Comparison of Gastrulation in Frogs and Fish 1

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1 AMER. ZOOL., 34: (1994) Comparison of Gastrulation in Frogs and Fish 1 J. A. BOLKER 2 Department of Molecular and Cell Biology, 315 Life Sciences Addition, and Museum of Vertebrate Zoology, University of California, Berkeley, California SYNOPSIS. Comparative embryological studies of frogs and fish provide valuable information about the mechanisms and evolution of vertebrate development. First, by mapping developmental data from a range of species onto a cladogram, one can distinguish general features of a ground plan from variation within it. Two studies illustrate this: comparison of gastrulation mechanisms in sturgeon and Xenopus, and morphogenesis of the dorsal mesoderm in five species of anurans. Second, phylogenetic analysis of developmental data makes it possible to identify radical departures from the ground plan among related groups. Teleost gastrulation is a highly derived process that appears to have little in common with the ancestral version. However, teleost gastrulation may have evolved as a result of two specific developmental changes: loss of bottle cells in the surface layer, and changes in the yolk. The phylogenetic distribution of developmental characters forms the basis for mechanistic hypotheses about the origins of major evolutionary changes in development. INTRODUCTION Comparative studies both enhance our understanding of developmental processes, and (together with phylogenetic hypotheses) help define developmental ground plans, the broad themes upon which variations evolve. Much recent molecular and genetic work has documented the widespread conservation of developmental genes, but even where genes are conserved, their expression patterns, functions, and epigenetic regulation evolve. The same (or a closely related gene) can assume different functions in different contexts, either within an organism, or between species. The first kind of evolution is illustrated by the dual role of genes related to engrailed in Drosophila blastoderm segmentation and in neurogenesis: the gene functions in neurogenesis in most taxa examined, and appears to have been coopted to participate in blastoderm segmentation in Drosophila (Patel et ai, 1989). The role of homeobox-containing genes in pattern- 1 From the Symposium Conserved Genes and Developmental Mechanisms in Embryos of Divergent Species presented at the Annual Meeting of the American Society of Zoologists, December 1992, at Vancouver, British Columbia. - Current address: Department of Biology, 142 Jordan Hall, Indiana University, Bloomington, IN ing different regions of the vertebrate embryo is an example of a set of conserved genes whose function within individual embryos has expanded: they are involved in pattern formation not only along the body axis, but also within the limb bud (McGinnis and Krumlauf, 1992; Tabin, 1992). In each of these cases, discovering the changes and variation in the function of highly conserved genes required studying them in several organisms, and in different developmental stages and regions. A similar comparative approach can be applied to conserved developmental mechanisms, including those of gastrulation. Until we compare the function of common processes in embryos of different species, we have no way to determine how widely conserved such developmental mechanisms are, or how and why they may vary. Comparative studies of gastrulation provide data on the variety of mechanisms in different species, and can help explain the evolution of developmental patterns. Such studies also complement the model-systems approach, broadening our knowledge of fundamental developmental processes by testing our understanding of these processes in different organisms. Comparing gastrulation in frogs and fish is feasible because, in contrast to teleosts, 313

2 314 J. A. BOLKER the chondrostean fishes (sturgeons and paddlefishes) closely resemble amphibians during early development. Chondrosteans have large eggs which cleave holoblastically, and gastrulate by involution of surface material through a blastopore. Phylogenetic analyses suggest that chondrosteans and amphibians retain many general features of the likely developmental plan of their common osteichthyan ancestor (although developmental details in sturgeons and amphibians do not represent those in their ancestor). Thus, data from these organisms may provide insight into conserved, fundamental developmental processes. Along with data from other taxa and a phylogenetic hypothesis, this information can be used to recognize developmental ground plans, as discussed below, which provide a basis for identifying evolutionary shifts in ontogenetic patterns. Phylogenetic analyses and developmental ground