Development 1994 Supplement, 125-133 (1994) 125Printed in Great Britain @ The Company of Biologists Limited 1994

Gene duplications and the origins of vertebrate development

Peter W. H. Hollandl'*, Jordi Garcia-Fernindezl't, Nic A. Williamsl'* and Arend Sidow2l Department of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3PS, UK2Department of Molecular and Cellular Biology, 401 Barker Hall, University of California, Berkeley, CA 94720, USA*Present address: Department of Pure and Applied Zoology, University of Reading, Whiteknights, PO Box 228, Reading, RG6 2AJ, UKtPresent address: Departament de Gendtica, Facultat de Biologia, Universitat de Barcelona, Avda. Diagonal 645, 08071 Barcelona, Spain

SUMMARY

All vertebrates possess anatomical features not seen in theirclosest living relatives, the protochordates (tunicates andamphioxus). Some of these features depend on develop-mental processes or cellular behaviours that are againunique to vertebrates. We are interested in the geneticchanges that may have permitted the origin of these inno-vations. Gene duplication, followed by functional diver-gence of new genes, may be one class of mutation thatpermits major evolutionary change. Here we examine thehypothesis that gene duplication events occurred close tothe origin and early radiation of the vertebrates. Genomesize comparisons are compatible with the occurrence ofduplications close to vertebrate origins; more preciseinsight comes from cloning and phylogenetic analysis ofgene families from amphioxus, tunicates and vertebrates.

Comparisons of Hox gene clusters, other homeobox genefamilies, Wnt genes and insulin-related genes all indicatethat there was a major phase of gene duplication close tovertebrate origins, after divergence from the amphioxuslineage; we suggest there was probably a second phase ofduplication close to jawed vertebrate origins. Fromamphioxus and vertebrate homeobox gene expressionpatterns, we suggest that there are multiple routes bywhich new genes arising from gene duplication acquire newfunctions and permit the evolution of developmental inno-vations.

Key words: gene duplication, evolution, amphioxus, tunicate,homeobox, Wnt genes

INTRODUCTION

The origin of vertebrates has been the subject of conjecture anddebate for over a century. Discussion has centred on the affini-ties of the vertebrates, the nature of their ancestors and theanatomical changes that must have occurred during vertebrateevolution. (There is also disagreement concerning usage of theterm 'vertebtate'; here we include mammals, birds, reptiles,amphibians, true fish, lampreys and, unlike some authors,hagfish). Many attempts have been made to derive vertebratesfrom either extant invertebrate taxa or hypothetical ancestralforms; each scenario suggests various morphological changesto the body plan, but only rarely have authors considered theunderlying genetic causes or the plausibility in a developmen-tal context. In this regard, comparative genome analysis, phy-logenetic studies of developmental regulator genes and com-parative developmental biology of vertebrates and their extantrelatives has much to offer, since it could reveal how genes anddevelopmental processes have changed in evolution.

One influential hypothesis for vertebrate origins, which didtake a developmental perspective, was proposed ten years agoby Gans and Northcutt (for a recent review see Gans, 1993).These authors proposed an evolutionary scenario in which theorigin of a suite of novel vertebrate characters (including thesensory and cranial ganglia, three paired special sense organs,sensory capsules, and cartilaginous gill arches), were

dependent on the origin of neural crest cells and ectodermalplacodes. These have important developmental roles in thecranial region of vertebrates, and the structures derived fromthem (and through their interactions with other cells) dominatethe vertebrate head. In this sense, much or all of the vertebratehead was proposed to be an evolutionarily new structure (orneomorph): an innovation of the vertebrates. Other significantmorphological changes are proposed to have occuffed earlieror later in chordate evolution; for example, the origin of seg-mentation within early chordates, and the origin of vertebraeand jaws during early vertebrate radiation (Fig. 1).

How did each of these developmental changes occur inevolution? What kinds of genetic changes allowed the originof the vertebrate developmental program? For example, didnew genes permit the evolution of new cell behaviours (seen

in neural crest cell migration and differentiation)? Answers toquestions such as this may come from considering the geneticbasis for evolutionary changes in development. Specifically,we must ask what sort of mutations were potentially, andactually, responsible for particular changes in developmentalcontrol during vertebrate origins.

One type of mutation that may have played an important roleis gene duplication. The great potential of gene duplication inthe evolution of increasing complexity was discussed bySusumu Ohno in his classic book (Ohno, 1970). He argued thattandem duplication of genes, and polyploidy, could create

126 P. W. H. Holland and others

neuralcrestplacodes

paired sense organs

Chordates

Fig. L. Possible phylogenetic relationships between chordatesshowing origin of developmental and anatomical innovations.

redundant genes that were then able to diverge, relativelyunchecked by purifying selection, until co-opted for newfunctions. In Ohno's words "natural selection merely modified,while redundancy created". Since Ohno's insight, the hypoth-esis that gene duplications are a major force in the generationof organismal complexity has been put on a sound populationgenetic basis (Ohta, 1989).

With respect to the origin and radiation of the vertebrates,little data on gene duplications were available at the time ofOhno's book. Even so, he was able to make some specula-tions, based on allozyme data and genome sizes within thechordates. Ohno suggested that at least one round oftetrapl oidization occurred in the lineage leading to amniotes(reptiles, birds and mammals), probably in our Devonian fishor amphibian ancestors, and that independent tetraplotdizationevents occurred in other fish and amphibian lineages (see alsoOhno, 1993). He also suggested that, much earlier inevolution, genome expansion (by either tetraploidy or tandemgene duplication) occurred in the common ancestor ofamphioxus and vertebrates (after divergence from tunicates);he did not explicitly propose significant genome changes at

the origin of vertebrates. More recently, Holland (1992) spec-

ulated that multiple gene duplications may have occulred at

vertebrate origins; new genes could then have been co-optedto new roles, facilitating the evolution of new developmentalpathways.

These hypotheses make predictions concerning the diversi-fication of gene families that are testable. For example, thenumber of related genes in a particular gene family can be

estimated by application of the polymerase chain reaction(PCR) using degenerate primers; although this technique maynot detect all related genes, it does have the advantage of beingapplicable to multiple species (essential for the comparativeapproach needed). Furthermore, even if only a subset of genes

within a gene family are cloned, from a few key species,

molecular phylogenetic analyses can reveal relationshipsbetween genes and hence the pathways and timings of gene

duplication. Linkage analysis by genomic walking or chromo-some in situ hybridization is also now widely applicable, and

can be used to distinguish tandem duplication from polyploidy.Of course, it should not be expected that all duplicated genes

are retained in the genome after duplication; unused genes

could be deleted or scrambled during evolution with little con-

sequence. In addition, it seems possible that even genes thatwere once essential could be secondarily lost. Nonetheless,applying PCR, genomic library screening and molecular phy-logenetic analysis to multiple gene families, in multiplechordate species, should allow the general patterns of genomeevolution to be elucidated.

