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AMER. ZOOL., 38:647-658 (1998)
Developmental Gene Expression in Amphioxus: New Insights into the
Evolutionary Origin of Vertebrate Brain Regions,
Neural Crest, and
Rostrocaudal Segmentation1
LINDA Z. HOLLAND 2 AND NICHOLAS D.
HOLLAND
Marine Biology Research Division, Scripps Institution of Oceanography,
La Jolla, California 92093-0202
SYNOPSIS. Amphioxus is widely held to be the closest invertebrate relative of the
vertebrates and the best available stand-in for the proximate ancestor of the vertebrates. The spatiotemporal expression patterns of developmental genes can help
suggest body part homologies between vertebrates and amphioxus. This approach
is illustrated using five homeobox genes (AmphiHoxl, AmphiHox2, AmphiOtx,
AmphiDll, and AmphiEri) to provide insights into the evolutionary origins of three
important vertebrate features: the major brain regions, the neural crest, and rostrocaudal segmentation. During amphioxus development, the neural expression
patterns of these genes are consistent with the presence of a forebrain (detailed
neuroanatomy indicates that the forebrain is all diencephalon without any telencephalon) and an extensive hindbrain; the possible presence of a midbrain requires
additional study. Further, during neurulation, the expression pattern of AmphiDll
as well as migratory cell behavior suggest that the epidermal cells bordering the
neural plate may represent a phylogenetic precursor of the vertebrate neural crest.
Finally, when the paraxial mesoderm begins to segment, the earliest expression of
AmphiEn is detected in the posterior part of each nascent and newly formed somite.
This pattern recalls the expression of the segment-polarity gene engrailed during
establishment of the segments of metameric protostomes. Thus, during animal evolution, the role of engrailed in establishing and maintaining metameric body plans
may have arisen in a common segmented ancestor of both the protostomes and
deuterostomes.
INTRODUCTION
nerve cord, segmented axial muscles, and a
The amphioxus (or lancelet) belongs to Pharynx perforated with gill slits). This onsubphylum Cephalochordata of the phylum togenetic pattern (Fig. 1) impressed many of
Chordata and is represented by about 30 liv- Kowalevsky's contemporaries as reflecting
ing species, all superficially much the same, t h e phylogenetic origin of the vertebrates
resembling small, colorless fish without ob- f r o m t h e invertebrates and helped insure a
vious anterior sense organs. Cephalochor- W l d e acceptance of Haeckel's law of recadates inhabit coastal marine sediments, Pitulation—i.e., that ontogeny recapitulates
where they spend most of their time in shal- phylogeny.
low burrows filter feeding on small particles.
Haeckel predicted that the true topograIn the 1860s, Kowalevsky discovered that P h y o f t h e t r e e o f l i f e c o u l d b e ^vealed by
the early embryology of amphioxus is in- describing the embryologic development of
vertebrate-like (a hollow blastula invaginates a 1 1 t h e P h y l a - Thu^ d u n n § t h e l a s t t h i r d o f
to form a gastrula of the sea urchin sort), but t h e nineteenth century, his law of recapitthe later development is vertebrate-like (with u l a t i o n stimulated a research program that
the production of a notochord, dorsal hollow n r m l y u m t e d t h e fields o f development and
evolution. The embryology of phylum after
phylum was described, but, disappointingly,
1
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2
E-mail: [email protected]
in
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disrepute, and development and evolution separated and went their own ways.
647
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L. Z. HOLLAND AND N. D. HOLLAND
8.0 hr
FIG. 1. Diagram of amphioxus embryology (after Conklin, 1932). Animal pole or anterior is toward the left
and dorsal is at the top. Times of development at 24°C for Branchiostoma floridae; cv, cerebral vesicle; np,
neuropore; nt, notochord; ps, first pigment spot; so, somite.
Embryologists, by then concerned with experimentation, lost interest in evolution.
And most evolutionary biologists became
microevolutionists who distrusted development as a spawning ground for heretical
ideas about macroevolution and saltation.
Development and evolution remained divorced for much of the twentieth century,
but were quickly reunited about a decade
ago when molecular genetics revealed a re-
markable, and unexpected, conservation of
the molecular machinery underlying development throughout the animal kingdom.
