AMER. ZOOL., 39:650-663 (1999)
Out on a Limb: Parallels in Vertebrate and Invertebrate Limb Patterning
and the Origin of Appendages'
CLIFFORD J. TABIN,* SEAN B. CARROLL,! AND GRACE PANGANIBAN:):2
*Department of Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts
^Howard Hughes Medical Institute and Laboratory of Molecular Biology, University of Wisconsin,
Madison, Wisconsin 53706
^.Department of Anatomy, University of Wisconsin Medical School, Madison, Wisconsin 53706
Recent discoveries of similarities in the developmental genetics underlying the formation of insect and vertebrate eyes, hearts, segments, and other structures have fueled new speculation and debate about the origins of these features
and the morphological complexity of early bilaterians. The pivotal issue concerning
these developmental similarities is whether they represent convergence of patternforming mechanisms or reveal developmental regulatory mechanisms or even
physical characteristics derived from a common ancestor. Here, we set forth an
explicit hierarchical set of criteria for assessing developmental genetic similarities
among animals. We suggest that interpretations of convergence versus descent
from common ancestors should be weighed by the number, type, and phylogenetic
distribution of genetic regulatory similarities. We then apply these criteria to the
analysis of appendage evolution. We conclude that there has been no continuity of
any structure from which the insect and vertebrate appendages could be derived,
i.e., they are not homologous structures. However, there is abundant evidence for
continuity in the genetic information for building body wall outgrowths and/or
appendages in several phyla which must date at least to the common, potential
appendage-bearing pre-Cambrian ancestor of most protostomes and deuterostomes. In order to further trace the origin of this genetic information and of
appendages, it will be essential to analyze more primitive taxa such as the Cnidaria
and to obtain a much better fossil record of pre-Cambrian animals.
SYNOPSIS.
INTRODUCTION
One of the most profound impacts of the
last decade of research in developmental biology has been the realization that a large
number of analogous processes in Drosophila and model vertebrates are regulated by
homologous genes. For example, in both
groups, homeotic genes specify positional
differences along the anterior-posterior axis
of the body, Pax-6 plays an essential role
in eye development, tinman/Nkx2.5 is required for heart development, etc. (for review see de Robertis, 1997). These striking
developmental parallels are providing an
entirely new source of comparative data for
formulating and testing hypotheses about
the pattern and process of morphological
evolution.
In
particular, long-standing ideas about
the origins and evolution of various anatomical structures are being re-examined in
a new light. For example, the function of
Pax-6 and other key regulatory genes in the
developing anlage of both vertebrate and
invertebrate eyes begs the question of just
how independent was the origin and evolution of these structures? The subsequent
discovery of Pax-6 expression during eye
development in a variety of phyla suggests
that Pax-6 could have been involved in the
formation of some form of primitive eye or
type of photoreceptor organ in a common
ancestor of many animals (reviewed in
1
From the Symposium Developmental and Evolu- Haider et al., 1995). These findings Seem to
tionary Perspectives on Major Transformations in forcefully exclude the notion that eyes
Body Organization presented at the Annual Meeting arose absolutely de novo, in either a genetic
of the Society for Imegrative and Comparative Biolog u l a t o r y o r c e l l biological sense, in these
gy, 3-7 January 1998, at Boston, Massachusetts.
-corresponding author: E-mail: gePangan@facstaff.
wisc.edu
fe
'
&
>
lineages. Even more problematic is the interpretation of the large number of genetic
650
APPENDAGE DEVELOPMENT AND EVOLUTION
parallels which have been discovered in the
formation and patterning of vertebrate and
arthropod limbs (reviewed in Shubin et al.,
1997). Clearly, since phylogenetically intermediate taxa do not possess comparable
structures, insect and vertebrate appendages
are not homologous in any historical or
morphological sense. Yet, there are developmental similarities among them, some of
which are shared with the appendages of
animals in other phyla.
These sorts of findings present difficulties in describing relationships among morphologically divergent structures whose
formation and patterning depend upon homologous genes. Their interpretation, however, is profoundly important to understanding the origin and evolution of structures
and for drawing inferences about the potential developmental and morphological complexity of animal ancestors. It has been argued that an integrated approach, encompassing both new developmental and traditional historical comparative methods that
recognizes homologies at different levels of
biological organization (genes, embryonic
origins, morphological structures) is necessary for assessing the relationships between
structures (e.g., Van Valen, 1982; Abouheif,
1997; Abouheif et al., 1997).