Here we examine the evidence for gene duplications duringchordate evolution, comparing the conclusions drawn fromgenome size comparisons to the insights now possible fromgene cloning. A11 protein-coding genes reported to date fromamphioxus are reviewed in an evolutionary context, and twoamphioxus Wnt genes are reported. Tunicate genes are

compared where relevant; we also report the PCR cloning ofa Hox gene from an appendicularian. We then consider alter-native ways in which duplication of developmental controlgenes could contribute to the evolution of vertebrate develop-ment, and assess these alternatives in the light of in situ hybrid-tzation analyses of amphioxus homeobox gene expression.

CAN GENOME SIZE GIVE ANY EVOLUTIONARYINSIGHTS?

Since the cephalochordates (amphioxus) are generally thoughtto be the closest extant relatives of the vertebrates (Fig. 1),

genome comparisons between amphioxus and vertebrates mayyield clues to the genetic events that accompanied the

evolution of developmental innovations at the origin of verte-brates. Atkin and Ohno (1967) reported the haploid genome ofthe amphioxus Branchiostoma lanceolatum to be approxi-mately 0.6 pg, about 17% of the value for placental mammals.This is considerably larger than seen in many tunicates (forexample, Ciona at0.2 pg), but similar to the smallest vertebrategenomes (for example, puffer fish at 0.5 pg; see also Brenneret al., 1993). This led Ohno (1970) to suggest that genome

enlargement by tandem gene duplications and/or polyploidyoccurred in a common ancestor of amphioxus and vertebrates,but not significantly in the immediate vertebrate ancestors.Taking into account the genome sizes of mammals, birds andreptiles, he also suggested the occurrence of one or more addi-tional rounds of tetraploidy in our Devonian fish or amphibianancestors (Ohno, 1970, 1993).

These proposed timings of genome expansion do notcorrelate with vertebrate origins. Does this mean that gene

duplications did not play an important role in the origin of the

complex vertebrate body plan? Not necessarily, since genome

size may be only a very approximate indicator of gene number:for example, repetitive DNA comprises from 20Vo to over 50Vo

of metazoan genomes, and this fraction is prone to dramaticchanges in evolution, probably without concomitant changes ingene number (Lewin, 1990). Furtherrnore, the distribution ofgenome sizes within the fishes, together with phylogenetic con-siderations, make it very unlikely that the extremely compactpuffer fish genome is representative of early vertebrates. Pufferfish, being members of the order Tetraodontiformes, occupy avery derived phylogenetic position within the ray-finned fish,and have genome sizes well below the modal value for fishes;

this unusually small genome size must be secondarily derived,unless one is willing to accept the occuffence of very frequent,but independent, genomic expansion events in many divergentfish lineages (P. E. Ahlberg, unpublished analyses). If

jawspaired fins? Gnathostomes or

jawed vertebrates(mammals, birds,reptiles, amphibia,fish)

Lampreys

Hagfish

Amphioxus

Appendicularians

Ascidians

amphioxus is compared instead to the living members of theearliest vertebrate lineages, hagfish and lampreys, significantlylarger genomes are indeed seen in vertebrates (haploid valuesI.4-2.8 pg). Furtherrnore, it has been shown that the brooklamprey genome (at l.a pg rs not complicated by very recenttetraploidy (Ward et al., 1981); hence, it may be valid to use itas an approximate guide to early vertebrate genome size. Ofcourse, modern lampreys could have secondarily expanded orcompacted genomes, in which case it would not be valid to inferearly vertebrate genome size from them.

The assumption would be testable if genome sizes could be

measured from representatives of other (now extinct) jawless

vertebrate lineages. Perhaps surprisingly, this may be feasiblesince the outlines of cells are preserved in some fossils. Celloutlines give an estimate of cell volume, which in turn is an

approximate indicator of genome size within vertebrates (if thesame cell type is compared between species). The feasibilityof this approach was demonstrated by Conway Morris and

Harper (1988), who estimated genome size in extinctconodonts (thought to be an ancient lineage of jawless ver-tebrate; Sansom et a1., 1992). These analyses need extendingto other lineages; at present, however, the data from both livingand fossil jawless vertebrates support the contention that sig-nificant genome enlargement occurred at vertebrate origins(after divergence of the amphioxus lineage).

EVOLUTIONARY INSIGHTS FROM AMPHIOXUSHOMEOBOX GENES

More accurate insight into the evolution of vertebrate genome

organization will undoubtedly come from cloning and phylo-genetic analysis of gene families in representatives of severalprotochordate and vertebrate lineages. Phylogenetic consider-ations make amphioxus a particularly important protochordatefor gene family analysis, since its lineage diverged after theurochordates but before the diversification of vertebrates (Fig.1). Of particular interest in these analyses will be multigenefamilies implicated in the control and coordination of devel-opmental processes (for example homeobox and Wnt genes),

since their molecular evolution may give insight into the originof vertebrate developmental control. Relatively few protein-coding genes have been cloned from amphioxus, but these

include genes related to vertebrate transcription factors, growthfactors, signalling molecules, structural proteins and enzymes.In this section and the next, we look at every examplepublished to date.

One group of homeobox genes for which comparativesurveys have been undertaken in the chordates is the Msx gene

family. Three members of this gene family were cloned fromthe mouse genome by PCR (Holland, 1991b); two of these are

known to be expressed in cranial neural crest-derived mes-enchymal tissue and in complementary patterns at many sites

of tissue interaction during development (including duringbranchial arch development, palate development, tooth mor-phogenesis, and development of the paired eyes; Davidson and

Hill, l99I). Aspects of the gene expression patterns are

certainly functional; for example, a point mutation in thehomeobox of the human MSX2 gene is thought be one cause

of a skull morphology abnorm ality , craniosynostosis (Jabs et

&1., 1993), whilst deletion of mouse Msxl by gene targeting

Gene duplications and vertebrate origins 127

causes a range of cranial defects (Satokata and Maas, 1994).

Many of the expression sites of the Msx-l and Msx-2 genes,

although not all, are vertebrate-specific features (Holland,1992); hence it is intriguing to ask whether amphioxus has

homologues of these genes.To date, we have succeeded in isolating only a single

member of the Msx homeobox gene family from the genomeof Branchiostoma floridae, both by PCR (Holland et al., 1994)and by genomic library screening (A. Sharman and P. W. H.H., unpublished data). This parallels the results from an

ascidian, but contrasts with the multiple Msx genes present ina teleost fish, Brachydanio rerio (Holland, I991b). These

results are consistent with the hypothesis that gene duplicationsin this gene family occurred in the vertebrate lineage afterdivergence of amphioxus; however, a wider survey of verte-brates must be completed before the timing of duplication can

be ascertained.Comparative data are more sparse for three other homeobox

gene families analyzed in amphioxus: the En, Cdx and the

XlHbox8-related genes. In the latter two cases, PCR has iden-tified a single homologue to date in B. floridae (Holland et aL.,

1994); the Cdx genes, at least, form a multigene family inmammals (Gamer and Wright, 1994). The size of the XlHbox8gene family is unknown in any species; within vertebrates, rep-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

need to be analyzed (including jawless vertebrates) before the

timing and extent of gene duplications can be ascertained.

