AMER. ZOOL., 39:630-640 (1999)
Amphioxus and the Utility of Molecular Genetic Data for Hypothesizing
Body Part Homologies between Distantly Related Animals1
NICHOLAS D. HOLLAND2 AND LINDA Z. HOLLAND
Marine Biology Research Division, Scripps Institution of Oceanography, La Jolla,
California 92093-0202
SYNOPSIS. Expression domains of developmental genes can indicate body part homologies between distantly related animals and give insights into interesting evolutionary questions. Two of the chief criteria for recognizing homologies are relative position with respect to surrounding body parts and special quality (for instance, a vertebrate testis, regardless of its location, is recognizable by its seminiferous cysts or tubules). When overall body plans of two animals are relatively
similar, as for amphioxus versus vertebrates, body part homologies can be supported by developmental gene expression domains, which have properties of special
quality and relative position. With expression patterns of AmphiNk2-l and
AmphiPax2ISI8, we reexamine the proposed homology between the amphioxus endostyle and the vertebrate thyroid gland, and a previously good homology is made
better. When body plans of animals are disparate, body part homologies supported
by molecular genetic data are less convincing, because the criterion of relative
position of gene expression domains becomes uncertain. Thus, when expression of
amphioxus AmphiBMP2l4 is used to compare the dorsoventral axis between amphioxus and other animals, a comparison between amphioxus and vertebrates is
more convincing than comparison between amphioxus and other invertebrates with
disparate body plans. In spite of this difficulty, the use of developmental genetic
evidence comparing animals with disparate body plans is currently putting the big
picture of evolution into new perspective. For example, some molecular geneticists
are now suggesting that the last common ancestor of all bilaterian animals might
have been more annelid-like than fiatworm-like.
INTRODUCTION
In 1830, there was a famous debate between Geoffroy Saint-Hilaire, who dared to
propose body part homologies over a wide
spectrum of animals, and Cuvier, who
»u
u*
u u
i
u u u
J
thought such homologies should be made
only between very closely related animals
(for instance, between two kinds of mammal). In the long run, the debate had no
clear winner (Appel, 1987; Corsi, 1988)
and started a controversy that is still with
us. The attitude of Geoffroy Saint-Hilaire is
needed for approaching big-picture questions like the phylogenetic relations among
the animal phyla, but tends to be plagued
by errors due to such problems as conver1
From the Symposium Developmental and Evolutionary Perspectives on Major Transformations in
Body Organization presented at the Annual Meeting
of the Society for Integrate and Comparative Biology. 3-7 January 1998. at Boston. Massachusetts.
2
E-mail: [email protected]
gence. In comparison, homologies that Cuyier would have applauded are much more
llkel
y t o b e <; orrect ' b u t f e s u l t e d o n l y f o r
mo
J * n a r r °wly conceived questions.
P u r i n S t! ? e l a s t f d e c * d e ' a d v a n c e s i n d e "
velopmental genetics
have been Pproviding
^
*f e y i d e n c e
a J^
fof
the sizing
homologies between animals. It
bo(J
was ' d f S covered, quite unexpectedly, that the
m o l e c u l a r machinery directing developm e n t h a s b e e n r e m a r k a b l y conserved duri n g m e t azoan evolution. Most development a j g e n e s encode proteins containing highly
conserved amino acid sequences that can be
used to identify homologous genes among
different organisms. In addition, homologous genes tend to be expressed in similar
places and at similar developmental stages
in different kinds of animals. These cons e r v e d expression domains
d e v e l o p m e n t a l genes, along
of homologous
with more tra...
, ,
• .
. .
dltional data, Can help Suggest body part
homologies between distantly related Species.
630
AMPHIOXUS, HOMOLOGY, DEVELOPMENT, AND EVOLUTION
We have been using this approach to
study body part homologies between amphioxus and other animals. Amphioxus
(also called the lancelet) is an invertebrate
chordate of the subphylum Cephalochordata (Stokes and Holland, 1998), the sister
group of the vertebrates (Wada and Satoh,
1994). As the closest living invertebrate relative of the vertebrates, amphioxus is a useful stand-in for the proximate ancestor of
the vertebrates. Holland and Holland (1998)
have already summarized developmental
genetic studies of amphioxus that have given new insights into the evolutionary origin
of vertebrate brain regions, neural crest, and
rostrocaudal segmentation.
Detailed molecular biology techniques
with special reference to amphioxus have
been published by L. Z. Holland et al.
(1996). In brief, developmental genes are
isolated from genomic or cDNA libraries,
sequenced, and compared to related genes
on the basis of sequence structure and
sometimes intron positions. The genes are
named to reflect their relationship with homologous genes in other animals. (For the
genes considered in the present paper, this
step was straightforward, but paralogy and
domain shuffling can sometimes confuse
the issue, as discussed by Abouheif, 1997.)
For each developmental gene, an antisense
riboprobe is synthesized and used for in situ
hybridization in whole mounts or sections
of amphioxus embryos and larvae to visualize the expression domains for the gene.
These spatiotemporal patterns of gene transcription can then help characterize the
body parts at the phenotypic level.
There have been some criticisms of this
appproach (e.g. Miiller and Wagner, 1996;
Davidson, 1997), and the present paper is
an opportune place to discuss them. We first
review the barest essentials of homology
and amphioxus development. Then we
summarize some recent work on the developmental genetics of amphioxus: the first
example, comparing the amphioxus endostyle with the vertebrate thyroid gland, concerns animals with relatively similar overall
body plans; the second example, comparing
dorso-ventral body axes throughout the bilaterian animals, ranges over animals with
very disparate body plans. In a final section,
631
we discuss the strengths and weaknesses of
using developmental genetic data for indicating body part homologies; special attention is given to the effect of body plan disparity on the analysis.
