by which the genetic networks controlling vertebrate embryogenesis have been pieced together during evolution, will this reveal an underlying logic to vertebrate developmental control mechanisms?

Summary A wide range of anatomical features are shared by all vertebrates, but absent in our closest invertebrate relatives. The origin of vertebrate embryogenesis must have involved the evolution of new regulatory pathways to control the development of new features, but how did this occur? Mutations affecting regulatory genes, including those containing homeobox sequences, may have been important: for example, perhaps gene duplications allowed recruitment of genes to new roles. Here I ask whether comparative data on the genomic organization and expression patterns of homeobox genes support this hypothesis. I propose a model in which duplications of particular homeobox genes, followed by the acquisition of gene-specific secondary expression domains, allowed the evolution of the neural crest, extensive organogenesis and craniofacial morphogenesis. Specific details of the model are amenable to testing by extension of this comparative approach to molecular embryology. Introduction In the past decade dramatic progress has been made towards the identification of genes involved in the control of early vertebrate development. Families of genes encoding transcription factors have been implicated, and there are a multitude of reports describing complex spatiotemporal gene expression patterns, families of closely related genes within and between species, and experimental results suggesting partial functional redundancy between genes. At first sight the complcxity seems bewildering: it is difficult to interpret any simple logic behind thc control networks being postulated. For example, why are genes of the Hox clusters apparently utilized in the development of the spinal cord and hindbrain. but not the midbrain or forebrain? Why are related genes from different Hox clusters similar in some aspects of their expression but different in others? Why are the related mouse Hox-7 and Hox-8 genes expressed during such seemingly dissimilar processes as branchial arch, limb, eye and tooth development? In this article 1ask whether a solution to this problem of apparent complexity can be found by taking an evolutionary perspective. If we can unravel the pathway

Structural Evolution of Hox Gene Clusters Vertebrate homeobox genes of the Antennapedia (Antp)-class have been best characterized in the mouse and human. The emerging picture is that there are at least 38 members of this gene family, arranged in four gene clusters, Hox-1 to Hox-4, on different chromo50mes(l”) (Fig. 1). Molecular phylogenetic analyses applied to the homeobox and flanking sequences, plus comparisons of gene cluster organization, suggest that the clusters arose in two stages. First, tandem duplication of an ancestral Antp-like gene resulted in up to 13 linked Hox genes; then the resulting cluster duplicated as part of a larger genomic region, yielding 13 ‘cognate groups’ or ‘subfamilies’ each containing Hox genes related by cluster d ~ p l i c a t i o n ( ~Although -~). the genomic organization is similar between the resultant four mammalian Hox clusters, they do not contain identical numbers of genes. This suggests that some of the duplication events did not copy the entire cluster, or that gcnes have been subsequently lost. Hox genes have been cloned from other vertebrate species. primarily chick, zebrafish and Xenopus laevis, and in each case there is good evidence for multiple Hox clusters of similar organization. The Hox cluster duplication events, therefore, must have occurred early in vertebrate evolution, and the general features subsequently conserved. However, a wide range of vertebrates have not yet been analyzed in sufficient detail to reveal whether the precise gene organization of the Hox clusters differs between divergent vertebrate lineages. Clustered homeobox genes of the Amp-class are also present in the genomes of several invertebrates, including Drosophila melanogaster (principally the homeotic selector genes of the ANT-C and BX-C), other insects and a nematode@,’). It is now apparent that these gene clusters are evolutionarily homologous to the mammalian Hox clusters; implying that the genome of the distant common ancestor of insects, vertebrates, and probably nematodes, contained a cluster of Hox genes. For a few Drosophila homeotic selector genes (Scr, D f d , lab) sequence analyses suggest a direct relationship to single cognate groups of mammalian genes; in other cases, it seems that tandem duplication events took different courses in the two lineages (e.g., the AbdB-related genes; Fig. 1). Further insight into the evolution of the vertebrate Hox clusters may be gained by analysis of taxa more closely related to vertebrates than are the insects and nematodes. It is widely believed, and supported by molecular phylogenetic evidence, that these key invertebrate taxa include the echinoderms, hemichordates, tunicates and cephalochordates(11-’3). The published sequences of Antp-class genes from the echinoderm Tripneustes gratilla are consistent with the

-AbdB

abdA

Ubx

Antp

Scr

Dfd

22

21

pb

lab

Drosophila

I - I I - I

Common

ancestor 1.10

1.9

1.8

1.7

1.1

1.2

1.3

1.4

1.5

1.11

1.6

23

22

21

26

27

28

29

33

34

35

M~~~~ Hox-1

25

24

32

31

HOX-2

< 48

47

46

45

44

43

42

HOX-3

41 HOX-4

I

II

111

IV

V

VI

VII

Vlll

IX

X

XI

XI1

Xlll

Cognate group

Fig. 1. Genomic organization of mammalian Antp-class Hox genes (based on mouse and human) and their inferred evolutionary relationships to Drosophilu homeobox genes of thc BX-C and ANT-C. Also shown is thc possible Hox gene organization of the most recent common ancestor of insects and vertebrates. Not shown are Drosophila ftz and bcd, which are not clearly implicated in regional specification of cell fate. The mammalian Hox genes are assigned to cognate groups rclated by cluster duplication; thc possibility that the clusters contain additional Hox genes has not been not ruled out. Modified from refs. 1. 3, 6 , 10, 30, 34.