Developmental genes typically have motifs
coding for conserved amino acid sequences
that can be used to help support homologies
between genes of different animals. Because of this conservation, comparative developmental genetics can enrich phylogenetic discussions in several ways. One can
AMPHIOXUS GENES AND VERTEBRATE EVOLUTION
use sequence homologies to construct developmental gene trees for interpretation in
a phylogenetic context (Schubert et al,
1993) and one can study developmental
gene duplication in evolutionary lineages
(Garcia-Fernandez and Holland, 1994). In
addition, homologous genes tend to be transcribed in similar places and developmental
stages in different kinds of animals. Thus,
conserved expression domains of developmental genes can help suggest body part
homologies between distantly related organisms (Holland, 1996; L. Z. Holland et
al., 1996).
In recent years we, along with our colleagues Peter Holland and Nic Williams,
have been using developmental gene expression in amphioxus to address questions
about the origin of the vertebrates from the
invertebrates. Amphioxus is probably the
closest living invertebrate relative of the
vertebrates (Wada and Satoh, 1994) and, in
spite of half a billion years of independent
evolution, it is a useful stand-in for the
proximate invertebrate ancestor of the vertebrates. Although the amphioxus body
plan is vertebrate-like in many ways, it
lacks such vertebrate features as paired anterior sense organs, a complete endothelium
in the vascular system, and a cartilaginous
and/or bony skeleton. Furthermore, amphioxus has not undergone the numerous gene
multiplications that occurred at the base of
the vertebrates and are correlated with the
relatively abrupt evolution of complex
structures early in the vertebrate lineage
(Holland and Garcia-Fernandez, 1996).
Our approach is first to use conserved
amino acid sequences to identify amphioxus homologs of developmental gene
products and then to demonstrate the embryonic expression patterns of these genes
to help reveal body part homologies between amphioxus and other organisms. The
present review considers the following amphioxus genes: AmphiHox3 (Holland et al.,
1992), AmphiHoxl (Holland and GarciaFernandez, 1996), AmphiDll (N. D. Holland
et al., 1996), AmphiOtx (Williams and Holland, 1996), and AmphiEn (Holland et al.,
1997). All of these genes are classified as
homeobox genes (the homeobox is a 180base pair sequence encoding a stretch of 60
649
amino acids that binds to DNA). The homologies indicated by the expression domains of these genes have provided insights
into the evolutionary origins of three salient
features of vertebrates: the major brain
regions, the neural crest, and rostrocaudal
segmentation.
MATERIALS AND METHODS
Since our work requires a reliable source
of amphioxus embryos and larvae, we
study the Florida amphioxus, Branchiostoma floridae, which is very abundant in
Tampa Bay, Florida USA and spawns from
late spring through late summer (Stokes and
Holland, 1996). Ripe adults are collected by
shovel and sieve in waist-deep water and
electrically stimulated to spawn (Holland
and Holland, 1989). Embryos and larvae
can be raised in the laboratory through
metamorphosis in about a month (Stokes
and Holland, 1995).
Amphioxus developmental genes have
been isolated either from genomic libraries
or from cDNA libraries constructed from
mRNA of specific developmental stages.
Usually, a probe for library screening is obtained by using the polymerase chain reaction to amplify a short portion of genomic
or cDNA with degenerate primers based on
conserved stretches of amino acid sequences of homologs from other species. Once a
gene is isolated and sequenced, we use
Southern blot analysis to determine if the
amphioxus genome contains closely related
genes. This analysis suggests that each of
the amphioxus genes described here is the
sole representative of its group {e.g.,
AmphiDll is the only Distal-less related
gene in the amphioxus genome), which
simplifies the interpretation of the results.
For each gene, antisense riboprobes are
used to visualize its transcripts by in situ
hybridization to whole mounts or sections
of embryos and larvae. Details of experimental techniques are available with special
reference to amphioxus in L. Z. Holland et
al. (1996).
EVOLUTIONARY ORIGIN OF VERTEBRATE
BRAIN REGIONS
Vertebrate brains are divisible into forebrain (comprising telencephalon and dien-
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L. Z. HOLLAND AND N. D. HOLLAND
cephalon), midbrain, and hindbrain. In the
most basal groups of vertebrates, the agnathan fishes, these major subdivisions of
the brain were already present—as seen in
brain casts of extinct ostracoderms and in
the brain structure of extant hagfishes and
lampreys (Forey and Janvier, 1994). Therefore, the question of the origin of the major
brain regions must be addressed by considering the sort of brain present in the proximate invertebrate ancestor of the vertebrates, or in its stand-in, amphioxus.
