Beruflich Dokumente
Kultur Dokumente
SUMMARY
All vertebrates possess anatomical features not seen in their Comparisons of Hox gene clusters, other homeobox gene
closest living relatives, the protochordates (tunicates and families, Wnt genes and insulin-related genes all indicate
amphioxus). Some of these features depend on develop- that there was a major phase of gene duplication close to
mental processes or cellular behaviours that are again vertebrate origins, after divergence from the amphioxus
unique to vertebrates. We are interested in the genetic lineage; we suggest there was probably a second phase of
changes that may have permitted the origin of these inno- duplication close to jawed vertebrate origins. From
vations. Gene duplication, followed by functional diver- amphioxus and vertebrate homeobox gene expression
gence of new genes, may be one class of mutation that patterns, we suggest that there are multiple routes by
permits major evolutionary change. Here we examine the which new genes arising from gene duplication acquire new
hypothesis that gene duplication events occurred close to functions and permit the evolution of developmental inno-
the origin and early radiation of the vertebrates. Genome vations.
size comparisons are compatible with the occurrence of
duplications close to vertebrate origins; more precise
insight comes from cloning and phylogenetic analysis of Key words: gene duplication, evolution, amphioxus, tunicate,
gene families from amphioxus, tunicates and vertebrates. homeobox, Wnt genes
amphioxus is compared instead to the living members of the causes a range of cranial defects (Satokata and Maas, 1994).
earliest vertebrate lineages, hagfish and lampreys, significantly Many of the expression sites of the Msx-l and Msx-2 genes,
larger genomes are indeed seen in vertebrates (haploid values although not all, are vertebrate-specific features (Holland,
I.4-2.8 pg). Furtherrnore, it has been shown that the brook 1992); hence it is intriguing to ask whether amphioxus has
lamprey genome (at l.a pg rs not complicated by very recent homologues of these genes.
tetraploidy (Ward et al., 1981); hence, it may be valid to use it To date, we have succeeded in isolating only a single
as an approximate guide to early vertebrate genome size. Of member of the Msx homeobox gene family from the genome
course, modern lampreys could have secondarily expanded or of Branchiostoma floridae, both by PCR (Holland et al., 1994)
compacted genomes, in which case it would not be valid to infer and by genomic library screening (A. Sharman and P. W. H.
early vertebrate genome size from them. H., unpublished data). This parallels the results from an
The assumption would be testable if genome sizes could be ascidian, but contrasts with the multiple Msx genes present in
measured from representatives of other (now extinct) jawless a teleost fish, Brachydanio rerio (Holland, I991b). These
vertebrate lineages. Perhaps surprisingly, this may be feasible results are consistent with the hypothesis that gene duplications
since the outlines of cells are preserved in some fossils. Cell in this gene family occurred in the vertebrate lineage after
outlines give an estimate of cell volume, which in turn is an divergence of amphioxus; however, a wider survey of verte-
approximate indicator of genome size within vertebrates (if the brates must be completed before the timing of duplication can
same cell type is compared between species). The feasibility be ascertained.
of this approach was demonstrated by Conway Morris and Comparative data are more sparse for three other homeobox
Harper (1988), who estimated genome size in extinct gene families analyzed in amphioxus: the En, Cdx and the
conodonts (thought to be an ancient lineage of jawless ver- XlHbox8-related genes. In the latter two cases, PCR has iden-
tebrate; Sansom et a1., 1992). These analyses need extending tified a single homologue to date in B. floridae (Holland et aL.,
to other lineages; at present, however, the data from both living 1994); the Cdx genes, at least, form a multigene family in
and fossil jawless vertebrates support the contention that sig- mammals (Gamer and Wright, 1994). The size of the XlHbox8
nificant genome enlargement occurred at vertebrate origins gene family is unknown in any species; within vertebrates, rep-
(after divergence of the amphioxus lineage). resentatives have been cloned from Xenopus (Wright et al,,
1989), mouse (Ohlsson et a1., 1993) and rat (Miller et al. , 1994).
For both the Cdx and XlHboxS gene families, additional species
EVOLUTIONARY INSIGHTS FROM AMPHIOXUS need to be analyzed (including jawless vertebrates) before the
HOMEOBOX GENES timing and extent of gene duplications can be ascertained.