presence of a single set of Hox genes (including enes representing the AbdB, Dfd and 'core' abdAfAnrp subfamilies), suggesting that Hox cluster duplication (and probably also cluster expansion) occurred later than the divergence of echinoderms and chordate~('~>''). Preliminary evidence from a cephalochordate suggests these events may have occurred even more recently (P. W. H. Holland, L. Z. Holland and N. D. Holland, unpublished data).

Functional Evolution of Hox Gene Clusters It is valid to ask why vertebrate Hox clusters have the organization and expression described. Detailed expression analyses in chick, Xenopus and zebrafish(16-19) have revealed that Hox gene expression patterns are remarkably consistent between vertebrate species, suggesting functional constraints have conserved these patterns. Minor exceptions include differences in the precise anteroposterior (AP) limits to expression of chick and mouse Hox-2. 9("), and also of Xenopus and zebrafish Hox-3.3 products(''). Intraspecific comparison of Hox genes from different clusters can suggest whcther genes acquired different functions after cluster duplication. For mouse Hox genes, cognate genes often respect similar, if not identical, anterior limits within the early mesoderm and neurectoderm, and later i n the developing central nervous system (CNS)(2,20,'1).This generalisation does not apply, however, to the most 3' cognate group (the lub-related genes) at mid-gestation, or indeed the most 5' for which there are sufficient data. For the 5' genes Hox-2.5 and Hox-4.4, the anterior boundaries in the CNS are very different, being at the level of the 1st and 17th prevertebrae respectivelyi2'. For the 3' genes Hox-

1.6 and Hox-2.9, a difference arises via a temporal shift in expression domains: thc earliest expression patterns in the neurectoderm are similar, extending into the presumptive hindbrain, but by 8.5 days of development only Hox-2. 9 has exprcssion in the hindbrain (in presumptive rhombomerc 4)(22). Similar comparisons made for expression in mesodermal derivatives reveal more gene-specific differences. For example, within cognate group X (Dfd-related genes), mouse Hox-7.4 and Hox-2.6 are abundantly expressed in the lung and stomach but not the testis at 12.5 days ost coitum, whilst the opposite is true for H0x-4.2(~' . Mesodermal expression differences are seen within other cognate groups, but there is no simple pattern to suggest co-ordinate regulation of Hox genes within a cluster(21). Cognate genes also differ in the dorsoventral limits to thcir expression in the CNS at mid to late gestation, although here the data hint at coordinate regulation within a cluster. Thus Hox-I genes have ventral restriction, IIox-2 genes dorsal, and Hox-3 genes ventral and Is there an evolutionary rationalization for these similarities and differences in the expression patterns of closely related Hox genes? Many authors have speculated that a large number of Hox genes, generated in part by cluster duplication, provides additional regulatory genes necessary for controlling the highly complex and integrated events which characterize vertebrate e r n b r y o g e n e ~ i s ( ~ ~ ~I ~ suggest ~ ~ ~ ~ ~ ~this ~). functional divergence of related genes, following their origin by duplication, was dominated by gain of genespecific controls, particularly for deployment during the development of newly evolving features. This hypothesis makes a clear prediction: all similarities in expression between cognate genes should correlate with

