f’larenta (1992), 13, 513-522

Current Topic: Hex Genes: Losses, Gains and Targets

P. LOUISE COLETTA, SEBASTIAN M. SHIMELD & PAUL T. SHARPE .+folecular

Emb yoloa Group, Department of Cell and Structural Biology, Stopford Building, University of Manchester, Manchester 12113YPT, UK

It is now 8 years since the discovery of the homeobox, a highly conserved DNA sequence motif first identified in Drosophila homeotic genes and subsequently shown to encode a helixturn-helix DNA binding domain (McGinnis et al, 1984; Scott and Weiner, 1984; Kissinger et al, 1990). Since then, the homeobox has been identified in a wide range of genes from both invertebrates and vertebrates. In the mouse at least 35 Hox genes have been isolated which contain a homeobox showing greatest sequence homology with that of the Drosophila Antennapedia @ntp) gene (Scott, Tamkun and Hartzell, 1989). These are arranged in four clusters, Hox-1 to -4, each located on a different chromosome (Figure 1). The arrangement of the genes in the clusters resembles that of the Drosophila HOM-C locus and the four Hox clusters are thought to have arisen by serial duplication of a single ancestral locus (Akam, 1989). Using in situ hybridization and reporter gene constructs in transgenic mice it has been shown that the Hox genes are expressed in spatially and temporally restricted domains along the anterior-posterior (A-P) axis during embryogenesis. From these experiments and by analogy with their Drosophila counterparts it has becomes widely accepted that the Hox genes play a role in specifying positional identity in the vertebrate embryo (for a review see McGinnis and Krumlauff, 1992). However, how this specification is achieved by a family of transcription factors whose target genes are mostly unknown, remains a mystery. It is the aim of this review therefore to assess the role of Hox genes in embryonic development and to address the question of ‘What do homeobox gene products do?’ To answer this question we have considered the affect of Hox gene expression on two different but related aspects of development namely on embryonic phenotype and on cell biology and speculated as to how these effects are related to Hox gene function.

HOX GENE AND EMBRYONIC PHENOTYPE: LOSSES AND GAINS In the mouse, information relating to Hox gene expression and embryonic phenotype has come from the analysis of mutant embryos in which expression has been disrupted either by deletion or by overexpression of a given gene. To date, only three Hox genes, two from the Hox-1 cluster and one from the Hox-3 cluster, have been functionally deleted via 0143-4004/92/060513

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Figure 1. The murine Antp-like Hox genes are organized in four clusters, Hox-1, -2, -3 and -4. As shown in the

diagram, the four clusters can be aligned such that genes of the most similar sequence identity (paralogues) occupy the same relative position in each cluster. The Hox-1.4 paralogue group members are shown in the dashed box. This alignment also extends to the Drosophilahomeotic genes (HOW6) such that genes encoding homeodomains most similar to, for instance, that of the Lkjkmed (DjZ) gene are positioned under Dfd. This suggests that the Hox complexes have been produced by duplication of a single ancestral cluster present before the divergence of the arthropod and vertebrate lineages. All the genes within a Hox cluster are transcribed in the same 5’to 3’direction. The more 5’a gene in the cluster, the more posterior its embryonic expression domain (colmearity), thus 5’genes are often referred to as posterior genes and 3’genes as anterior genes. Abbreviations used are Abd-B, AbdomittaZ-B; abd-A, abdominal-A; Ubx, Ultrabithorax;Antp, Antennapedia; scr, sex combs reduced; Dfd, Deformed; pb, proboscipedia; zenl/zen2, zerkniillt I and 2; lab, labial.

homologous recombination in pluripotent embryonic stem (ES) cells (Chisaka and Capecchi, 1991; Lufkin et al, 1991; Chisaka, Musci and Cappechi, 1992; Le Mouellic, Lallemand and Brtilet, 1992). The Hox-3.1 null mutant, unlike the Hox-I.5 and -1.6 mutants, was created by replacing the endogenous gene with the 1ucZgene thus allowing expression of the targeted gene to be monitored. Deletion of both the Hox-1.5 and Hox-1.6 genes result in spatially restricted developmental defects in embryos homozygous for the mutant locus, although heterozygotes appear normal. The defects do not result in a transformed or homeotic phenotype (a mutation in which a body structure is replaced by one appropriate to a different axial level) as seen in some loss-of-function mutants in Drosophila. Hox-2.F mutants exhibit several defects in the thoracic region including reduction of the thyroid and submaxillary tissue and loss of the thymus and parathyroid gland. In addition to these defects, Hox-Z..5- mutants frequently show craniofacial abnormalities and defects of the heart and arteries. A comparison of the Hox-1.5- mutant phenotype with that of the Hox-1.C mutant shows that deletion of these Hox genes, from different paralogous groups, results in different defects. Hox-Z.6- mutants have defects of the outer, middle and inner ear, in specific hindbrain nuclei and in the cranial nerves and ganglia (L&kin et al, 1991; Chisaka, Musci and Cappechi, 1992). Lufkin et al (199 1) also reported delayed neural tube closure. Both the Hox-1.5- and -2.6- mutant phenotypes exhibit defects which originate at the level of the