The central nervous system of amphioxus consists of a dorsal, hollow neural cord
lacking constrictions along its rostrocaudal
axis. Even so, there is a regional difference
at the anterior end, where the neural canal
is slightly dilated into a cerebral vesicle
(Fig. 1, cv) at the level of somite 1 plus the
anterior part of somite 2. At the cellular level, the comprehensive microanatomy of the
cerebral vesicle in late larvae has only recently been described from computer-assisted, 3D reconstructions of serial transmission electron micrographs (Lacalli et al,
1994; Lacalli, 1996). Because of the lack of
gross anatomical landmarks, the structure
of the amphioxus central nervous system
has long been contentious. At one extreme,
Rathke (1841) proclaimed amphioxus completely brainless, and, at the other, Huxley
(1874) proposed that amphioxus had a huge
brain comprising the anterior 20% of the
central nervous system. Between these extremes, a small to medium-sized brain has
often been proposed, with much disagreement over which of the major brain subdivisions are present (Lonnberg, 1924; Arie'ns
Kappers, 1929; Guthrie, 1975; Gans and
Northcutt, 1983; Olsson, 1986; Lacalli,
1996).
We began molecular genetic identification of amphioxus brain regions with a
search for amphioxus homologs of Hox
genes known to be expressed in the developing vertebrate hindbrain (diagrammed in
Fig. 2). The first gene studied was
AmphiHox3, the amphioxus homolog of
mouse Hoxb3. The mouse gene is expressed
in the spinal cord and posterior hindbrain
with a rostral limit at the boundary between
rhombomeres 4 and 5. Correspondingly,
AmphiHox3, is expressed in the dorsal
ps
B
FIG. 2. (A) Anterior part of 30-hr embryo of Branchiostoma floridal (cerebral vesicle region enlarged in
top diagram) showing numbered somites and first pigment spot (ps). (B) Anterior portion of mouse embryo
showing forebrain (0, midbrain (m) and numbered
hindbrain rhombomeres. Codes for gene expression are
AmphiHox3 and Hoxb-3, small dots; AmphiHoxl and
Hoxb-3; triangles; AmphiEn and En-2, large dots; forward hatching, AmphiOtx and Otx; backward hatching,
Dll-2 and AmphiDll.
nerve cord with a rostral expression limit at
the level of the boundary between somites
4 and 5 (Holland et al., 1992). It should be
emphasized that, for comparing the expression domains of most Hox genes, it is only
the anterior boundary and not the whole domain that signifies; this distinction has
sometimes been misunderstood (e.g.,
Blackburn, 1998). Next, the expression of
AmphiHoxl, an amphioxus homolog of
Hoxbl was determined. The mouse gene is
expressed in the hindbrain only in rhombomere 4. In the amphioxus nerve cord,
AmphiHoxl is expressed in a stripe at the
level of somites 4 and 5 (Fig. 3A) (Holland
and Garcia-Fernandez, 1996). Taken together, the expression patterns of these two
Hox homologs suggest that amphioxus has
at least a relatively extensive hindbrain (cf.
Fig 2A with Fig. 2B) that joins the spinal
cord at a yet undetermined level posterior
to the first pigment spot (Figs. 1, 2, ps).
AMPHIOXUS GENES AND VERTEBRATE EVOLUTION
651
The question of whether amphioxus has markers of the midbrain-hindbrain boundhomologs of the vertebrate forebrain and/or ary or with genes specific for the vertebrate
midbrain was addressed with the amphi- midbrain, such as amphioxus homologues
oxus homologs of Otx (known to mark ver- of FGF-8 (Crossley et al, 1996) or her-5
tebrate telencephalon, diencephalon and (Miiller et al., 1996a), respectively.
midbrain) and Dlx (known to mark vertePOSSIBLE EVOLUTIONARY PRECURSOR OF
brate telencephalon and diencephalon).
VERTEBRATE NEURAL CREST
During amphioxus development, AmphiOtx
Neural crest is responsible for much of
is expressed in two regions of the cerebral
vesicle (Fig. 2)—at its extreme anterior end the structural complexity setting vertebrates
and, a little more posteriorly, in its ventral apart from invertebrates (Gans and Northand lateral walls (Williams and Holland, cutt, 1983; Northcutt and Gans, 1983). Dur1996). Similarly, AmphiDH is expressed at ing vertebrate development, neural crest dethe extreme anterior end of the cerebral ves- rives from cells located near the junction of
icle and, more posteriorly, in its dorsal wall the neural plate and adjacent epidermis.