plans Phylogenetic analyses are a critical part of evolutionary developmental studies. They provide an organizing framework for observed diversity in developmental processes, and a way to distinguish general, frequently plesiomorphic, developmental patterns from derived, synapomorphic ones (which may be very widespread, like the teleost pattern of gastrulation). We can then recognize developmental ground plans, and variations upon or flexibility within them, in groups of related organisms. A developmental ground plan represents the general theme upon which variations evolve. It must be defined in a particular phylogenetic and developmental context for example, there is a ground plan for gastrulation in vertebrates, which is characterized by involution of tissues to form multiple germ layers. Recognizing the broad, deep similarities in the early development of different vertebrates is intuitive, but defining them as a "ground plan" adds implications of their significance: it is an "act of explanation," in contrast to the initial recognition of similarities, or "act of discovery" (Riedl, 1989). In what follows I will use phylogenetic analyses and developmental ground plans as a framework for discussing developmental themes and variations in three examples from frogs and fishes. The first two examples (a comparison of gastrulation mechanisms in sturgeons and frogs, and a study of axial mesoderm morphogenesis in several species of frogs) illustrate variations within conserved developmental ground plans. The third example, the evolution of teleost gastrulation, illustrates a radical departure from an ancestral ground plan. In this case, phylogenetic analysis suggests some specific mechanisms by which this departure could have evolved. FIRST EXAMPLE: GASTRULATION MECHANISMS IN STURGEONS AND XENOPUS The comparison of gastrulation in Acipenser transmontanus, the white sturgeon, and the clawed frog Xenopus laevis reveals changes in the use of conserved morphogenetic processes within a developmental ground plan. Cell- and tissue-level mechanisms (radial and mediolateral cell intercalation; Keller et ai, 1985; Keller, 1980) are conserved in these organisms, as is the overall plan of gastrulation (dorsal extension, involution through a blastopore, etc.) (Bolker, 1993a). The mechanical context in which the shared mechanisms operate is different in the two species, and changes in the relative timing of conserved processes act to maintain their morphogenetic function in these different contexts (Bolker, 1992, 19936). Detailed comparative studies of morphogenesis are most informative when the systems compared are relatively similar at the level of interest. Comparisons between frogs and a chondrostean fish, such as the white sturgeon, are particularly appropriate for studying the mechanical basis of gastrulation. Sturgeon gastrulation closely resembles that of Xenopus, which has been thoroughly studied using a variety of techniques that transfer readily to sturgeons. Chondrosteans are an excellent taxonomic outgroup for the Amphibia. Outgroups are taxa phylogenetically outside the primary group whose members are being compared with each other (as in studies of development in different amphibians). If a

3 GASTRULATION IN FROGS AND FISH 315 character varies within the primary group, one can look at the state of that character in the outgroup to determine the direction of its evolutionary change, with the state seen in the outgroup inferred to be that of the common ancestor (Hennig, 1966; Wiley, 1981). (Teleost fishes and amniotes are also outgroups for Amphibia, but it is difficult to make direct comparisons between embryonic structures and processes in organisms that gastrulate as differently as do amphibians and amniotes or teleosts.) The overall mechanism of gastrulation in sturgeons and most amphibians is extremely similar, and probably represents an original developmental ground plan for gastrulation in vertebrates. Bottle cells form on the dorsal side, initiating the involution of surface material that will form the mesodermal and endodermal lining of the gastrocoel. Involution correlates with, and may in part be driven by, extension of the dorsal side (comprising both involuted prospective axial mesoderm, and non-involuted prospective neural tissues) (Bolker, 19936; Keller et al, 1991). These processes of extension and involution have been studied in Xenopus by Keller and colleagues, who have described the cell- and tissue-level processes that generate morphogenetic movements (see Keller et al, 1991 for review). The major mechanisms producing extension in Xenopus are radial intercalation of cells within a tissue, in which the tissue becomes flatter and longer but maintains its width, and mediolateral intercalation, in which a tissue