Download Developmental Gene Regulation and the

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-
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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-
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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
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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