HOMOLOGY
Homology is a contentious and complex
issue, and what follows is no more than a
selective outline. Comprehensive coverage
of the subject can be found in Hall (1994).
At the outset, it should be made clear that
no homology can be proven in an absolute
sense. Homologies should be thought of as
hypotheses that are always open to further
testing and possible rejection. There is no
international commission for approving homologies—instead, the scientific community either rejects them or accepts them tentatively as hypotheses.
There are currently several concepts of
homology (neo-idealistic, cladistic, biological, and historical), which are explained in
the chapters in Hall (1994). For our present
purposes, we will stay within the framework of the dominant concept, which is historical. Thus, characters are homologous
because they correspond to an equivalent
character in a common ancestor. There are
three main criteria by which homology is
recognized (Remane, 1971; Riedl, 1978).
First is positional equivalence within the
overall body plan. Second is special quality
(=compositional similarity) (this criterion
is sometimes misunderstood, so we will return to it in the next paragraph); one brief
example would be that all vertebrate testes
agree in special features, like seminiferous
cysts or tubules, without necessarily having
equivalent positions in the body. The third
criterion is transition, such that structures,
although appearing dissimilar in two species, are recognized as homologous if united in a paleontological or developmental
series by transitional structures. To these
criteria, a fourth may be added (considered
subsidiary by some, but very important by
others): this is congruence, which means
that each proposed homology should be examined within the context of all the other
evidence to see if it fits.
The criterion of special quality has provided an opportunity to consider homolo-
632
N. D. HOLLAND AND L. Z. HOLLAND
gies at diverse levels of biological organization {e.g., behavior, physiology, and biochemistry) in addition to traditional anatomy. As discussed by Striedter and Northcutt
(1991), characters do not always map oneto-one between different levels of organization, and it is valid to make hypotheses
of homology at one organizational level
without reference to higher or lower ones
(this illustrates the hierarchical nature of
homology). For instance, one can study
purely molecular homologies for insulin
and insulin-related peptides (Chan et ai,
1990). Even so, when comparing body
parts, the hierarchical nature of homology
should not be interpreted as preventing one
from including characters at other levels of
organization as criteria of special quality.
For instance, for studying pancreatic homologies, it is relevant to consider the presence of islets of Langerhans (histological
level) predominantly comprising alpha and
beta cells (cytological and fine structural
levels) which are rich in glucagon and insulin, respectively (molecular level) and express Nkx2-2 in the developing and differentiated state (genetic level) (Rudnick et al.,
1994).
To use developmental genetic results in
evolutionary discussions, one must make an
initial homology decision at the molecular
genetic level to identify and name a particular gene. Subsequently, on the basis of the
developmental expression patterns of that
gene, one makes a second homology decision at the phenotypic level to relate body
parts of different animals (our main concern). When considering these second stage
homologies, it is necessary to stipulate the
level of biological organization under consideration (as discussed by Dickinson,
1995, and by Bolker and Raff, 1996). For
example, there is a big difference between
using Pax-6 expression domains to compare
photoreceptors at the cell level and using
them to compare eyes at the organ level.
BACKGROUND ON AMPHIOXUS EMBRYOLOGY
FIG. 1. Diagrams of important features of amphioxus
development. In side views (A—C, G). anterior is to the
right and dorsal is up. A. Side view of mid gastrula.
B. Side view of late gastrula. C. Side view of early
neurula. D. Cross section of early neurula (through level X in C). E. Cross section of mid neurula. F. Cross
section of late neurula. G. Side view of early larva;
the arrow indicates the endostyle, which is the thickened right wall of the pharynx; X in G is explained in
caption of Figure 2. Abbreviations, in alphabetical order, are: dm, dorsal mesoderm; en, endoderm; mo,
mouth; nc, nerve cord; no. notochord; np, neural plate;
pno, presumptive notochord; vm, ventral mesoderm.
ing the summer breeding season, gametes
are obtained from ripe males and females
by electrical stimulation, and the embryos
and larvae are cultured in the laboratory
(Holland and Holland, 1993; Stokes and
Holland, 1995). The early stages of development are sea urchin-like: cleavage produces a hollow blastula, which becomes
converted into a gastrula by invagination
(Fig. 1A). In contrast, subsequent development is vertebrate-like, with a dorsal nerve
cord, a notochord, and muscular somites
appearing during the neurula stage (Fig.
1C—F). During the neurula stage of amphioxus, the left-right axis of the body becomes asymmetrical; for example, the
mouth opens on the left side of the head
(Fig. 1G), presumably as an adaptation for
larval feeding (van Wijhe, 1919; Bone,
1958).
Most of the recent work on amphioxus
THE AMPHIOXUS ENDOSTYLE VERSUS THE
development has been done on BranchiosVERTEBRATE THYROID GLAND
toma floridae, which is extremely abundant
In the amphioxus neurula, an early morin Tampa Bay, Florida, and can be collected
in hip-deep water by shovel and sieve. Dur- phological manifestation of the left-right
AMPHIOXUS, HOMOLOGY, DEVELOPMENT, AND EVOLUTION
FIG. 2. A. Early larva of Branchiostoma floridae:
cross section through level X in Figure 1G and viewed
from the tail end of the animal; prominent features
include the nerve cord (nc), notochord (no), and pharyngeal lumen (pi); the endostyle (arrow), which is the
thickened right wall of the pharynx, is conspicuously
expressing AmphiNk2-l\ no expression is detectable in
the remaining tissues, which have been lightly counterstained to make them visible; the scale line is 20
\x,m. B. Late gastrula of Branchiostoma floridae: optical section through level X in Figure 1C; AmphiBMPH
4 expression is detectable throughout the epiblast (ep)
and hypoblast (hy), but is strongest in the dorsolateral
regions of the hypoblast where the dorsal mesoderm
will later give rise to somites; the scale line is 50 p.m.
asymmetry is an increase in the height of
the cells comprising the right anterior wall
of the pharynx. This thickened patch of endoderm becomes the larval endostyle (Figs.
