Homeobox genes and the vertebrate head

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 of embryonic development contain a characteristic sequence of DNA, known as the homeobox. Homeobox sequences are also present in a family of vertebrate genes, which may therefore have regulatory roles during vertebrate embryogenesis. In this article, data concerning the spatial patterns of vertebrate homeobox gene expression are discussed in relation to recent descriptive and experimental analyses of head development. It is concluded that the patterns of gene expression are consistent with homeobox genes having roles in anteroposterior positional specification within the developing brain and possibly the neural crest. The data are not clearly consistent with these genes having' direct roles in controlling the patterns of cranial segmentation, although further studies may reveal whether vertebrate segments are units of developmental specification. Key words: homeobox, vertebrate head, specification, segmentation. Introduction The vertebrate head is a complex assemblage of cranial specializations, involving the central and peripheral nervous systems, axial skeleton, musculature and connective tissue. Most of the morphological differences between vertebrates and other chordates relate to the organization of the head; its evolution can therefore be considered fundamental to the origin of the vertebrates (Gans & Northcutt, 1983). In addition to its evolutionary significance, the spatial complexity of the head has made it a challenging area for embryological investigations. Such studies have led to considerable progress being made in understanding cell fate, tissue interactions, morphogenetic movements, mechanical factors and the roles of extracellular matrix, in head development (for reviews see Meier, 1982; Noden, 1984; Jacobson & Meier, 1986; Thorogood, 1987). In contrast, little progress has been made towards understanding the genetic control of head development, particularly at the molecular level. In this paper, I wish to consider whether this understanding may be gained from analysis of a recently identified gene family, the vertebrate homeobox genes. Possible roles of these genes in vertebrate embryogenesis are discussed and data concerning patterns of homeobox gene expression in the head are reviewed. These data are discussed in relation to two fundamental aspects of head development, the control of spatial organization and the nature of cranial segmentation. Homeobox genes Genetic mutations that affect the establishment of correct spatial organization have been identified in a range of organisms, most notably in the fruitfly Drosophila. Many of the Drosophila genes identified by mutation have been cloned, and their expression analysed in normal and mutant embryos. Genes have been analysed that have critical roles in establishing the body axes, metameric patterning (segmentation genes) and regional specialization (homeotic genes). In many of these cases, the patterns of expression in normal embryos correlate with predictions of function made from descriptions of mutant embryos. Furthermore, these studies are leading to a detailed understanding of the genetic control of Drosophila embryogenesis (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 a conserved 183 base pair sequence, the homeobox, within their protein-coding region (McGinnis et al. 1984; Scott & Weiner, 1984). It has since become apparent that a homeobox is present in many essential Drosophila developmental genes, including at least six homeotic genes, five segmentation genes and a 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 the extensive Antennapedia (Antp)-\\ke class, the engrailed (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 the genomes of a range of segmented and unsegmented animals besides Drosophila, including other arthropods, annelids, molluscs, echinoderms, urochordates, cephalochordates and vertebrates (McGinnis, 1985; Holland & Hogan, 1986). This conservation between disparate taxa means that homeobox genes are candidates for genes controlling embryogenesis in a range of metazoa, including vertebrates. More specific hypotheses concerning the function of vertebrate homeobox genes have often been proposed from mutant phenotypes associated with Drosophila homeobox genes. For example, Slack (1984) proposed a role in anteroposterior positional specification, since homeotic genes can be considered to control this process in Drosophila. An alternative hypothesis is that vertebrate homeobox genes control segmentation, since several Drosophila genes involved in this process have homeoboxes, and since homeotic genes can act within metameric boundaries (Struhl, 1984). However, Drosophila homeobox genes are involved in several different, but interacting, embryonic events (Gehring, 1987), and the phylogenetic distribution of homeobox genes does not correlate with any specific developmental strategy (Holland & Hogan, 1986). At present, therefore, hypotheses concerning the roles of vertebrate homeobox genes must be based primarily on their patterns of expression. Over twenty homeobox genes have so far been identified in the mouse, the species that has received most attention among the vertebrates. Sequence comparisons indicate that most of these genes (given the