For the largest homeobox gene family, the Hox genes, moreextensive cloning and phylogenetic surveys have been under-taken. Mammals (and probably all higher vertebrates) have

four similar clusters of Hox genes, homologous to the singleHox or HOM-C cluster of arthropods and nematodes (reviewed

by Holland, 1992; Burglin and Ruvkun, 1993). Elucidating the

number of Hox clusters in amphioxus and lower vertebrates is

crucial to determining the time of Hox cluster duplication. Inaddition to cluster duplication, there is the question of tandemduplications within a cluster. The 38 mammalian Hox genes

are divisible between 13 paralogous groups (containing genes

related by the cluster duplication events); many of these groupsare not present in arthropods and nematodes (Holland, 1992;

Burglin and Ruvkun, 1993). Phylogenetic reconstructionssuggest that the pre-duplication Hox cluster organization was

not identical to any of the clusters of mouse or human (Kappen

and Ruddle, 1993); hence, tandem duplications anilor gene

losses must have occuffed after cluster duplication. AmphioxusHox genes could give clues to the timing of these events.

The first amphioxus Hox gene published was AmphiHox3(Holland et a1., 1992) from Branchiostoma floridae; completegene sequence showed this gene is homologous to parulagous

group 3 of mammalian genes. This assignment suggests thatthe tandem duplication event that yielded paralogous groups 2

and 3 (both related to the Drosophila pb gene) predated the

divergence of amphioxus and vertebrates; it cannot be dated

more accurately at present. The number of Hox clusters in the

amphioxus genome has been estimated in two studies usingPCR (Pendleton et al. , 1993; Holland et al., 1994). Both studies

utilized the same species (8. floridae) and identified multipleHox genes. From analysis of the deduced translation productsof 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 clustermodel"; however, from similar data Holland et al. (1994)conclude that there is "probably a single Hox cluster". The dif-ficulty in determining the number of clusters stems partly fromthe fact that PCR primers capable of amplifying a broadspectrum of Hox genes can only yield up to 82 nucleotides ofunique sequence from each gene. This is often insufficient toassign a gene accurately to a paralogous group (Garcia-Ferndndez and Holland, unpublished data). To overcome thisproblem, and resolve the discrep ancy, we have isolatedgenomic clones of ten amphioxus Hox genes and mapped theirgenomic organisation. We find there is a single cluster of Hoxgenes in the amphioxus genome (Garcia-Ferndndez andHolland, unpublished data).

It is interesting to compare our one cluster model foramphioxus Hox genes (Holland et al., 1994) with PCR resultsobtained for a lamprey (Pendleton et al. ,1993). Despite the dif-ficulty in assigning PCR clones to paralogous groups, the 19

Hox genes identified in Petromyzon marinus are consistentwith lampreys having at least two, and perhaps three or four,Hox clusters. This suggests the initial Hox cluster duplica-tion(s) occurred in the lineage leading to the first vertebrates,after the divergence of amphioxus.

EVOLUTIONARY INSIGHTS FROM OTHERAMPHIOXUS GENES

The first clues to gene family complexity in amphioxus, predatingthe homeobox results discussed above, came from Chan et al.(1990). These authors reported that Branchiostoma califurnien-sis has a single insulin-like gene (ILP), homologous to three genefamily members in mammalian genomes (insulin, IGF-L, IGF-2); the deduced mature protein sequence shares equal identitywith each of the three human proteins. The simplest explanationis that amphioxus retains a single member of this gene family,and that insulin gene duplications occurred on the vertebratelineage, after divergence of amphioxus and vertebrates. One ofthe duplication events occuffed very early on the vertebrate (orpre-vertebrate) lineage, since both hagfish and lampreys possess

an insulin gene and at least one IGF gene (Nagamatsu et al.,I99I). Remarkably, evidence for this very ancient duplicationmay still be present in the human genome: IGF-L maps to chro-mosome 12, within a region of paralogy to chromosome 11 thatcontains the insulin and IGF-2 genes (Brissenden et al., 1984;Lundin, 1993). It should be possible to test if this paralogy isgenuinely the result of a very early duplication event (of a chro-mosome, chromosomal region or entire genome) by examinationof the genes linked to the ILP gene in amphioxus.

The Mn superoxide dismutase (Mn SOD) gene and an inter-mediate filament Qn gene have also been cloned fromamphioxus (Smith and Doolittle, 1992; Riemer et a1., 1992).The former appears to be a single copy gene in all animalsstudied, implying that gene duplications during vertebrateancestry did not affect every gene (or subsequent gene loss hasreturned some gene families to singletons). The intermediatefilament genes could be an informative source of data on dupli-cations, since in mammals they form five subfamilies (types Ito V), each containing multiple genes. Exhaustive surveys havenot been caffied out in amphioxus; the one gene reported todate is clearly a type III gene (vimentin/desmin family), con-sistent with the idea that initial subdivision of the IF gene

superfamily predated the divergence of amphioxus and verte-brates (Riemer et a1., 1992).

It would be interesting to know the number of amphioxusgenes in each IF gene subfamily, particularly since mammaliangene mapping studies reveal that some of the 'within group'duplications almost certainly coincided with Hox cluster dupli-cations. For example, within both the type I (acidic cytoker-atin) and the type II (basic cytokeratin) gene families, there arerelated genes very closely linked to the HOXB and HOXCgene clusters on human chromosomes 12 and 17 (Bentley etal., 1993; Lundin, 1993).

There are several other cases where members of a gene familyare chromosomally linked to more than one marnmalian Hoxcluster; in each case, their origin by chromosomal or genomeduplication may have coincided with Hox cluster duplication.Possible examples of 'co-duplicated' gene families include (inaddition to the cytokeratins): collagen genes, retinoic acidreceptor genes, Evx homeobox genes, erythrocyte band 3 relatedgenes, glucose transporter genes, actin genes, GLVciD zincfinger genes, myosin light chain genes, some Wnt genes (but see

below) and the neuropeptide Y/pancreatic polypeptide genes(gene mapping data from Bentley et al., 1993; Lundin, 1993).Extrapolating from data on the timing of Hox cluster duplica-tions (Pendleton et aI., 1993; Holland et al., 1994; Garcia-Ferndndez and Holland, unpublished data), we suggest thatexpansion of many of these gene families occuffed close to ver-tebrate origins. We do not, however, discount the possibility thatadditional duplication events occurred in these gene familiesduring the subsequent evolutionary radiation of the vertebrates.

