Peter and Davidson, 2009 and Peter and Davidson, in pressSmith and Davidson, 2009 and Davidson, 2010). Though multiple such devices lead to the given overall developmental outcome, on principle they cannot be redundant, and in fact they never are when tested experimentally. That is, 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 ( Peter and Davidson, 2010 and Peter and Davidson, in press). 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 (e.g., Ben-Tabou de-Leon and Davidson, 2010 and Smith and Davidson, 2008). 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 ( Peter and Davidson, 2009 and Ben-Tabou de-Leon and Davidson, 2010). 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 ( Ransick and Davidson, 2006). 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( Oliveri et al., 2008). 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.


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 (Peter and Davidson, in press), 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.
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1) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3135751/
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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.



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Molecular Genetics and the evolution of animal design,

Sean B.Carroll,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.

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Biological systems demonstrate engineering principles, which point to design

http://reasonandscience.heavenforum.org/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://sci-hub.bz/http://www.nature.com/nrg/journal/v8/n10/abs/nrg2192.html

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

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