prefix 'Hox') are related to the Antp-\\ke genes of Drosophila, whilst two (En-1 and En-2) are more closely related to engrailed. Other divergent vertebrate homeobox genes have been identified, but not extensively studied. The organization of the vertebrate homeobox genes is described in several recent reviews (Colberg-Poley et al. 1987; Fienberg et al. 1987; Martin er al. 1987). In situ hybridization has been used to analyse the patterns of expression of several homeobox genes during vertebrate embryogenesis. These experiments have revealed that the expression of each gene is localized to particular regions of the embryo. Gaunt (1987) has shown that these 'region-specific' patterns can be apparent from the onset of detectable expression, in the presomite embryo. Slightly later, at the early somite stage, transcripts are detected in the neurectoderm, with characteristic anterior, and sometimes posterior, limits for each gene. For example, the distribution of mouse Hox 2.1 RNA has an anterior limit within the presumptive myelencephalon (Holland & Hogan, 1988#), whilst mouse En-2 expression is limited to a more anterior band of neurectoderm (Davis et al. 1988). Several homeobox genes are also expressed in an anteroposterior spatial domain of the mesoderm at the early somite stage, although the limits of this expression are not necessarily coincident with those in the neurectoderm (Carrasco & Malacinski, 1987; Gaunt, 1987; Holland & Hogan, 1988a). The spatial pattern of expression at later stages has been analysed for at least twelve homeobox genes in the mouse (reviewed by Holland & Hogan, 1988b; Stern & Keynes, 1988) and one in Xenopus (Carrasco & Malacinski, 1987). All vertebrate homeobox genes studied to date are expressed in the central nervous system (CNS), as is also characteristic of many Drosophila homeobox genes (Doe & Scott, 1988). Transcripts from each gene are restricted to a characteristic anteroposterior region of the CNS, and several are also restricted along the dorsoventral axis. Many homeobox genes are also expressed in ganglia of the peripheral nervous system (PNS), in mesodermal derivatives, or in both. As with the CNS, this expression can often be described in terms of simple axial limits characteristic for each gene. This is most clear in the developing vertebral column, where several genes have overlapping domains of expression. The most anterior of these domains is that of Hox 1.5, which is expressed in all presumptive vertebrae, including the atlas and axis (Gaunt, 1987). Homeobox gene expression has not been reported in derivatives of paraxial mesoderm in the occipital region (where somites contribute to the skull) or in more anterior mesoderm. Thus vertebrate homeobox genes are expressed in region-specific, as well as tissue-specific, patterns. Tissue specificity becomes more pronounced at later stages of embryogenesis, and may reflect roles in cell determination or differentiation (Holland & Hogan, 1988 >). The theme that dominates homeobox gene expression during vertebrate development, however, is a restriction to spatial domains, primarily along the anteroposterior body axis. These axial domains have

been described in the CNS, PNS, somitic mesoderm and visceral organs (Holland & Hogan, 1988b). It is tempting to speculate that these patterns of expression reflect roles of homeobox genes in the specification of axial position. This suggestion will be discussed in the next section, with particular reference to the control of spatial organization in the head. It is also interesting that many of the tissues in which these domains have been described are segmented. It will therefore be useful to consider if the patterns of homeobox gene expression correlate in any way with segmentation, and whether segmentation is linked to the control of positional specification. Homeobox genes and the vertebrate head 19 Pciruxial mesoderm CNS Homeobox gene expression in CNS En-1 En-2 Hox 1.5 Spatial organization of the head Development of the vertebrate head involves complex tissue interactions, cell migrations and morphogenesis. Nevertheless, the basic spatial organization of the head can be traced to anteroposterior specializations of the major axial structures (including the CNS, mesoderm and neural crest). Furthermore, the developmental control of these axial specializations may be less complex than first assumed, since it is possible that the cells of only some structures will be intrinsically specified in terms of their axial position, while others rely on extrinsic positional cues. The existence of intrinsic positional specification can be investigated by experimental manipulation, but its molecular basis is more elusive. To assess if homeobox genes may be involved in this process, the experimental evidence for positional specification will be considered in relation to the expression patterns of vertebrate homeobox genes. This analysis will consider, in turn, the CNS, the mesoderm and the neural crest. Many classical experiments have demonstrated that regions of the embryonic brain are involved in several important tissue interactions during craniofacial development (reviewed by Jacobson, 1966; Balinsky, 1981; Nieuwkoope/a/. 