In this paper, we describe an explicit hierarchical set of genetic, anatomical, embryological, and phylogenetic criteria for
assessing the potential evolutionary relationships among structures. We apply these
to the analysis of appendage evolution to
elucidate whether the observed genetic parallels reflect some level of homology with
developmental processes in an ancestral
structure, the convergent use of similar genetic circuits, or the independent and coincidental co-option of the same individual
genes. We argue that the number, type, and
phylogenetic distribution of genetic similarities suggests that the genetic circuits in extant appendages patterned some outgrowth
of the body wall in a common ancestor of
appendage-bearing phyla. Finally, we submit that further progress on this question
will require studies of more primitive taxa
and, ultimately, unambiguous corroborating
physical evidence from the fossil record.
651
CRITERIA FOR ASSESSING THE
RELATIONSHIPS AMONG STRUCTURES
The rapid explosion of information concerning the various types of genes, signaling pathways, spatially regulated gene expression patterns, and regulatory hierarchies that govern the development of cells,
tissues, organs, and embryos in a wide variety of animals has provided a new framework for comparative embryology. This information can be integrated with traditional
morphological and historical approaches to
test hypotheses about the potential relatedness of structures. This requires a scheme
that considers developmental genetic similarities at some different hierarchical levels
of organization in order to weigh the likelihood that any one or set of characters may
represent convergence or conservation of
developmental regulatory mechanisms. We
outline below the genetic and historical criteria for our comparisons.
Levels of genetic similarity in
developmental operations
Genes. The first question concerning any
similarity is whether homologous genes are
present in the animals being compared. If
they are present, one needs to determine
whether the genes are orthologous (the
same genes that have diverged via speciation) or paralogous (genes derived from duplication events prior to lineage divergence). Clearly, the conservation of a gene
does not necessitate the conservation of its
function since the evolution of genes will
predate the evolution of particular structures. In addition, while it seems logical to
give weight only to orthologous genes, cases are known where homologous structures
deploy different paralogs (see Wnt3a below). Thus, the presence or absence of any
one gene within an animal's genome is not
sufficient to determine the evolutionary relationships among structures or organisms.
Pathways. Communication between cells
involves signaling ligands, receptors, and
intracellular transduction molecules that
carry signals to target genes in the nucleus.
There is tremendous conservation of signal
transduction pathways such that conserved
usage of a ligand will almost always neces-
652
CJ. TABIN ETAL.
sitate conserved usage of the same receptor
and transducers. Thus, while the discovery
of the same signaling molecule in different
animals or structures is potentially significant, the utilization of the rest of the pathway should not be taken as additional evidence for relatedness. Extracellular components may also be obligately linked to
pathways. For example, the secreted factors
encoded by the fringe (fng) gene family
modulate the Notch (N) pathway (Panin et
al., 1997) so the expression of N pathway
genes at sites of fng expression may not
constitute an independent parallel in the genetic regulation of two structures.
Spatiotemporal deployment. Most of the
new data that is provoking novel hypotheses about evolution is emerging at the level
of gene expression in developing animals.
When homologous genes are deployed in
similar ways in structures or processes that
serve similar functions, this is positive evidence for potential relationships among the
developmental mechanisms and the structures or processes themselves. Furthermore,
if there is a conserved spatial geometry
such that homologous genes regulate the
same patterning axes (e.g., Hox genes on
the rostrocaudal axis, shortened gastrulationlchordin and bone morphogenetic proteins(BMPs)/decapentaplegic(dpp) on the
dorsoventral axis), this is an additional potential level of conservation. While comparison of the expression patterns of a single gene between two phyla are not sufficient to assess the relatedness of structures,
expression data can be informative in one
increases the number of taxa or genes under
analysis.
Genetic regulatory circuits
Signals and their target genes, and transcription factors and their target genes are
connected by regulatory interactions that
can evolve independently. Thus, when regulatory interactions among the same two or
more components are found in two structures, this potentially represents a level of
conservation that may not be apparent morphologically. For example, the BMP-2,4/
Dpp-type signaling proteins are often but
not always expressed as secondary signals
induced by Hedgehog (Hh) proteins (Bit-
good and McMahon, 1995), including in
appendages as discussed below. However,
they have no requisite biochemical relationship and do not always function together.
