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AMER. ZOOL., 38:609-620 (1998) Developmental Gene Regulation and the Evolution of Large Animal Body Plans'2 R. ANDREW CAMERON, 3 KEVIN J. PETERSON, AND ERIC H. DAVIDSON Division of Biology, Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena, California 91125 A diverse assemblage of invertebrate animals, some of which basically resemble the forms found in modern oceans, appears in the fossil record soon after the advent of the Cambrian period, though the first large multicellular animals clearly arose even earlier. How this occurred is among the intellectually challenging mysteries of biology. The solution to this mystery is likely to emerge, in part, from an understanding of the molecular processes by which modern animals use their genetic information to construct their body plans during embryonic development. We discuss a mechanistic hypothesis that was presented earlier as an explanation of the causal events underlying the "Cambrian explosion," and thus the divergence of large animal body plans. SYNOPSIS. such as walking or flying appendages, air breathing apparatus, body structures strong One of the most exciting and fundamento preserve motility and form withenough tally important discoveries of paleontology is that remains of many animal phyla ap- out the buoyant support of sea water, senpear in the fossil record almost at once, dur- sory apparatuses that operate in air, etc. ing the geological period known as the This article is focused on the initial apCambrian. The lower boundary of the Cam- pearance of large animals, i.e., of marine brian period has now been dated very ac- invertebrate forms, rather than on the later curately, as it occurs in two distant regions evolutionary burst in which originated land of the earth, to about 544 million years vertebrates and arthropods. Despite the very dramatic and very sud(my) before the present (Bowring et al., den appearance of disparate body plans ev1993; Grotzinger et al., 1995). Within about 20 my thereafter a tremendous increase in ident in Cambrian fossils, so dramatic that the diversity of the body plans of the or- it is frequently referred to as the "Cambrian ganisms represented in the fossil record is explosion," it is clear that at least some observed. Some examples of remarkably evolutionary lineages of large animals had well preserved Cambrian fossils are shown already appeared before the Precambrianin Figure 1. The Cambrian period ended Cambrian boundary (e.g., Gehling, 1987; about 505 my ago, and by then most of the Gehling and Rigby, 1996; Waggoner, 1996; major branches of the Animal Kingdom Conway Morris, 1993; Fedonkin and Wagwere probably present except for some ter- goner, 1997; see Runnegar, [1995] and restrial clades. Land masses were initially Gehling, [1991] for discussion). Indeed, colonized by animal forms about 80 my lat- molecular estimates extend the divergence er (Gray and Boucot, 1994; Shear et al., times for these major groups to even earlier 1996). This required highly specialized new times (Runnegar, 1982; Wray et al., 1996). biological inventions for terrestrial life, Precambrian fossil forms that are unmistakably those of animal organisms are given the general name Ediacaran or Vendian fau1 From the Symposium The Evolution of Development: Patterns and Process presented at the Annual na, and they extend back in time some milMeeting of the Society for Integrative and Compara- lions of years before the Cambrian and fortive Biology, 26-30 December 1996, at Albuquerque, ward to this boundary (Horodyski et al., New Mexico. 1994; Grotzinger et al., 1995). Some ex2 An earlier version of this paper appeared in the amples are also included in Figure 1. Many French journal La Recherche. 3 of the Vendian forms evidently represent E-mail: [email protected] INTRODUCTION 609 R. A. CAMERON ETAL. C E FIG. 1. Cambrian and possible Precambrian representatives of three modern animal groups: a and b: cnidarians; c and d: echinoderms; e and f: arthropods, a. Thaumaptilon, a sea pen from the Middle Cambrian of British Columbia (Conway Morris, 1993). b. Charniodiscus, a sea pen from the Late Precambrian of South Australia (Jenkins and Gehling, 1978). c. Camptostroma, a primitive echinoderm from the Early Cambrian of Pennsylvania (Paul and Smith, 1984). d. Arkarua, a possible echinoderm from the Late Precambrian of South Australia (Gehling, 1987). e. Opabinia, a primitive arthropod from the Middle Cambrian of British Columbia (Whittington, 1975). f. Bomakellia, a possible primitive arthropod from the Late Precambrian of Russia (Waggoner, 1996). Scale bars in a—c, e—f = 2 cm; scale bar in d = 2 mm. primitive cnidarians, the first examples of which may have arisen as much as 50 my earlier (Hofmann et ah, 1990). However, the differences between modern cnidarians and bilaterally organized animals run so deep, that it is not clear that the processes by which cnidarians arose directly illuminate the origins of bilateral animals, all of which include more cell types and much more highly organized multicellular organs than possessed by cnidarians. Sponges are even more remotely related to the bilateral invertebrates (Nielsen, 1995), and in the following our focus is on the latter rather than on cnidarians, sponges (or plants, fungi or other forms). The Ediacaran fauna includes some animals that appear very much like primitive, bilateral organisms (Fedonkin and Waggoner, 1997); if that is what they were, the evolution of organisms of this grade must extend deep into the Precambrian. Furthermore, there is reason to be suspicious that some of the suddenness of appearance of Cambrian fossils could be due to the evolutionary onset of characteristics which greatly improve the chances of fossilization, for example, large size, shells, carapaces, skeletons. Precambrian ancestors representing some Cambrian (and modern) body plans could have existed earlier, but have fossilized poorly. Indeed some such organisms apparently left traces of themselves in Vendian age deposits, in the form of burrows and tracks that only a bilaterally symmetrical animal could have produced (see, for example, Fedonkin, 1994). Thus the great mystery that we would all like to understand is not quite so lurid as implied by the term "Cambrian explosion," but a fundamental mystery it remains. The solution to this mystery cannot consist of elucidations of changes in environmental conditions that permitted large animal forms to exist, for instance an increase in dissolved O2; such conditions are indeed necessary but their enumeration tells us nothing about how different forms of organisms are actually generated, or how the different features of these organisms arise biologically. What we need to understand, and what we might refer to as the internal mechanisms of evolution, is the nature of the genetic changes that potentiated the appearance of animals. For what animals bequeath to their descendants is only their genetic systems, and all biological evolution- 611 GENE REGULATION AND EVOLUTION ary change that affects the properties of animals is ultimately change in DNA. We believe that the mystery of animal origins can be solved, at least in principle, by understanding the mechanisms by which body plans are formed in embryological development, during the life cycle of every individual. This point was made by Britten and Davidson (1971), but only now that we are beginning to understand how development really works at the molecular and genetic level can such an approach be applied to understanding evolution. GENE BATTERIES REGULATORY GENES SB F ^ . PROTEIN A1 A 0 — | I ^ _ PROTEIN A2 09 [-> PROTEIN A3 A4 PROTEIN B1 | - * * - PROTEIN B2 PROTEIN 83 Genetic regulatory programs specify the adult body plans of complex animals The structures of which an animal is built—organs, tissues, body parts—are heritable characteristics of each species. Therefore the regulatory program for the assembly of these structures, as well as the proteins of which they are composed, are encoded in the genome and are genetically transmitted to successive generations. For the problem of understanding the origin of animal body plans, the essential aspects of the genetic developmental program are regulatory. A very complex network of gene control circuitry determines what genes are turned on, and when and where they are turned on in a developing organism (Arnone and Davidson, 1997). The genetic regulatory program thus directs the whole process of development, step by step, controlling the formation of each element of the body plan. We believe that some layers of the developmental regulatory system encoded in the genomes of modern animals are more basic, more ancient in terms of evolution, than others. The hypothesis that we have presented earlier provides a mechanistic way of thinking about the origins of animals, but not a scenario for why these changes took place (Davidson et al., 1995). It is rooted in new concepts of how the developmental regulatory systems of modern animals work. The necessary essentials lie in three aspects of the genetic regulatory circuitry that controls development. An elemental level of the genetic regulatory network is the gene battery (Morgan, 1934). A gene battery is a set of coordinately controlled genes which encode pro- PROTEIN B4 FIG. 2. Regulation of gene batteries. Regulatory genes encode transcription factors (shaded polygons) which in various combinations control the coordinated expression of different gene batteries. For example, one regulatory gene encodes a factor depicted as a hatched square that regulates all the downstream genes of one battery while another factor (a stippled inverted triangle) regulates the genes of a second downstream battery. teins that are required in concert. Britten and Davidson (1971) considered gene batteries as molecular regulatory systems in which the individual genes are subject to the control of common regulatory factors. As we indicate in the cartoon shown in Figure 2a, expression of each gene of the battery {i.e., transcription of RNA) is caused to occur when particular regulatory activators appear in the nucleus of the cell, bind to the target DNA sequences in the control region of the gene, and by means of interactions with one another and with the transcription machinery, cause the synthesis of RNA molecules that encode the protein. These regulatory activators are transcription factors, proteins that have the capacity to recognize certain specific target DNA sequences present in the control regions of every gene of the battery. Thus when the regulatory genes encoding the right transcription factors are expressed, and these factors appear in the cell nucleus, all the genes of the battery may be turned on. In life each gene requires many different transcription factors, often more than ten, and many genes belong to several different batteries. In mammals, more than 10% of all genes in the genome are likely to be regu- 612 R. A. CAMERON ET AL. latory genes encoding transcription factors (Henikoff et al., 1997). The definitive property of multicellular life is the use of different genes in different cells, so that given sets of genes are expressed in each cell type and in each developmental circumstance; no one cell expresses the totality of gene batteries encoded in the whole genome. Gene batteries encode cell type-specific properties, for example the sets of proteins required to make a contractile muscle cell or a blood cell. In these cases, and in many others, at least some of the key transcription factors are known, and their DNA target sites are indeed found in the cw-regulatory regions of the genes of the battery, often in multiple copies. For example, the cis-regulatory sequences involved