P

individual Hox genes have essential but non-coincident the development of ancient anatomical features, whilst roles in subpopulations of the neural crest, possibly to most differences should correlate with more advanced specify cell fate prior to migration from the hindcharacters. Further, we may predict partial functional brain( 28): for example, Hox-I.5 may specify branchial redundancy between cognate genes during the developmesenchymal crest and Hox-1.6 branchial neurogenic ment of ancient, but not of advanced, characters. crest. Homozygous Hox-1.6- mutant mice also lack at A dorsal hollow nerve cord is an ancient feature least some of the motor nuclei and nerve roots normally which evolved in the chordate lineage prior to located in rhombomeres 4 to 7, and have malformations vertebrate origins. The similar anterior expression of the inner ear. These defects suggest additional cell limits in the CNS seen within many cognate groups of fate specification roles for Hox-1.6 within the posterior mouse Hox genes is, therefore, consistent with the hindbrain: a region which correlates with the most above model. The domains of Hox gene expression rostral part of the early expression domain of this within the CNS are confined to the spinal cord and gene. hindbrain: they do not extend into the embryonic The association between the mutant phenotypes and midbrain or forebrain. This limit to expression can also neural crest specification is clearly compatible with the be interpreted within the evolutionary scenario, since a predictions made by the evolutionary model above: the major characteristic which sets vertebrates apart from presence of neural c r e ~ tand , its extensive utilization in other chordates is extensive elaboration of the head: head morphogenesis and ganglion formation, are both including, of course, the complex subdivided brain. vertebrate-specific characters. A correlation also holds Gans and NorthcutttZ5)interpret this morphological for the other malformations caused by lack of H O X - 7 . 6 difference as reflecting the evolution of a ‘new head’; in function, since a complex hindbrain and the formation effect, they suggest that the extreme anterior of the of paired special sense organs (otic, optic, olfactory) are body became vastly expanded and elaborated during similarly unique to vertebrates. vertebrate origins. It is unclear precisely where the Pronounced mediolateral specialisation of the mesodelineation between ’ancestral trunk’ and ‘new head’ derm and extensive organogenesis are also advanced lies along the AP axis. Lonai and Orr-Urtreger(26)have vertebrate features. Consistent with this, the aspects of made the intriguing suggestion that this position may mesodermal exprescion shared between cognate Hox correlate with the rostral limit to Hox gene expression genes are the early AP domains, whilst the differences domains. If this proposal is accurate, then the often relate to the development of specific visceral confinement of Hox gene expression (within the CNS) organs (described above). The gene cluster-specific to the hindbrain and spinal cord is another ancestral dorsoventral expression patterns in the developing CNS feature retained by cognate genes following Hox cluster do not obviously correlate with advanced features, duplication. since the origin, and spatial segregation, of many In contrast to the shared expression patterns, the neuronal cell types within the CNS predates vertebrate unusual, single rhombomere restricted, anterior exorigins(29).Analysis of Hox gene expression patterns at pression domain of Hox-2.9 may be a more recently the single cell level during vertebrate CNS development acquired feature, perhaps reflecting the evolution of will be necessary to distinguish cryptic complexity from additional regulatory signals involved in hindbrain inheritance of ancestral roles. patterning; whilst the diverging expression limits of 5‘ Taken as a whole, therefore, the evidence favours the cognate genes may reflect the evolution of more fine hypothesis that early in embryogenesis there is partial grained molecular coding of AP positional information functional overlap between Hox genes (probably in the vertebrate CNS. between cognate genes active in the early postcranial Experimental evidence for partial functional redunmesoderm and neurectoderm): this property may dancy between Hox genes in the developing CNS has reflect the functions of an ancestral Hox gene cluster. recently been obtained. Homologous recombination in Gene-specific and gene cluster-specific expression ES cells has been used to engineer mice homoz gous for null mutations in either Hox-7.5 or H o x - I . ~In ~ ~ .patterns ~ ~ ) .mostly reflect roles acquired uftw the Hox clusters duplicated during vertebrate evolution. These both cases the mice die just after birth, following the roles include the control of neural crest cell fate, and development of dramatic (but different) anatomical probably also of organogenesis, hindbrain differendefects. In each case, however, it is striking that the tiation and otic morphogenesis: all of which are anatomy of the spinal cord, and the overt segmentation advanced vertebrate features. of the hindbrain, is normal, suggesting that any major roles for these genes in primary CNS patterning can be substituted by other (Hox?) genes. Evolution of Vertebrate AbdB-related Hox Genes For both mutants, many of the defects are associated with structures receiving neural crest input in the It is intriguing that multiple AbdB-related genes lie at hindbrain region: mice lacking Hox-1.5 function have the 5’ end of three of the mouse and human Hox clusters(’”) (and at least two of the chick Hox defects in derivatives of the branchial arches and clusters(”)) apparently without direct homologues in pharyngeal pouches and mice lacking Hox-1.6 have missing or malformed cranial ganglia associated with the Drosophiln BX-C. Sequence comparisons suggest These phenotypes suggest these genes arose by tandem duplication from an rhombomeres 4 to 7(27,28).

ancestral AhdB-related gene, before the Hox clusters duplicated. Insight into the evolutionary rationale for the existence of these genes has come from some detailed expression anal ses recently performed for the mouse Hox-4 genest31-‘1. These studies revealed that the 5‘ most 1T-lo.x-4genes (Hox-4.4 to Hox-4.8) are expressed within overlapping domains in the extreme posterior parts of the embryo, respecting the ‘structural colinearity’ rule established for other vertebrate Hox genes (the more 3’ in a cluster the gene is located, the more anterior the expression extends). This applies to expression in the CNS, in mesodermal derivatives (including prevertebrae and in structures forming the urinogenital tract(32)),and is also reflected in the time of activation of each gene (temporal colinearity). In addition, the genes have quantitatively different expression levels in the developin genital bud (more 5’ genes have higher RNA levelst3 ) and possess nested domains of expression in developing limb buds (transcripts from more 5’ genes are restricted to more posterior and distal regions). The latter pattern at least is conserved in the chick; in this species similar (but not identical) nested patterns are also shown by the AbdBhomologues of the Hox-1 clusted3”). What do these patterns suggest about the evolutionary history of the AbdB-related Hox genes? IzpisuaBelmonte et al .(34) point to the strikingly different strategies used by Drosophila and mammalian embryos to produce differential homeobox gene expression in posterior body regions. Whilst the mouse expresses tandem AbdB-related genes in this region, Drosoplzila produces a set of spatially regulated alternative transcripts from a single AbdB gene, Izpisua-Belmonte et al. suggest these patterns may represent the endpoints of two alternative pathways to the evolution of increased gene complexity and hence potential for positional coding along the AP axis. An alternative. but not mutually exclusive, interpretation is that the origin of these extra genes facilitated the evolution of paired limbs or genital ducts; both of which evolved during early vertebrate radiation and express vertebrate AbdB-related Hox-4 genes during development. Analysis of other species will be required to ascertain which of the expression characteristics is evolutionarily most ancient, and hence which may have provided the initial selective pressure for maintenance of the newly evolved genes.