Indeed, the two classes of factors act in opposition in patterning the vertebrate neural
tube (reviewed in Sporle and Schughart,
1997) and somites (e.g., Hirsinger et al.,
1997). Similarly, while individual transcription factors are often expressed at more
than one site in the developing animal, the
regulation of the same target genes in comparable sites in different animals could be
a very significant indication of conserved
developmental mechanisms. Thus, while
Pax-6 deployment in vertebrate and insect
eyes could be coincidental, Pax-6 regulation of homologous downstream genes such
as sine oculis, etc. in developing eyes in
both taxa is compelling evidence of a conserved genetic regulatory circuit.
Anatomical and embryological similarities
The interpretation of genetic regulatory
similarities is most useful when coupled
with consideration of more traditional criteria concerning the structures being compared. These include both morphological
and phylogenetic information. The detail of
morphological features, the position of
structures and characters, and their embryonic origin are all established means of
evaluating relationships among structures.
This has usually served to identify both historically homologous and serially homologous structures. Yet, it is difficult to apply
in the case of some of the potential relationships among either long diverged structures or taxa. For example, the relationship
of the paired pectoral and pelvic fins to tetrapod limbs is well-established. But how do
we describe the relationship of unpaired
fins, which are no doubt developmentally
related to other fins, to tetrapod limbs?
Phylogenetic distribution of structures and
genetic similarities
Similarities in genetic mechanisms and
structures identified through the above criteria must be considered in a phylogenetic
context in order to draw inferences about
the direction of change and the potential origin of a trait. Furthermore, the best test of
APPENDAGE DEVELOPMENT AND EVOLUTION
whether a particular feature may be convergent is to examine more independent
taxa. For example, the discovery of Pax-6
expression in the developing light-sensing
organs of animals representing phyla other
than arthropods and vertebrates vanquishes
the probability of convergence.
The interpretation of the relationships
among structures and underlying developmental processes should be weighed on the
basis of the number, type, and phylogenetic
distribution of genetic similarities and integrated in the context of morphological
and historical data. The central issue that
will be confronted in the analyses of appendages and other structures is how to interpret continuity in genetic regulatory information in the absence of evidence for
continuity in structures and in morphology.
THE EVOLUTION OF APPENDAGES
Possible explanations for the similarities
between arthropod and vertebrate limb
development
In a general sense, there are four classes
of explanation one could entertain for the
existence of genetic parallels among arthropod and vertebrate limb patterning:
653
been an early outgrowth patterned coordinated by these genes. Rather the modern
appendages may be patterned by a number
of sets of genes which were linked together,
but used for different purposes and structures in the ancestral organism. These genetic "cassettes" of multiple genes would
have been reused for different purposes in
the embryo because they proved useful and
flexible when working in concert. Thus the
use of the same cassettes to pattern the
same axes in insect and vertebrate appendages would be convergent and coincidental.
(4) Finally there may be no meaningful
genetic cassettes coopted in the evolution of
animal appendages. In both invertebrate and
vertebrate lineages, genes may have been individually recruited (including their obligate
partners such as ligands and receptors) in independent evolutionary steps as the patterning of appendages evolved. In this model,
parallels between insect and vertebrate appendages are purely coincidental.
Similarities between arthropod and
vertebrate limb patterning
DlxlDistal-less. One of the most important parallels between the development of
Drosophila and chick wings is the expres(1) The formation of limbs of vertebrates sion of the homeodomain transcription facand arthropods are regulated by similar sets tor Distal-less {Dll; Dlx in vertebrates) at the
of genes because both types of limbs distal tip of each developing appendage
evolved from a common ancestral append- (Panganiban et al., 1997). In both the Droage which was formed from the action of sophila and chick wings this takes the shape
those genes. This possibility can be safely of a stripe of gene expression running along
the distal leading edge of the outgrowth
excluded.
(2) It is possible, however that the set of (Carroll et al., 1994, Panganiban et al.,
genes currently utilized to pattern append- 1995). DillDlx expression is also found in a
ages was already linked into a three-dimen- group of cells at the tip of other Drosophila
sional patterning network in the formation appendages (Cohen and Jiirgens, 1989; Panof a distinct structure present in the com- ganiban et al., 1994). Moreover Dll has been
mon ancestor of vertebrates and insects. shown to be essential for appendage outThis structure, likely some sort of out- growth in Drosophila (Cohen et al., 1989),
growth from the body wall, was not ho- and is capable of inducing the formation of
mologous to modern appendages in either limbs at novel locations when ectopically
group, but the genetic network that pat- expressed (Gorfinkiel et al., 1997).