in the regulation of vertebrate muscle-specific genes including those encoding muscle actins, myosin heavy and light chains, troponin T, muscle creatinine kinase and acetylcholine receptor subunits have been identified (reviewed in Arnone and Davidson, 1997). These regulatory regions share binding sites for a suite of transcription factors. For example, mouse muscle creatine kinase, rat myosin light chain, chicken actin proximal element and chicken cardiac actin proximal element all contain sites for the myogenic determination factor, a basic helix-loop-helix protein. Other kinds of gene batteries encode sets of proteins needed to execute particular functions, e.g., the proteins needed to carry out cell division; to build particular subcellular structures such as ribosomes or cilia; or to produce secreted, extracellular structures that again require the products of many different genes, such as bone, or the external shells of insect eggs. Transcription factors can diffuse freely within the cell nucleus and thus the genes of a battery may be widely separated within the genome, both from one another and from the regulatory genes that control them. Many experiments in which genes are inserted experimentally at random locations in the genome indicate that genes can be virtually anywhere in the genome, and providing they have the necessary cw-regulatory signals associated with them they will work properly. The flexibility of gene regulation has enormous evolutionary conse- quences. Thus genes can be added to preexistent gene batteries or combined to create new ones by transposition processes that move old genes to new places, where they may come under the control of some other gene's regulatory apparatus, or transposition processes may copy and move the regulatory apparatus of one gene to the vicinity of a different gene (Britten and Davidson, 1971). Transposons that move elements of DNA around the genome occur in all known genetic systems, and these and other mechanisms causing DNA rearrangements have probably been a major source of evolutionary novelty (Britten, 1996). A second fundamental aspect of developmental gene regulatory circuitry that is essential to keep in mind when thinking about the evolution of developmental regulatory programs is the way expression of genes is controlled by signals from other cells. Gene regulatory systems that respond to signals from other cells are essential to all forms of metazoan development (e.g., nematodes: Lambie and Kimble, 1991; Sternberg, 1993; sea urchins: Ransick and Davidson, 1995; Drosophila: Venkatesh and Bodmer, 1995; Gonzalez et al., 1995; mouse: Williams et al., 1995). They are required for cells of a given tissue to communicate with one another; they allow cells in different parts of the organism to change their states of gene expression in a coordinated fashion as development proceeds; and they provide the mechanism by which cells expressing given genes establish spatial boundaries with cells expressing other genes. The most direct mechanism by which signals from outside a cell may affect expression of specific genes is diagrammed in Figure 3. This mechanism is particularly potent when the affected gene encodes a transcription factor which in turn controls many other genes. All developing organisms use signaling to place cells which are to express certain genes in the right locations in the developing organism. Many signaling pathways that play a role in the establishment of axes or embryonic regions have been identified from the signal ligand to the transcription factor that ultimately transfers this information to the nucleus GENE REGULATION AND EVOLUTION TRANSCRIPTION /\ FACTOR NUCLEAR ENVELOPE RECEPTOR FIG. 3. A schematic representation of the mechanism by which an external chemical signal can influence gene expression. The receptor transduces the binding of an external ligand to a second messenger by chemical changes. The second messenger in turn activates a transcription factor which binds to the regulatory region of a transcription unit to influence the transcription of that particular gene. (e.g., dorsoventral axis in Xenopus [reviewed in Miller and Moon, 1996]). Thus signaling systems that affect gene regulation serve as major determinants of the form of a developing organism and of its parts. Evolutionary changes in gene regulatory circuitry which bring new batteries of genes under control of given signal systems can create new spatial organizations of differentially functioning cells (Shubin et al., 1997). This can mean new morphological boundaries, new cell types in given regions of an animal, new patterns of development, and downstream, new variations in the resulting structure of the organism. The third organizational feature of gene regulatory systems that we focus on here is that which (in our view) endows the genomes of modern organisms with the capacity to program the development of their body plans, and the complex structures of which they are composed (see Davidson, 1994; Davidson et al, 1995). The way these systems function is to set out spatial domains that will give rise to future body parts and structures, using a step-wise process of subdivision so that growing cells can be allocated to the diverse parts of each structure. In initial stages, genes encoding transcription factors are expressed in certain spatial domains of the embryo, thus defining the progenitor field for the structure that 613 is to be formed. The downstream targets of these genes are other genes encoding other transcription factors, and perhaps genes encoding elements of signal systems. There may be several levels of this hierarchical genetic "superstructure" (e.g., the Drosophila wing; Gomez-Skarmeta and Modolell, 1996). The boundaries of the spatial domains of the organism that are defined by regional regulatory systems of this kind are set by signal systems. Growth