For the largest homeobox gene family, the Hox genes, more
More accurate insight into the evolution of vertebrate genome extensive cloning and phylogenetic surveys have been under-
organization will undoubtedly come from cloning and phylo- taken. Mammals (and probably all higher vertebrates) have
genetic analysis of gene families in representatives of several four similar clusters of Hox genes, homologous to the single
protochordate and vertebrate lineages. Phylogenetic consider- Hox or HOM-C cluster of arthropods and nematodes (reviewed
ations make amphioxus a particularly important protochordate by Holland, 1992; Burglin and Ruvkun, 1993). Elucidating the
for gene family analysis, since its lineage diverged after the number of Hox clusters in amphioxus and lower vertebrates is
urochordates but before the diversification of vertebrates (Fig. crucial to determining the time of Hox cluster duplication. In
1). Of particular interest in these analyses will be multigene addition to cluster duplication, there is the question of tandem
families implicated in the control and coordination of devel- duplications within a cluster. The 38 mammalian Hox genes
opmental processes (for example homeobox and Wnt genes), are divisible between 13 paralogous groups (containing genes
since their molecular evolution may give insight into the origin related by the cluster duplication events); many of these groups
of vertebrate developmental control. Relatively few protein- are not present in arthropods and nematodes (Holland, 1992;
coding genes have been cloned from amphioxus, but these Burglin and Ruvkun, 1993). Phylogenetic reconstructions
include genes related to vertebrate transcription factors, growth suggest that the pre-duplication Hox cluster organization was
factors, signalling molecules, structural proteins and enzymes. not identical to any of the clusters of mouse or human (Kappen
In this section and the next, we look at every example and Ruddle, 1993); hence, tandem duplications anilor gene
published to date. losses must have occuffed after cluster duplication. Amphioxus
One group of homeobox genes for which comparative Hox genes could give clues to the timing of these events.
surveys have been undertaken in the chordates is the Msx gene The first amphioxus Hox gene published was AmphiHox3
family. Three members of this gene family were cloned from (Holland et a1., 1992) from Branchiostoma floridae; complete
the mouse genome by PCR (Holland, 1991b); two of these are gene sequence showed this gene is homologous to parulagous
known to be expressed in cranial neural crest-derived mes- group 3 of mammalian genes. This assignment suggests that
enchymal tissue and in complementary patterns at many sites the tandem duplication event that yielded paralogous groups 2
of tissue interaction during development (including during and 3 (both related to the Drosophila pb gene) predated the
branchial arch development, palate development, tooth mor- divergence of amphioxus and vertebrates; it cannot be dated
phogenesis, and development of the paired eyes; Davidson and more accurately at present. The number of Hox clusters in the
Hill, l99I). Aspects of the gene expression patterns are amphioxus genome has been estimated in two studies using
certainly functional; for example, a point mutation in the PCR (Pendleton et al. , 1993; Holland et al., 1994). Both studies
homeobox of the human MSX2 gene is thought be one cause utilized the same species (8. floridae) and identified multiple
of a skull morphology abnorm ality , craniosynostosis (Jabs et Hox genes. From analysis of the deduced translation products
&1., 1993), whilst deletion of mouse Msxl by gene targeting of short Hox clones, Pendleton et al. ( 1993) conclude that "the
128 P. W. H. Holland and others
amphioxus data are in good agreement with a two cluster superfamily predated the divergence of amphioxus and verte-
model"; however, from similar data Holland et al. (1994) brates (Riemer et a1., 1992).
conclude that there is "probably a single Hox cluster". The dif- It would be interesting to know the number of amphioxus
ficulty in determining the number of clusters stems partly from genes in each IF gene subfamily, particularly since mammalian
the fact that PCR primers capable of amplifying a broad gene mapping studies reveal that some of the 'within group'
spectrum of Hox genes can only yield up to 82 nucleotides of duplications almost certainly coincided with Hox cluster dupli-
unique sequence from each gene. This is often insufficient to cations. For example, within both the type I (acidic cytoker-
assign a gene accurately to a paralogous group (Garcia- atin) and the type II (basic cytokeratin) gene families, there are
Ferndndez and Holland, unpublished data). To overcome this related genes very closely linked to the HOXB and HOXC
problem, and resolve the discrep ancy, we have isolated gene clusters on human chromosomes 12 and 17 (Bentley et
genomic clones of ten amphioxus Hox genes and mapped their al., 1993; Lundin, 1993).