The expression of Dll/Dlx is particularly
terned that outgrowth was coopted to pattern the appendages as they evolved inde- important for the consideration of appendage
pendently. In this view, the modern ap- relationships because it is also expressed at
pendages evolved in both the early verte- the distal tips of developing appendages in
brate and arthropod lineages using similar a wide variety of organisms, including the
genetic programs as the original outgrowth. ampullae and siphons of tunicates, tube feet
(3) Alternatively, there may not have and spines of echinoderms, parapodia of an-
654
C.J. TABIN ETAL.
nelids and lobopodia of Onychophora (Panganiban et al., 1997). With the possible exception of the Onychophora and arthropod
legs, it is clear that these are independently
evolved, non-homologous structures. The
expression of the same transcription factor
(out of the thousands available to be exploited in evolution) specifically in distal cells of
appendages in six distinct phyla would represent a truly remarkable convergence. The
more parsimonious explanation is that a Dill
Dlx gene was already involved in regulating
body wall outgrowth in a common ancestor
of these taxa. As a transcription factor, Dll/
Dlx may have recognized target genes present in that ancestor which were required for
the outgrowth. This provided a simple mechanism by which additional outgrowths from
the body wall could have been generated.
When new appendages arose independently
in the different phyla, they were established
by novel patterns of Dll/Dlx expression,
which in turn triggered the expression of the
downstream genes required for outgrowth.
If one accepts the existence of Dll/Dlx in
a common ancestral appendage as, at least,
a plausible hypothesis, then one has to consider the possibility that the ancestral outgrowth may have been patterned and hence
that the genes which organize pattern along
the three cardinal axes in modern appendages could have also been coopted from the
ancient structure.
Sonic hedgehog/hedgehog and bone
morphogenetic proteinsldecapentaplegic
The key organizing signal of the anteriorposterior axis of the chick wing is Sonic
hedgehog (Shh) (Riddle et al., 1993). Shh
expression is localized to the posterior margin of the limb bud (Fig. 1A). Misexpression of Shh in the anterior causes mirrorimage duplications of posterior structures.
Thus Shh appears to be responsible for instructing differential anterior-posterior fates
in the developing wing. The patterning response to Shh occurs in a dose-dependent
graded fashion such that higher levels of
Shh define more posterior structures (Yang
et al., 1997). The details of how Shh patterns the anterior-posterior axis are still being worked out, but at least part of its activity is attributable to secondary signals
such as BMP-2, which is induced in the
posterior mesenchyme by Shh (Laufer et
al, 1994).
The anterior-posterior axis of the Drosophila wing is established in a remarkably
similar way. The anterior-posterior axis of
the wing imaginal disc (the larval precursor
of the adult appendage, analogous to the
vertebrate limb bud) is divided into two
compartments (reviewed in (Struhl, 1982)).
The cells in the posterior half of the disc
express the gene hedgehog (Lee et al.,
1992; Tabata et al., 1992) (Fig. IB). Like
its vertebrate homologue shh, hedgehog encodes the key signal initiating anterior-posterior patterning. Like Shh, misexpression
of Hedgehog in the anterior causes mirrorimage anterior-posterior pattern duplications (Basler and Struhl, 1994; Ingham and
Fietz, 1995; Kojima et al., 1994). The patterning effects of Hedgehog are dose dependent, as in the chick wing, and like that
structure the response is due to the induction of a secondary signal of the BMP family, called decapentaplegic (dpp) (Basler
and Struhl, 1994; Ingham and Fietz, 1995).
Dpp is induced by Hedgehog in a thin line
of cells along the border of the anterior and
posterior halves of the disc, and acts as a
long range signal providing positional information and hence differential anteriorposterior cell fates throughout the disc
(Capdevila et al., 1994; Lecuit et al., 1996;
Nellen et al., 1996; Posakony et al., 1990;
Sanicola et al., 1995).
These parallels are indeed remarkable.
Not only do the two factors Hedgehog and
BMP play analogous roles in patterning the
anterior-posterior axis of vertebrate and insect wings, but they do it with a similar
geometric relationship such that, in both
cases, Hedgehog is expressed in cells at the
posterior of the appendage primordium. A
key difference, however, is that the Drosophila wing is primarily an ectodermal
structure. Vertebrate appendages, in contrast, consist of both ectodermal and mesodermal components, and Shh and BMP2 are expressed within the posterior mesoderm. Thus if the signaling systems were
originally utilized in an ectodermal structure present in the arthropod/vertebrate ancestor, the production of Shh must have
APPENDAGE DEVELOPMENT AND EVOLUTION
been secondarily taken over by the mesoderm as the vertebrate appendages evolved.