1985). Furthermore, analysis of mutant mice (Hogan et al. 1986 and this volume) and cats (Noden & Evans, 1986) has suggested that anteroposterior differences within the embryonic brain are critical for tissue interactions during craniofacial development. In view of this conclusion, it is particularly interesting that different homeobox genes are expressed in characteristic anteroposterior regions of embryonic brain tissue. For example, at 12-5 days post coitum, mouse En-2 is expressed in a band of cells within the mesencephalon and metencephalon (Davis et al. 1988), Hox 1.5 expression in the CNS extends posteriorly from a boundary in the anterior myelencephalon (Fainsod et al. 1987; Gaunt, 1987), whilst Fig. 1. Spatial relationship between the domains of homeobox gene expression in the CNS and theories of segmentation in the paraxial mesoderm and CNS. Somitomeres in the paraxial mesoderm are numbered (Meier, 1982; Jacobson & Meier, 1984, 1986). Numbers in the CNS refer to the neuromeres of the mouse embryo described by Sakai (1987), although their relationship to the metencephalon-myelencephalon boundary is uncertain. The axial limits shown for homeobox gene expression in the CNS are approximate (see text for references), 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 posterior myelencephalon (Holland & Hogan, 1988a; Fig. 1). Mouse En-1 has the most extensive expression domain yet detected, covering most of the developing brain and spinal cord (M. Frohman and G. Martin, personal communication; Fig. 1). These patterns of expression are consistent with a role for homeobox genes in the specification of axial position in the brain. A plausible model is that expression of a specific set of homeobox genes may act as a molecular code for anteroposterior position, intrinsic to cells of the CNS. This suggestion has been made by several authors, usually with specific reference to the spinal cord (for example, Toth et al. 1987; Utset et al. 1987; Holland & Hogan, I988a,b). Present evidence suggests that the mesoderm of the head may be regionally specified via more indirect Hox 2.1

20 P. W. H. Holland means than is the CNS. For example, transplantation experiments suggest that the spatial arrangement of many mesoderm-derived voluntary muscles of the head is not intrinsic to the muscle precursors, but is controlled by the neural crest-derived connective tissue (Noden, 1983, 1984). Hence it is tempting to speculate that at least some cells from the cranial paraxial mesoderm do not have an intrinsic molecular code for position. In this context, it may be significant that no homeobox gene expression has yet been reported in cranial mesoderm derivatives. The mechanisms that control the spatial organization of neural crest derivatives are poorly understood. This is particularly so in the head region, where neural crest cells contribute to cranial ganglia, skeletal elements and much connective tissue. The experimental evidence that is available, however, does indicate that intrinsic specification occurs in at least some cranial neural crest. In a particularly informative experiment performed by Noden, the premigratory neural crest which would normally populate the first branchial arch was transplanted in place of presumptive second arch crest. This resulted in a duplication of first arch skeletal elements, indicating that the spatial pattern of morphogenesis was intrinsically specified in the premigratory neural crest (Noden, 1983, 1984). Further evidence for intrinsic specification derives from description of a mutation in the Burmese cat, in which the inherited facial abnormalities are consistent with incorrect spatial programming of presumptive frontonasal neural crest cells (Noden & Evans, 1986). For this population of cells, it is suggested that final specification of spatial characteristics occurs during migration over the prosencephalon, rather than prior to crest cell dispersal. In view of the suggestion that vertebrate homeobox genes have roles in region-specific spatial programming, it may be expected that neural-crest-derived cranial mesenchyme would express combinations of these genes. However, although incomplete, the analyses carried out to date have not revealed such expression. It may be speculated, therefore, that transient expression may be sufficient for specification, or that homeobox genes are not the only molecular codes for position. However, before it is concluded that homeobox genes have no role in the specification of neural crest cell fate, it is worth considering the expression of homeobox genes in sensory ganglia. Several homeobox genes are expressed in the spinal ganglia associated with a characteristic set of segments (for example, Hox 1.1 is expressed in spinal ganglia posterior to the second cervical segment; K. Mahon, personal communication). Due to embryological and functional differences, it is unclear whether cranial ganglia represent an anterior extension of the segmental series of ganglia in the trunk (Goodrich, 1958; Le Douarin, 1986). Nevertheless, it is interesting that the patterns of homeobox gene expression seen in cranial ganglia are consistent with these being a continuation of the spinal ganglia series. For example, at 12-5 days post coitum, mouse Hox 2.1 is expressed in spinal ganglia (including the first cervical) and in the inferior ganglion of the Xth cranial nerve (the nodose ganglion), but