1G, 2A), which is thought to supply some
of the food-trapping secretions of the larvae
(Gilmour, 1996). At larval metamorphosis,
the endostyle shifts to a ventral position and
grows posteriad, giving rise to the adult endostyle, a midventral groove running along
the floor of the pharynx. As in the larva,
the most obvious function of the endostyle
of the adult is to produce extracellular secretions to trap and transport ingested particulate food.
An homology between the adult amphioxus endostyle and the vertebrate thyroid
gland was first proposed by Miiller (1873)
on the basis of positional equivalence (both
were structures running down the ventral
midline of the pharynx) and transition (in
developing lampreys, the larval endostyle is
converted to a postlarval thyroid gland).
Recently, this homology has been supported on the basis of special quality. Biochemical data show that both the endostyle and
the thyroid metabolize iodine to form iodothyronines, and both synthesize a similar
633
thyroglobulin and a peroxidase (Thomas,
1956; Barrington, 1958; Covelli et al,
1960; Tong et al, 1962; Monaco et al,
1981; Tsuneki et al, 1983; Fredriksson et
al, 1984).
Cumulatively, the data above favor an
homology between the iodine-fixing regions of the amphioxus endostyle and the
follicular epithelium of the vertebrate thyroid. Even so, the available information
leaves room for some doubt, because there
is no clear evidence that the amphioxus endostyle has an endocrine function (Ericson
and Fredriksson, 1990). In recent years,
most biologists have looked favorably on
the homology, but some have not. For instance, in the opinion of Burrow (1989, p.
11), "evidence of thyroid evolution from
prevertebrate ancestry is inconclusive."
For the last ten or fifteen years, the homology of the amphioxus endostyle and the
vertebrate thyroid has been in the category
of "good but could be better." Starting at
this point, we and our colleagues studied
amphioxus homologs of vertebrate genes
involved in thyroid development. Vertebrate Pax-8 (Plachov et al, 1990) and
Nkx2-1 (also called thyroid transcription
factor-1) (Lazzaro et al, 1991) are expressed in the early rudiment of the thyroid
and are probably involved in the commitment of endodermal thyrocytes there. Later
in thyroid development, both genes also
function as transcriptional regulators by
turning on the thyroglobulin gene and the
thyroid peroxidase gene, which are thyrocyte-specific (van der Kallen et al, 1996).
The amphioxus genome includes
AmphiNk2-l, an homolog of vertebrate
Nkx2-1 (Venkatesh et al, 1999), as well as
AmphiPax2/5/8, an homolog of the collective group of vertebrate Pax2, Pax5 and
Pax8 genes (Kozmik et al, 1999). During
amphioxus development, AmphiNk2-l expression is first detected in the central nervous system and ventral endoderm during
the neurula stage. Subsequently, expression
is progressively restricted until it remains
detectable only in cells of the right anterior
wall of the pharynx—namely, in the rudiment of the endostyle (Fig. 2A)—where it
persists until the larva is about a week old.
Expression of amphioxus AmphiPax2/5/
634
N. D. HOLLAND AND L. Z. HOLLAND
homologs, BMP2 and BMP4 of vertebrates,
encode extracellular morphogens from the
anti-neural side of the embryo, which are
antagonized, respectively, by short gastrulation and chordin proteins; the latter are
homologs of one another and are produced
on the neural side of the embryo (Nellen et
al, 1996; Piccolo et al, 1996). In both
Drosophila and vertebrates, the signals
from the neural side of the body combine
with and thereby inactivate signals from the
anti-neural side. These antagonistic morphogens play a crucial role in the initial
dorsoventral patterning of the body. This
patterning is limited to the ectoderm of
Drosophila, but occurs nearly simultaneously in the ectoderm and mesoderm of
vertebrates (Wilson and Hemmati-Brivanlou, 1995; Holley et al, 1996).
The polarities of the morphogenetic systems establishing dorsoventral axes correspond between Drosophila and vertebrates.
In contrast, the body plan topographies of
Drosophila and vertebrates are reversed
dorsoventrally, such that the neural side of
the former is ventral and the neural side of
the latter is dorsal. This pattern has favored
the idea that the animal body became inverted dorsoventrally with respect to the
substratum in some invertebrate ancestor of
the vertebrates (Arendt and Niibler-Jung,
1994; Holley et al, 1995; De Robertis and
Sasai, 1996). These modern studies are reviving a venerable idea about dorsoventral
inversion that Geoffroy Saint-Hilaire originally proposed in 1822, nearly a decade before his debate with Cuvier.