The Wnt gene family is an interesting case from the per-spective of gene duplications. These genes encode an extensivefamily of secreted proteins implicated in cell-cell signallingduring vertebrate and invertebrate embryogenesis (Nusse andVarmus, 1992). Sidow (1992) investigated the diversificationand molecular evolution of the Wnt gene family, by phyloge-netic analysis of 72 partial Wnt gene sequences isolated froma diversity of vertebrates, echinoderms and Drosophila. Theresults suggested that Wnt-L, -3, -5, and -7, and one or moreancestors of Wnt-2, -4, -6, and -10 were probably present inthe genome of the last common ancestor of arthropods and ver-tebrates. Later duplications of Wnt-3, -5, -7, -8 and -10 (givingrise to, for example, Wnt-3a and -3b) occuffed before the diver-sification of jawed vertebrates, perhaps after divergence of thehagfish lineage.

We used PCR to search for Wnt genes in amphioxusgenomic DNA, since no protochordate genes were included inthe original analysis. After cloning of the amplified band,sequence analysis of 12 recombinants revealed just twoamphioxus Wnt genes (Fig. 2). The phylogenetic position ofamphioxus and the history of gene duplications in the Wntfamily (Sidow, 1992) imply that amphioxus should have addi-tional Wnt genes, unless they were lost during its evolution. Itwill be particularly interesting to determine if amphioxus hashomologues of those genes that are duplicated in jawed verte-brates (Wnt-3, -5, -7, -& and -10).

EVOLUTIONARY INSIGHTS FROM TUNICATEGENES

In the examples discussed above, the assumption is made that

AmphionrsWnt-A A1 GGCGTGTC@ GATCCTGCGA GCTCqAGACC TGCTGGCGGG CCATGCCGCC51 TTTCCG@AG GTCEGGGCGA GGCTGAAGGA ryUU\TTCGAC GGCGCCACCG

101 AGGTGCAACA GAJ\TU{J{GATC GGCAGCAGGA GAGAACTCGT GCCGCTCAAT151 TCTGACTTCA AGCCGCACTC GAGTTCCGAC CTGGTGTATC TGGATGCTTCzOL CCCAGACTTT TcCGTcCccc ACACCAAGGT GGGGTCGATG GGTACAGTCG fi125L GGAGGGTGTG CAACAAGACT TCCAAGGCCA TCGATGGCTG CGAACTTCTG301 TGCTGTEGGA GAG@TACAA CACCCATACC CGCGAAGTAG TGGAGAGATG351 TAGCTGTAAG

trnnslationGVSGSCE LKTCT{RAITIPPFREVGARLKEKFDGAT EVQQKK I GSRRE LVP LNSDFKPHS SSD LVYT,DAS PDFCVRDTKVGSMGTVGRVCNKTSKA I DGCE L LCCGRGYNTHTREV\TERCSCK

Amphioxus Wnt-B

Gene duplications and vertebrate origins 129

stomacfr

trt91 CCACTACAACAAATACCTGACGAGAGCGAG

HYNKYLTRAR

3 1 AAGGGTGGAj{ATCGCGTCGAACTTGGCTCTRVEIASNLAL

61 CAACGAAACTCAAGTGAAGATTNETOVKI

15110115120L25L301351

GGACTGTCTG ECTCATGCGC AGTAJU{GACG TGTTGGNUU{ AGATGCCGATATTCCGGGAG GTCGGAGTTC GGCTAJU{GGA GAGGTTTAAC GGTGCCTTCCAAGTCATGGG CTCCAACAAC GGCAAATATC TCATACCCGT CGGGGACACTATCAJU{GCTC CTACGGCAGA GGACCTCGTG TATACGAACG AGTCGCCGAATTTTTGCNU{ AccAACAcN{ AIU{CA@GTC C'CAAGGGACC AiU{GGGCGGG

CCTGTAACGC CACGTCCATG GGGATTGGCG GCTGTGACTT GTTGTGTTGTGGGAGAGGGT ACAAGGAGAG ACAGGTGGTC GTGGAGGAGA ACTGCAAGTGTCGC

translationcLscscAvKTctfKKMp I FREVGVRLKERFNGAFQWTGSNNGKYL I PVGDT r KAPTAED LVYTNE S PNFCKRNRKTGSOGTKGRACNATSMG I GGCD L LCCGRGYKERO

Fis. ,. il,::;:and deduced amino acid sequences of two partialWnt genes cloned by PCR from amphioxus (Branchiostoma

floridae). PCR primers were as described by Gavin et al. (1990);thermal cycling and recombinant cloning was performed as

described by Holland (1993). Molecular phylogenetic analysessuggest amphioxus Wnt-A rs orthologous to vertebrate Wnt-4.EMBL/GenBank/DDBJ accession numbers 234213 and 234274.

gene family organization in amphioxus is primitive withrespect to the vertebrate condition. To test this assumption,comparison can be extended to an outgroup. The Urochordates(tunicates) may be useful for this comparison, since they are

thought to be the sister group to the clade comprisingamphioxus plus vertebrates (Fig. 1). In addition, this compar-ison could help evaluate the hypothesis that, after divergenceof the tunicates, substantial gene duplications occulred on thelineage leading to amphioxus plus vertebrates (Ohno, 1970).

The majority of tunicates belong to the Ascidiacea: a groupof animals widely used in developmental studies. Conse-quently, many genes and gene families have been cloned fromascidians. For the present purposes, however, we ate onlyconcerned with members of those gene families also analyzedin amphioxus and several vertebrates. One example alluded toabove was the Msx homeobox gene family; PCR analyses

suggest that the ascidian Ciona intestinalis probably has asingle member of this gene family (Holland, I991b), as alsofound for amphioxus. Hence in this example, Msx gene dupli-cations postdated the amphioxus-vertebrate divergence.

Perhaps surprisingly, at the time of writing, few homeoboxgenes from the Hox family have been reported from ascidians.Single Hox genes have been isolated from Phallusiamammilata (W. Gehring and Paul Baumgartner, personal com-munication) and, by PCR, from several other ascidians (Ruddleet aI., this volume). In addition, in the ascidian Halocynthiaroretzi, screening of genomic Southern blots and a cDNAlibrary using Antp as a probe yielded only the divergenthomeobox gene, AHoxl (Saiga et al., l99I)

We decided to examine complexity of the Hox gene familyin a group of tunicates related to the ascidians, the appendicu-larians. These are small (1.5 mm) pelagic tunicates with an

adult morphology similar to ascidian tadpole larvae (Fig. 3A);

Fig. 3. (A) Schematic diagram of an appendicularian (modified fromAlldridge, 1976), plus (B) the nucleotide and deduced amino acidsequence of a parttal Hox gene cloned by PCR from Oikopleuradioica. PCR primers were based on those of Frohman et al. (1990).

