Development 103 Supplement, 17-24 (1988)Printed in Great Britain © The Company of Biologists Limited 1988

17

Homeobox genes and the vertebrate head

PETER W. H. HOLLAND

Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3 PS, UK

Summary

Several Drosophila genes important in the control ofembryonic development contain a characteristic se-quence of DNA, known as the homeobox. Homeoboxsequences are also present in a family of vertebrategenes, which may therefore have regulatory rolesduring vertebrate embryogenesis. In this article, dataconcerning the spatial patterns of vertebrate homeo-box gene expression are discussed in relation to recentdescriptive and experimental analyses of head devel-opment. It is concluded that the patterns of geneexpression are consistent with homeobox genes having

roles in anteroposterior positional specification withinthe developing brain and possibly the neural crest.The data are not clearly consistent with these geneshaving' direct roles in controlling the patterns ofcranial segmentation, although further studies mayreveal whether vertebrate segments are units of devel-opmental specification.

Key words: homeobox, vertebrate head, specification,segmentation.

Introduction

The vertebrate head is a complex assemblage ofcranial specializations, involving the central and per-ipheral nervous systems, axial skeleton, musculatureand connective tissue. Most of the morphologicaldifferences between vertebrates and other chordatesrelate to the organization of the head; its evolutioncan therefore be considered fundamental to the originof the vertebrates (Gans & Northcutt, 1983). Inaddition to its evolutionary significance, the spatialcomplexity of the head has made it a challenging areafor embryological investigations. Such studies haveled to considerable progress being made in under-standing cell fate, tissue interactions, morphogeneticmovements, mechanical factors and the roles ofextracellular matrix, in head development (for re-views see Meier, 1982; Noden, 1984; Jacobson &Meier, 1986; Thorogood, 1987).

In contrast, little progress has been made towardsunderstanding the genetic control of head develop-ment, particularly at the molecular level. In thispaper, I wish to consider whether this understandingmay be gained from analysis of a recently identifiedgene family, the vertebrate homeobox genes. Poss-ible roles of these genes in vertebrate embryogenesisare discussed and data concerning patterns of homeo-

box gene expression in the head are reviewed. Thesedata are discussed in relation to two fundamentalaspects of head development, the control of spatialorganization and the nature of cranial segmentation.

Homeobox genes

Genetic mutations that affect the establishment ofcorrect spatial organization have been identified in arange of organisms, most notably in the fruitflyDrosophila. Many of the Drosophila genes identifiedby mutation have been cloned, and their expressionanalysed in normal and mutant embryos. Genes havebeen analysed that have critical roles in establishingthe body axes, metameric patterning (segmentationgenes) and regional specialization (homeotic genes).In many of these cases, the patterns of expression innormal embryos correlate with predictions of func-tion made from descriptions of mutant embryos.Furthermore, these studies are leading to a detailedunderstanding of the genetic control of Drosophilaembryogenesis (reviewed by Gehring & Hiromi,1986; Anderson, 1987; Akam, 1987; Scott & Carroll,1987).

Initial molecular analysis of two homeotic genes(Antennapedia and Ultrabithorax) and one segmen-

18 P. W. H. Holland

tation gene (fushi tarazu) revealed that each had aconserved 183 base pair sequence, the homeobox,within their protein-coding region (McGinnis et al.1984; Scott & Weiner, 1984). It has since becomeapparent that a homeobox is present in many essen-tial Drosophila developmental genes, including atleast six homeotic genes, five segmentation genes anda gene involved in specifying the dorsoventral axis(Gehring, 1987; Rushlow et al. 1987; Scott & Carroll,1987). On the basis of DNA sequence comparisons,several subfamilies can be recognized, including theextensive Antennapedia (Antp)-\\ke class, theengrailed (ert)-like class and the paired-Yike class(Laughon et al. 1985; Poole et al. 1985; Bopp et al.1986).

Homeobox sequences have been detected in thegenomes of a range of segmented and unsegmentedanimals besides Drosophila, including other arthro-pods, annelids, molluscs, echinoderms, urochor-dates, cephalochordates and vertebrates (McGinnis,1985; Holland & Hogan, 1986).