Y

8

Evolution of Vertebrate En-class Homeobox Genes En-class homeobox genes have been cloned and analyzed in representatives of scvcral vertebrate lineages, plus many invertebrates, giving potential for interspecies comparison. Tnterpretation is complicated, however, because the number and relationships of Enclass genes in different species is not resolved. Mouse, human, chick, Xenopus. zebrafish and hagfish each possess at least two En gene^(^^-^'); whilst lamprey, sea

urchin and brachiopod may posses5 only These data suggest that duplications of an ancestral Enclass gene have occurred and been fixed in evolution on at least one, and probably more, occasions in vertebrate radiation. Expression of the two mouse genes (En-I and En-2) has been analyzed by in sifu hybridiza t i ~ n ( 43), ~ ’ but accurate comparison to other species is difficult because most expression studies have utilized antibodies not specific for the products of single genes. One site of vertebrate En expression clearly conserved between all vertebrates analyzed is the ‘unction between cmbryonic midbrain and hindbraini39,44,4i): interestingly, this is also the only shared site of expression for mouse En-1 and En-2(’8’4’). Other sites of En gene expression conserved between vertebrate species include a paired ventrolateral stripe in the spinal cord of mouse, chick, Xenopus and zebrafish (known to the distal be En-l expression in mouse)(39’43244), ectoderm of limb or fin buds in mouse, chick and zebrafish(” 44), and specific mandibular arch muscles in zebrafish and Expression in somite derivatives is less consemed: for example, expression in muscle pioneer cells of zebrafish, but in vcrtebral condensations and dermatome of r n o u ~ e ( ~ ~ ,Dif‘~~~~). ferences between vertebrates with essentially similar body plans could reflect transcription without essential developmental roles: hence, direct tests of function are particularly critical for the vertebrate En class genes. The production of mutant mice lacking En-2 gene function has demonstrated a role for this gene in one site which normally expresses En-2 but not En-1: the cerebellum(47).The lack of defects in the embryonic midbrain-hindbrain junction, where both En-l and En2 are expressed, suggests there is functional overlap between these two related genes. These comparative and experimental data are consistent with the hypothesis that an ancestral site of vertebrate En gene deployment, which has since been conserved in all vertebrates, is the region of the CNS forming the midbrain-hindbrain junction. After En genes duplicated, gene regulation diverged and additional roles evolved: one of these is a requirement for En-2 in the mammalian cerebellum. Other gcnespecific roles could relate to neuronal differentiation, limb bud formation, specification of jaw muscle identity and somite differentiation. Elucidation of when and how these gene-specific roles may have evolved during vertebrate radiation will require more detailed molecular characterization of the En gene family, and analysis of further expression patterns using gene-specific probes. Evolution of Vertebrate msh-like Homeobox Genes Vertebrate genes relatcd to Drosophila mcisscle segment homeobox (msh) gene have provoked much interest recently, from both developmental and evolutionary perspectives (reviewed by Davidsoii and Hill(3s)). Three msh-like genes have been cloned from mouse