not in more anterior cranial ganglia (Holland & Hogan, 1988a; Graham et al. 1988; Fig. 2). Other mouse homeobox genes expressed 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 the cranial ganglia, are derived from neural crest (Le Douarin, 1986). It is therefore a possibility that homeobox genes have roles in coding for positional identity in at least some neural crest derivatives. However, enthusiasm regarding this suggestion must be restrained until the expression in cranial ganglia is better characterized with respect to cell-type specificity and timing of onset. Furthermore, recent transplantation experiments of trunk neural crest have revealed no evidence for stable regional specification in this population of cells (Lim et al. 1987). Cranial segmentation The definition of segmentation has often been the subject of debate (discussed by Hyman, 1951). The term is usually used to describe a body plan based on the serial repetition of morphological units along the anteroposterior body axis. In the trunk region of vertebrate embryos, a coincident segmental pattern is shared by several structures, primarily the paraxial mesoderm (somites or their derivatives), intermediate mesoderm (nephrotome), spinal cord and peripheral 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 consider the head as comprising at least four categories of repeating structure; namely the paraxial mesoderm, neuromeres of the brain, cranial ganglia and branchial arches. Several important questions relate to these repeated cranial structures. Have they evolved from a single segmental series, or have separate series been superimposed? Are any metameric units in the head continuous with those in the trunk? How many segments comprise each series and what are their fates? Are the different segmental series developmentally interdependent? The segmental nature of the branchial arches has often 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 the cervical region of a 12-5 days post coitum mouse embryo hybridized with a Hox 2.1 antisense probe and photographed under 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, fourth ventricle; hn, hypoglossal nerve; mn, mandible; ng, nodose ganglion; ot, otic vesicle. From Holland & Hogan, 1988a. segmentation consider the branchial arch series to be coincident with mesodermal and neuronal segmentation (de Beer, 1937; Goodrich, 1958). However, such a view is inconsistent with the observed variability in branchial arch number and their time of development (Meier, 1982). Furthermore, comparison between the gill structure of several vertebrates strongly suggests that branchial arches evolved secondarily to mesodermal segmentation (Mallat, 1984). Perhaps the most important questions relate to segmentation in the cranial mesoderm and central nervous system. Scanning electron microscopy of embryos from several vertebrate groups has revealed that the anterior paraxial mesoderm is patterned into a series of paired units, called cranial somitomeres (reviewed by Meier, 1982; Jacobson & Meier, 1986). These are often considered to be part of the same metameric series as the somites of the trunk, with which they share several morphological and developmental features. However, it should be realized that serial homology between somites and somitomeres has not been proven; thus the number of mesodermal segments anterior to the first somite is uncertain. Furthermore, in many vertebrates the most anterior true somites also contribute to head structures; the precise number of these occipital segments being variable between species (de Beer, 1937). Therefore, the number of cranial mesodermal segments will depend on both the number of occipital somites and the number of segments in the cranial somitomeric region. Representative species of mammals, birds, reptiles and fish have been found to have seven somitomeres anterior to the first distinct somite, whilst amphibia only have four (Jacobson & Meier, 1984, 1986). One explanation for this difference is that a secondary doubling up of anterior segments has occurred during the evolution of the amphibia. To evaluate this hypothesis, however, it will be necessary to investigate the somitomeric organization of many more vertebrates and to elucidate the precise fate of somitomeres and anterior somites in different species. This latter information, to date only available for the chick (Noden, 1984), is critical if homologous segments 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 sensory capsules has been used to draw comparisons between species (Jacobson & Meier, 1984). However, this is only valid if there is a consistent developmental relationship between somitomeres and neuronal segmentation. Such a relationship is certainly plausible, since, in the mouse, chick, turtle and newt, the cranial somitomeres develop directly adjacent to the primary neuromeres of the brain (Meier, 1982; Jacobson & Meier, 1984, 1986; Fig. 1). Nevertheless, since the pattern of neuromeres in the brain is dynamic and complex (Sakai, 1987; Fig. 1), it is still unclear whether neuronal segments and mesodermal segments are part of a coincident metameric series. As a result, the number of segments in each series is uncertain. In Drosophila, metameric boundaries have been revealed by the patterns of expression of several