(Figs. 2, 3B) (N. D. Holland et al, 1996). About the time of neural cord formation,
The expression patterns of these amphioxus neural crest cells detach from their neighgenes suggest that the cerebral vesicle is bors and migrate from the dorsal portion of
largely homologous to the vertebrate fore- the neural cord to destinations throughout
brain, but cannot rule out a midbrain ho- the body. These cells ultimately differentimolog. Microanatomical studies of the am- ate and contribute to cranial ganglia, pephioxus cerebral vesicle have strongly sup- ripheral nervous system, branchial arch
ported homologies inferred from gene ex- mesenchyme, melanocytes, adrenal chropression. These studies revealed numerous maffin cells, and bone and cartilage of the
structural homologies with the diencephalic skull and face (Hall and Horstadius, 1988).
part of the vertebrate forebrain but found It is generally believed that the neural crest
no evidence for a homolog of the telen- arose in the vertebrate line and has no preccephalon (Lacalli et al, 1994; Lacalli, edents in any invertebrate. Nevertheless,
1996). These findings, together with the some of our recent work suggests that amgene expression data are strong evidence phioxus—and, by extension, the proximate
that the anterior % of the amphioxus cere- invertebrate ancestor of the vertebrates—inbral vesicle is homologous to the vertebrate cludes a cell population with intriguing
diencephalon.
similarities to vertebrate neural crest (N. D.
The fine structure of the central nervous Holland et al., 1996).
Before discussing the possible evolutionsystem of larval amphioxus also indicted
that the posterodorsal extremity of the ce- ary precursor of vertebrate neural crest, it
rebral vesicle might be homologous to the is important to describe neurulation, which
vertebrate tectum—a midbrain structure. To occurs in deuterostomes above the level of
test this possibility, we studied the neural echinoderms and is the overall process
expression of an engrailed gene in amphi- leading to the formation of the dorsal nerve
oxus {AmphiEn). In vertebrates, engrailed cord. In tunicates and most vertebrates,
genes are expressed in a stripe at the mid- neurulation is essentially an invagination of
brain-hindbrain boundary (Fig. 2). Discon- the neural plate followed by a mid-dorsal
certingly, AmphiEn is expressed in the ce- fusion of the neural folds (Nicol and Meirebral vesicle in a region considerably an- nertzhagen, 1988; Moury and Schoenwolf,
terior to the proposed tectal region (Figs. 2, 1995). Neurulation in amphioxus, instead
3C). This pattern could mean that AmphiEn of being a relatively continuous invaginais not a reliable marker for the midbrain- tion, occurs in two distinct phases. The first
hindbrain boundary in amphioxus or that is a rapid epidermal overgrowth of the neuthe proposed tectal homology is incorrect ral plate, which remains relatively flat (Fig.
and amphioxus lacks a midbrain. One way 4A-C), and the second is a slow rolling up
to resolve this question is further mapping of the neural plate into the neural cord (Fig.
of the amphioxus brain with additional 4D-E). It is not known whether such bi-
FIG.3. Whole mount in siru hybridization of embryos of BrunrhiostomaJloridae, anterior at left except in D.
Dorsal at top in A-C. All scale lines 5 0 pm, except in D (20 pm). ( A ) 9 hr neuruia with AmphiHox-l expressed
in a stripe in the nerve cord and in posterior presomitic mesoderm (arrow): neuropore is labeled np. ( B ) 2.5 day
larva with AmphiDIl expressed strongly in the cerebral vesicle (arrows) and more weakly in the epidermis (out
of the plane of focus except at the anterior end and dorsal surface of the larva). ( C ) 28 hr embryo with AmphiEn
is expressed in a ventral stripe in the cerebral vesicle; neuropore is labeled np. (D) Cross section through 9 hr
embryo at the same stage as Fig. 4C showing AmphiDll expressed strongly in the cells at the leading edges of
the sheets of epidermis (arrows) that are migrating over the neural plate (n): arrowheads indicate presomitic
grooves. (E) Dorsal view of 12 hr embryo showing somites arising from presomitic groove on either side of the
midline: AmphiEt~is expressed in the posterior portion (arrows) of each forming and nascent somite. ( F ) Dorsal
view of 19 hr embryo showing AmphiEn expression in the posterior portion of each of the first six comites on
either side of the midline.