Diagnostic amino acids suggest affinity to the lab group of Hoxgenes. EMBL/GenBanklDDBJ accession number 234284.

they do not metamorphose into a sessile stage. We reasoned

that these animals may be a good outgroup for comparison toamphioxus and vertebrates, since recent studies of sperm mor-phology suggest they derive from a more basal lineage withinthe tunicates than do the ascidians (Fig. 1), and they possiblyhave a less highly modified morphology and life cycle(Holland et a1., 1988; Holland, 1991a).

Using degenerate primers (complementary to Hox genes

from paralagous groups 1 to 10), we employed PCR to searchfor Hox genes in genomic DNA of Oikopleura dioica. Aftercloning of the amplified band, we determined the DNAsequence of 19 recombinant clones. All clones were identical(or with up to one nucleotide difference), and presumed toderive from the same Oikopleura Hox gene (Fig. 3B). Failureto clone additional genes does not disprove their presence inthe genome, but similar PCR conditions have yielded multipleHox genes in many other metazoan species (Averof and Akam,1993; Pendleton et al.,1993; Holland et al., 1994).It would be

surprising if Oikopleura dioica, or other tunicates, have only a

single Hox gene. An alternative possibility is that some aspectof genome organisation, codon usage or sequence divergencein tunicates causes inefficient cloning or PCR amplification.Further work is necessary to resolve these alternatives. Pos-

session of a single Hox gene cannot be the primitive state

nerve cord

130 P. W. H. Holland and others

within the chordates, since wider comparisons to arthropods,echinoderms and nematodes indicate that a cluster of at leastfive Hox genes predated the origin of the chordates (Burglinand Ruvkun, 1993). Hence, despite the phylogenetic positionof tunicates, it may be difficult to address satisfactorily thequestion of exactly when genome duplications occutred duringthe very early phases of chordate radiation.

TIMING OF GENE DUPLICATIONS

Table 1 summarizes the data on timing of gene duplicationsdirectly inferred from cloning of amphioxus genes. These data,and the above discussions, suggest that different gene familiesfollowed different patterns of diversification during the evolu-tionary radiation of chordates. Some gene families apparentlyshowed stability without duplication (Mn SOD gene); whilstsome gene duplications occurred after vertebrate origins (eg:

some Wnt gene duplications; one IGF gene duplication). Acommon theme found is expansion of gene families on theancestral lineage of vertebrates, occurring after the lineageleading to amphioxus had diverged. Examples include dupli-cation of the Hox cluster, Msx gene, Cdx gene and the ancestralinsulin/IcF gene, plus probably duplication of several genes

chromosomally linked to the Hox clusters.These gene family analyses can be used to evaluate previous

hypotheses concerning the evolution of vertebrate genomes.

Ohno suggested that there were two principal phases of gene

duplication on the lineage leading to higher vertebrates (Ohno,1970, 1993; see also Lundin, 1993). He postulated that genomeexpansion occurred (a) before vertebrate origins (predatingdivergence of amphioxus and vertebrates), and (b) during ver-tebrate radiation (in Devonian fish or amphibia).

We believe that the available comparative data fromamphioxus, tunicates and jawless vertebrates (see above)suggest either of two different scenarios. Either (1) there wasone major phase of genome expansion, involving two roundsof extensive gene duplication (perhaps by complete or partialtetraplotdtzation), close to the origin of the vertebrates; or (2)there were indeed two phases, but the first was close to theorigin of the vertebrates and the second was close to the originof the jawed vertebrates or gnathostomes (Fig. 4). At presentwe favour the second of these models, since it is compatiblewith data from the Hox, Wnt, insulin/IcF and En gene families.

We propose that the first phase of gene duplication, close tovertebrate origins, included the initial Hox cluster duplicationand an insulin gene duplication. Evidence for the former comesfrom our demonstration of a single Hox gene cluster inamphioxus (Holland et aI., 1994; Garcia-Fernhndez andHolland, unpublished data), coupled with PCR data suggestingmore than one cluster in lampreys (Pendleton et aI., 1993).Together, these date the initial Hox cluster duplication to afterdivergence of the amphioxus lineage, but before emergence oflampreys. Suggestive evidence for similar timing of the firstinsulin gene duplication stems from the single ILP gene inamphioxus (Chan et al. ,1990), but two genes (insulin and IGF)in lampreys and hagfish (Nagamatsu et al., I99I).

We suggest the second phase of duplication was close tognathostome origins, and probably included further Hoxcluster duplication, a second IGF duplication, duplication ofthe En gene, and expansion of the Wnt gene family. Pendleton

Table L. Gene number within gene families in amphioxusand selected vertebrates

Msx Hox Cdxgenes clusters genes Mn SOD

Insulin/IGF

Amphioxus 1

Agnatha nltMammal 3

1

>24

111nltl2>4 13

Results from hagfish and lampreys are amalgamated under agnatha,although we do not suggest this is a monophyletic group. Not shown are theEn, XlHbox8, IF and Wnt gene families, for which insufficient data are

available. nlt, not tested. See text for further details and references.

Gnathostomes orjawed vertebrates(mammals, birds,reptiles, amphibia,fish)

Lampreys

Hagfish

Amphioxus

Appendicularians

Ascidians

Fig. 4. Phylogenetic tree of the chordates showing the timing ofmultiple gene duplications proposed in this paper. See text for detailsand alternative scenarios.

et al. (1993) suggest lampreys have three Hox clusters; webelieve their datacould reflect a two-cluster model. Either way,a second Hox cluster duplication event is implied, after diver-gence of the lamprey lineage, possibly around the origin ofjawed vertebrates. The second IGF duplication is implied bythe existence of a single IGF gene in lampreys and hagfish(Nagamatsu et al., I99I), but two in jawed vertebratesanalyzed. Evidence that several Wnt gene duplications mayhave also occurred close to jawed vertebrate origins comesfrom PCR surveys of hagfish and gnathostomes (Sidow, 1992).The best placement of duplications affecting the Wnt-3, -5, -7,

and -10 genes (in each case giving a and b paralogues) wasafter the divergence of the hagfish lineage on the ancestrallineage of jawed vertebrates, although this is not at statisticalsignificance. Timing of an En homeobox gene duplication isinferred by the isolation of one En gene in lampreys, but twoto three in teleosts, amphibia and mammals (Holland andWilliams, 1990). The two En genes in hagfish could be theresult of a separate duplication event, as suggested by Hollandand Williams (1990); this may be related to recent, indepen-dent, tetraploidy in this lineage (see Ward et a1., 1981). TheCdx and Msx homeobox gene families also expanded on theancestral lineage of the vertebrates, but there are fewer cluesto timing (see above).