segmentation genes, including the homeobox genes fushi tarazu, even-skipped and engrailed (Lawrence et al. 1987). However, a comparable approach cannot be taken in vertebrates at present, since none of the known vertebrate homeobox genes are expressed with segmental periodicities along the entire length of the body. In addition, although several are expressed in the developing brain, no expression has yet been reported in the segmented cranial paraxial mesoderm (see previous section). Leaving aside the question of segment numbers, it is also important to ask whether the segmental units in the head have any developmental role. In other words, do regional specification mechanisms act within segmental boundaries? In Drosophila, analysis of homeotic mutant embryos and expression patterns of homeotic genes suggests that several genes controlling anteroposterior specialization do respect metameric boundaries (Lewis, 1978; Martinez-Arias & Lawrence, 1985; Martinez-Arias et al. 1987). The segmentation gene fushi tarazu has a role in this interaction, being essential for the correct expression of the homeotic genes Antp, Ultrabithorax and Sex combs reduced (Ingham & Martinez-Arias, 1986). However, in the unsegmented common ancestor of insects and vertebrates, regional specialization must have been controlled in the absence of metamerism, and hence there is no a priori reason for assuming a link between these processes in vertebrates. One approach to investigate whether a link does exist between segmentation and regionalization is based on analysis of the vertebrate homeobox genes. In the previous section, it was argued that the patterns of homeobox gene expression during vertebrate embryogenesis are consistent with these genes having roles in specifying positional values along the anteroposterior body axis. Hence, if the axial limits of homeobox gene expression consistently coincide with metameric features, this would suggest that positional specification in vertebrates does act within segmental boundaries. At present, this analysis cannoi be applied to the segmented cranial mesoderm, since, as previously mentioned, no homeobox gene expression has been detected in paraxial mesoderm anterior to the cervical region. However, several expression boundaries do occur within the developing brain (Dony & Gruss, 1987; Fainsod etal. 1987; Gaunt, 1987; Toth et al. 1987; Davis et al. 1988; Graham etal. 1988; Holland & Hogan, 1988a; Sharpe et al. 1988). The boundaries of expression reported to date do not correlate with any gross subdivisions of the brain (prosencephalon, mesencephalon, metencephalon, myelencephalon). For example, the mouse En-2 gene is expressed in the posterior mesencephalon and anterior metencephalon at 12-5 days post coitum (Davis et al. 1988). However, since these regions are further subdivided into neuromeres (Sakai, 1987), a correlation with segmental boundaries cannot be ruled out. Many homeobox genes have anterior limits of expression in the myelencephalon, but for at least one gene (Hox 7.5) the boundary does not coincide with a constriction between neuromeres (Gaunt, 1987). Although no direct correlation has been revealed between segmental and expression boundaries, it is clear that several genes will need to be examined in detail if any consistent trend is to be revealed. Conclusions Vertebrate homeobox genes are candidates for regulatory genes involved in the control of embryonic development. By analogy with the fruitfly Drosophila, specific hypotheses have been proposed for the role of homeobox genes, including potential roles in anteroposterior regional specification or in segmentation. These hypotheses can be evaluated, and modified, by consideration of the patterns of expression of vertebrate homeobox genes. A universal feature of the genes so far analysed is that, within a given tissue, expression is restricted to precise anteroposterior domains, characteristic for each gene. Since the axial limits of expression are defined by position, not cell type, the patterns are consistent with homeobox genes having roles in the control of regional specification. Axially restricted patterns of expression have been described in the CNS, PNS and mesoderm; hence it is possible that all these tissues utilize homeobox genes for specifying anteroposterior position. In the head, however, region-specific domains have only been detected to date in the CNS and the cranial ganglia. This may indicate that 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 further analyses. No vertebrate homeobox genes have been found to be expressed with segmental periodicities, thus there is no evidence that these genes control vertebrate segmentation. At present, therefore, studies of these genes cannot accurately reveal the number and arrangement of cranial segments. Analyses of expression should, however, give insight into the possible developmental role of cranial segmentation. For example, it should be possible to assess if segments, or groups of segments, act as developmental units of regional 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 helpful discussions. References AKAM, M. E. (J987). The molecular basis for metameric pattern in Drosophila embryos. Development 101, 1-22. ANDERSON, K. V. 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