AMPHIOXUS GENES AND VERTEBRATE EVOLUTION
653
FIG. 4. Cross sections of amphioxus embryos showing neurulation. (A) 8-hr gastrula with endoderm (en),
epidermis (ep), and neural plate (np). (B) Early neurula (9 hr) with the epidermis beginning to overgrow the
neural plate and the somites (so) and notochord (nt) starting to form. (C) Early neurula (9.3 hr) with epidermal
overgrowth of the neural plate in progress. (D) Mid-neurula (12 hr) with completed epidermal overgrowth of
the neural plate (E) Late neurula (16 hr) after the neural plate has curled up into a dorsal, hollow neural cord
(nc).
phasic neurulation is unique to amphioxus
or also occurs in hemichordates, as suggested by Morgan (1891). For now, the possibility remains open that amphioxus neurulation represents the primitive type of
neurulation in deuterostomes and that neurulation by invagination is derived.
Just before the first phase of amphioxus
neurulation (Fig. 4A), expression of
AmphiDll is upregulated in the epidermis
immediately bordering the neural plate. Expression in these cells (Figs. 3D, 4B, C)
remains strong during epidermal migration
over the neural plate, where expression is
undetectable (Fig. 3D). During migration,
many of the cells at the leading edge of the
epidermis have lamellipodia splayed out on
the surface of the neural plate (Fig. 5A, B),
indicating that epidermal migration is impelled at least in part by lamellipodial traction. The first phase of neurulation ends as
the epidermal cells from either side meet
mid-dorsally and re-establish the integrity
of the epidermis (Fig. 4D); soon thereafter,
epidermal expression of AmphiDll is downregulated.
During the first phase of amphioxus neu-
rulation, the epidermal cells bordering the
neural plate exhibit three features suggestive of vertebrate neural crest. First, these
amphioxus cells are at the neural plate border, which is the general region in vertebrates where neural crest cells are first specified (Selleck and Bronner-Fraser, 1995).
Second, Distal-less homologs are expressed
in premigratory and migratory epidermal
cells in amphioxus and also in migratory
and differentiating neural crest cells of vertebrates (Dirksen et ai, 1994; Akimenko et
ai, 1994; Robinson and Mahon, 1994; Qiu
et al., 1995)—however, the amphioxus cells
begin transcribing AmphiDll before cell migration begins, whereas, in vertebrates, cells
derived from neural crest do not express
Distal-less homologs until after migration
has commenced. Third, the apparent lamellipodial traction by the epidermal cells leading the overgrowth of the neural plate of
amphioxus has some similarity with migrating neural crest of vertebrates. However, unlike vertebrate neural crest cells,
which usually migrate individually in the
interior of the embryo, the amphioxus cells
migrate at the edge of a cell sheet and fol-
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L. Z. HOLLAND AND N. D. HOLLAND
FIG. 5. Scanning electron micrographs of a 9.5 hr early neurula. (A) Dorsal surface with anterior at bottom
left. The epidermis is beginning to overgrow the neural plate (np). Scale, 20 u.m. (B) Enlargement of cells at
edge of advancing epidermal sheet in A. Lamellipodia (arrowhead) extend on surface of the neural plate (np).
Scale, 5 (jim.
low a path over the exterior of the embryo.
Moreover, the migrating cells of amphioxus
do not differentiate into the wide variety of
cell types known to originate from the vertebrate neural crest, but evidently remain
part of the epidermis. During subsequent
amphioxus development, no epidermal neuronal cells differentiate in the dorsal midline, although several kinds of specialized
sensory cells have been described from other epidermal regions (Stokes and Holland,
1995).
Because the amphioxus epidermal cells
leading the overgrowth of the neural plate
have some of the attributes of vertebrate
neural crest, it is tempting to speculate that
the proximate vertebrate ancestor had similar cells that constituted the starting point
for evolution of definitive neural crest. Such
an ancestor may have formed the dorsal
nerve cord by amphioxus-like neurulation
instead of invagination. Later, with the advent of vertebrate neurulation by invagination, motile epidermal cells no longer
played a role in roofing over the neural
plate, but remained contiguous with it while
invagination carried them into the dorsal
part of the nerve cord. From there, at least
some of these cells could have emerged
from the nerve cord to migrate through the
remnant of the blastocoel as definitive neural crest cells. If this scenario is valid, the
advent of neurulation in higher deuterostomes can be regarded not only as a key
innovation in itself, but also as setting the
stage and providing raw material for the
subsequent evolution of vertebrate neural
crest. Consistent with this view, Wada et al.