converges, or narrows, and extends (Keller, 1980; Keller et al, 1985). Radial intercalation produces extension with thinning. In Xenopus, this process occurs mainly during epibolic expansion of the animal cap before gastrulation, while in sturgeons extension with thinning continues through early gastrulation. Convergent extension is the major driving force for involution during gastrulation in Xenopus (Keller et al, 1985), and the mediolateral cell intercalation that produces convergent extension is expressed autonomously in explanted Xenopus dorsal tissues (Keller and Danilchik, 1988). Dorsal explants of A. transmontanus also converge and extend, and express morphogenetic behaviors closely resembling those in Xenopus (Bolker, 19936). A critical difference between sturgeons and Xenopus is the location at which the dorsal bottle cells form and involution begins: at the equator in sturgeons, but nearer the vegetal pole in Xenopus. The importance of this difference in blastopore location is that initial extension based on circumferential convergence is not feasible in sturgeons, because the blastopore forms at the widest part of the embryo. This constraint can be confirmed experimentally by removing the blastocoel roof and upper marginal zone from a sturgeon embryo when the pigment line forms at the start of gastrulation (Bolker, 19936). This operation prevents the initial phase of extension normally correlated with the thinning of the blastocoel roof and upper marginal zone. Convergence still occurs, as predicted by the demonstration in explants that this process is intrinsic to dorsal marginal zone tissues, but it happens in the wrong place. Instead of contributing to dorsal extension and blastopore closure, convergence at the equator produces an equatorially constricted embryo, with a ring of bottle cells at the line of constriction. Sturgeons gastrulate using a slightly different strategy than Xenopus. In sturgeons convergent extension is preceded by an early phase of thinning and extension (possibly generated by radial intercalation of cells in the thick blastocoel roof and upper marginal zone; Bolker, 1993a) (Fig. 1A). This initial extension without convergence serves to move the marginal zone below the equator before it begins to converge, and thus constrict the embryo. During the subsequent phase of extension (Fig. IB) the marginal zone lengthens by converging, much as it does in Xenopus (Fig. 1C). The two extension mechanisms (extension with thinning, and convergent extension based on mediolateral cell intercalation) are apparently the same as in Xenopus, but their temporal organization is different: the early phase of dorsal thinning and extension is longer and more important in sturgeons, where the marginal zone must be moved below the equator before convergence begins. This example illustrates variation within a ground plan: the overall pattern of gastru-

4 J. A. BOLKER FIG. 1. Comparison of gastrulation processes in Acipenser (A, B) and Xenopus (C) (not drawn to scale). Large arrows indicate broad morphogenetic movements, and small arrows show direction of cell intercalation within tissues. The marginal zone is stippled. (A) and (C) show embryos at the start of gastrulation: (B) shows a later stage in Acipenser. Abbreviations: ar, archenteron; be, bottle cells; bl, blastocoel. Reprinted with permission from Bolker,

5 GASTRULATION IN FROGS AND FISH 317 lation is conserved, as are cell- and tissuelevel morphogenetic behaviors, but the time and place of expression of these conserved behaviors is different. In fact, the difference in timing is responsible for maintaining the morphogenetic function of conserved mechanisms in an altered geometric and mechanical context. SECOND EXAMPLE: MESODERM MORPHOGENESIS IN ANURANS Recent comparative studies of mesoderm morphogenesis in anurans (Purcell, 1992a, b; Purcell and Keller, 1993) illustrate how related organisms may use diverse means to a given developmental end. In many vertebrates, including nearly all amphibians, gastrulation occurs by involution of surface material through a blastopore. This involuting surface material usually includes some prospective axial mesoderm (notochord and somites), which poses a topological problem. After involution, this prospective mesoderm is part of the lining of the gastrocoel, but it must eventually come to lie in the deep layer, between the dorsal endodermal lining of the archenteron and the prospective neural plate (Fig. 2). A widelyconserved mechanism to deploy the mesoderm into the deep layer is the formation of bottle cells from prospective mesoderm lining the gastrocoel; these cells then ingress from zones in the gastrocoel roof. (No such process occurs in Xenopus, where the mesoderm is all located in the deep layer before