and zebrafish, and related genes have also been cloned from quail, chick, X e n o p ~ s ( ~ ' , ~a~~)c,i d i a n ( ~sea ~), urchin and amphioxus (P. W. H. Holland, S. Webb and R. Zelenka, unpublished data). Two of the mouse genes, Hox-7.1 and HOX-8.1,are expressed in complex spatiotemporal patterns during embryogenesis; possibly reflecting both cell lineage-dependent expression (in cranial mesenchymal neural crest cells) and position dependent expression in response to local inductive signals (during histogenesis of particular structures, including the eyes, teeth, heart and early limb buds)(35). The expression patterns do not respect particular axial limits: instead, a feature common to the predominant sites of expression is that they are vertebrate-specific features. This correlation is particularly interesting since phylogenetic reconstructions using msh-like homeobox sequences suggest that msh gene duplications occurred around the time of vertebrate origins: a conclusion also supported by the report that an ascidian apparently has only one msh-like gene(37). Considered together, the expression and comparative data suggest that msh gene duplications, followed by regulatory divergence and functional recruitment, occurred during the origin of the vertebrate body plan. Evolution of Vertebrate Homeobox Genes Related to Dll, ofdand ems Many other vertebrate homeobox genes have been identified, in addition to those comprising the Antp-, En- and msh-classes. Sequence comparisons of the homeodomain allow these to be assi ned to additional gene classes; for example the cadJ8), H2.0(49),eve, ~ r d ( ~ ' )Dll, , otd, and ems class genes. Interspecific and intraspecific comparisons of expression patterns are being undertaken for several of these genes: for the Dll, otd and ems classes, these have yielded data of particular interest in an evolutionary context. Two mouse genes containing homeoboxes related to Drosophila Distal-less (DlZ) are expressed in specific in an spatial regions of the developing k~rain("~~~), analogous way to the Hox cluster genes. However, these Dlx genes are expressed considerably more rostra1 than either the Hox or En genes, in the presumptive diencephalon. Other mammalian genes expressed within the forebrain are otd, a homologue of Drosophila orthodenticle (otd), and Emxl and Emx2, two genes containing homeoboxes related to that of Drosophila empty spiracles (ems; E. Boncinelli, personal communication). Consideration of the Drosophila homologues of these genes gives clues to the evolution of these patterns and their putative roles in the vertebrate forebrain. Dll is involved in specifying pattern along the proximodistal axis of the Drosophila limb: it is not involved in AP positional specification. This suggests that Dll-class genes have been recruited for different roles in different animal lineages. As previously mentioned, anatomical

- -Posterior

Anterior

Rho

Hox En

Mes

Di

Tel

Emx

~

Dlx

Genetic co-option during cranial evolution?

Fig. 2. Expression domains of mouse homeobox genes from different classes, together with a hypothesis for their origins during cranial evolution. The anteroposterior limits to expression in the developing CNS at 9 to 10 days post coitum are shown by black bars, relative to a schematic diagram of the anterior region of the vertebrate CNS. Thicker bars indicate where multiple genes within a homeobox class havc overlapping expression domains. Abbreviations: Rho, rhombencephalon; Mes, mesencephalon; Di, diencephalon; Tel, telencephalon.

elaboration of the brain occurred around the time of vertebrate origins: presumably this resulted in a need for new genes to direct region-specific development. This may have been achieved, in part, by recruitment of homeobox genes for new functions, perhaps following Dll gene duplication. For the ems- and otd-class genes, comparison with Drosophila suggests they evolved in a different manner. Unlike Dll, Drosophila ems and otd do play roles in positional specification of extreme anterior body regions(54).This similarity to their vertebrate homologues suggests that the common ancestor of insects and vertebrates utilized ems- and otd-class homeobox genes for controlling the development of very anterior regions, and that these roles have been evolutionarily conserved. Since Emxl and Emx2 are both expressed in the presumptive telencephalon (E. Boncinelli, personal communication), this may correspond to an ancestral ems expression domain. Such a conclusion implies that the telencephalon is an 'ancestral' region, spatially homologous to the anterior nerve cord of a prevertebrate animal. Expression of Emx2, but not Emxl, in the presumptive diencephalon and various sites outside the CNS may reflect recruitment of this gene to secondary functions required as new anatomical structures arose during vertebrate evolution (Fig. 2). One mammalian otd homologue, otx, is expressed in embryonic telencephalon, diencephalon and mesencephalon (E. Boncinelli, personal communication) ; however, further characterization of the vertebrate otd homeobox gene class will be necessary for insight into the ancestral expression domain of this gene family. A Model: Co-evolution of Molecules and Morphology A question asked earlier was whether the complex

embryonic expression patterns described for vertebrate homeobox genes can be rationalized from an evolutionary perspective. The approach taken relied principally on intraspecific and interspecific comparison of homeobox gene expression patterns (and in a few cases mutant phenotypes) to distinguish putative ancestral and derived roles. These could then be compared with hypotheses concerning vertebrate evolution, to elucidate how the structure and function of the homeobox gene family has co-evolved with morphology. Several principal conclusions can be derived from these analyses, as detailed below. 1. The complex and varied patterns of gene expression displayed by the different classes of homeobox gene are remarkably conserved between mammals, birds, amphibia and fish. This implies that most sites of homeobox gene expression are likely to be of functional significance to developmental processes shared by higher vertebrates. 2. Three classes of homeobox genes, Antp, otd and ems, have been implicated in positional specification along the AP axis in both Drosophila and vertebrates. In each case, evidence points to common ancestry: emsand otd-class genes from both species are implicated in the development of similar body regions, whilst both Hox and honieotic genes respect ‘structural colinearity’ of gene organization and expression. The common ancestor of insects and vertebrates, therefore, may have used genes from these three classes to specify cell fate along the AP axis. 3. I proposed that expression characteristics shared by related homeobox genes within a species reflect roles of an ancestral gene, whilst most differences relate to functions acquired after gene duplication. Examples of the former include the expression of: Hox genes in AP domains of the early postcranial mesoderm, early neurectoderm, and later hindbrain and spinal cord; ems-like genes in the telencephalon; and En genes at the midbrain-hindbrain junction. Examples of secondarily derived roles are: Hox and msh-like genes in neural crest specification and differentiation, plus in ear and eye development respectively; msh-like genes in tooth development; Hox genes in organogenesis; abdBrelated genes in urinogenital tract and limb development; and En-2 in the cerebellum. These putative secondary roles are associated with the development (and possibly evolution) of features either specific to vertebrates, or particularly advanced in vertebrates. 4. Comparing these secondary expression patterns with the genes’ putative ancestral roles suggests two alternative modes of functional evolution. In the first, the putative secondary functions operate within the same overall spatial co-ordinates as do the ancestral roles. For example, Hox genes respect similar (or more restricted) AP boundaries within the neural crest and visceral organs as they do within the CNS. An alternative situation is seen for vertebrate genes of the En-, ems- and msh-classes, where gene-specific expression patterns are more extensive than the features shared by related genes. This implies that when new