This two-phase model for gene family expansion duringearly vertebrate evolution is testable by analysis of other genefamilies. Resolution will require a combination of careful phy-logenetic analysis of extant gene sequences, combined withgene family analysis and genome analysis in protochordates,jawless vertebrates, teleost fish and other chordate taxa.

FROM DUPLICATION TO INNOVATION

The hypothesis that mutations in regulatory genes could

underlie evolutionary change in embryonic development is

now widely accepted (Ohno, I9l0; Raff and Kaufman, 1983;

Arthur, 1988; Holland, 1992). But different types of mutationcould play different roles in developmental evolution. For

example, it seems unlikely that minor alterations to the codingsequence or expression pattern of a regulatory gene couldallow the origin of completely new morphological features or

developmental processes, at least in the majority of cases.

Duplication of regulatory genes (followed by divergence ofone or both daughter genes), seems more likely to precede the

origin of such developmental innovations. Gene duplicationwould not necessarily cause major developmental alteration;rather, it is considered permissive to subsequent phenotypic

change. This hypothesis, therefore, does not propose the

creation of radically altered 'hopeful monsters' in a single gen-

eration, but envisages new genes being made available to the

gradual modifying effects of natural selection and genetic drift.The hypothesis predicts a coffelation between the origin of

new regulatory genes and the emergence of new cell behav-

iours, body regions or structures (Holland, 1992); furthermore'significant changes to body organisation may correlate withsignificant genome changes simultaneously affecting several

gene families. The scenario presented in the previous section

for the timing of gene duplications during vertebrate evolutionsuggested there may have been two phases of gene duplica-tion: one close to vertebrate origins and a second close to jawed

vertebrate origins. Both stages of chordate evolution involvedsignificant developmental changes; in addition, both phases ofgenome expansion involved duplication of developmental reg-

ulatory (and other) genes.

The origin of vertebrates involved the evolution of several

innovations in developmental strategy, notably the involve-ment of neural crest and placodes in craniofacial morphogen-

esis, elaboration of the brain (origin of the midbrain?), and spe-

cialisation of the segmented mesoderm (Holland, 1992; Gans,

1993; Holland and Graham, 1994). In terms of anatomy, the

differences between extant jawed vertebrates and jawless ver-

tebrates are less dramatic than between vertebrates and proto-

chordates, but important developmental transformations can be

inferred. These include the origin of paired appendages and a

remodelling of the anterior branchial arches to form the jaws

and jaw support apparatus (these transformations need not have

occutred simultaneously).It seems at least plausible, therefore, that multiple new genes

originating close to vertebrate origins, and close to jhwed ver-

tebrate origins, permitted the evolution of these developmen-

tal innovations. Without new sets of genes, developmentalcontrol may have been constrained from further elaboration;significant gene duplication may have allowed release fromthese genetic constraints, allowing rapid adaptive radiation ofthe first vertebrates and, later, the jawed vertebrates.

These hypotheses require new genes to be recruited to new

roles after duplication. How could this occur? One possible

route would be evolutionary modification of the codingsequence of a gene, such that it is optimi zed for a differentfunction to that of the ancestral gene. This may be accompa-

nied by changes in gene regulation to allow deployment at anew site or new time. An example that shows the feasibility of

Gene duplications and vertebrate origins 131

this pattern of evolution (although not relating to the early ver-

tebrates) is the lys ozyme gene family in mammals. Lysozymegene duplication in the ruminant mammal lineage was

followed by changes in protein sequence and expression,

allowing lysozyme to be co-opted to a digestive role in the

foregut of cows, sheep and relatives (Irwin et &1., 1992). Incontrast, arecent experimental analysis of the prd gene familyin Drosophila, demonstrates that adaptive divergence ofprotein-coding sequences does not always accompany func-tional divergence after duplication (Li and Noll, 1994). The

related genes prd, gsb and gsbn apparently encode function-ally equivalent proteins; their divergent roles may have

evolved solely by changes in deployment. We suggest this

latter route to functional diversification may have occulred fre-quently.

At present, there are limited clues to the mechanisms that

allowed functional divergence within regulatory gene familiesduring vertebrate evolution. From the inferred timing of gene

duplication, and the expression patterns of the mammalian

gene family members, a hypothesis can be proposed regarding

functional evolution of the vertebrate Msx homeobox genes

(Holland, I99Ib, 1992). As described above, two of the three

mammalian Msx genes resultant from duplication are predom-

inantly expressed in vertebrate-specific tissues, including cran-

iofacial neural crest derivatives, developing teeth and eyes. Wesuggest that these expression characteristics reflect co-optionto new functions at vertebrate origins; the origin of Msx-l and

Msx-2 might even have permitted the evolution of new patterns

of cell behaviour and differentiation and new developmentalprocesses. This hypothesis implies that Msx- I and Msx-2

acquired new control elements after gene duplication, leavingMsx-3 to persist with an ancestral function. Further insight willcome from analysis of the expression pattern of the thirdmammalian paralogue, Msx-3, and comparison to the single

amphioxus Msx gene. These analyses ate in progress (S.

Shimeld and P. Sharpe, personal communication; A. Sharman

and P. W. H. H., unpublished data).

The evolution of Hox gene function following cluster dupli-cation is discussed in detail by Gaunt (1991) and Holland(1992). The expression patterns of mouse (and other ver-

tebrate) Hox genes suggests that Hox and Msx genes followeddifferent courses of functional diversification. Each

mammalian Hox gene is expressed within a precise, regionallyrestricted, domain along the anteroposterior axis of the devel-

oping neural tube, plus often in a subset of tissues from somiticor lateral mesoderm and/or neural crest cells (for reviews, see

Holland and Hogan, 1988; Shashikant et 41., 1990; Gaunt,

1991). Hox genes related by cluster duplication have similar(but not always identical) expression patterns in the develop-

ing neural tube, but there are dramatic differences in whichmesodermal and neural crest derivatives express these par-

alogues. Furthermore, gene targeting of Hox genes often

causes more severe disruption in mesodermal and neural crest

derivatives than in the neural tube, suggesting partial func-tional overlap in the latter. This suggests that most Hox genes

retained ancestral roles in neural patterning after Hox cluster

duplication, but added to these roles were secondary

expression sites and functions (perhaps by acquisition of addi-

tional cis-regulatory elements; Holland, 1992).