programs are called into play in each region, and when the regionalization process nears completion differentiation gene batteries are activated. The genetic regulatory hierarchies that define the major elements of the adult body plans of modern animals are evidently shared properties of large groups of evolutionarily related organisms. For example, all tetrapods probably define the territories that will give rise to their limbs (the limb buds) by expression of the same initial set of upper level transcriptional regulators (Molven et al., 1990). Even teleost fishes use these same regulators for regional specification of their pectoral fin buds (Sordino et al., 1995). Thus, different tetrapods produce from these similarly specified limb buds many diverse structures that differ in morphological detail, though their appendages are obviously homologous in general structural organization (see Fig. 4). The limb buds can be considered a "morphospace," which is filled in during development by calling forth different regulatory subprograms following the initial definition of the limb bud region, and the subdivision of the forming limb into its different prospective regions. The same is true of the insect wing, which in diverse insects develops from a similarly specified disc of undifferentiated cells, but is then patterned differently by species-specific regulatory functions. Some of the same upper level transcription factors are used to pattern wings that end up looking very different, as illustrated in fascinating comparative studies of butterfly and fly wings (Warren et al., 1994; Carroll, 1994; Carroll et al., 1994). In our view (Davidson et al., 1995), evolution of animal body plans may have involved repeated bursts of change that occur 614 R. A. CAMERON ETAL. FISH AMPHIBIAN BIRD MAMMAL FIG. 4. Regional specification and diversification of limbs during vertebrate evolution. Early in limb development, before a distinct limb bud is formed, both fish and tetrapods express the same transcription factor, Hoxc6 (arrow in a [Molven el ai, 1990]). As these limb buds grow, another set of transcription factors are turned on which demarcate the posterior region of the limb bud (arrow in b [Sordino et al, 1995]). Tetrapod limbs become distinguished from those of fish when they newly express some of these same posterior genes in the region of the limb bud that will become the hand or foot (c). These structures have no homologues in fish. Thus, even though the final morphological outcomes are distinct among the limbs of mammals, birds, amphibians and fish, these upper level transcription factors define a set of possible morphologies that can be generated from the limb bud region. when a new "morphospace" is created by the institution of a novel genetic regionalization program. The new morphospace would be quickly filled in with the assembly of many different downstream regulatory programs, resulting in many different morphological variants. The process of indirect development still utilized in many groups of marine invertebrates may indicate the regulatory mechanisms that preceded the evolutionary appearance of large animals It is a fascinating thought that in the seas of today there survives an extremely com- mon form of embryonic development that might long predate the dawn of the Cambrian. We think that the immediate developmental products of this basic but elegant process of embryogenesis may resemble in their cellular organization the earliest types of animal, those that were present before large animals evolved. These products are the marine larvae of modern indirectly developing species. Such larvae are small, free-living organisms usually less than 1 mm across. They consist of only a few thousand cells, and they generate only a very modest repertoire of differentiated cell GENE REGULATION AND EVOLUTION 615 FIG. 5. Three life phases of the sea urchin Strongylocentrotus purpuratus. The upper right panel shows the bilaterally symmetrical larval body plan of the pluteus at the end of embryogenesis (72 hr after fertilization) when it consists of about 1800 cells (150 microns longest dimension). The left panel depicts the larva just before metamorphosis to the adult (1 mm longest dimension). At this stage there are about 150,000 cells of which 90% are in the sea urchin rudiment which lies beside the stomach. The lower right panel illustrates the radially symmetrical adult body plan in a recently metamorphosed juvenile sea urchin crawling on a coralline algal substrate. The body is about 500 microns in diameter. types. Usually they have a few neurons, a few muscle cells, a functional gut with mouth and anus, and a ciliated epidermis. They feed on microalgae and bacteria in the ocean, and in modern forms they serve essentially as life support systems which nourish and protect the slowly developing rudiments from which the adult body plans of their species arise. A good example of a species that develops by this entirely indirect process is the familiar sea urchin. As Figure 5a shows, the embryo of the sea urchin produces a small larva with the features just described, that displays bilateral symmetry. The five-fold radially symmetric adult body plan of the sea urchin develops within the feeding larva, and Figure 5b shows both the elaboration of the larva as it feeds, and the growing adult rudiment that it carries within. At metamorphosis the larval structures per se are jettisoned, and the juvenile sea urchin emerges. Certain aspects of indirect development are in our view extremely significant for the problem of animal origins. First, indirect development occurs in many distantly related groups of animals. Not only sea urchins, but representatives from many invertebrate phyla develop indirectly, generating ciliated larvae essentially similar in grade of organization to that shown in Figure 5 (see Peterson et al., 