To shed more light on the evolution of
the dorsoventral body axis of animals generally, Panopoulou et al. (1998) studied
AmphiBMP2/4, an amphioxus gene homologous to decapentaplegic of Drosophila
and to BMP2 and BMP4 of vertebrates. The
presence of a single BMP2/4-related gene
in amphioxus and duplicate members of
this gene family in vertebrates is yet another instance of extensive gene multiplication
during early vertebrate evolution (Holland
et al, 1994). Presumably, during vertebrate
COMPARISON OF THE DORSOVENTRAL BODY
evolution, the functions of the original
AXIS BETWEEN AMPHIOXUS AND OTHER
ANIMALS
BMP2/4-related gene were parcelled out beAround the time of gastrulation, the de- tween BMP2 and BMP4, although with
capentaplegic gene of Drosophila and its some overlap and possible redundancy (as
8 is first seen during the neurula stage in
the central nervous system, kidney rudiment, and gut. By the early larval stage,
there is strong expression in pharyngeal endoderm, both in the endostyle and also
where gill slits will form. Transcripts are no
longer detectable by in situ hybridization by
the time the larvae are about a week old.
For the examples of AmphiNk2-l and
AmphiPax2/5/8, the expression data, when
added to the existing morphological and
biochemical data, fit very well (i.e., satisfy
the criterion of congruence). In this instance, homologous morphological structures appear to develop under the control of
homologous genes (and by extension homologous genetic pathways). Such a correspondence between genotype and phenotype is not always found. Abouheif (1997)
has discussed non-homologous structures
controlled by homologous genetic pathways
and, conversely, homologous phenotypic
structures that develop under the control of
very different genes. The most striking examples of discordance between the genetic
and phenotypic levels have been noted
where pre-phylotypic stages of development (as defined and discussed by Raff,
1996) differ markedly (as between insects
with long germ bands and short germ
bands) and where individual organs—like
the nematode vulva (more correctly the vagina)—could well be homoplastic innovations at low taxonomic levels.
In spite of this caution, the addition of
molecular genetic data considerably
strengthens the previously proposed homology between the amphioxus endostyle and
the vertebrate thyroid. As already mentioned, however, the comparison here is between two animals with relatively similar
overall body plans. In addition, it is only
fair to consider how effectively developmental genetic data can support body part
homologies between animals with markedly
disparate body plans. In the following section, we will consider an homology that
ranges over a wide spectrum of the animal
kingdom.
AMPHIOXUS, HOMOLOGY, DEVELOPMENT, AND EVOLUTION
discussed for chordate Distal-less genes by
N. D. Holland et al., 1996).
Detectable expression of AmphiBMP2/4
begins ubiquitously throughout the epiblast
and hypoblast of the mid gastrula stage. By
the late gastrula stage (Fig. IB), the gene is
expressed most conspicuously in regions of
the hypoblast where the paraxial mesoderm
will soon form (Fig. 2B). Thus AmphiBMP2/4 may be involved in the earliest
stages of somite formation, but not in the
initial dorsoventral patterning of the mesoderm as a whole. Indeed, before the late
neurula stage, amphioxus lacks any ventral
mesoderm to pattern; all the mesoderm runs
paraxially along the dorsal side (Fig. 1D,E),
and it is only at the end of the neurula stage
that the dorsal mesoderm (collectively, the
myogenic somites) gives rise to the ventral
mesoderm (the mesothelia of the perivisceral coelom) as diagrammed in Figure IF.
At the very early neurula stage, as neurulation is beginning, AmphiBMP2/4 expression is detectable everywhere in the ectoderm except dorsally in the neural plate
and immediately adjacent ectoderm. This
pattern indicates that the morphogenic system establishing the dorsoventral axis of
amphioxus acts within the ectoderm. For
our present purposes, it is not necessary to
go into details about the additional domains
where AmphiBMP2/4 is expressed during
later developmental stages of amphioxus
(these are the tail bud mesoderm, pharynx,
hindgut, possible olfactory epithelium, anterior central nervous system, and heart primordium). The important point is that
AmphiBMP2/4 is expressed in several different spatiotemporal patterns during the
course of development: this is a common
feature of developmental genes, and we will
return to it below.
If one is willing to consider homologies
between animals with markedly disparate
overall body plans, one can compare the
early expression of amphioxus AmphiBMP2/4 and its homologs in other animals.
A comparison among amphioxus, tunicates
(Miya et al., 1997); and Drosophila
(Frasch, 1995; Brehs et al, 1998) suggests
that, during most of bilaterian evolution, the
ectoderm has apparently been the only
germ layer in which BMP-like morphogens
635
from the antineural side interact with antagonistic proteins from the neural side to establish the initial dorsoventral axis of the
body. Only the vertebrates appear to have
evolved a parallel BMP-based system in the
mesoderm (Graff, 1997; Hemmati-Brivanlou and Melton, 1997) in addition to the
one in the ectoderm, thereby establishing
dorsoventral polarity in both germ layers simultaneously. The new data on amphioxus
alone are insufficient to suggest when dorsoventral inversion took place during animal evolution; to shed more light on this
problem, comparative molecular genetic
studies would be required over a much wider spectrum of animal phyla.
GENE EXPRESSION DOMAINS AND
HOMOLOGY: PROBLEMS
As discussed by Reeck et al. (1987) and
by Bolker and Raff (1996), developmental
geneticists have sometimes encountered
difficulties when attempting to deal with
subjects like homology and evolution. We
will discuss two admonitions here before
going on to more substantive problems.
First, it is generally (although by no means
universally, e.g., Meyer, 1998) agreed that
a given feature is either homologous or it
is not; the majority view is that there is no
in-between state of partial homology. Second, body part homologies should not be
based solely on molecular genetic evidence,
which should be considered as an adjunct
to more traditional data and not in isolation.
Thus, expression domains should comprise
just another column in a larger data matrix.
Used in this way, developmental genetic
data can potentially strengthen already proposed homologies, as in the example of the
amphioxus endostyle versus the vertebrate
thyroid. In other words, when the evidence
for homology is pretty good in the first
place, you can make it much better by adding developmental genetic evidence (Dickinson, 1995).