This evolutionary scenario, which was based primarily on

data from mouse Hox genes, made testable predictions

132 P. W. H. Holland and others

B

- -

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 wasvisualised by whole-mount in situ hybridization using a digoxigenin-labelled riboprobe (Holland et al ., 1992,, 1994); embryos were obtained byin vitro fertilization (Holland and Holland,1993). (A) Reproduced from Holland et al. (1992). Juvenile amphioxus were collected by finesieving of sand from Old Tampa Bay,Florida: AmphiHox3 expression in juveniles (C,D) was examined using radioactive in situ hybridizationto wax sections, following the protocol of Wilkinson (1993). C and D are bright-field photographs. Arrows in A and B indicate the anteriorexpression 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 theancestral function of chordate Hox genes, prior to cluster dupli-cation, was to encode positional information within the devel-oping neural tube, then this should be the predominant (oronly) expression site in amphioxus, which retains a single Hoxcluster. Consistent with this prediction, the AmphiHox3 genewas found to be expressed predominantly in the developingamphioxus neural tube, where it respects a stable anteriorboundary at the level of somite five. Expression is also seen inposterior mesoderm, but this does not respect a stable boundaryand remains posteriorly localised through development(Holland et al., 1992, 1994; Fig. 5A).

Signals were not detected by whole-mount in situ hybridiz-ation on amphioxus larvae older than 5 days, perhaps due totechnical problems relating to probe penetration (Holland et al.,1992). We therefore used radioactive in situ hybridization tohistological sections to assess if additional, secondaryexpression sites appear later in development. These experi-ments revealed that the predominant site of expression remainsthe dorsal nerve cord in adult and juvenile amphioxus; we findno consistent evidence for secondary expression sites (Fig. 58).

Acquisition of completely new roles (as proposed for Msxgenes) or the supplementation of ancestral roles with secondary

roles (Hox genes) are just two of many routes possible for func-tional diversification of duplicated genes. It seems likely that,even if gene duplication events affected many (or all) genefamilies simultaneously in evolution, different gene familieswill have followed quite different routes of functional diversi-fication. These patterns of evolution need to examined in muchmore detail (including analysis of coding sequences, regulatoryelements and function), and in many more gene families, ifstrong correlations are to be found between particular geneticand phenotypic changes in vertebrate evolution. Correlationsshould be tested by examination of multiple taxa, but cannot beconsidered proof of causality in evolution. Even so, by analysisof multiple gene families in many taxa, it should be possible toassess the hypothesis that gene duplications have played animportant permissive role in the evolution of vertebrate devel-opment. Is it unrealistic to hope that insight will eventually begained into the mutations that permitted the evolution ofspecific innovations, such as the origin of neural crest cells andplacodes, or the transition from branchial arches to jaws?

We thank Walter Gehring, Frank Ruddle, Anna Sharman, PaulSharpe and Seb Shimeld for communication of results prior to publi-cation; Per Ahlberg for discussions; and Linda Holland, Nick Holland

and the rest of Team Amphioxus for help with specimen collection.The authors' research in this field was supported by the SERC (P. W.H. H., N. A. W.), a Royal Society Research Fellowship to P. W. H.H. and a Human Frontiers Fellowship to J. G. F.

REFERENCES

Altdridge, A. (197 6) Appendicularian s. S c ientific Ame ric an 235 (1 ), 95 - 102.Atkin, N. B. and Ohno, S. (1967) DNA values of four primitive chordates.

Chromosoma 23, 10-13.Arthur, W. (1988) A Theory of the Evolution of Development. Chichester:

Wiley.Averof, M. and Akam, M. E. (1993) HOM/FIox genes of Artemia:

implications for the origin of insect and crustacean body plans. CurrentBiology 3,73-78.

Bentley, K. L., Bradshaw, M. S. and Ruddle, F. H. (1993) Physical linkage ofthe murine Hox-b cluster and nerve growth factor receptor on yeast artificialchromosomes. Genomics L8, 43-53.

Brenner, S., Blgar, G., Sandford, R., Macrae, A., Venkatesh, B. andAparicio, S. (1993). Characterisation of the pufferfish (Fugu) genome as a

compact vertebrate genome. Nature 366, 265-268.Brissenden, J. E., Ullrich, A. and Francke, U. (1984) Human chromosomal

mapping of genes for insulin-like growth factors I and II and epidermalgrowth factor. Nature 310, 781-784.

Burglin, T. R. and Ruvkun, G. ( 1993)The Caenorhabditis elegans homeoboxgene cluster. Current Opinion in Genetics and Devel.3, 615-620.

Chan, S. J., Cao, Q. -P. and Steiner, D. F. ( 1990) Evolution of the insulinsuperfamily: cloning of a hybrid insulin/insulin-like growth factor cDNAfrom amphioxus. Proc. Natl. Acad. Sci. U9A87,9319-9323.

Conway Morris, S. and Harper, E. (1988) Genome size in conodonts(Chordata): inferred variations during 270 mrllion years. Science 241, 1230-1232.

Davidsoro D. R. and Hill, R. E. (199I) Msh-Iike genes: a family of homeoboxgenes with wide-ranging expression during vertebrate development.Seminars Dev. Biol. 2,405-412.

Frohman, M. A., Boyle, M. and Martin, G. R. (1990) Isolation of the mouse

Hox-2. 9 gene; analysis of embryonic expression suggests that positional

information along the anteroposterior axis is specified by mesoderm.

Development 1L0, 589-607 .

Gamer, L. \ry. and Wright, C. V. E. (1994). Murine Cdx-4 bears strikingsimilarities to the Drosophila caudal gene in its homeodomain sequence and

early expression pattern . Mech. Dev. 43, 7 1-81.

Gans, C. (1993) Evolutionary origin of the vertebrate skull. InThe Skull Vol. 2

(ed. J. Hanken and B. K. Hall), pp. 1-35. Chicago: University of ChicagoPress.

Gaunt, S. J. (J991) Expression patterns of mouse Hox genes: clues to an

understanding of developmental and evolutionary strategies. BioEssays 13,

505-513.Gavin, B. J., McMahon, J. A. and McMahon, A. P. (1990) Expression of

multiple Wnt-l/int-1-related genes during fetal and adult mouse

developm ent. Gene s D ev. 4, 23 19 -2332.Holland, L. Z. (1991a) The phylogenetic significance of tunicate spenn

morphology. In Comparative Spermatology, 20 years after (ed. B. Baccetti).S erono Sympo sium 5, 961 -965 .

Holland, P. \ry. H. (1991b) Cloning and evolutionary analysis of msh-llkehomeobox genes from mouse, zebrafish and ascidian. Gene 98,253-257 .

Holland, P. \ry. H. (1992) Homeobox genes in vertebrate evolution. BioEssays14,267 -273.