This conservation between disparate taxa meansthat homeobox genes are candidates for genes con-trolling embryogenesis in a range of metazoa, includ-ing vertebrates. More specific hypotheses concerningthe function of vertebrate homeobox genes haveoften been proposed from mutant phenotypes associ-ated with Drosophila homeobox genes. For example,Slack (1984) proposed a role in anteroposterior pos-itional specification, since homeotic genes can beconsidered to control this process in Drosophila. Analternative hypothesis is that vertebrate homeoboxgenes control segmentation, since several Drosophilagenes involved in this process have homeoboxes, andsince homeotic genes can act within metamericboundaries (Struhl, 1984). However, Drosophilahomeobox genes are involved in several different, butinteracting, embryonic events (Gehring, 1987), andthe phylogenetic distribution of homeobox genesdoes not correlate with any specific developmentalstrategy (Holland & Hogan, 1986). At present, there-fore, hypotheses concerning the roles of vertebratehomeobox genes must be based primarily on theirpatterns of expression.

Over twenty homeobox genes have so far beenidentified in the mouse, the species that has receivedmost attention among the vertebrates. Sequencecomparisons indicate that most of these genes (giventhe prefix 'Hox') are related to the Antp-\\ke genes ofDrosophila, whilst two (En-1 and En-2) are moreclosely related to engrailed. Other divergent ver-tebrate homeobox genes have been identified, but notextensively studied. The organization of the ver-tebrate homeobox genes is described in several recentreviews (Colberg-Poley et al. 1987; Fienberg et al.1987; Martin er al. 1987).

In situ hybridization has been used to analyse thepatterns of expression of several homeobox genesduring vertebrate embryogenesis. These experimentshave revealed that the expression of each gene islocalized to particular regions of the embryo. Gaunt(1987) has shown that these 'region-specific' patternscan be apparent from the onset of detectable ex-pression, in the presomite embryo. Slightly later, atthe early somite stage, transcripts are detected in theneurectoderm, with characteristic anterior, andsometimes posterior, limits for each gene. Forexample, the distribution of mouse Hox 2.1 RNA hasan anterior limit within the presumptive myelen-cephalon (Holland & Hogan, 1988#), whilst mouseEn-2 expression is limited to a more anterior band ofneurectoderm (Davis et al. 1988). Several homeoboxgenes are also expressed in an anteroposterior spatialdomain of the mesoderm at the early somite stage,although the limits of this expression are not necess-arily coincident with those in the neurectoderm(Carrasco & Malacinski, 1987; Gaunt, 1987; Holland& Hogan, 1988a).

The spatial pattern of expression at later stages hasbeen analysed for at least twelve homeobox genes inthe mouse (reviewed by Holland & Hogan, 1988b;Stern & Keynes, 1988) and one in Xenopus (Carrasco& Malacinski, 1987). All vertebrate homeobox genesstudied to date are expressed in the central nervoussystem (CNS), as is also characteristic of manyDrosophila homeobox genes (Doe & Scott, 1988).Transcripts from each gene are restricted to a charac-teristic anteroposterior region of the CNS, and sev-eral are also restricted along the dorsoventral axis.

Many homeobox genes are also expressed inganglia of the peripheral nervous system (PNS), inmesodermal derivatives, or in both. As with the CNS,this expression can often be described in terms ofsimple axial limits characteristic for each gene. This ismost clear in the developing vertebral column, whereseveral genes have overlapping domains of ex-pression. The most anterior of these domains is thatof Hox 1.5, which is expressed in all presumptivevertebrae, including the atlas and axis (Gaunt, 1987).Homeobox gene expression has not been reported inderivatives of paraxial mesoderm in the occipitalregion (where somites contribute to the skull) or inmore anterior mesoderm.

Thus vertebrate homeobox genes are expressed inregion-specific, as well as tissue-specific, patterns.Tissue specificity becomes more pronounced at laterstages of embryogenesis, and may reflect roles in celldetermination or differentiation (Holland & Hogan,1988£>). The theme that dominates homeobox geneexpression during vertebrate development, however,is a restriction to spatial domains, primarily along theanteroposterior body axis. These axial domains have

been described in the CNS, PNS, somitic mesodermand visceral organs (Holland & Hogan, 1988b). It istempting to speculate that these patterns of ex-pression reflect roles of homeobox genes in thespecification of axial position. This suggestion will bediscussed in the next section, with particular refer-ence to the control of spatial organization in the head.It is also interesting that many of the tissues in whichthese domains have been described are segmented. Itwill therefore be useful to consider if the patterns ofhomeobox gene expression correlate in any way withsegmentation, and whether segmentation is linked tothe control of positional specification.