Having praised the procedure of adding
developmental genetic evidence to already
existing data, we should also point out that
contemplation of an isolated expression domain may sometimes suggest a previously
unsuspected homology, which then requires
further testing with additional anatomical,
636
N. D. HOLLAND AND L. Z. HOLLAND
physiological, and biochemical data. For
example, gene expression data gave the first
hint of a possible phylogenetic origin of the
vertebrate neural crest in lower chordates
(Baker and Bronner-Fraser, 1997). Therefore, isolated information on developmental
gene expression has its place in the generation of hypotheses and should not be deprecated (as by Miiller and Wagner, 1996).
A more serious complaint about using
gene expression domains to help establish
body part homologies is that a given developmental gene is almost always transcribed in more than one spatiotemporal domain in a given ontogeny; in other words,
these genes have pleiotropic effects. Their
gene products retain structure/function aspects while becoming associated with various organ systems. Nature is lazy and has
a propensity for reusing (co-opting) existing
developmental genes and combinations
thereof for new purposes instead of inventing new genes from scratch (Holland et al,
1994; Gans, 1997; Duboule and Wilkins,
1998).
As a concrete example of the pleiotropy
problem, amphioxus AmphiNk2-l is expressed in both the endostyle and in the
hindgut—so why not consider those two
structures somehow homologous? This difficulty seems to arise from regarding a gene
expression pattern as one of the irreducible
criteria of homology (Bolker and Raff,
1996); to us, the way out of the problem is
to consider a given gene expression domain
as a phenotypic character having (1) a relative position within the overall body plan
and (2) a special quality (namely, the presence of cells containing mRNAs made by
that gene). From this viewpoint, a developmental geneticist would no more make
an homology between the endostyle and the
hindgut on the basis of similar gene expression than a morphologist would make
an homology between the femur and the
ulna on the grounds that the two structures
are both composed of endochondral bone.
The quality of relative position, which is
built into every gene expression domain,
prevents ridiculous homologies from being
proposed between parts of a given organism
or between parts of two organisms—pro-
vided that the animals being compared
share relatively similar body plans.
In contrast, animals with markedly disparate body plans must be compared whenever one attempts to reconstruct the broad
outline of the history of animal life on
earth. It is at this point that one begins to
encounter the substantive problems of using
molecular genetic data (or any data for that
matter) for supporting body part homologies. Ever since the 1830 debate, some biologists have leaned towards the conservatism of Cuvier, while others have preferred
to contemplate the broad, but seemingly
less reliable, vistas of Geoffroy Saint-Hilaire. Those who use developmental genetics to support homologies over vast stretches of the animal kingdom obviously must
have a bit of the Geoffroy Saint-Hilaire in
them: good examples are Arendt and Niibler-Jung (1994) and De Robertis and Sasai
(1996) with their scenario for dorsoventral
axis inversion and Gehring (1996) with his
master gene scenario for eye evolution.
Others, more toward Cuvier's end of the
spectrum have reservations and council
moderation (Bolker and Raff, 1996; Averof
and Patel, 1997; some commentators in
Pennisi and Roush, 1997).
As one compares animals with increasingly disparate body plans, it becomes more
and more difficult to recognize the positional equivalence of the component body
parts. Eventually, the expression domain of
a single gene retains its special quality, but
its positional equivalence becomes unreliable. The problem is greatly compounded
because a given developmental gene is typically expressed in a multiplicity of spatiotemporal domains during a given ontogeny.
There have been recent discussions about
whether elucidating entire networks of developmental genes {i.e., data at the epigenetic level of organization) might be especially useful for supporting homologies between body parts of distantly related animals (Miiller and Wagner, 1996; Laufer et
al, 1997; Rodriguez-Estaban et al, 1997).
In theory, detailed information on a whole
network of genes should be more reliable
than data on a single gene, which might be
subject to convergence (Swanson et al,
1991) or might change its linkage with up-
AMPHIOXUS, HOMOLOGY, DEVELOPMENT, AND EVOLUTION
stream promoters or downstream targets
(Carroll, 1994). In practice, however, very
few developmental gene networks have
been adequately elucidated (Akam, 1998),
and we are decades away from having
enough information to test this idea adequately. The problem of where to delimit
one gene network from the next has yet to
be faced. Moreover, in the absence of
enough relevant data, it is not at all clear
what level of difference between two gene
networks would divide a decision of homology from a decision of non-homology.
It even remains possible that no amount of
detail at the gene network level will suffice
to sort out signalling pathways that have
been co-opted for building non-homologous
structures during the ontogeny of a given
organism or in distantly related organisms
(Gaunt, 1997; Panganiban et al, 1997; Shubin et al, 1997). Moreover, as pointed out
by Abouheif et al., (1997), the possibility
remains open that even similar gene networks might be assembled by convergence.
GENE EXPRESSION DOMAINS AND
HOMOLOGY: PROSPECTS
A recent study of developmental genetics
of echinoderms (Lowe and Wray, 1997)
was interpreted by Davidson (1997) as a
sort of Waterloo for the entire approach of
using gene expression domains for hypothesizing body part homologies and for gaining interesting evolutionary insights. Davidson's line of argumentation was: (1) the
expression domains of three genes did not
give any insights into the evolution of radial
symmetry in echinoderms, (2) therefore,
this sort of evidence tells us little that we
want to know about homologies and evolution in any animals, so (3) we should turn
our attention to working out networks of
interacting developmental genes in exquisite detail. In answer to the last point, which
was discussed above, it is by no means certain that a thorough description of developmental gene networks will ever lead to
widely acceptable body part homologies between animals with very disparate body
plans. For now, we can only hope that this
approach will work.