(1996) have suggested, on the basis of gene
expression patterns, that neural crest-like
tissue may be present in neurulating ascidian larvae.
INSIGHTS INTO EVOLUTION OF VERTEBRATE
SEGMENTATION
There are two main ways of explaining
the evolutionary source of vertebrate metamerism: first, as an inheritance from some
metameric protostome, often an arthropod
or annelid (e.g., Leydig, 1864; Dohrn,
1875; Patten, 1912), and second, as an inheritance from some invertebrate deuterostome (e.g., Bateson, 1886; Garstang, 1928;
Jefferies, 1986). Ideas in the latter class,
which connote independent origins for vertebrate and protostome segmentation, have
been widely accepted through most of the
twentieth century.
During development of metameric protostomes, engrailed homologs are segmentation genes involved in the specification of
the nascent metameres. In contrast, during
vertebrate development, engrailed homologs and other segmentation genes may be
AMPHIOXUS GENES AND VERTEBRATE EVOLUTION
expressed in segments (for instance in muscle pioneer cells), but only after these structures are already morphologically well defined (Patel, 1994; De Robertis and Sasai,
1996). An exception is her-1, a vertebrate
homolog of the insect pair-rule gene hairy:
in zebrafish, her-1 is expressed in the forming somites in a pattern strikingly like that
of its homolog in the forming segments of
short germ insects (Miiller et al., 1996ft).
This tantalizing similarity led Kimmel
(1996) to revive the old suggestion that
deuterostomes and protostomes have
evolved from a common segmented ancestor, thus raising the question of why other
segmentation genes do not seem involved
in establishing the metameric body plan of
vertebrates.
To address this question we studied the
early expression of AmphiEn, the amphioxus homolog of engrailed (the later expression domain of AmphiEn in the cerebral
vesicle has already been discussed). In the
early neurula of amphioxus, the first somites form by a rostrocaudal pinching off
from presomitic grooves in the paraxial mesoderm (Figs. 3D-F; 4D, E). Each pinchedoff somite is a monolayered sphere of cells
surrounding a hollow lumen, which is an
enterocoel. At the stage when two somites
are present on either side of the midline,
AmphiEn is expressed in the posterior wall
of the first (most anterior) somite, in the
posterior cells of the second somite as it is
forming, and in the wall of the presomitic
groove where the posterior part of the third
somite will form (Fig. 3E). Subsequently,
shortly before each succeeding somite
pinches off, expression begins in its future
posterior region.
Up until the stage of eight somites per
side, the posterior wall of each somite expresses AmphiEn (Fig. 3F), but expression
in all the somites is then down-regulated.
Subsequently, additional somites continue
to form—not by enterocoely from the gut,
but by budding off from the posterior mesoderm—and they do not express detectable
AmphiEn. This pattern brings to mind the
speculation of Gilland and Baker (1993)
that the late gastrula of amphioxus is homologous with the head of vertebrates, and
posterior growth by the neurula subsequent-
655
ly generates regions homologous with the
vertebrate trunk and tail.
During amphioxus development, the early expression of AmphiEn resembles the expression of engrailed homologs in metameric protostomes—expression is in nascent segments, suggesting that AmphiEn
plays a role in establishing and maintaining
posterior segment boundaries. Amphioxus
resembles some metameric protostomes in
that engrailed homologs act in the mesoderm of forming segments {e.g., Whittington et al., 1991). Thus, we suggest that the
role of engrailed in establishing and maintaining a metameric body plan arose only
once in evolution and originally functioned
in the mesoderm of a common segmented
ancestor of the metameric protostomes and
deuterostomes. Certainly, there is a pressing
need for studying more of the segmentation
genes of amphioxus.
If deuterostomes were metameric from
the start of their evolution, there must have
been a tendency for segmentation to be lost
in some advanced groups. For echinoderms,
a segmented ancestor may have given rise
to the extinct segmented homalozoans and
also, by reduction of segmentation, to living
species, which retain only embryonic trimery. Similarly, modern hemichordates
could have reduced the original segmentation to trimery and, in some species, iterated gill slits. For tunicates, the possible
segmentation of the tail musculature in larval ascidians and appendicularians is controversial (Bone, 1989; Nielsen, 1995);
however, Wada et al. (1996) recently found
iterated ectodermal and neural expression
of ascidian Pax3l7, indicating tunicate descent from more elaborately segmented ancestors.