Consistent with this view, other vertebrate
embryonic outgrowths, such as the branchial arches, also display localized posterior
expression of Shh (Marigo et al., 1996;
Wall and Hogan, 1995) and BMPs (Wall
and Hogan, 1995. However, in the branchial
arches they are synthesized by cells in the
ectodermal layer.
Lmx-IIapterous
There are also parallels in the patterning
of the dorsal-ventral axis of vertebrate and
arthropod wings. In the vertebrate limb bud
there is a LIM-homeodomain transcription
factor, Lmx-1, which is expressed throughout the dorsal half of the limb bud, and is
sufficient to specify dorsal cell fate (Riddle
et al., 1995; Vogel et al., 1995) (Fig. 2A).
In Drosophila, a related LIM-homeodomain
protein, Apterous, is expressed throughout
the dorsal disc compartment and is similarly necessary and sufficient for dorsal-specific wing patterning (Fig. 2B) (Diaz-Benjumea and Cohen, 1993).
While it is amazing that highly related
transcription factors are playing equivalent
roles in establishing dorsal-ventral pattern
in the two systems it is essential to take
note of the fact that Lmx-1 and apterous are
not direct orthologs. On the one hand this
would seem to point towards convergence,
that similar but distinct genes were recruited to play the same role in the two appendages. However an alternative possibility is
that, since they are highly related genes,
one member of the LIM-homeodomain
family might have secondarily taken over
for another. The plausibility of this scenario
is suggested by the observation that a similar substitution of one member of a gene
family for another does appear to have taken place between different Wnt genes, as
discussed below.
Radical fringe/fringe, Notch and Serrate
The outgrowth and patterning of the proximal-distal axis of the vertebrate appendages
is driven by a specialized group of ectodermal cells called the apical ectodermal ridge
(AER) (Todt and Fallon, 1984). The AER
forms at the boundary of what will be the
655
dorsal and ventral faces of the limb, and can
be seen as a morphologically distinct thickening of the ectoderm into a pseudostratified
epithelium extending along that border. The
AER is established at its proper location under the influence of a gene encoding a secreted factor, called Radical fringe (R-fng),
which is expressed throughout the dorsal
half of the wing bud prior to formation of
the AER (Fig. 3A) (Laufer et al., 1997; Rodriguez-Esteban et al., 1997). As noted above,
frig genes are believed to act by modulating
N signaling (Panin et al., 1997). As a consequence of R-fng expression in the dorsal
cells, a N ligand Serrate-2 (Ser-2) is induced
at the dorsal-ventral boundary (Fig. 3A) and
the AER forms. Loss of the R-fng expression boundary prevents AER formation, and
ectopic R-fng can induce an additional AER
on the ventral surface of the wing bud (Fig.
3A).
The outgrowth of the Drosophila wing
blade is similarly driven by a specialized set
of cells running along the dorsal-ventral
border of the wing imaginal disk, called the
wing margin. The wing margin forms, similarly to the AER, at the border between
dorsal cells which express the gene frig
(Fig. 3B) and ventral cells which do not
(Irvine and Wieschaus, 1994). Loss of the
border of frig expression prevents formation
of wing margin, and experimental creation
of a second Fng-non Fng boundary in the
wing disk produces an ectopic wing margin
(Fig. 3B). As in vertebrates a key downstream effector of Fng activity is Ser
(Speicher et al., 1994). Ser expression is
induced in response to Fng activity (Fig.
3B) leading to activation of a genetic cascade which ultimately establishes the wing
margin (Couso et al., 1995; Diaz-Benjumea
and Cohen, 1995; Kim et al., 1995).
There is thus a striking parallel between
chick and Drosophila wings in the formation of a proximal-distal organizing structure at the dorsal-ventral boundary by the
action of dorsally expressed frig genes acting to modulate N signaling by Ser. However, as noted above, the use of fringe, N
and Ser are collectively a single example of
a parallel. As a genetic cassette they fall
into the "obligately linked" category, as
Fng acts biochemically to influence N sig-
656
or
wild type
wild type
hh misexpression Shh misexpression
657
APPENDAGE DEVELOPMENT AND EVOLUTION
Lm\l
FIG. 2. Expression patterns of Apterous/Lmx-1 and wingless/Wnt3a in developing Drosophila and chick limbs.