genes of these classes arose by gene duplication, they were able to escape ancestral spatial constraints to expression: something which may have been necessary for extensive morphological evolution and the origin of essentially new spatial regions. 5. It has been proposed that the head is a new structure, characterizing the vertebrates(25). If the above model for homeobox gene evolution is accepted, then the expression patterns of vertebrate homeobox genes allow insight into the molecular changes which accompanied cranial evolution. I suggest that the AP expression domains of the vertebrate Hox genes, plus the very anterior shared expression domain of Emxl and Emx2, may together have comprised the ancestral nerve cord. At the origin of the vertebrates, an additional AP domain evolved in between these regions: a domain which required new regulatory activities to control positional identity. These activities may have been provided, in part, by Dll-, ems- and Enclass genes, perhaps recruited after gene duplication (Fig. 2 ) . Finally, duplication and functional divergence of Hox and msh-like genes may have given the potential for evolution of structures associated with the CNS such as the ear and eye, and for the origin and extensive utilization of the cranial mesenchyrnal neural crest. It should be realised that this scenario is certainly incomplete, extremely speculative and quite possibly wrong. Nonetheless, it does indicate that intraspecific and interspecific comparisons of regulatory gene deployment, interpreted in a phylogenetic context, can lead to new hypotheses concerning old evolutionary problems: hypotheses that make specific predictions for comparative studies. Acknowledgements I thank Anthony Graham and Adam Wilkins for helpful comments and discussion, and Edoardo Boncinelli and Nick Holland for communicating data prior to publication. I also thank anonymous referees for critical comments on a previous version of this article.

References 1 Holland, P. W. H. (1990). Homeobox gencs and scgmcntation: co-option, coevolution and convcrgcncc. Seminars Deuel. Bid. 1, 135-145. 2 Shashikant, C. S., Utset, M. F., Violette, S. M., Wise, T. L., Eimat, P., Pendleton, J. W., Schughart, K. and Ruddle, F. H. (1990). Homcobox gencs in mouse development. Critical Reviews in Eukuryotic Gene Expression I , 207-

245. 3 Wright, C. V. E. (1991). Vertebrate homeobox genes. Curr. Op. Cell Biol. 3, 976-982. 4 Ihhoule, D. and Dolle, P. (1989). The structural and fimctional organization of the murine Hox gene family resembles that of Drosophilu. EMBO J. 8,14971505. 5 Graham, A., PdpalOpUhI, N. and Krumlauf, R. (1989). ‘The murine and Drosophilu homeobox gene complexes have common features of organization and expression. Cell 57, 367-378. 6 Kappen, C., Schughart, K. and Ruddle, F. H. (1989). Two steps in the cvolution of Antennupedia-class vertebrate homeobox genes. Proc. Nut1 Acad. Sci. USA 86, 5459-5463. 7 Schughart, K., Kappen, C. and Rnddle, F. H. (1989). Duplication of large genomic regions during thc evolution of vertebrate homeobox genes. Proc. Nutl Acad. Sci. USA 86, 7067-7071. 8 Stuart, J. J., Brown, S. J., Beeman, K. W. and Denell, R. E. (1991). A deficiency of the horneotic complex of the beetle Tribolium. Nufure 350, 72-74.