gastrulation begins; Keller, 1976). Purcell (1992a, b; Purcell and Keller, 1993) has compared the morphogenesis of axial mesoderm in five species of frogs, representing a broad taxonomic sample within the Anura. She concludes that while ingression of surface mesoderm is widely conserved among anurans, the timing and spatial pattern of ingression vary among species. In most of the anuran species studied, portions of the prospective somitic, notochordal, and tailbud mesoderm ingress from the epithelium of the gastrocoel roof, often in a pattern of tissue-specific zones (Fig. 2D). Earlier studies of urodeles reveal a similar process of notochord ingression, as well as ingression of prospective somitic mesoderm from the sides of the blastopore immediately following involution (Lundmark, 1986; Pasteels, 1942; Smith and Malacinski, 1983). The two pipid frogs for which data are available, X. laevis and Hymenochirus boettgeri, are exceptions to this rule: in these species all prospective mesoderm is in the deep layer before gastrulation, and none ingresses from the gastrocoel roof after involution (Purcell, 1992a, b). PurcelFs work, along with older studies (Lundmark, 1986; Pasteels, 1942; Smith and Malacinski, 1983), clearly establishes that there are two general ways of getting axial mesoderm into the deep layer: either prospective mesoderm forms exclusively in the deep layer, as in pipids, or it ingresses from the gastrocoel roof during or after involution, as in most other species. The distribution of these traits on a cladogram of the Anura (Fig. 3) implies that ingression is plesiomorphic within the Anura. Studies of many amphibians describe surface prospective mesoderm that ingresses into the deep layer during or after involution. A cladogram including data from urodele and gymnophionan amphibians (Brauer, 1897; Delarue^a/., 1992; Lundmark, 1986; Smith and Malacinski, 1983; Vogt, 1929) and a chondrostean fish (Bolker, 1993a), together with the information on anurans, suggests that ingressing surface mesoderm is probably plesiomorphic not only for anurans, but for all amphibians (see Fig. 10 in Purcell and Keller, 1993). Comparing data from a range of species within a phylogenetic framework makes it possible to identify a generally conserved developmental ground plan within amphibians (ingression of prospective mesoderm from the gastrocoel after involution), variations within that plan (differences in timing and pattern of ingression in different frogs), and substantial departures from it (such as the pipid pattern of having all mesoderm in the deep layer before gastrulation, and no ingression from the gastrocoel roof). THIRD EXAMPLE: THE EVOLUTION OF TELEOST GASTRULATION The evolution of the teleost pattern of gastrulation, which phylogenetic analyses imply is derived from an ancestral pattern

6 d - early gastrula early neurula Fio. 2. The general amphibian pattern of mesoderm morphogenesis. A-C: Mid-sagittal (top) and dorsal horizontal (bottom) cross-sections of embryos at early and late gastrula, and early neurula stages, showing prospective mesoderm in the deep (stippled) and superficial (hatched) layers. The broken line in the upper diagram indicates the level of the horizontal cross-section shown below. Black pointers indicate bottle cells. D: Scanning electron

7 GASTRULATION IN FROGS AND FISH 319 resembling that of amphibians and sturgeons, has long been regarded as a radical and largely inexplicable evolutionary transition. In this section, I present a mechanistic scenario describing one possible route this transition could have taken (Collazo et ai, 1994). What can comparative phylogenetic analyses say about apparently radically different modes of vertebrate development, such as gastrulation in frogs and teleost fish? In contrast to differences between sturgeon and Xenopus gastrulation, or among anurans with respect to mesoderm morphogenesis, this is not variation within conserved plan: it is a radical departure. Nevertheless, knowing the osteichthyan ground plan (as seen in chondrostean fishes and in amphibians), this departure can be explained in terms of specific developmental changes: loss of surface bottle cells, and changes in yolk composition. In contrast to sturgeons and most amphibians, teleosts have meroblastic cleavage (formation of a blastodisc on top of an uncleaved yolk), and no involution of the surface layer. Until a few years ago it was thought they had no involution at all (Ballard, 1966, 1968), but recent work by Wood and Timmermans (1988) and Warga and Kimmel (1990) has demonstrated that at least in Barbus conchonius and Brachydanio rerio, cells in the deep layer do involute. (Part of the difficulty in analyzing teleost gastrulation stems from the assumption that all teleosts are identical: contradictory results are often derived from different species.) Nevertheless, gastrulation in teleosts is radically different from that in sturgeons and amphibians. Meroblastic cleavage produces a multilayer blastoderm of small cells, which overlies an acellular yolk syncytial layer (Fig. 4). The yolk syncytial layer is continuous with the anucleate yolk cytoplasmic layer which surrounds the yolk. Together these three layers constitute the blastodisc, which rests at the animal pole of the uncleaved FIG. 3. Cladogram of selected taxa within the Anura. indicates taxa known to have surface mesoderm. Modified from Purcell, yolk droplet. The outermost layer of the blastodisc, the enveloping layer, produces the periderm, an outer epithelium shed at hatching. The "deep" (actually middle) layer of the blastodisc gives rise to all germ layers and adult structures. During gastrulation the yolk syncytial layer expands epibolically, replacing the yolk cytoplasmic layer. The enveloping layer also expands. Deep cells form a thickened, two-layer ring at the periphery of the expanding blastoderm, and then the loose cell population involutes and nu FIG. 4. Diagram of a teleost gastrula (sagittal section). Abbreviations: ep, epiblast; evl, enveloping layer; hyp, hypoblast; nu, nuclei; ycl, yolk cytoplasmic layer; ysl, yolk syncytial layer. micrograph of the gastrocoel roof of Ceratophrys ornata, showing zones of ingressing cells (compare B). Scale bar = 100 jim. Solid arrows: prospective notochord; open arrows: prospective tailbud mesoderm; doubled open arrows: prospective somitic mesoderm. Abbreviations: a, anterior; d, dorsal; n, notochord; p, posterior; s, somites; v, ventral.

8 320 J. A. BOLKER FIG. 5. Cladogram of selected teleost taxa (based on Lauder and Liem, 1983, and Collazo et a/., 1994), showing the appearance of two changes proposed to lead to the teleost pattern of gastrulation: (1), loss of bottle cells in the surface layer; (2), change in yolk structure. converges dorsally to form the embryonic shield (Lentz and Trinkaus, 1967; Trinkaus, 1984; Warga and Kimmel, 1990). The shift from the ancestral vertebrate pattern of gastrulation to the teleost pattern may have occurred in two steps (Collazo et ai, 1994). The first step, which occurred in the common ancestor of Amia and the teleosts, was the loss of bottle cells in the surface layer (the enveloping layer of teleosts) (Fig. 5). As a result, the surface layer ceased to involute during gastrulation (although the deep layer, or hypoblast, continued to do so), and instead formed the non-embryonic enveloping layer. Deep layer involution was not greatly affected by the loss of bottle cells in the surface layer; a modern analogue may be the experimental result obtained in Xenopus showing that extirpation of the surface bottle cells before gastrulation has little effect on the involution of the deep layer (Keller, 1981). The second step in this scenario is a change in the yolk, from enclosed platelets within cells to a solid, uncleaved oil droplet (Soin, 1981; Wallace, 1985). This structural change was probably related to biochemical changes in yolk composition, and occurred in the common ancestor of teleosts, after the group represented by Amia diverged. Drastic alteration of the yolk structure required a reorganization of the pattern of gastrulation to accommodate a new set of mechanical constraints. Cleavage became meroblastic, a change that has evolved independently five times within vertebrates (and is associated with an increase in egg size in all these cases except the teleosts), with the formation of an embryonic disk atop a large fluid yolk. Gastrulation took the form of epibolic spreading of a non-embryonic enveloping layer over the whole egg, together with conserved types of tissue movements within the blastodisc. Despite the radical changes in the geometry of the entire embryo and in the nature of the cell populations within it (which are generally more mesenchymal and less highly organized into epithelia in teleosts as compared to amphibians and sturgeons), ancestral morphogenetic patterns such as involution and convergent extension were conserved (Warga and Kimmel, 1990; Wood and Timmermans, 1988). Phylogenetic analysis of developmental patterns shows that the teleost pattern of gastrulation departs radically from the ancestral ground plan and is highly derived, even though it is the commonest among vertebrate species (more than half of which are teleosts). It also makes possible the formulation of a mechanistic hypothesis about the transition from the vertebrate ground plan to the teleost pattern of gastrulation. This hypothesis is based on the phylogenetic distribution of characters that are important in morphogenesis, together with an understanding of their function derived from experimental and descriptive embryological studies in a range of species. DISCUSSION Combining comparative studies of development (especially detailed observations and experimental manipulations that elucidate mechanisms) with phylogenetic analyses provides several different kinds of information about development and its evolution. First, such studies enable us to recognize and organize existing variation in developmental patterns. While the model systems approach provides detailed informa-