1997). The detailed shape and structural organization of these larvae of course depends on the group to which they belong. The important point is that the process of indirect development from a ciliated larva, which itself bears little or no relation in structure to the adult body plan to which it will give rise, is a character shared by the majority of animal phyla. Therefore we postulate that the latest common ancestor of bilaterians minimally had a larval stage similar to what is found in modern indirectly developing marine organisms. This brings us to the nature of the embryonic 616 R. A. CAMERON ETAL. TABLE 1. Characteristics of lineage based (Type I) embryogenesis. A. The position of cleavage planes for each species is invariant and thus predictable in normal development. The fate assignments of blastomeres are therefore also generally invariant, and the embryo displays a canonical cell lineage. B. One embryonic axis is pre-specified in oogenesis. The egg is radially symmetrical with the second axis specified after or during the fertilization process. C. Lineage founder cells are specified during cleavage, with respect to the plane of cleavage. Some are specified autonomously at the poles of the embryonic axes, while others are specified conditionally by interblastomere signaling. D. The embryo has the capacity to regulate developmental programs after manipulation in direct relationship to the degree of conditional specification which occurs during normal development. E. The specification of cell types precedes any largescale embryonic cell migrations and thus occurs in situ where the founder cells arise during cleavage. F. Zygotic transcription is active at least as early as cleavage and there is no early embryonic period of transcriptional quiescence. processes by which larvae such as shown in Figure 5 arise. In terms of the genetic regulatory circuitry discussed above, this type of embryonic process is much simpler than those by which the adult body plans of most metazoan phyla develop, and it is even more widespread than is the extreme form of indirect development exemplified in Figure 5. We call this process Type 1 embryogenesis (Davidson, 1991), and we consider it likely to be the original form of embryonic development to have evolved (Davidson et al, 1995). Type 1 embryogenesis proceeds essentially by dividing the egg up into founder cells that generate specific cell lineages, each of which soon begins to express certain genes and ultimately generates certain parts of the completed embryo. Only about 10 ± 2 cell divisions occur before embryogenesis is completed. Some properties of the Type 1 process of embryogenesis are listed in Table 1. The key point, which for us unlocks the whole problem we are considering, is that Type 1 embryogenesis seems to require only the first two of the three aspects of developmental gene regulatory circuitry that we discussed in the preceding section of this article. To build an embryo by the Type 1 process requires the operation of differentiation gene batteries, and it requires that cells be able to affect each other's genetic activities in specific ways by signaling, but it involves little or no use of upper level regulatory hierarchies, or of the regional specification processes that we di-cussed above. For example, in the nematode Caenorhabditis elegans, the gene, Hn39, a HOMIHox gene, is completely unnecessary for early embryogenesis, but is absolutely required to progress through larval development (Wang et al, 1993). Thus, in Type 1 embryogenesis fixed cell lineages are utilized to put cells doing the right things in the right places in the embryo. All indirectly developing animals thus far examined that produce ciliated feeding larvae generate them by Type 1 embryonic processes, and in these cases we can see very clearly what the Type 1 process is good for: it suffices to build a small and simply constructed little animal consisting of only a few thousand cells, which is self-sufficient because it can feed and is motile. In many species that display Type 1 embryogenesis, the larval stage has been lost by an evolutionary process in which the mechanisms producing the adult body plan are telescoped directly onto the preceding embryonic process, and this is termed direct development (Jagersten, 1972; Davidson et al, 1995). This happens often and easily in evolution, as shown by the frequent occurrence of close relatives of indirect developers which display direct development, to various extents depending on the species (Raff, 1987; Wray, 1994; Wray and Bely, 1994). Some whole groups, the embryos of which develop by classic Type 1 processes, such as nematodes, develop only by direct means, and they display no signs of ancestral forms that developed by means of ciliated larvae. Nonetheless, if we consider both direct and indirect developers that utilize Type 1 embryogenesis, we see that the category of Type 1 developers includes almost all animal groups, with the important and interesting exceptions of vertebrates and arthropods (Davidson, 1991). Another interesting exception is the cephalopod GENE REGULATION AND EVOLUTION molluscs which in many ways develop as do vertebrates (Davidson, 1991). In summary then, we argue that Type 1 embryogenesis is primitive for bilaterians and resulted in organisms resembling the larvae of modern marine invertebrates. The larval forms of modern indirect developing marine species indicates to us the probable nature of these ancestral organisms, except that they then constituted the terminal or adult stage of development; thus they must have generated reproductive cells, i.e., eggs and sperm. In their modern descendants reproductive systems of course appear only in the course of development of the adult body plans (Ransick et al., 1996). Recent molecular evidence from both 18S rDNA sequences and Hox gene duplications suggest that