The second point of Davidson (1997) is
that developmental gene expression do-
637
mains are inadequate for suggesting homologies and for gaining evolutionary insights. When it comes to studying body part
homologies in animals that have relatively
similar body plans {e.g., amphioxus versus
vertebrates or even versus hemichordates),
we think Davidson is wrong. In comparisons of such animals, the approach has
firmed up old homologies and suggested
new ones that have given insights into such
outstanding evolutionary questions as the
origin of the major regions of the vertebrate
brain and the origin of the vertebrate neural
crest (Holland and Holland, 1998).
When one begins to consider body part
homologies of animals that have extremely
disparate body plans, Davidson's criticisms
need to be taken seriously, although, as already mentioned, it is far too soon to know
whether his proposed remedy (intensive
study of gene networks) will solve the problem. From our point of view, comparisons
of animals with disparate body plans are
still worth attempting—even if based on
only a few genes per developmental expression domain. Such comparisons are
forcing us to question traditional versions
of animal evolution that have not been seriously challenged for over a century. For
example, there have been recent suggestions (e.g., Kimmel, 1996; De Robertis,
1997; Akam, 1998) that the last common
ancestor of all the bilaterian phyla might
have been a creature that was rather annelid-like instead of flatworm-like. We like to
think that Geoffroy Saint-Hilaire would be
gratified by this new turn of events.
ACKNOWLEDGMENTS
We are deeply indebted to John
Lawrence and Ray Wilson of the University
of South Florida for their generous hospitality and laboratory space during the summer breeding season of Branchiostoma floridae. Our research was supported by NSF
research grant IBN96-309938.
REFERENCES
Abouheif, E. 1997. Developmental genetics and homology: A hierarchical approach. TREE 12:405408.
Abouheif, E., M. Akam, W. J. Dickinson, P. W. H.
Holland, A. Meyer, N. H. Patel, R. A. Raff, V. L.
638
N. D. HOLLAND AND L. Z. HOLLAND
Roth, and G. A. Wray. 1997. Homology and developmental genes. TIG 13:432-433.
Akam, M. 1998. The yin and yang of evo/devo. Cell
92:153-155.
Appel, T. 1987. The Cuvier-Geoffroy debate. Oxford
Univ. Press, Oxford.
Arendt, D. and K. Nubler-Jung. 1994. Inversion of dorsoventral axis? Nature 371:26.
Averof, M. and N. H. Patel. 1997. Crustacean appendage evolution associated with changes in Hox
gene expression. Nature 388:682-686.
Baker, C. V. H. and M. Bronner-Fraser. 1997. The origin of the neural crest. Part II: An evolutionary
perspective. Mech. Dev. 69:13-29.
Barrington, E. J. W. 1958 The localization of organically bound iodine in the endostyle of Amphioxus.
J. Mar. Biol. Assoc. U. K. 35:117-129.
Bolker, J. A. and R. A. Raff. 1996. Developmental
genetics and traditional homology. BioEssays 18:
489-494.
Bone, Q. 1958. Observations upon the living larvae of
amphioxus. Pubbl. Staz. Zool. Napoli 30:458471.
Brehs, B. V. Francois, and E. Bier. 1996. The Drosophila short gastrulation gene prevents dpp from
autoactivating and supporting neurogenesis in the
neuroectoderm. Genes Dev. 10:2922-2934.
Burrow, G. N. 1989. Thyroid hormone biosynthesis. In
G. N. Burrow, J. H. Oppenheimer, and R. Volpe
(eds.), Thyroid Function and Disease, pp. 11—40
Saunders, Philadelphia.
Carroll, S. B. 1994. Developmental regulatory mechanisms in the evolution of insect diversity. Development 1994 Suppl.:217-223.
Chan, S. J., Q. Cao, and D. F. Steiner. 1990. Evolution
of the insulin superfamily: Cloning of a hybrid
insulin/insulin-like growth factor cDNA from amphioxus. Proc. Natl. Acad. Sci. U.S.A. 87:93199323.
Corsi, P. 1988. The age of Lamarck: Evolutionary theories in France. Univ. Calif. Press, Berkeley.
Covelli, I., G. Salvatore, L. Sena, and J. Roche. 1960.
Sur la formation de hormones thyroidiennes et de
leurs pr^curseurs par Branchiostoma lanceolatum.
C. R. Soc. Biol. Paris 154:1165-1169.
Davidson, E. H. 1997. Insights from echinoderms. Nature 389:679-680.
De Robertis, 1997. The ancestry of segmentation. Nature 387:25-26.
De Robertis, E. M. and Y. Sasai. 1996. A common
plan for dorsoventral patterning in Bilateria. Nature 380:37-40.
Dickinson, W. J. 1995. Molecules and morphology:
Where's the homology? TIG 11:119-121.
Duboule, D. and A. S. Wilkins. 1998. The evolution
of "bricolage." TIG 14:54-59.
Ericson, L. E. and G. Fredriksson. 1990. Phylogeny
and ontogeny of the thyroid gland. In M. A. Greer
(ed.). The thyroid gland, pp. 1-35. Raven Press,
New York.
Frasch, M. 1995. Induction of visceral and cardiac mesoderm by ectodermal Dpp in the early Drosophila embryo. Nature 374:464-467.
Fredriksson, G., L. E. Ericson, and R. Olsson. 1984
Iodine binding in the endostyle of larval Branchiostoma lanceolatum (Cephalochordata). Gen.