Spatial organization of the head

Development of the vertebrate head involves com-plex tissue interactions, cell migrations and morpho-genesis. Nevertheless, the basic spatial organizationof the head can be traced to anteroposterior specializ-ations of the major axial structures (including theCNS, mesoderm and neural crest). Furthermore, thedevelopmental control of these axial specializationsmay be less complex than first assumed, since it ispossible that the cells of only some structures will beintrinsically specified in terms of their axial position,while others rely on extrinsic positional cues. Theexistence of intrinsic positional specification can beinvestigated by experimental manipulation, but itsmolecular basis is more elusive. To assess if homeo-box genes may be involved in this process, theexperimental evidence for positional specification willbe considered in relation to the expression patterns ofvertebrate homeobox genes. This analysis will con-sider, in turn, the CNS, the mesoderm and the neuralcrest.

Many classical experiments have demonstratedthat regions of the embryonic brain are involved inseveral important tissue interactions during cranio-facial development (reviewed by Jacobson, 1966;Balinsky, 1981; Nieuwkoope/a/. 1985). Furthermore,analysis of mutant mice (Hogan et al. 1986 and thisvolume) and cats (Noden & Evans, 1986) has sugges-ted that anteroposterior differences within the em-bryonic brain are critical for tissue interactions duringcraniofacial development.

In view of this conclusion, it is particularly interest-ing that different homeobox genes are expressed incharacteristic anteroposterior regions of embryonicbrain tissue. For example, at 12-5 days post coitum,mouse En-2 is expressed in a band of cells within themesencephalon and metencephalon (Davis et al.1988), Hox 1.5 expression in the CNS extends pos-teriorly from a boundary in the anterior myelen-cephalon (Fainsod et al. 1987; Gaunt, 1987), whilst

Homeobox genes and the vertebrate head 19Pciruxial mesoderm CNS Homeobox gene expression in CNS

En-1

En-2

Hox 1.5

Hox 2.1

Fig. 1. Spatial relationship between the domains ofhomeobox gene expression in the CNS and theories ofsegmentation in the paraxial mesoderm and CNS.Somitomeres in the paraxial mesoderm are numbered(Meier, 1982; Jacobson & Meier, 1984, 1986). Numbers inthe CNS refer to the neuromeres of the mouse embryodescribed by Sakai (1987), although their relationship tothe metencephalon-myelencephalon boundary isuncertain. The axial limits shown for homeobox geneexpression in the CNS are approximate (see text forreferences), cs, cervical somite; d, diencephalon; ms,mesencephalon; mt, metencephalon; my,myelencephalon; op, optic vesicle; os, occipital somite;oi, otic vesicle; t, telencephalon.

for Hox 2.1 the anterior boundary is in the posteriormyelencephalon (Holland & Hogan, 1988a; Fig. 1).Mouse En-1 has the most extensive expressiondomain yet detected, covering most of the developingbrain and spinal cord (M. Frohman and G. Martin,personal communication; Fig. 1). These patterns ofexpression are consistent with a role for homeoboxgenes in the specification of axial position in thebrain. A plausible model is that expression of aspecific set of homeobox genes may act as a molecularcode for anteroposterior position, intrinsic to cells ofthe CNS. This suggestion has been made by severalauthors, usually with specific reference to the spinalcord (for example, Toth et al. 1987; Utset et al. 1987;Holland & Hogan, I988a,b).

Present evidence suggests that the mesoderm of thehead may be regionally specified via more indirect

20 P. W. H. Holland

means than is the CNS. For example, transplantationexperiments suggest that the spatial arrangement ofmany mesoderm-derived voluntary muscles of thehead is not intrinsic to the muscle precursors, but iscontrolled by the neural crest-derived connectivetissue (Noden, 1983, 1984). Hence it is tempting tospeculate that at least some cells from the cranialparaxial mesoderm do not have an intrinsic molecularcode for position. In this context, it may be significantthat no homeobox gene expression has yet beenreported in cranial mesoderm derivatives.