9 Burglin, 1'. R., Ruvkun, G., Coulson, A., Hawkins, N. C., McGhee, J. D., Schaller. D., Wittmann, C., Miiller, F. and Waterston, R. H. (1991). Nematode homeobox cluster. Nature 351. 703. 10 S i e o n e , A., Acampora, D., Nigro, V., Faiella, A., D'Espnsito, M., Stornaiulo, A., Mavilio, F. and Boncinelli, E. (1991). Differential regulation by retinoic acid of the homeobox genes of the four HOX loci in human embryonal carcinoma cells. Mech. Devel. 33; 215-227. 11 Field, K. G., Olsen, G. J., Lane, D. J., Giovannoni, S. J., Ghiselin, M. T., Raff, E. C., Pace, N. R. and Raff, R. A. (1988). Molecular phylogeny of the animal kingdom. Science 239. 76.753. 12 Brusca, R. C. and Brusea, G . J. (1990). Invertebrates. Sinauer Associates. Sunderland, MA, USA. 13 Holland, P. U'.H., Hacker, A. M. and Williams, N . A. (1991). A molecular analysis of the phylogenetic affinities of Saccog/ossuscamhvenJib Rrambcll and Cole (Heniichordata). Phil. Trans. R. Soc. Loizd. B 33, 185-189. 14 Dolecki, G. J., Wannakrairoj, S., Lum, R., Wang, G . , Riley, H. D.. Carlos, R., Wmg, A. and Humphreys, T. (1986). Stage-specific expression of a homeobox-containing gene in tlie non-segmented sea urchin embryo. E M B O J. 5 , 925-930. 15 Dolecki, G. J., Wang, 6. and Humphreys, T. (1988). Stagc- and tissuespecific expression of two homeobox genes in sea urchin embryos and adults. ivucl. Acids Res. 16, 11543-11555. 16 Wedden, S. E., Pang, K. and Eichele, G. (1989). Gxpressiori pattern of homeobox-containing genes during chick embryogenesis. Devchpment 105, 639.650. 17 Molren, A., Wright, C. V. E.. BreMiller, R., De Robertis, E. M. and Kimmel, C. B. (1990). Expression of a homcobox gene product in normal and mutant zebrafish embryos: evolution of the tetrapod body plan. Dei,eZopment 109, 279-288. 18 Sundin, 0. H. and Eichele, G . (199n). A homcodomain protein reveals the metameric nature of thc developing chick hindbrain. Genes Dev. 4, 1267-1276. 19 De Rohertis, E.M., Morita, E. A. and Cho, K. W. Y.(1591). Gradient fields and homeobox genes. DeiJelopmpmenf112, 669-678. 20 Gaunt, S. J., Krumlauf, R. and Duhoule, D. (1989). Mouse honieogencs within a subfamily, Hox-1.4, -2.6 and -5. I . display similar anteroposterior domain, of expression in the embryo, but show stagc- and tissue-dependent differences in their regulatjon. Development 107, 131-141. 21 Gaunt, S. J., Cdetta, P. L., Pravtcheva, D. and Sharpe, P. T. (1990). Mouse Hox-3.4: homeobox sequence and embryonic expression patterns compared with other members of the H o x gene network. Developmmt 109, 329-339. 22 Murphy, P. and Hill, R. E. (1991). Expression of the mouse lahial-like homeohox-containing genes, Hox-2.9 and Hox-1.6. during segmentation 01the hindbrain. Development 111, 61-74. 23 Graham, A., Maden, &I. and Krumlauf, R. (1991). The murine Hox-2 gene4 display dynainic dorsovcntral patterns of exprecsion during central nervous system development. Deve/opmenf 112. 255-264. 24 Gaunt, S. J. (1991). F.xprcssion patterns of mouse Hou genes: clues to an understanding of developmcntal and evolutionary btrategies. RmEssays 13. 505-513. 25 Gans. C. and Northcutt, K. G. (1983). Neural crest and the origin of the vertebrates: a new head. 26 Lnnai, P. and Orr-Urtreger, A. (1990). Homeogenes in mammalian dcvclopment and the evolution of the cranium and central nervouc Tystem. FASEB J. 4, 1436-143. 27 Chisaka, 0. and Capecchi. M. R. (1991). Regionally res(ricted developmental defects resulting from targcted disruption of the mowe homeobox gcnc Hox-1.5. Nature 350, 473-479. 28 Lufkin, T., Dierich, A., Lehlaur, M., Mark, M. and Chamhon, P. (1991). Disruplion of the Hox-1.6 homeobox gene results in dcfccts in a region corresponding to its rostra1 domain of expression. Cell 66, 1105-1119. 29 Jefferies, R. P. S. (1986). The ancestry of the vertebrates. British Museum (Natural History), London. 30 Yokouchi, Y., Saski, H. and Kuroiwa, A. (1991). Homeobox gene expression correlated with the bifurcation process of limb cartilage development. Nature 353, 443-445. 31 Doll& P., Izpisiia-Belmonte, J.-C.. Falkenstcin, IL, Renucci, A. and Duboule, D. (1989). Coordinate exprcssion of the murine Nor-5 complex homcobox-containing genes during limb pattern formation. NaturP 342. 767772. 32 Doll6, P.. Izpisua-Belmonte, J X . , Brown, J. M., Tickle, C. and Duboule, D. (1991a). Hox-4 genes and the morphogenesir of mammalian genitalia. Genes Dev. 5 , 1767-1776. 33 Dolle, P., Izpisua-Belmonte, JX.,Bonrinelli, E. and Duhoule, D. (1991b) The Hox-4.8 gene is localized at the 5' extremity of the Hox-4 complcx and is