Comp. Endocrinol. 56:177-184.
Gans, C. 1997. Book review of Before the Backbone
by H. Gee. Amer. Zool. 37:433-434.
Gaunt, S. J. 1997. Chick limbs, fly wings and homology at the fringe. Nature 368:324-325.
Gehring, W. J. 1996. Eye evolution. Science 272:468469.
Geoffrey Saint-Hilaire, E. 1822. Considerations generales sur la vertebre. Mem. Mus. His. Hist. Nat. 9:
89-119 + pi. V-VII.
Gilmour, T. H. J. 1996. Feeding methods of cephalochordate larvae. Israel J. Zool. Suppl. 42:87—95.
Graff, J. M. 1997. Embryonic patterning: To BMP or
not to BMP, that is the question. Cell 89:171-174.
Hall, B. K. 1994. Homology: The hierarchical basis
of comparative biology. Academic Press, San Diego.
Hemmati-Brivanlou, A. and D. Melton. 1997. Vertebrate embryonic cells will become nerve cells unless told otherwise. Cell 88:13-17.
Holland, L. Z. and N. D. Holland. 1998. Developmental gene expression in Amphioxus: New insights
into the evolutionary origin of vertebrate brain regions, neural crest, and rostrocaudal segmentation.
Amer. Zool. 38:647-658.
Holland, L. Z., P. W. H. Holland, and N. D. Holland.
1996. Revealing homologies between body parts
of distantly related animals: Amphioxus versus
vertebrates. In i. D. Ferraris and S. R. Palumbi
(eds.). Molecular zoology: Advances, strategies,
and protocols, pp. 267-282, 473-483. Wiley,
New York.
Holland, N. D. and L. Z. Holland. 1993. Embryos and
larvae of invertebrate deuterostomes. In C. D.
Stern and P. W. H. Holland (eds.), Essential developmental biology: A practical approach, pp.
21-32. IRL Press, Oxford.
Holland, N. D., G. Panganiban, E. L. Henyey, and L.
Z. Holland. 1996. Sequence and developmental
expression of AmphiDII, an amphioxus Distalless gene transcribed in the ectoderm, epidermis
and nervous system: Insights into evolution of
craniate forebrain and neural crest. Development
122:2911-2920.
Holland, P. W. H., J. Garcia-Fernandez, N. A. Williams, and A. Sidow. 1994. Gene duplications and
the origins of vertebrate development. Development 1994 Suppl.: 125-133.
Holley, S. A., P. D. Jackson, Y. Sasai, B. Lu, E. M. De
Robertis, F. M. Hoffman, and E. L. Ferguson.
1995. A conserved system for dorsal-ventral patterning in insects and vertebrates involving sog
and chordin. Nature 376:249-253.
Holley, S. A., J. L. Neul, L. Attisano, J. L. Wrana, Y.
Sasai, M. B. O'Connor, E. M. De Robertis, and
E. L. Ferguson. 1996. The Xenopus dorsalizing
factor noggin ventralizes Drosophila embryos by
preventing DPP from activating its receptor. Cell
86:607-617.
Kimmel, C. B. 1996. Was Urbilateria segmented? TIG
12:329-331.
Kozmik, Z., N. D. Holland, A. Kalousova, J. Paces,
AMPHIOXUS, HOMOLOGY, DEVELOPMENT, AND EVOLUTION
M. Schubert, and L. Z. Holland. 1999. Characterization of an amphioxus paired box gene,
AmphiPax-2/5/8: developmental expression patterns in optic support cells, nephridium, thyroidlike structures, and pharyngeal gill slits, but not
in the midbrain-hindbrain boundary region. Development 126:1295-1304.
Laufer, E., R. Dahn, O. E. Orozco, C. Y. Yeo, J. Pisenti, D. Henrique, U. K. Abbott, J. F. Fallon, and
C. Tabin. 1997. Expression of Radical fringe in
limb-bud ectoderm regulates apical ectodermal
ridge formation. Nature 386:366—373.
Lazzaro, D., M. Price, M. De Felice, and R. Di Lauro.
1991. The transcription factor TTF-1 is expressed
at the onset of thyroid and lung morphogenesis
and in restricted regions of the foetal brain. Development 113:1093-1104.
Lowe, C. J. and G. A Wray. 1997. Radical alterations
in the roles of homeobox genes during echinoderm evolution. Nature 389:718-721.
Meyer, A. 1998. We are devo-evo. TIG 14:482-483.
Miya, T., K. Morita, A. Suzuki, N. Ueno, and N. Satoh.
1997. Functional analysis of an ascidian homologue of vertebrate Bmp-2/Bmp-4 suggests its role
in the inhibition of neural fate specification. Development 124:5149-5159.
Monaco, F, R. Dominici, M. Andreoli, R. De Pirro,
and J. Roche. 1981. Thyroid hormone formation
in thyroglobulin synthesized in the amphioxus
(Branchiostoma lanceolatum Pallas). Comp.
Biochem. Physiol. B 70:341-343.
Muller, G. B. and G. P. Wagner. 1996. Homology, Hox
genes, and developmental integration. Amer. Zool.
36:4-13.
Muller, W. 1873. Uber die Hypobranchialrinne der
Tunicaten und deren Vorhandensein bei Amphioxus und den Cyklostomen. Jena. Z. Med. Naturw. 7:327-332.
Nellen, D., R Burke, G. Struhl, and K. Balser. 1996.
Direct and long range action of a DPP morphogen
gradient. Cell 85:357-368.
Panganiban, G., S. M. Irvine, C. Lowe, H. Roehl, L.