9 GASTRULATION IN FROGS AND FISH 321 tion about a few systems, only comparative studies can address variation and its significance. Knowledge of variation is essential to testing common assumptions about the universality of developmental mechanisms. In addition, analyzing the variation in developmental processes among different organisms yields insights into how morphogenetic mechanisms function, by testing their behavior and effects in different contexts (for example gastrulae ofacipenser and Xenopus). Second, a comparative approach permits the identification of developmental ground plans, sets of shared developmental processes in related organisms. Once the general features that constitute a ground plan are known, variations on the theme can be recognized. Such variations can take the form of relatively slight changes in timing of conserved processes (such as the two forms of extension in sturgeons and Xenopus), or more substantial reorganizations, such as the pattern of mesoderm morphogenesis in pipid frogs as compared with other anurans. Third, identifying ground plans and their phylogenetic distribution using a comparative and evolutionary approach makes it clear when they have changed, for example in the lineage leading to teleosts. Comparison of different ground plans occurring within a lineage reveals what constraints are broken, or "universal" elements altered, and can suggest scenarios by which such changes may evolve. Finally, looking at development in this way contributes to our understanding of the way development itself evolves. Detailed comparisons between related species reveal at what level developmental mechanisms and processes are evolving: for example, in the case of sturgeon and Xenopus gastrulation the morphogenetic mechanisms that produce extension are highly conserved, but the pattern in which they are expressed has changed along with the geometry of the embryos. In other systems, evolutionarily critical changes have evolved during the earliest stages of development (Wray and Raff, 1989; Raff, 1992). There is more and more data from molecular biology about the extraordinary conservation of genes and molecules important in development. If so much is conserved at this level, the proximate causes of phenotypic diversity may lie at higher levels of organization. Morphogenesis and other epigenetic processes are a good place to look, and detailed comparative analyses are a powerful tool for doing so (Miiller, 1991; Bolker, 1992). ACKNOWLEDGMENTS I would like to thank Susan Purcell, Andres Collazo, and Ray Keller for permission to present unpublished results (including Fig. 2D, provided by S. Purcell); David Wake for helpful discussions; and David Wake, John Gerhart and Steve Minsuk for comments on the manuscript. Sturgeon embryos were provided by Sea Farms of California (Herald, Calif.) and the California Sturgeon Project at U.C. Davis. This work was supported by NSF DCB and NIH to R. Keller. REFERENCES Ballard,W.W Origin of the hypoblast in Salmo. I. Does the blastodisc edge turn inward? J. Exp. Zool. 161: Ballard, W. W History of the hypoblast in Salmo. J. Exp. Zool. 168: Bolker, J. A Constancy and variation in developmental mechanisms: An example from comparative embryology. In J. Mittenthal and A. Baskin (eds.), Principles of organization in organisms, pp Addison-Wesley, Menlo Park, California. Bolker, J. A. 1993a. Gastrulation and mesoderm morphogenesis in the white sturgeon, Acipenser transmontanus. J. Exp. Zool. 266: Bolker, J. A The mechanism of gastrulation in the white sturgeon. J. Exp. Zool. 266: Brauer, A I. Beitrage zur Kenntnis der Entwicklungsgeschichte und der Anatomie der Gymnophionen. Zool. Jahrbuch. Anat. 10: Collazo, A., J. A. Bolker, and R. E. Keller A phylogenetic perspective on teleost gastrulation. Amer. Nat. 144 (In press) Delarue, M., S. Sanchez, K. E. Johnson, T. Darribere, andj.c. Boucaut A fate map of superficial and deep circumblastoporal cells in the early gastrula of Pleurodeles waltl. Development 114: Hennig, W Phylogenetic systematics. University of Illinois Press, Urbana. Keller, R. and M. Danilchik Regional expression, pattern and timing of convergence and extension during gastrulation of Xenopus laevis. Development 103:

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