The mechanisms that control the spatial organiz-ation of neural crest derivatives are poorly under-stood. This is particularly so in the head region,where neural crest cells contribute to cranial ganglia,skeletal elements and much connective tissue. Theexperimental evidence that is available, however,does indicate that intrinsic specification occurs in atleast some cranial neural crest. In a particularlyinformative experiment performed by Noden, thepremigratory neural crest which would normallypopulate the first branchial arch was transplanted inplace of presumptive second arch crest. This resultedin a duplication of first arch skeletal elements, indi-cating that the spatial pattern of morphogenesis wasintrinsically specified in the premigratory neural crest(Noden, 1983, 1984). Further evidence for intrinsicspecification derives from description of a mutation inthe Burmese cat, in which the inherited facial abnor-malities are consistent with incorrect spatial program-ming of presumptive frontonasal neural crest cells(Noden & Evans, 1986). For this population of cells,it is suggested that final specification of spatial charac-teristics occurs during migration over the prosen-cephalon, rather than prior to crest cell dispersal.

In view of the suggestion that vertebrate homeoboxgenes have roles in region-specific spatial program-ming, it may be expected that neural-crest-derivedcranial mesenchyme would express combinations ofthese genes. However, although incomplete, theanalyses carried out to date have not revealed suchexpression. It may be speculated, therefore, thattransient expression may be sufficient for specifi-cation, or that homeobox genes are not the onlymolecular codes for position.

However, before it is concluded that homeoboxgenes have no role in the specification of neural crestcell fate, it is worth considering the expression ofhomeobox genes in sensory ganglia. Several homeo-box genes are expressed in the spinal ganglia associ-ated with a characteristic set of segments (forexample, Hox 1.1 is expressed in spinal gangliaposterior to the second cervical segment; K. Mahon,personal communication). Due to embryological andfunctional differences, it is unclear whether cranialganglia represent an anterior extension of the seg-

mental series of ganglia in the trunk (Goodrich, 1958;Le Douarin, 1986). Nevertheless, it is interesting thatthe patterns of homeobox gene expression seen incranial ganglia are consistent with these being acontinuation of the spinal ganglia series. Forexample, at 12-5 days post coitum, mouse Hox 2.1 isexpressed in spinal ganglia (including the first cervi-cal) and in the inferior ganglion of the Xth cranialnerve (the nodose ganglion), but not in more anteriorcranial ganglia (Holland & Hogan, 1988a; Graham etal. 1988; Fig. 2). Other mouse homeobox genesexpressed in cranial ganglia include Hox 2.6 (A.Graham, personal communication) and En-1 (M.Frohman and G. Martin, personal communication).

The spinal ganglia, as well as some cells of thecranial ganglia, are derived from neural crest (LeDouarin, 1986). It is therefore a possibility thathomeobox genes have roles in coding for positionalidentity in at least some neural crest derivatives.However, enthusiasm regarding this suggestion mustbe restrained until the expression in cranial ganglia isbetter characterized with respect to cell-type speci-ficity and timing of onset. Furthermore, recent trans-plantation experiments of trunk neural crest haverevealed no evidence for stable regional specificationin this population of cells (Lim et al. 1987).

Cranial segmentation

The definition of segmentation has often been thesubject of debate (discussed by Hyman, 1951). Theterm is usually used to describe a body plan based onthe serial repetition of morphological units along theanteroposterior body axis. In the trunk region ofvertebrate embryos, a coincident segmental pattern isshared by several structures, primarily the paraxialmesoderm (somites or their derivatives), intermedi-ate mesoderm (nephrotome), spinal cord and periph-eral nervous system (Hogan et al. 1985; Keynes &Stern, 1985).

The nature of segmentation in the head is less clear,and certainly more complex. It is helpful to considerthe head as comprising at least four categories ofrepeating structure; namely the paraxial mesoderm,neuromeres of the brain, cranial ganglia and bran-chial arches. Several important questions relate tothese repeated cranial structures. Have they evolvedfrom a single segmental series, or have separate seriesbeen superimposed? Are any metameric units in thehead continuous with those in the trunk? How manysegments comprise each series and what are theirfates? Are the different segmental series develop-mentally interdependent?