genomic organisation. We find there is a single cluster of Hox There are several other cases where members of a gene family
genes in the amphioxus genome (Garcia-Ferndndez and are chromosomally linked to more than one marnmalian Hox
Holland, unpublished data). cluster; in each case, their origin by chromosomal or genome
It is interesting to compare our one cluster model for duplication may have coincided with Hox cluster duplication.
amphioxus Hox genes (Holland et al., 1994) with PCR results Possible examples of 'co-duplicated' gene families include (in
obtained for a lamprey (Pendleton et al. ,1993). Despite the dif- addition to the cytokeratins): collagen genes, retinoic acid
ficulty in assigning PCR clones to paralogous groups, the 19 receptor genes, Evx homeobox genes, erythrocyte band 3 related
Hox genes identified in Petromyzon marinus are consistent genes, glucose transporter genes, actin genes, GLVciD zinc
with lampreys having at least two, and perhaps three or four, finger genes, myosin light chain genes, some Wnt genes (but see
Hox clusters. This suggests the initial Hox cluster duplica- below) and the neuropeptide Y/pancreatic polypeptide genes
tion(s) occurred in the lineage leading to the first vertebrates, (gene mapping data from Bentley et al., 1993; Lundin, 1993).
after the divergence of amphioxus. Extrapolating from data on the timing of Hox cluster duplica-
tions (Pendleton et aI., 1993; Holland et al., 1994; Garcia-
EVOLUTIONARY INSIGHTS FROM OTHER Ferndndez and Holland, unpublished data), we suggest that
AMPHIOXUS GENES expansion of many of these gene families occuffed close to ver-
tebrate origins. We do not, however, discount the possibility that
The first clues to gene family complexity in amphioxus, predating additional duplication events occurred in these gene families
the homeobox results discussed above, came from Chan et al. during the subsequent evolutionary radiation of the vertebrates.
(1990). These authors reported that Branchiostoma califurnien- The Wnt gene family is an interesting case from the per-
sis has a single insulin-like gene (ILP), homologous to three gene spective of gene duplications. These genes encode an extensive
family members in mammalian genomes (insulin, IGF-L, IGF- family of secreted proteins implicated in cell-cell signalling
2); the deduced mature protein sequence shares equal identity during vertebrate and invertebrate embryogenesis (Nusse and
with each of the three human proteins. The simplest explanation Varmus, 1992). Sidow (1992) investigated the diversification
is that amphioxus retains a single member of this gene family, and molecular evolution of the Wnt gene family, by phyloge-
and that insulin gene duplications occurred on the vertebrate netic analysis of 72 partial Wnt gene sequences isolated from
lineage, after divergence of amphioxus and vertebrates. One of a diversity of vertebrates, echinoderms and Drosophila. The
the duplication events occuffed very early on the vertebrate (or results suggested that Wnt-L, -3, -5, and -7, and one or more
pre-vertebrate) lineage, since both hagfish and lampreys possess ancestors of Wnt-2, -4, -6, and -10 were probably present in
an insulin gene and at least one IGF gene (Nagamatsu et al., the genome of the last common ancestor of arthropods and ver-
I99I). Remarkably, evidence for this very ancient duplication tebrates. Later duplications of Wnt-3, -5, -7, -8 and -10 (giving
may still be present in the human genome: IGF-L maps to chro- rise to, for example, Wnt-3a and -3b) occuffed before the diver-
mosome 12, within a region of paralogy to chromosome 11 that sification of jawed vertebrates, perhaps after divergence of the
contains the insulin and IGF-2 genes (Brissenden et al., 1984; hagfish lineage.
