AMER. ZOOL., 34:313-322 (1994)
Comparison of Gastrulation in Frogs and Fish1
J. A. BOLKER2
Department of Molecular and Cell Biology, 315 Life Sciences Addition, and
Museum of Vertebrate Zoology, University of California, Berkeley, California 94720
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.
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
1
From the Symposium Conserved Genes and Devel- approach, broadening our knowledge of
opmental Mechanisms in Embryos of Divergent Species fundamental developmental processes by
presented at the Annual Meeting of the American Soci- testing our understanding of these processes
ety of Zoologists, 27-30 December 1992, at Vancouin different organisms.
ver, British Columbia.
Comparing gastrulation in frogs and fish
- Current address: Department of Biology, 142 Jordan Hall, Indiana University, Bloomington, IN 47405. is feasible because, in contrast to teleosts,
313
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-
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
GASTRULATION IN FROGS AND FISH
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
315
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-
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, 19936.
GASTRULATION IN FROGS AND FISH
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
317
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).
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
THIRD EXAMPLE: THE EVOLUTION OF
in a pattern of tissue-specific zones (Fig. 2D).
Earlier studies of urodeles reveal a similar
TELEOST GASTRULATION
process of notochord ingression, as well as
The evolution of the teleost pattern of
ingression of prospective somitic meso- gastrulation, which phylogenetic analyses
derm from the sides of the blastopore imply is derived from an ancestral pattern
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
GASTRULATION IN FROGS AND FISH
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
319
FIG. 3. Cladogram of selected taxa within the Anura.
§ indicates taxa known to have surface mesoderm.
Modified from Purcell, 19926.
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.
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-
GASTRULATION IN FROGS AND FISH
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 con-
321
servation 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 89052
and NIH 25594 to R. Keller.
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