The segmental nature of the branchial arches hasoften been debated. Most classical models of head

Homeobox genes and the vertebrate head 21

Fig. 2. Expression of Hox 2.1 RNA in the nodose ganglion, revealed by in situ hybridization. (A) Section through thecervical region of a 12-5 days post coitum mouse embryo hybridized with a Hox 2.1 antisense probe and photographedunder bright-field illumination. (B,C) Higher magnification of the nodose ganglion from another hybridized section,photographed under bright-field (B) and dark-ground (C) illumination. Bars, 100^m. bv, blood vessel; fv, fourthventricle; hn, hypoglossal nerve; mn, mandible; ng, nodose ganglion; ot, otic vesicle. From Holland & Hogan, 1988a.

segmentation consider the branchial arch series to becoincident with mesodermal and neuronal segmen-tation (de Beer, 1937; Goodrich, 1958). However,such a view is inconsistent with the observed varia-bility in branchial arch number and their time ofdevelopment (Meier, 1982). Furthermore, compari-son between the gill structure of several vertebratesstrongly suggests that branchial arches evolved sec-ondarily to mesodermal segmentation (Mallat, 1984).

Perhaps the most important questions relate tosegmentation in the cranial mesoderm and centralnervous system. Scanning electron microscopy ofembryos from several vertebrate groups has revealedthat the anterior paraxial mesoderm is patterned intoa series of paired units, called cranial somitomeres(reviewed by Meier, 1982; Jacobson & Meier, 1986).These are often considered to be part of the samemetameric series as the somites of the trunk, withwhich they share several morphological and develop-mental features. However, it should be realized thatserial homology between somites and somitomereshas not been proven; thus the number of mesodermalsegments anterior to the first somite is uncertain.

Furthermore, in many vertebrates the most anteriortrue somites also contribute to head structures; theprecise number of these occipital segments beingvariable between species (de Beer, 1937). Therefore,the number of cranial mesodermal segments willdepend on both the number of occipital somites andthe number of segments in the cranial somitomericregion.

Representative species of mammals, birds, reptilesand fish have been found to have seven somitomeresanterior to the first distinct somite, whilst amphibiaonly have four (Jacobson & Meier, 1984, 1986). Oneexplanation for this difference is that a secondarydoubling up of anterior segments has occurred duringthe evolution of the amphibia. To evaluate thishypothesis, however, it will be necessary to investi-gate the somitomeric organization of many morevertebrates and to elucidate the precise fate ofsomitomeres and anterior somites in differentspecies. This latter information, to date only availablefor the chick (Noden, 1984), is critical if homologoussegments are to be recognized between species.

In the absence of fate-mapping data, the positional

22 P. W. H. Holland

relationship of somitomeres to neuromeres and sen-sory capsules has been used to draw comparisonsbetween species (Jacobson & Meier, 1984). However,this is only valid if there is a consistent developmentalrelationship between somitomeres and neuronal seg-mentation. Such a relationship is certainly plausible,since, in the mouse, chick, turtle and newt, thecranial somitomeres develop directly adjacent to theprimary neuromeres of the brain (Meier, 1982; Jacob-son & Meier, 1984, 1986; Fig. 1). Nevertheless, sincethe pattern of neuromeres in the brain is dynamic andcomplex (Sakai, 1987; Fig. 1), it is still unclearwhether neuronal segments and mesodermal seg-ments are part of a coincident metameric series. As aresult, the number of segments in each series isuncertain.

In Drosophila, metameric boundaries have beenrevealed by the patterns of expression of severalsegmentation genes, including the homeobox genesfushi tarazu, even-skipped and engrailed (Lawrence etal. 1987). However, a comparable approach cannotbe taken in vertebrates at present, since none of theknown vertebrate homeobox genes are expressedwith segmental periodicities along the entire length ofthe body. In addition, although several are expressedin the developing brain, no expression has yet beenreported in the segmented cranial paraxial mesoderm(see previous section).