Lundin, 1993). It should be possible to test if this paralogy is We used PCR to search for Wnt genes in amphioxus
genuinely the result of a very early duplication event (of a chro- genomic DNA, since no protochordate genes were included in
mosome, chromosomal region or entire genome) by examination the original analysis. After cloning of the amplified band,
of the genes linked to the ILP gene in amphioxus. sequence analysis of 12 recombinants revealed just two
The Mn superoxide dismutase (Mn SOD) gene and an inter- amphioxus Wnt genes (Fig. 2). The phylogenetic position of
mediate filament Qn gene have also been cloned from amphioxus and the history of gene duplications in the Wnt
amphioxus (Smith and Doolittle, 1992; Riemer et a1., 1992). family (Sidow, 1992) imply that amphioxus should have addi-
The former appears to be a single copy gene in all animals tional Wnt genes, unless they were lost during its evolution. It
studied, implying that gene duplications during vertebrate will be particularly interesting to determine if amphioxus has
ancestry did not affect every gene (or subsequent gene loss has homologues of those genes that are duplicated in jawed verte-
returned some gene families to singletons). The intermediate brates (Wnt-3, -5, -7, -& and -10).
filament genes could be an informative source of data on dupli-
cations, since in mammals they form five subfamilies (types I
to V), each containing multiple genes. Exhaustive surveys have EVOLUTIONARY INSIGHTS FROM TUNICATE
not been caffied out in amphioxus; the one gene reported to GENES
date is clearly a type III gene (vimentin/desmin family), con-
sistent with the idea that initial subdivision of the IF gene In the examples discussed above, the assumption is made that
Gene duplications and vertebrate origins 129
Amphioxus Wnt-B
within the chordates, since wider comparisons to arthropods, Table L. Gene number within gene families in amphioxus
echinoderms and nematodes indicate that a cluster of at least and selected vertebrates
five Hox genes predated the origin of the chordates (Burglin Msx Hox Cdx Insulin/
and Ruvkun, 1993). Hence, despite the phylogenetic position genes clusters genes Mn SOD IGF
of tunicates, it may be difficult to address satisfactorily the Amphioxus 1 111 1
question of exactly when genome duplications occutred during Agnatha nlt >2 nltl2
the very early phases of chordate radiation. Mammal 3 4 >4 13
Results from hagfish and lampreys are amalgamated under agnatha,
although we do not suggest this is a monophyletic group. Not shown are the
TIMING OF GENE DUPLICATIONS En, XlHbox8, IF and Wnt gene families, for which insufficient data are
available. nlt, not tested. See text for further details and references.
Table 1 summarizes the data on timing of gene duplications
directly inferred from cloning of amphioxus genes. These data,
and the above discussions, suggest that different gene families
Gnathostomes or
followed different patterns of diversification during the evolu- jawed vertebrates
tionary radiation of chordates. Some gene families apparently (mammals, birds,
showed stability without duplication (Mn SOD gene); whilst reptiles, amphibia,
some gene duplications occurred after vertebrate origins (eg: fish)
FROM DUPLICATION TO INNOVATION this pattern of evolution (although not relating to the early ver-
tebrates) is the lys ozyme gene family in mammals. Lysozyme
The hypothesis that mutations in regulatory genes could gene duplication in the ruminant mammal lineage was
underlie evolutionary change in embryonic development is followed by changes in protein sequence and expression,
now widely accepted (Ohno, I9l0; Raff and Kaufman, 1983; allowing lysozyme to be co-opted to a digestive role in the
Arthur, 1988; Holland, 1992). But different types of mutation foregut of cows, sheep and relatives (Irwin et &1., 1992). In
could play different roles in developmental evolution. For contrast, arecent experimental analysis of the prd gene family
example, it seems unlikely that minor alterations to the coding in Drosophila, demonstrates that adaptive divergence of
sequence or expression pattern of a regulatory gene could protein-coding sequences does not always accompany func-
allow the origin of completely new morphological features or tional divergence after duplication (Li and Noll, 1994). The
developmental processes, at least in the majority of cases. related genes prd, gsb and gsbn apparently encode function-
Duplication of regulatory genes (followed by divergence of ally equivalent proteins; their divergent roles may have
one or both daughter genes), seems more likely to precede the evolved solely by changes in deployment. We suggest this
origin of such developmental innovations. Gene duplication latter route to functional diversification may have occulred fre-
would not necessarily cause major developmental alteration; quently.