bilaterians can be divided into three groups: deuterostomes, lophotrochozoans and ecdysozoans (Aguinaldo et al, 1997; Balavoine, 1997). Of particular interest is the affinity of lophophorates with annelids and allies, although traditionally they were allied with the deuterostomes because of the particular characters they share (Halanych et al, 1995). This raises the interesting possibility that, contrary to our earlier speculations (Peterson et al., 1997), deuterostomy is primitive for bilaterians (Valentine, 1997). If, as Valentine suggests, lophophorates are basal within the lophotrochozoans, then this would lead to the conclusion that the latest common ancestor of bilaterians had a larval stage comparable to the hypothetical "tornaria" larva of Nielsen (1995). Hence both chordates, as direct developing deuterostomes, and all ecdysozoans, including arthropods, are derived from more primitive maximally indirect developers whose larval stage would be similar to recent echinoderms, enteropneusts and phoronids. The gene regulatory circuitry required for the development of large bilaterian animal body plans How are adult body plans formed in modern indirectly developing organisms? This largely ignored area of contemporary developmental biology suddenly becomes a matter of great interest for evolutionary biology. For the few organisms for which 617 FIG. 6. Set-aside cells in two different indirect developing invertebrate forms. The set-aside cells (shaded) are the groups of larval cells which after extensive division and developmental changes will contribute portions of the adult body plan. The cells which are lost at metamorphosis, that is, the strictly larval structures are unshaded, a. A cross section of the trochophore larva of the polychaete worm, Polygordius neopalitanus. b. A cross section of a typical sea urchin larva. Diagonal hatching, endoderm or gut; vertical hatching, adult epithelium; dark gray, mesoderm. In the sea urchin the water vascular system is shown stippled; there is no equivalent structure in the polychaete. there is any detailed knowledge, however, a rather striking generalization can be made. This is that certain patches of cells sequestered during the process of embryogenesis produce most of the adult body plan, while most of the larval cells have a dead-end fate—-they are unable to divide much more after embryogenesis is completed, and they retain the state of differentiation that was originally imposed on their lineage during that process. The patches of cells that give rise to the adult body plan have remarkably different properties. They have an essentially unlimited division capacity, and they produce new populations of cells that are organized into the parts of the adult body plan by developmental processes clearly distinct from those of Type 1 embryogenesis. We call these cell patches "set-aside cells," because they are developmentally set aside from the embryo-larva differentiation process (Davidson et al., 1995; Peterson et al., 1997). Figure 6 illustrates the location of set-aside cells in two different kinds of indirectly developing larvae. Developmental mechanisms leading to adult body plan formation have so far been studied primarily in terrestrial direct developers, mainly insects, vertebrates, and nematodes. We assume that the same stepwise 618 R. A. CAMERON ETAL. genetic regionalization mechanisms are required for the development of the adult body plans of indirect developers, but applied to their larval set-aside cell populations rather than directly to the embryo. Unlike the larva per se, the size of an organism developing from the set-aside cells is not limited by a fixed potential for cell division. A theory for the evolutionary origins of large animals whose body plans are of the complexity of modern species must provide a proposition for the nature of their predecessors. We think the arguments and evidence all point in the same direction: the evolutionary predecessor was a grade of organism equivalent to that of modern ciliated larvae that would have developed by the ancient Type 1 mechanism of embryogenesis (Davidson et al., 1995). By virtue of the in-built limitations of this mechanism these organisms would have been small, and though they have left no trace in the fossil record, they must have existed long before even the advent of the Ediacaran fauna, the first unequivocal large animals to appear in the fossil record. Thus we believe the genetic regulatory circuits necessary for Type 1 embryogenesis, viz at least some differentiation gene batteries and genetic signal response systems, are probably of far greater evolutionary antiquity than are the Ediacaran metazoans. The key element of evolutionary novelty that potentiated all the rest, then, would have been the appearance of genetic regulatory circuitry that underlies the development of set-aside cell populations. These cells must be dissociated from the mechanisms that assign immediate terminal fates to all the other cells of the embryo; and they must be endowed with new growth and cell division control systems. New regulatory mechanisms must be installed to permit regional organization and morphogenesis within the populations of growing cells to which they give rise. This clearly implies a great increase in the complexity of gene regulatory systems, particulary in respect to the processes required for the progressive definition of the spatial elements of body plans. These essential changes in the "deep structure" of the genetic control systems that underlie devel- opment preceded the appearance of adult metazoan body plans. Perhaps changes that could have had these consequences occurred many times in the genome, long before those that ultimately took over the animal world, but the conditions were not permissive until the later Vendian period; it is impossible to say. Nor is it very useful to try to