Leaving aside the question of segment numbers, itis also important to ask whether the segmental unitsin the head have any developmental role. In otherwords, do regional specification mechanisms actwithin segmental boundaries? In Drosophila, analysisof homeotic mutant embryos and expression patternsof homeotic genes suggests that several genes control-ling anteroposterior specialization do respect meta-meric boundaries (Lewis, 1978; Martinez-Arias &Lawrence, 1985; Martinez-Arias et al. 1987). Thesegmentation gene fushi tarazu has a role in thisinteraction, being essential for the correct expressionof the homeotic genes Antp, Ultrabithorax and Sexcombs reduced (Ingham & Martinez-Arias, 1986).However, in the unsegmented common ancestor ofinsects and vertebrates, regional specialization musthave been controlled in the absence of metamerism,and hence there is no a priori reason for assuming alink between these processes in vertebrates.

One approach to investigate whether a link doesexist between segmentation and regionalization isbased on analysis of the vertebrate homeobox genes.In the previous section, it was argued that thepatterns of homeobox gene expression during ver-tebrate embryogenesis are consistent with thesegenes having roles in specifying positional valuesalong the anteroposterior body axis. Hence, if theaxial limits of homeobox gene expression consistently

coincide with metameric features, this would suggestthat positional specification in vertebrates does actwithin segmental boundaries. At present, this analy-sis cannoi be applied to the segmented cranial meso-derm, since, as previously mentioned, no homeoboxgene expression has been detected in paraxial meso-derm anterior to the cervical region. However, sev-eral expression boundaries do occur within the devel-oping brain (Dony & Gruss, 1987; Fainsod etal. 1987;Gaunt, 1987; Toth et al. 1987; Davis et al. 1988;Graham etal. 1988; Holland & Hogan, 1988a; Sharpeet al. 1988). The boundaries of expression reported todate do not correlate with any gross subdivisions ofthe brain (prosencephalon, mesencephalon, meten-cephalon, myelencephalon). For example, the mouseEn-2 gene is expressed in the posterior mesencepha-lon and anterior metencephalon at 12-5 days postcoitum (Davis et al. 1988). However, since theseregions are further subdivided into neuromeres(Sakai, 1987), a correlation with segmental bound-aries cannot be ruled out. Many homeobox geneshave anterior limits of expression in the myelen-cephalon, but for at least one gene (Hox 7.5) theboundary does not coincide with a constriction be-tween neuromeres (Gaunt, 1987). Although no directcorrelation has been revealed between segmental andexpression boundaries, it is clear that several geneswill need to be examined in detail if any consistenttrend is to be revealed.

Conclusions

Vertebrate homeobox genes are candidates for regu-latory genes involved in the control of embryonicdevelopment. By analogy with the fruitfly Dros-ophila, specific hypotheses have been proposed forthe role of homeobox genes, including potential rolesin anteroposterior regional specification or in seg-mentation. These hypotheses can be evaluated, andmodified, by consideration of the patterns of ex-pression of vertebrate homeobox genes.

A universal feature of the genes so far analysed isthat, within a given tissue, expression is restricted toprecise anteroposterior domains, characteristic foreach gene. Since the axial limits of expression aredefined by position, not cell type, the patterns areconsistent with homeobox genes having roles in thecontrol of regional specification. Axially restrictedpatterns of expression have been described in theCNS, PNS and mesoderm; hence it is possible that allthese tissues utilize homeobox genes for specifyinganteroposterior position. In the head, however, re-gion-specific domains have only been detected to datein the CNS and the cranial ganglia. This may indicatethat intrinsic and stable regional specification occurs

Homeobox genes and the vertebrate head 23

only in the CNS and certain neural crest cells.However, firm conclusions must clearly await furtheranalyses.

No vertebrate homeobox genes have been found tobe expressed with segmental periodicities, thus thereis no evidence that these genes control vertebratesegmentation. At present, therefore, studies of thesegenes cannot accurately reveal the number and ar-rangement of cranial segments. Analyses of ex-pression should, however, give insight into the poss-ible developmental role of cranial segmentation. Forexample, it should be possible to assess if segments,or groups of segments, act as developmental units ofregional specification.

I thank M. Frohman, A. Graham, K. Mahon and G.Martin for communicating data prior to publication, and B.Hogan, P. Ingham, J. Slack and P. Thorogood for helpfuldiscussions.

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