rather, it is considered permissive to subsequent phenotypic At present, there are limited clues to the mechanisms that
change. This hypothesis, therefore, does not propose the allowed functional divergence within regulatory gene families
creation of radically altered 'hopeful monsters' in a single gen- during vertebrate evolution. From the inferred timing of gene
eration, but envisages new genes being made available to the duplication, and the expression patterns of the mammalian
gradual modifying effects of natural selection and genetic drift. gene family members, a hypothesis can be proposed regarding
The hypothesis predicts a coffelation between the origin of functional evolution of the vertebrate Msx homeobox genes
new regulatory genes and the emergence of new cell behav- (Holland, I99Ib, 1992). As described above, two of the three
iours, body regions or structures (Holland, 1992); furthermore' mammalian Msx genes resultant from duplication are predom-
significant changes to body organisation may correlate with inantly expressed in vertebrate-specific tissues, including cran-
significant genome changes simultaneously affecting several iofacial neural crest derivatives, developing teeth and eyes. We
gene families. The scenario presented in the previous section suggest that these expression characteristics reflect co-option
for the timing of gene duplications during vertebrate evolution to new functions at vertebrate origins; the origin of Msx-l and
suggested there may have been two phases of gene duplica- Msx-2 might even have permitted the evolution of new patterns
tion: one close to vertebrate origins and a second close to jawed of cell behaviour and differentiation and new developmental
vertebrate origins. Both stages of chordate evolution involved processes. This hypothesis implies that Msx- I and Msx-2
significant developmental changes; in addition, both phases of acquired new control elements after gene duplication, leaving
genome expansion involved duplication of developmental reg- Msx-3 to persist with an ancestral function. Further insight will
ulatory (and other) genes. come from analysis of the expression pattern of the third
The origin of vertebrates involved the evolution of several mammalian paralogue, Msx-3, and comparison to the single
innovations in developmental strategy, notably the involve- amphioxus Msx gene. These analyses ate in progress (S.
ment of neural crest and placodes in craniofacial morphogen- Shimeld and P. Sharpe, personal communication; A. Sharman
esis, elaboration of the brain (origin of the midbrain?), and spe- and P. W. H. H., unpublished data).
cialisation of the segmented mesoderm (Holland, 1992; Gans, The evolution of Hox gene function following cluster dupli-
1993; Holland and Graham, 1994). In terms of anatomy, the cation is discussed in detail by Gaunt (1991) and Holland
differences between extant jawed vertebrates and jawless ver- (1992). The expression patterns of mouse (and other ver-
tebrates are less dramatic than between vertebrates and proto- tebrate) Hox genes suggests that Hox and Msx genes followed
chordates, but important developmental transformations can be different courses of functional diversification. Each
inferred. These include the origin of paired appendages and a mammalian Hox gene is expressed within a precise, regionally
remodelling of the anterior branchial arches to form the jaws restricted, domain along the anteroposterior axis of the devel-
and jaw support apparatus (these transformations need not have oping neural tube, plus often in a subset of tissues from somitic
occutred simultaneously). or lateral mesoderm and/or neural crest cells (for reviews, see
It seems at least plausible, therefore, that multiple new genes Holland and Hogan, 1988; Shashikant et 41., 1990; Gaunt,
originating close to vertebrate origins, and close to jhwed ver- 1991). Hox genes related by cluster duplication have similar
tebrate origins, permitted the evolution of these developmen- (but not always identical) expression patterns in the develop-
tal innovations. Without new sets of genes, developmental ing neural tube, but there are dramatic differences in which
control may have been constrained from further elaboration; mesodermal and neural crest derivatives express these par-
significant gene duplication may have allowed release from alogues. Furthermore, gene targeting of Hox genes often
these genetic constraints, allowing rapid adaptive radiation of causes more severe disruption in mesodermal and neural crest
the first vertebrates and, later, the jawed vertebrates. derivatives than in the neural tube, suggesting partial func-
These hypotheses require new genes to be recruited to new tional overlap in the latter. This suggests that most Hox genes
roles after duplication. How could this occur? One possible retained ancestral roles in neural patterning after Hox cluster
route would be evolutionary modification of the coding duplication, but added to these roles were secondary
sequence of a gene, such that it is optimi zed for a different expression sites and functions (perhaps by acquisition of addi-
function to that of the ancestral gene. This may be accompa- tional cis-regulatory elements; Holland, 1992).