imagine what the earliest structures deriving from setaside cells looked like or how at the earliest stages they served the organisms on which they grew. But what one can imagine is how each new form of set-aside cell population, and each new upstream regulatory system for regional specification created what we termed above a new morphospace. Rapid diversification, an evolutionary burst of new forms, would follow as the morphospace was utilized in different ways, and this we believe is the explanation for the apparent explosion of organismal novelty that we see repeatedly in the fossil record. The "Cambrian explosion" is the most impressive of these occurrences because the novelty was at the body plan level, rather than, e.g., at the appendage plan level. All of the most powerful and in some ways most interesting developmental regulatory genes that have recently been discovered are expressed both in arthropods and chordates. In fact patterning genes of this kind are used in the development of every kind of complex animal thus far examined. For example, the Hox genes function as hierarchical regionalization regulators that are required universally for specification of the anterior-posterior body axis of bilateral animals (Holland and Garcia-Fernandez, 1996; Ledouarin et al. 1996; Maconochie et al., 1996; Sengupta and Bargmann, 1996). We see that such genes (of which there are many kinds besides Hox genes), all function in hierarchical processes of adult body plan formation. These genes themselves are certainly older than are the adult body plans of bilateral animals (Balavoine, 1997). Perhaps they were used in the predecessors of these animals just to run simple gene batteries. The co-optation of these genes to the hierarchical functions of building the adult body plan (see Grenier et al., 1997, for arthropods; Lowe and Wray, GENE REGULATION AND EVOLUTION 1997, for echinoderms) was among the changes that made organisms able to use set-aside cells. Since the genes are all still there, the genomes of living animals may hold within them the key evidence as to their own evolutionary origins. One day we will understand specifically what must have happened to erect the regulatory networks that characterize each branch of animal evolution. ACKNOWLEDGMENTS We thank the following for kindly providing illustrations: Drs. James Gehling, Simon Conway Morris, Andrew Smith and Benjamin Waggoner. This work was supported by NIH grant (HD-05753) to EHD and by NSF grant (IBN 9604454)to RAC. REFERENCES Aguinaldo, A. M. A., J. M. Turbeville, L. S. Linford, M. C. Rivera, J. R. Garey, R. A. Raff, and J. A. Lake. 1997. Evidence for a clade of nematodes, arthropods and other molting animals. Nature 387: 489-493. Arnone, M. and E. H. Davidson. 1997. The hardwiring of development: Organization and function of genomic regulatory systems. Development 124: 1851-1864. Balavoine, G. 1997. The early emergence of platyhelminths is contradicted by the agreement between 18S rRNA and Hox genes data. C. R. Acad. Sci. Paris, Sciences de la vie/Life Sciences 320:83-94. Bowring, S. A., J. P. Grotzinger, C. E. Isachsen, A. H. Knoll, S. M. Pelechaty, and P. Kolosov. 1993. Calibrating rates of Early Cambrian evolution. Science 261:1293-1298. Britten, R. J. 1996. DNA sequence insertion and evolutionary variation in gene regulation. Proc. Natl. Acad. Sci. U.S.A. 93:9374-9377. Britten, R. J. and E. H. Davidson. 1971. Repetitive and non-repetitive DNA sequences and a speculation on the origins of evolutionary novelty. Quart. Rev. Biol. 46:111-138. Carroll, S. B. 1994. Developmental regulatory mechanisms in the evolution of insect diversity. In M. Akam, P. Holland, P. Ingham, and G. Wray (eds.), The evolution of developmental regulatory mechanisms, Development 1994 Supplement, pp. 217— 223. The Company of Biologists Limited, Cambridge. Carroll, S. B., J. Gates, D. N. Keys, S. W. Paddock, G. E. F. Panganiban, J. E. Selegue, and J. A. Williams. 1994. Pattern formation and eyespot determination in butterfly wings. Science 265:109— 114. Conway Morris, S. 1993. Ediacaran-like fossils in Cambrian Burgess Shale-type faunas of North America. Palaeontology 36:593-635. Davidson, E. H. 1991. Spatial mechanisms of gene 619 regulation in metazoan embryos. Development 113:1-26. Davidson, E. H. 1994. Molecular biology of embryonic development: How far have we come in the last ten years? BioEssays 16:603-615. Davidson, E. H., K. J. Peterson, and R. A. Cameron. 1995. Origin of adult bilaterian body plans: Evolution of developmental regulatory mechanisms. Science 270:1319-1325. Fedonkin, M. A. 1994. Vendian body fossils and trace fossils. In S. Bengtson, (ed.), Early life on earth. Nobel Symposium No. 84, pp. 370—388. Columbia University Press, New York. Fedonkin, M. A. and B. M. Waggoner. 1997. The Late Precambrian fossil Kimberella is a mollusc-like bilaterian organism. Nature 388:868—871. Gehling, J. G. 1987. Earliest known echinoderm—a new Ediacaran fossil from the Pound Subgroup of South Australia. Alcheringa 11:337—345. Gehling, J. G. 1991. The case for Ediacaran fossil roots to the metazoan tree. Geol. Soc. India, Mem. 20: 181-224. Gehling, J. G. and J. K. Rigby. 1996. Long-expected sponges from the Neoproterozoic Ediacaran fauna of South Australia. J. Paleontol. 70:185-195. Gomez-Skarmeta, J. L. and J. Modolell. 1996. araucan and caupolican provide a link between compartment subdivisions and patterning of sensory organs and veins in the Drosophila wing. Genes Dev. 10:2935-2945. Gonzalez, A., H. Elliot, and D. St. Johnston. 1995. Polarization of both major body axes in Drosophila by gurken-torpedo signaling. Nature 375:654658. Gray, J. and A. J. Boucot. 1994. Early Silurian nonmarine animal remains and the nature of the early continental ecosystem. Acta Palaeon. Polon. 38: 303-328. Grenier, J. K., T. L. Garber, R. Warren, P. M. Whitington, and S. Carroll. 