Compared to the reduction of segmentation in some invertebrate deuterostomes,
the evident loss of engrailed participation
in forming the segmented body plan of vertebrates is a more complicated problem.
Among vertebrates, the single known instance where an engrailed gene is expressed in the posterior region of a forming
somite is in the head cavity of the mandibular arch of the embryonic lamprey (Holland et al., 1993). This head cavity, which
arises by enterocoely, is a hollow vesicle
656
L. Z. HOLLAND AND N. D. HOLLAND
with a monolayered wall of mesoderm
cells—the parallels with an amphioxus somite are striking. Yet the pattern of engrailed expression in the lamprey embryo
is not iterated in the more posterior forming
somites. Perhaps one explanation is that
most of the somites of lampreys and of
higher vertebrates form only after an epithelium-to-mesenchyme transition (Keynes
and Stern, 1988). It may be that the genetic
controls needed for such a transition are in
some way incompatible with the establishment of metamery by segmentation genes.
If so, engrailed expression in the mesoderm
of the mandibular somite of lampreys might
be the last vestige of the involvement of
engrailed in vertebrate somite formation.
CONCLUSION
The present review illustrates how the
spatiotemporal expression patterns of developmental genes can help establish homologies between body parts of animals
that are relatively distant relatives. Homology is a hierarchical concept (Bolker and
Raff, 1996), and it is important to stress that
we are comparing body parts and not deeper homologies (like transcriptional regulation). Whenever possible, our homology
decisions are based not only on expression
patterns of developmental genes but on other information as well—the more kinds of
data pointing to the same answer, the better
(Dickinson, 1995; Bolker and Raff, 1996;
Galis, 1996). For example, the excellent
agreement between the neural expression
domains of AmphiDll, AmphiOtx, and the
microanatomy of the amphioxus cerebral
vesicle is gratifying. A discrepancy in two
kinds of data, for example, between
Amp/i/£vi-expression in the cerebral vesicle
of amphioxus and the microanatomy, points
up the need for additional data before a
conclusion can be reached. Importantly,
where the detailed anatomy has yet to be
described (e.g., for the presumed hindbrain
region), the gene expression patterns highlight the exact regions of the amphioxus
central nervous system that should be described next with serial electron microscopy
and computer-assisted, 3D reconstructions.
When the overall body plans of the animals compared are relatively similar, as
they are in amphioxus and the vertebrates,
expression data for one or two genes can
suffice to give important new insights (for
instance, as illustrated here, into the possible evolutionary precursor of vertebrate
neural crest). In contrast, when expression
domains of only a few developmental genes
are used to compare body parts between animals with markedly different body plans,
the suggested homologies may not be entirely convincing. Such homologies may
become more widely acceptable when they
can be supported by at least partial cascades
of interacting developmental genes (Holland, 1996).
Gee (1996) concludes his recent book on
the origin of the vertebrates by suggesting
that the true topography of the tree of life
may finally be resolved by matching the order of genes (establishing the details of synteny) in genomes. This approach may well
give an accurate branching diagram for the
tree of life, but it will not provide a detailed
scenario of how one body plan might have
evolved into another. However, if a reliable
cladogram of the animal phyla were to become available, the additional analysis of
body part homologies (based partly on
comparing expression domains of developmental genes) should go a long way toward
providing a connected historical narrative
of life on earth.
ACKNOWLEDGMENTS
It is a pleasure to acknowledge the cordial hospitality of John Lawrence and Ray
Wilson who have generously shared their
facilities with us at the University of South
Florida. This is also an opportune place to
acknowledge the following colleagues who
have waded with us in Tampa Bay collecting amphioxus: Claus Andersen, Torea
Bent-van Every, Meriko Blink, Jakob BroJorgensen, Anna Byskov, Anne-Marie Coriat, Archie Ellwood, Garietta Falls, David
Ferrier, Christian Garcia-Canestro, Jordi
Garcia-Fernandez, Tom Gilmour, Anders
Hay-Schmidt, Erika Henyey, Michael Hobbins, Amanda Horsfall, Peter Holland, Bill
Jackman, Mamata Kene, Hiromichi Koyama, Thurston Lacalli, Gary LaFleur, Jim
Langeland, John Lawrence, Mike Lares,
Peter Luykx, Stuart Miller, Georgia Pano-
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657
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Corresponding Editor: Gregory A. Wray