nied by changes in gene regulation to allow deployment at a This evolutionary scenario, which was based primarily on
new site or new time. An example that shows the feasibility of data from mouse Hox genes, made testable predictions
132 P. W. H. Holland and others
-
-
t
Fig. 5. Distribution of AmphiHox3 RNA in the developing neural tube of B. floridae. Expression in l3 hour (A) and 20 hour (B) embryos was
visualised by whole-mount in situ hybridization using a digoxigenin-labelled riboprobe (Holland et al ., 1992,, 1994); embryos were obtained by
in vitro fertilization (Holland and Holland,1993). (A) Reproduced from Holland et al. (1992). Juvenile amphioxus were collected by fine
sieving of sand from Old Tampa Bay,Florida: AmphiHox3 expression in juveniles (C,D) was examined using radioactive in situ hybridization
to wax sections, following the protocol of Wilkinson (1993). C and D are bright-field photographs. Arrows in A and B indicate the anterior
expression limit in the neural tube; in C the arrow simply points to the neural tube. Pigment granules (p), ventrally located in the neural tube,
are visible in D. Scale bars: 50 pm (A,B,D) and 500 Fm (C).
regarding the expression of amphioxus Hox genes. If the roles (Hox genes) are just two of many routes possible for func-
ancestral function of chordate Hox genes, prior to cluster dupli- tional diversification of duplicated genes. It seems likely that,
cation, was to encode positional information within the devel- even if gene duplication events affected many (or all) gene
oping neural tube, then this should be the predominant (or families simultaneously in evolution, different gene families
only) expression site in amphioxus, which retains a single Hox will have followed quite different routes of functional diversi-
cluster. Consistent with this prediction, the AmphiHox3 gene fication. These patterns of evolution need to examined in much
was found to be expressed predominantly in the developing more detail (including analysis of coding sequences, regulatory
amphioxus neural tube, where it respects a stable anterior elements and function), and in many more gene families, if
boundary at the level of somite five. Expression is also seen in strong correlations are to be found between particular genetic
posterior mesoderm, but this does not respect a stable boundary and phenotypic changes in vertebrate evolution. Correlations
and remains posteriorly localised through development should be tested by examination of multiple taxa, but cannot be
(Holland et al., 1992, 1994; Fig. 5A). considered proof of causality in evolution. Even so, by analysis
Signals were not detected by whole-mount in situ hybridiz- of multiple gene families in many taxa, it should be possible to
ation on amphioxus larvae older than 5 days, perhaps due to assess the hypothesis that gene duplications have played an
technical problems relating to probe penetration (Holland et al., important permissive role in the evolution of vertebrate devel-
1992). We therefore used radioactive in situ hybridization to opment. Is it unrealistic to hope that insight will eventually be
histological sections to assess if additional, secondary gained into the mutations that permitted the evolution of
expression sites appear later in development. These experi- specific innovations, such as the origin of neural crest cells and
ments revealed that the predominant site of expression remains placodes, or the transition from branchial arches to jaws?
the dorsal nerve cord in adult and juvenile amphioxus; we find
no consistent evidence for secondary expression sites (Fig. 58). We thank Walter Gehring, Frank Ruddle, Anna Sharman, Paul
Acquisition of completely new roles (as proposed for Msx Sharpe and Seb Shimeld for communication of results prior to publi-
genes) or the supplementation of ancestral roles with secondary cation; Per Ahlberg for discussions; and Linda Holland, Nick Holland
Gene duplications and vertebrate origins 133
and the rest of Team Amphioxus for help with specimen collection. Holland ,L.2., Gorsky, G. and Fenaux, R. ( 1988) Fertilization in Oikopleura
The authors' research in this field was supported by the SERC (P. W. dioica (Tunicata, Appendicularia): acrosome reaction, cortical reaction and
H. H., N. A. W.), a Royal Society Research Fellowship to P. W. H. sperm-egg fusion . Zoomorphology 108, 229-243.
Holland, P. \ry. H., Holland, L.Z.rWilliams, N. A. and Holland, N. D. (1992)
H. and a Human Frontiers Fellowship to J. G. F.
An amphioxus homeobox gene: sequence conservation, spatial expression
during development and insights into vertebrate evolution. Development
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