Hottand, P. \ry. H. (1993) Cloning genes using the polymerase chain reaction.In Essential Developmental Biology: A Practical Approach. (ed. C. D. Stern

and P. W. H. Holland), pp. 243-255. Oxford: IRL Press at Oxford UniversityPress.

Holland, P. \ry. H. and Graham, A. ( 1994) Evolution of regional identity inthe vertebrate nervous system. Perspectives on DevelopmentalN eurobiolo gy. In press.

Holland, P. \ry. H. and Hogan, B. L. M. (1988) Expression of homeo boxgenes during mouse development: a review. Genes Dev.2,773-782.

Holland, N. D. and Holland, L. Z. (1993) Embryos and larvae of invertebratedeuterostomes. In Essential Developmental Biology: A Practical Approach.(ed. C. D. Stern and P. W. H. Holland), pp. 2I-32. Oxford: IRL Press at

Oxford University Press.

Holland, P. \ry. H. and Williams, N. A. (1990) Conservation of engrailed-likehomeobox sequences during vertebrate evolutton. FEBS Len. 277, 250-252.

Gene duplications and vertebrate origins 133

Holland ,L.2., Gorsky, G. and Fenaux, R. ( 1988) Fertilization in Oikopleuradioica (Tunicata, Appendicularia): acrosome reaction, cortical reaction and

sperm-egg fusion . Zoomorphology 108, 229-243.Holland, P. \ry. H., Holland, L.Z.rWilliams, N. A. and Holland, N. D. (1992)

An amphioxus homeobox gene: sequence conservation, spatial expression

during development and insights into vertebrate evolution. Development116, 653-661.

Holland, P. \ry. H., Garcia-Fernindez, J., Holland, L. 2., Williams, N. A.and Holland, N. D. (1994) The molecular control of spatial patterning inamphioxus. "/. Marine Biol. Assoc. UK74,49-60.

Irwin, D. M., Prager, E. M. and Wilson, A. C. (1992) Evolutionary genetics

of ruminant lysozymes. Animal Genetics 23,, 193-202.

Jabs, E. \ry., Miiller, U., Li, X., Ma, L., Luo, W., Haworth, I. S., Klisak, I.,Sparkes, R., Warma[, I., Mulliker, J. 8., Snead, M. L. and Maxson, R.(1993) A mutation in the homeodomain of the human MSX2 gene in a familyaffected with autosomal dominant craniosynosto sis. Cell 7 5, 443-450.

Kappen, C. and Ruddle, F. H. (1993) Evolution of a regulatory gene family:HOM/Hox genes. Curcent Opinion in Genetics and Devel. 3,931-938.

Lewin, B. (1990) Genes IV. Oxford University Press, Oxford.Li, X. and Noll, M. (1994) Evolution of distinct developmental functions of

three Drosophila genes by acquisition of different cis-regulatory regions.

Nature 367 ,83-87.Lundin, L. (1993) Evolution of the vertebrate genome as reflected in

paralogous chromosomal regions in man and the house mouse. Genomics 1.6,

1- 19.

Miller, C. P., McGehe€, R. E. and Habener, J. F. (1994) IDX-I: a new

homeodomain transcription factor expressed in rat pancreatic islets and

duodenum that transactivates the somatostatin gene. EMBO J. 13, tI45-1156.

Nagamatsu, S., Chan, S. J., Falkmer, S. and Steiner, D. F. (1991) Evolutionof the insulin gene superfamily: sequence of a preproinsulin-like growthfactor cDNA from the atlantic hagfish. J. Biol. Chem. 266,2391-2402.

Nusse, R. and Varmus, H. E. (1992) Wnt genes. Cell69,1073-1087.Ohlsson, H., Karlssor, K. and Bdlund, T. (1993) IPFI, a homeodomain-

containing transactivator of the insulin gene. EMBO J. 12, 425I-4259.Ohno, S. (1970) Evolution by Gene Duplication. Heidelberg: Springer-Verlag.Ohno, S. (1993) Patterns in genome evolution. Current Opinion in Genetics

and Devel. 3,911-914.Ohta, T ( 1989) Role of gene duplication in evolution. Genome 31,304-3 10.

Pendleton, J. W., Nagai, B. K., Murtha, M. T. and Ruddler F. H. (1993)

Expansion of the Hox gene family and the evolution of chordates. Proc. Natl.Acad. Sci USA 90, 6300-6304.

Raff, R. A. and Kaufman, T. C. (1983) Embryos, Genes and Evolution. NewYork: Macmillan.

Riemer, D., Dodemont, H. and Weber, K. (1992) Analysis of the cDNA and

gene encoding a cytoplasmic intermediate filament (IF) protein from the

cephalochordate B r an c hi o s t o ma I anc e o latum; implic ation s for the evolutionof the IF protein family . Eur. J. Cell Biol. 58, 128-135.

Saiga, H., Mizokami, A., Makabe, K. \ry., Satoh, N. and Mitar T. (1991)

Molecular cloning of a novel homeobox gene AHoxI of the ascidian,

H alo cy nthia ro r etzi. D ev e lo pme nt 1 1. L, 82I -828 .

Sansom, I. J., Smith, M. P., ArmstrotrB, H. A. and Smithr M. M. (1992)

Presence of the earliest vertebrate hard tissues in conodonts. Science 256,,

1308-1311.Satokata, I. and Maas, R. (1994) MsxI deficient mice exhibit cleft palate and

abnormalities of craniofacial and tooth development. Nature Genetics 6,

348-355.Shashikant, C. S., Utset, M. F., Violette, S. M., Wise, T. L., Eimat, P.,

Pendleton, J. W., Schughart, K. and Ruddle, F. H. (1990) Homeoboxgenes in mouse development. Critical Reviews in Eukaryotic Gene

Expression 1,207 -245.Sidow, A. ( 1992) Diversification of the Wnt gene family on the ancestral

lineage of vertebrates. Proc. Natl. Acad. Sci. USA 89, 5098-5210.Smith, M. 'W. and Doolittle, R. F. (1992) A comparison of evolutionary rates

of the two major kinds of superoxide dismutase. J. Molec. Evol.34,ll5-184.Ward, R. D., McAndrew, B. J. and Wallis, G. P. (1981) Enzyme variation in

the brook lamprey, Lampetra planeri (Bloch), a member of the vertebrate

group Agnatha . Genetica 55, 67 -13.

Wilkinson, D. G. ( 1993) In situ hybridization. In Essential DevelopmentalBiology: A Practical Approach. (ed. C. D. Stern and P. W. H. Holland), pp.

257 -27 4. Oxford: IRL Press at Oxford University Press.

Wright, C. V. E., Schnegelsberg, P. and DeRobertis, E. M. (1989) XlHbox 8

- a novel Xenopus homeoprotein restricted to a naffow band of endoderrn.

Development 105, 787 -794.