S. Corley, B. Sherbon, J. K. Grenier, J. F. Fallon,
J. Kimble, M. Walker, G. A. Wray, B. J. Swalla,
M. Q. Martindale, and S. B. Carroll. 1997. The
origin and evolution of animal appendages. Proc.
Natl. Acad. Sci. U.S.A. 94:5162-5166.
Panopoulou, G. D., M. D. Clark, L. Z. Holland, H.
Lehrach, and N. D. Holland. 1998. AmphiBMP2/
4, an amphioxus bone morphogenetic protein
closely related to Drosophila Decapentaplegic and
vertebrate BMP2 and BMP4: Insights into evolution of dorso-ventral axis specification, Dev. Dynam. 213:130-139.
Pennisi, E. and W. Roush. 1997. Developing a new
view of evolution. Science 277:34-37.
Piccolo, S., Y. Sasai, B. Lu, and E. M. De Robertis.
1996. Dorsoventral patterning in Xenopus: Inhibition of ventral signals by direct binding of chordin to BMP-4. Cell 86:589-598.
Plachov, V., K. Chowdhury, C. Walther, D. Simon, J.
L. Guenet, and P. Gruss. 1990. Pax8, a murine
paired box gene expressed in the developing ex-
639
cretory system and thyroid gland. Development
110:643-651.
Raff, R. A. 1996. The shape of life: Genes, development, and the evolution of animal form. Univ.
Chicago Press, Chicago.
Reeck, G. R., C. de Haen, D. C. Teller, R. F. Doolittle,
W. M. Fitch, R. E. Dickerson, P. Chambon, A. D.
McLachlan, E. Margoliash, T. H. Jukes, and E.
Zukerkandl. 1987. "Homology" in proteins and
nucleic acids: A terminology muddle and a way
out of it. Cell 50:667.
Remane, A. 1971. Die Grundlagen des natiirliche Systems, der vergleichenden Anatomie und der Phylogenetik 2nd. ed. Koeltz, Konigstein-Tanaus.
Riedl, R. 1978. Order in Living Organisms. Wiley,
Chichester.
Rodriguez-Esteban, C , J. W. R. Schwabe, J De La
Pefia, B. Eshelman, and J. C. Izpisua Belmonte.
1997. Radical fringe positions the apical ectodermal ridge at the dorsoventral boundary of the vertebrate limb. Nature 386:360-366.
Rudnick. A., T. Y. Ling, H. Odagiri, W. J. Rutter, and
M. S. German. 1994. Pancreatic beta cells express
a diverse set of homeobox genes. Proc. Natl.
Acad. Sci. U.S.A. 91:12202-12207.
Shubin, N., Tabin, C. and Carroll, S. 1997. Fossils,
genes and the evolution of animal limbs. Nature
388:693-648.
Stokes, M. D. and N. D. Holland. 1995. Embryos and
larvae of a lancelet, Branchiostoma floridae, from
hatching through metamorphosis: Growth in the
laboratory and external morphology. Acta Zool.
Stockh. 76:105-120.
Stokes, M. D. and N. D. Holland. 1998. The lancelet:
Also known as "amphioxus," this curious creature has returned to the limelight as a player in
the phylogenetic history of the vertebrates. Amer.
Sci. 86:552-560.
Striedter, G. F. and R. G. Northcutt. 1991. Biological
hierarchies and the concept of homology. Brain
Behav. Evol. 38:177-189.
Swanson, K. W., D. M. Irwin. and A. C. Wilson. 1991.
Stomach lysozyme gene of the langur monkey—
tests for convergence and positive selection. J.
Mol. Evol. 33:418-425.
Thomas, I. M. 1956. The accumulation of radioactive
iodine by Amphioxus. J. Mar. Biol. Assoc. U. K.
35:203-210.
Tong, W, P. Kerkof, and I. L. Chiakoff. 1962. Identification of labeled thyroxine and triiodothyronine
in amphioxus treated with 13II. Biochim. Biophys.
Acta 561:326-331.
Tsuneki, K., H. Kobayashi and M. Ouji. 1983. Histochemical distribution of peroxidase in amphioxus
and cyclostomes with special reference to the endostyle. Gen. Comp. Endocrinol. 50:188-200.
van der Kallen, C. J. H., D. C. J. Spierings, J. H. H.
Thijssen, M. A. Blankenstein, and T. W. A. Bruin.
1996. Disrupted co-ordination of Pax-8 and Thyroid transcription factor-/ gene expression in a
dedifferentiated rat thyroid tumor cell line derived
from FRTL-5. J. Endocrinol. 150:377-382.
van Wijhe, J. W. 1919. On the anatomy of the larva
of Amphioxus lanceolatus and the explanation of
640
N. D. HOLLAND AND L. Z. HOLLAND
its asymmetry. Proc. Kon. Nederl. Akad. Wetensch. Amsterdam 21:1013-1023.
Venkatesh, T. V., N. D. Holland, L. Z. Holland, M. T.
Su, and R. Bodmer. 1999. Sequence and developmental expression of AmphiNk2-l: Insights into
the evolutionary origin of the vertebrate thyroid
gland and forebrain. Dev. Genes Evol. 209:254259.
Wada, H. and H. Satoh. 1994. Details of the evolutionary history from invertebrates to vertebrates,
as deduced from the sequence of 18S rDNA. Proc.
Natl. Acad. Sci. U.S.A. 91:1801-1804.
Wilson, P. A. and A. Hemmati-Brivanlou. 1995. Induction of epidermis and inhibition of neural fate
b
y BMP-4. Nature 376:331-333.
Corresponding Editor: Gregory A. Wray