Apterous is expressed in the dorsal cells of the developing Drosophila wing (A). A vertebrate homolog, Lmx1, is expressed in dorsal cells of the developing chick limb (B). Wingless is expressed along the dorsal-ventral
compartment boundary of the developing Drosophila wing (A). A vertebrate homolog, Wnt3A, is expressed
similarly in the developing chick limb (B).
naling, and Ser and N form a ligand-receptor pair.
Wnt 3a wingless
The AER drives outgrowth of the vertebrate limb buds by producing members of
the fibroblast growth factor (FGF) family,
which act as signals to the underlying mesoderm. This inductive signaling is necessitated by the fact that, in contrast to the
arthropod appendages, vertebrate limb buds
must coordinate patterning of the mesoderm
with the ectoderm. Within the ectoderm, the
expression of FGFs is presaged by the expression of another secreted factor Wnt 3a
(Kengaku et al., 1998) (Fig. 2A). Indeed
Wnt3a is the earliest known AER marker.
Misexpression of Wnt3a induces expression
of FGF4 and FGF8 in the ectoderm. Mesodermally expressed genes implicated in
the outgrowth of the distal mesenchyme
(the so-called "Progress Zone") are induced indirectly by Wnt3a via ectodermal
FGF4/8 induction (Kengaku et al, 1998).
In addition blocking Wnt signaling with a
dominant-negative variant of the signal
transduction molecule LEF1 leads to a lack
of FGF expression and an absence of limb
bud outgrowth (Kengaku et al., 1998).
In Drosophila, wing outgrowth is also
driven by a member of the Wnt gene family
produced at the dorsal-ventral boundary.
Wingless (Wg) is the key signal produced
by the cells of the wing margin organizing
center (Fig. 2B) and is necessary for distal
growth of the forming wing (Couso et al.,
1995; Diaz-Benjumea and Cohen, 1995;
Kim et al., 1995).
As in the case of Lmx-1 and apterous,
wg and Wnt3a are related members of the
same gene family, but are not direct orthologues (wg is orthologous Wntl). However
before dismissing the possibility of evolutionary homology of the two patterning systems, it is informative to consider the comparison of Wnt genes expressed during limb
bud outgrowth in mice and chicks. There is
no question that the mouse and chick AERs
are homologous structures, present in both
because an AER existed, and organized the
proximal-distal axis of the limb buds, in a
common reptilian ancestor. Yet Wnt3a, apparently essential in chick AER function, is
not expressed in the mouse AER and tar-
FIG. 1. Ectopic expression of (S)hh in the anterior compartment of developing Drosophila and chick limbs
leads to mirror-image duplications of anterior-posterior pattern elements. Normal expression of (S)hh (A, B) and
dpp/BMP-2 (C, D) in developing Drosophila wings (A, C) and chick limbs (B, D). Normal adult Drosophila
(E) and chick (F) wings. Adult Drosophila (G) and chick (H) wings resulting from misexpression of (S)hh.
658
C.J. TABIN ETAL.
geted disruption of the Wnt3a gene has no
effect on limb patterning (Greco et al.,
1996; Roelink and Nusse, 1991). However
several other Wnt genes are expressed in the
murine AER, including WntlOb which is
expressed in a very similar pattern to that
of Wnt3a in the chick (Hardiman et al.,
1996; Wang and Shackleford, 1996). Thus
even in the evolutionary time separating the
mouse and chick, one member of the Wnt
gene family can apparently substitute functionally for another in patterning homologous structures. If Wnt3a was substituted
for WntlOb during the divergence of mice
and chickens, then it is entirely plausible
that a similar substitution of Wnt3a for
Wntl could have taken place during the divergence of arthropods and vertebrates.
Vertebrate versus arthropod limb axes
The remarkable thing about the parallels
between the signaling systems that pattern
the insect and chick wings is not just that
related genes are used, or even that they are
used for analogous purposes, but that they
are used for the same orthogonal axes in the
two cases: Shhlhh and BMPIdpp for the anterior-posterior axis; Lmx-IIapterous for the
dorsal-ventral axis; dorsal expression of Rfng/fng defining an organizing center at the
dorsal-ventral border that patterns the proximal-distal axis; Wnt3a/wg expression at
that margin driving distal outgrowth; and
Dlx/DH expression in the distal portion of
the outgrowths. The parallel in spatial organization of these patterning mechanisms
suggests the possibility that they may indeed have been recruited to the evolution
of both vertebrate and arthropod appendages as an integrated three-dimensional genetic patterning system rather than as independent genes.