expressed in the mort posterior parts of the body during development. Mech. Devel. 36, 3-13. 34 Izpisua-Belmonte, J.-C., Falkenstein, H., Doll6, P., Renucci, A. and Duboule, D. (1991) Murine genes related to the Drosophilu AbdB homeotic gene are sequentially expressed during development of tlic postenor part of the body. K M B O .I. 10, 2279-2289. 35 Davidson, D. R. and Hill, R. E. (1992). Mrh-like genes: a family of homcobox genes with wide-ranging expression during vertebrate development. Seminars D e v d Riol.,in press. 36 Takahashi, Y. and LeDouarin, N. (1990). cDNA cloning of a quail horncobox gene and its expression in ncural crest-derived mesenchyme and lateral plate mesoderm. Proc. Not/ Acad. Sri. USA 87. 7482-7486. 37 Holland, P. W. H. (1991). Cloning and evolutionary analysis of msh-like homeobox gene, from niouse, zebrafish and ascidian. Gene 98, 253-257. 38 Davis, C. A. and Jnyner, A. 1,. (1988). Expression patterns of thc homeohox-containing genes En- 1 and En-2 and the mouse protu-oncogene in/I diverge during mouse development. Gcnri nev. 2, 1736-1744. 39 Davis, C. A., Holmyard, D. P., Millen, K. J. and Joyner, A. L. (1991). Examining pattern formation in mousc, chicken and frog embryos with an En5pecitic antiserum. Development 111. 287-298. 40 Holland, P. W. H. and Williams, N. A. (1990). Conservation of engrailedlike homeobox sequences during vertebrate evolution. FEBS Letf. 277, 250252. 41 nolecki, G. J. and Humphreys, 1'. (1988). An engrailed claas homeobox gene in sea urchins. Gene 64. 21-31. 42 Holland, P. W. H., Williams, N. A. and Lanfear, J. (1991). Cloning of segment polarity gene homologues from the unscgmented brachiopod Terebratulinu retwa (Linnaeus). FEBS Lett. 291, 211-213. 43 Davidson, D., Graham, E., Sime, C. and Hill, R. (1988). A gene with sequence similarity to DrosoplziZu engrailed is expressed during the development of tlie neural tube and vertebrae in the mouse. Developrnenr 104, 305-316, 44 Hatta, K., BreMiller, R., Westerfield, M. and Kimmel, C. B. (1991). Diversity of expression of engrailed-like antigens in zcbrafish. Development 112. 821-832. 45 Holland, N. D.. Holland, L. Z . , Davis, C. A. and Honma, Y. (1991). Expression domains of engrailed gene in lamprey embryos. Anier. Zoo/. 31, 46A. 46 Hatta, K., Schilling, T. P., RreMiller, R. A. and Kimmel, C. B. (1990). Spccification of jaw muscle identity in zcbrafish: correlation with engrailedhonieoprotein expression. Science 250, 802-805. 47 Joyner, A. I,.. Herrup, K.? Auerbach, B. A., Ihvis, C. A. and Rossant, J. (1991). Subtle cerebellar phenotype in mice homorygous for a targeted deletion of the En-2 homeobox. Science 251, 1239-1243. 48 Frumkin, A., Rangini, Z., Ben-Yehuda, A., Gruenhaum, Y. and Fainsod, A. (1991). A chicken caudal homologue. CHox-cad. is expresscd in the epiblast wilh porlerior localization and in the early endodermal lineage. Developmenr 112, W7-219. 49 Rangini, Z., Ben-Yehuda, A., Shapka, E., Gruenhaum, Y. and Fainsod, A. (1991). CHox E . a chicken homeogene of the H2.0 type cxhibits dorso-ventral restriction in the proliferating region of the spinal cord. Mech. Devel. 35.13-24. 50 Rosenfeld, M. G. (1991). POU-domain transcription factors: pou-er-ful developmental rcgulators. Genes Dev. 5, 897-907. 51 Walther, C., Guenet, J.-L., Simon, D., Deutsch, U., Jostes, B., Goulding, M. D., Plachov, D., Balling, R. and Gruss, P. (1991). Pax: a murine multigene [amily of paircd box-containing genes. Genomics 11, 424-434. 52 Price, IM., Lemaistre, M., Pischetola, M., Di Lauro, R. and Duhoule, D. (1991). A mouse gene related to DIxal-Zess shows a restricted expression in the devcloping forebrain. Nature 351. 748-751. 53 Porteus, M. H., Rulfone, A., Ciaranello, R. D. and Knbenstein, J. 1.. R. (1991). Isolation and characterization of a novel cDNA clone encoding a honieodomain that is developmentally expressed in the ventral forebrain. $Neuron7. 221-229. 54 Ingham, P. W. (199Oj.The X,Y,Z of head development. zV&m 346. 412413.

Peter W. H. Holland is at the Department of Zoology. University of Oxford, South Parks Road, Oxford, OX1 3PS, UK.