1997. Evolution of the entire arthropod Hox gene set predated the origin and radiation of the onychophoran/arthropod clade. Curr. Biol. 7:547-553. Grotzinger, J. P., S. A. Bowring, B. Z. Saylor, and A. J. Kaufman. 1995. Biostratigraphic and geochronologic constraints on early animal evolution. Science 270:598-604. Halanych, K. M., J. D. Bacheller, A. M. A. Aguinaldo, S. M. Liva, D. M. Hillis, and J. A. Lake. 1995. Evidence from 18S ribosomal DNA that the lophophorates are protostome animals. Science 267: 1641-1643. Henikoff, S., E. Green, S. Pietrokovski, P. Bork, T. Attwood, and L. Hood. 1997. Gene families: The taxonomy of protein paralogs and chimeras. Science 278:609-614. Hofmann, H. J., G. M. Narbonne, and J. D. Aitken. 1990. Ediacaran remains from intertillite beds in northwestern Canada. Geology 18:1199-1202. Holland, P. W. H. and J. Garcia-Fernandez. 1996. Hox genes and chordate evolution. Dev. Biol. 173: 382-395. Horodyski, R., J. G. Gehling, S. Jensen, and B. Runnegar. 1994. Ediacaran fauna and earliest Cam- 620 R. A. CAMERON ETAL. brian trace fossils in a single parasequence set, southern Nevada. Abstracts with Programs of the Geological Society of America 26:60. Jenkins, R. J. F. and J. G. Gehling. 1978. A review of the frond-like fossils of the Ediacara assemblage. Rec. S. Aust. Mus. 17:347-359. Jagersten, G. 1972. Evolution of the metazoan life cycle. A comprehensive theory. Academic Press, London and New York. Lambie, E. J. and J. Kimble. 1991. Genetic control of cell interactions in nematode development. Ann. Rev. Genet. 25:411-436. Ledouarin, N. M., A. Grapinbotton, and M. Catala. 1996. Patterning of the neural primordium in the avian embryo. Sem. Cell Dev. Biol. 7:157-167. Lowe, C. J. and G. A. Wray. 1997. Radial alterations in the roles of homeobox genes during echinoderm evolution. Nature 389:718-721. Maconochie, M., S. Nonchev, A. Morrison, and R. Krumlauf. 1996. Paralogous HOX genes—function and regulation. Ann. Rev. Gen. 30:529-556. Miller, J. R. and R. T. Moon. 1996. Signal transduction through beta-catenin and specification of cell fate during embryogenesis. Gene Dev. 10:2527-2539. Molven, A., C. V. E. Wright, R. Bremiller, E. M. De Robertis, and C. B. Kimmel. 1990. Expression of a homeobox gene product in normal and mutant zebrafish embryos: Evolution of the tetrapod body plan. Development 109:279-288. Morgan, X H. 1934. Embryology and Genetics. Columbia Univ. Press, New York. Nielsen, C. 1995. Animal evolution: Interrelationships of the living phyla. Oxford University Press. Oxford. Paul, C. R. C. and A. B. Smith. 1984. The early radiation and phylogeny of echinoderms. Biol. Rev. Camb. Philos. Soc. 59:443-481. Peterson, K. J., R. A. Cameron, and E. H. Davidson. 1997. Set-aside cells in maximal indirect development: Evolutionary and developmental significance. Bioessays 19:623—631. Raff, R. A. 1987. Constraint, flexibility and phylogenetic history in the evolution of direct development in sea urchins. Dev. Biol. 119:6-19. Ransick, A. and E. H. Davidson. 1995. Micromeres are required for normal vegetal plate specification in sea urchin embryos. Development 121:32153222. Ransick, A., R. A. Cameron, and E. H. Davidson. 1996. Postembryonic segregation of the germ line in sea urchins, in relation to indirect development. Proc. Natl. Acad. Sci. U.S.A. 93:6759-6763. Runnegar, B. 1982. A molecular-clock date for the origin of the animal phyla. Lethaia. 15:199-205. Runnegar, B. 1995. Vendobionta or Metazoa? Devel- opments in understanding the Ediacara "fauna." N. Jb. Geol. Palaont. Abh. 195:303-318. Sengupta, P. and C. I. Bargmann. 1996. Cell fate specification and differentiation in the nervous system of Caenorhabditis elegans. Dev. Genet. 18:73-80. Shear, W. A., P. G. Gensel, and A. J. Jeram. 1996. Fossils of large terrestrial arthropods from the Lower Devonian of Canada. Nature 384:555-557. Shubin, N., C. Tabin, and S. Carroll. 1997. Fossils, genes and the evolution of animal limbs. Nature 388:639-648. Sordino, P., F. van der Hoeven, and D. Duboule. 1995. Hox gene expression in teleost fins and the origin of vertebrate digits. Nature. 375:678-681. Sternberg, P. W. 1993. Intercellular signaling and signal transduction in C. elegans. Ann. Rev. Genet. 27:497-521. Valentine, J. 1997. Cleavage patterns and the topology of the metazoan tree of life. Proc. Natl. Acad. Sci. U.S.A. 94:8001-8005. Venkatesh, T. and R. Bodmer. 1995. How many signals does it take? BioEssays 17:754-757. Waggoner, B. M. 1996. Phylogenetic hypotheses of the relationships of arthropods to Precambrian and Cambrian problematic fossil taxa. Syst. Biol. 45: 190-222. Wang, B. B., M. M. Muller-Immergluck, J. Austin, N. T. Robinson, A. Chisholm, and C. Kenyon. 1993. A homeotic gene cluster patterns the anteroposterior body axis of C. elegans. Cell 74:29-42. Warren, R. W., L. Nagy, J. Selegue, J. Gates, and S. Carroll. 1994. Evolution of homeotic gene regulation and function in flies and butterflies. Nature 372:458-461. Whittington, H. B. 1975. The enigmatic animal Opabinia regalis. Middle Cambrian, Burgess Shale, British Columbia. Phil. Trans. R. Soc. Lond. Ser. B Biol. Sci. 271:1-43. Williams, R., U. Lendahl, and M. Lardelli. 1995. Complementary and combinatorial patterns of Notch gene family expression during early mouse development. Mech. Dev. 53:357-368. Wray, G. A. 1994. The evolution of cell lineage in echinoderms. Amer. Zool. 34:353-363. Wray, G. A. and A. E. Bely. 1994. The evolution of echinoderm development is driven by several distinct factors. In M. Akam, P. Holland, P. Ingham, and G. Wray (eds.), The evolution of developmental mechanisms. Vol. Development 1994 Supplement, pp. 97—106. The Company of Biologists Limited. Cambridge. Wray, G. A., J. S. Levinton, and L. H. Shapiro, L. H. 1996. Molecular evidence for deep Precambrian divergences among metazoan phyla. Science. 274: 568-573. Corresponding Editor: Gregory A. Wray