This conserved geometry would seem to
be at odds with the idea, for which there is
molecular evidence (Holley et al., 1995),
that the dorsal-ventral axis is inverted in arthropods relative to chordates. One would
indeed expect to see a parallel reversal in
the dorsal-ventral orientation of the patterning mechanisms of the vertebrate limb if
vertebrate and insect limbs were structural
homologues. However, we know that the
vertebrate paired appendages evolved after
the dorsal-ventral inversion of the body
axis. If a three-dimensional appendage patterning system were recruited after that
time, it could not be predicted whether it
would be established in one orientation or
another relative to the body dorsal-ventral
axis.
PARALOGOUS STRUCTURES—CONTINUITY OF
GENETIC INFORMATION, DISCONTINUITY OF
FORM
As described above, there are four possible explanations for apparent similarities
between developing structures: (1) homology (derivation from a common ancestral
structure); (2) redeployment of an existing
developmental genetic program at a novel
location; (3) redeployment of genetic cassettes that are subsets of existing developmental programs; and (4) convergence. In
the second and third instances, there would
be continuity of the genetic information that
regulates the development of the similar
structures, without evolutionary continuity
in the structures themselves.
A scenario we favor is that an ancient
ancestor of tetrapods and arthropods had
primitive appendages, perhaps antennae,
whose formation was under the control of
a network of genes that included DUIDlx,
Shhlhh, BMPIdpp, and R-fnglfng. Our view
is based on the observation that the constellation of genes used in appendage development is uniquely linked in developing
outgrowths, despite the fact that each of
FIG. 3. Ectopic expression of (Radical) fringe induces an ectopic wing margin in developing Drosophila wings
and an ectopic AER in developing chick limbs. (A) Expression of fringe in dorsal cells of the developing
Drosophila wing. (B) Expression of Radical fringe in dorsal cells of the developing chick limb. (C) Expression
of Serrate at the dorsal-ventral boundary of the developing Drosophila wing. (D) Expression of Serrate-2 at the
dorsal-ventral boundary of the developing chick limb. (E) An ectopic wing margin induced by misexpression
of fringe in the developing Drosophila wing. (F) An ectopic AER induced misexpression of Radical fringe in
the developing chick limb bud.
659
APPENDAGE DEVELOPMENT AND EVOLUTION
Chick
Drosophila
fringe
Dorsal
Ventral
Ventral
Dorsal
serrate
Ser2
AER
J
ectopic
•**ving margin
p..
•••„_
t,
•-
ectopic
AER
660
CJ. TABIN ETAL.
TABLE 1. Genes expressed in both developing vertebrate limbs and branchial arches.
Gene
Branchial arch
expression
Limb expression
ectoderm'-2
posterior mesenchyme3
Dlx-1
mesenchyme4
apical ectodermal ridge4
5
Dlx-2
mesenchyme and ectoderm
apical ectodermal ridge5
ectoderm2
mesenchyme and ectoderm6
BMP2
ectoderm7
apical ectodermal ridge7
FGF-4
Gsc
mesenchyme8
mesenchyme8
9
R-fng
ectoderm
dorsal ectoderm9
1
2
3
4
Marigo et al., 1996; Wall and Hogan, 1995; Riddle et al., 1993; Dolle et ai, 1992; 5 Bulfone et at., 1993,
Robinson and Mahon, 1994; 6 Lyons et al., 1989, 1990; 7 Niswander and Martin, 1992; 8 Gaunt et al., 1993;
9
Laufer et al., 1997, Rodriguez-Esteban et ai, 1997.
Shh
these genes is used during the development sophila limb development (Cohen et al.,
of other types of tissues and structures. We 1989; Gorfinkiel et al., 1997) and that Shh
propose that in the protostome lineage that and the Bmps are expressed in the branchial
eventually gave rise to the arthropods, an arch ectoderm (Marigo et al., 1996; Wall
appendage whose development was under and Hogan, 1995), as are their homologs in
control of this network may have existed the Drosophila limb ectoderm (Masucci et
continuously. Consistent with this idea, al, 1990; Tabata et al, 1992). Nonetheless,
many protostomes, including molluscs, ar- there are significant differences between
thropods, and annelids, have antennae. branchial arches and limbs. For instance,
However, in the deuterostome lineage that some genes that are expressed in branchial
gave rise to tetrapod chordates, echino- arch ectoderm are expressed in limb mederms, and ascidians, there were probably soderm (Table 1). Furthermore, some genes
intermediate forms that lacked appendages. required for branchial arch development are
Nonetheless, the genes required to make not needed for limb formation {e.g., Dlx-1
outgrowths were retained, as probably were and -2; Qiu et al, 1995, 1997) and vice
critical regulatory sequences. It therefore versa {e.g., Hoxa9-13, Hoxd91-13; rewould have been possible to evolve ap- viewed in Shubin et al, 1997). Thus, if the
pendages at new sites by reactivating the tetrapod limb developmental program is dedevelopmental genetic program that regu- rived from a program previously used to
lates their formation. Thus, the develop- elaborate the branchial arches, it has underment of echinoderm tube feet, ascidian am- gone some substantive alterations during
pullae, and vertebrate limbs may operate chordate evolution.
Further tests of the veracity of the limb
via similar programs, i.e., they represent
continuity in genetic information, without evolution scenario proposed here will rethese structures actually being homologous. quire that we learn more about the genetic
So where was the outgrowth program networks controlling the formation of apfirst activated in the chordate lineage? And pendages in other phyla and from deepenhow was it subsequently modified prior to ing our understanding of limb patterning in
deployment in evolving limbs? A possible insects and vertebrates. It will be critical to
venue is the branchial arches. Several es- determine, for instance, how many genes
sential "limb-patterning" genes are ex- may have been linked together in a compressed in the branchial arches (Table 1), mon appendage-patterning regulatory netand branchial arches predate fins in the work prior to the arthropod/vertebrate dichordate fossil record (Gerhart and Kir- vergence.
schner, 1997). Also consistent with this idea
INFERENCES ABOUT ANCESTORS
are the observations that in vertebrates the
Dlx-1 and Dlx-2 genes are required for
The discoveries of underlying genetic
branchial arch development (Qiu et al., similarities in arthropod and vertebrate organ
1995, 1997), as is their homolog for Dro- formation and axial patterning (especially
APPENDAGE DEVELOPMENT AND EVOLUTION
the dorsal-ventral axis) led deRobertis and
Sasai to postulate that these shared features
were present in the common ancestor of
most bilaterians ("Urbilateria"). Similarly,
we have suggested that the genetic system
that governs the formation and patterning of
extant appendages arose in a common ancestor of appendage-bearing phyla (Panganiban et al., 1997; Shubin et al., 1997). Several issues surround the quality of inferences
one can make about the identity and morphological complexity of ancestors.
First, we must accept that developmental
genetics only tells us what characters might
have been possible. Possession of some genetic regulatory mechanisms for building
eyes or appendages does not mean that any
structure existed nor does it inform us of
the complexity of that structure. Second, as
our concepts of metazoan phylogeny
change with new molecular systematic data,
the evolutionary relationships of proposed
ancestors to various other taxa and to each
other also change. For example, we pictured a potential appendage-bearing ancestor as above the nematode, flatworm, and
Urbilaterian grade (see Fig. 3 Panganiban
et al., 1997). Recent work, however, from
Aguinaldo et al. (1997) have placed the
nematodes within a new clade, the Ecdysozoa, that includes the arthropods and excludes annelids and flatworms, while Balavoine (1997) has argued that flatworms
are not basal bilaterians but belong in a
large clade of protostomes that include the
annelids. Thus, it is now unclear what grade
either our hypothetical appendage-bearing
ancestor or Urbilateria would represent.
The only path out of this state of flux
requires additional data. For the questions
posed here we see three essential directions.
First, the phylogeny of extant metazoa remains a critical challenge. It is hoped that
new methods using gene orders or gene duplications might break the impasse of current 18S rRNA approaches (see Maley and
Marshall, 1998). Second, we must understand more about the genes, pathways, regulatory circuits and spatial deployment of
genes in primitive metazoa. It remains unclear what triploblasts would be representative of an arthropod/vertebrate ancestor.
However additional studies of diploblastic
661
groups such as the Cnidaria are likely to be
very rewarding in terms of identifying very
deeply conserved mechanisms. For the appendage question, a search for an unambiguous cnidarian DUIDlx ortholog and for ligands of signaling systems would be worthwhile, as would determination of their expression patterns. Finally, the ultimate
resolution of the actual characteristics of bilaterian ancestors from their possible states
would come from the fossil record. The discovery of exceptionally well preserved animals, perhaps even embryos, from preCambrian formations (Xiao et al., 1998)
provides new hope that primitive structures
may have been captured in fossil forms.
Progress on all three fronts would bring us
within reach of an integrated view of the
history of animals and the origin of new
structures.
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Corresponding Editor: Gregory A. Wray