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embryonic hindbrain, corresponding to the most anterior domain of expression of the genes. Disruption in the Hox-1.6- embryo occurs slightly more anterior to that in the Hox-1.T mutant, a phenomenon consistent with the rule of colinearity whereby the more 3 ’the gene in a cluster, the more anterior its boundary of expression (Gaunt, Sharpe and Duboule, 1988). The phenotypes of these loss-of-function mutants suggest that it is the anterior boundary of Hox gene expression, at least in the hindbrain, which is significant in determining axial coordinates and that posterior expression domains are relatively unimportant or indeed nonfunctional in this mechanism. Both Hox-1.5 and 1.6 are expressed in more posterior tissues to those affected in the mutant phenotypes. Why deletion of these Hox genes only manifests at their most anterior boundary of expression is not clear. However, the phenotypes of the deletion mutants can be interpreted as further evidence of a role for Hox genes in specifying positional identity. In contrast to the Hox-I.5- and -1.6- mutants described above, deletion of the more posterior gene, 110x-3. I, does result in a transformed phenotype. The most striking effect is the phenotypic alteration of the first lumbar vertebrae (Ll) to that of an extra thoracic vertebra (T14) complete with ribs, a change consistent with an anterior transformation. Hox3. I- mice also show an additional sternebra between the 6th and 7th ribs and often the 8th rib touches or is fused with the sternum. Unlike the Hox- 1 mutations, the phenotypic changes arising from deletion of the Hex-3. I gene do not occur at the most anterior boundary ofHox3.1 expression as determined by in situ hybridization or by expression of the targeted IQCZ reporter gene. Expression of Hox-3.2 has not been previously detected at the level of the lumbar vertebrae, where the major transformation occurs. The expression of the IucZ reporter which replaced the endogenous Hox-3.Z gene was identical in heterozygotes and homozygotes. This suggests that it is the identity of cells rather than their position which is altered by deletion of the Hox-3.1 gene, a fact which is not known for the Hox- 1 null mutants. The advantages of gene deletion using a functional marker gene, as opposed to targeted disruption of a gene, are therefore significant. A transformed embryonic phenotype has also been reported for a gain-of-function mutant. Ectopic expression of Hox-2.1 in transgenic mice results in the formation of the proatlas, a posterior transformation of the last occipital somite (Kessel, Balling and Gruss, 1990). In addition to vertebral defects, mutant mice exhibit extensive craniofacial abnormalities and die shortly after birth (Balling et al, 1989). Phenotype transformations along the vertebral column can also be induced by exposure of embryos to retinoic acid (RA) (Kessel and Gruss, 1991). Teratogenic doses of RA administered at day 7 of gestation results in anterior shifts of the spatial domains of Hox gene expression with posterior transformations of vertebrae along the entire A-P axis. Mice dosed at 8.5 days post coitum produced embryos with caudal defects only, suggesting a narrow window for anterior sensitivity to RA. These results have been interpreted to relate the spatial domains ofHoxgene expression to specification of positional identity. Kessel and Gruss (1991) suggested that it is the combination ofHox genes expressed in a vertebral segment and presumably therefore in each cell within that segment, that provides a Hox code which contains the information for specification along the A-P axis. Thus, alteration of the Hox code in a given segment results in a different segment identity. An alternative hypothesis to that of the combinatorial Hox code postulates that it is not the combination ofHox genes that encodes positional identity but that it is the most posteriorly expressed Hox gene i.e. most 5’ in a cluster, that is functional in encoding positional identity (posterior prevalence rule; Duboule, 199 1). The posterior prevalence rule can also be viewed as producing a Hox code via the expression of single genes rather than a combination of genes.

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The phenotypes of loss- and gain-of-function mutants can be explained in terms of both the combinatorial Hox code and the posterior prevalence rule (Figure 2). However, as all three null mutant phenotypes arise from disruption of a discrete region of the embryo rather than a single segment, it could be argued that the deletion experiments support the

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combinatorial Hox code theory as different posterior genes are expressed in the segments within the region. Alternatively, it is possible that the mechanism for specifying positional identity involves two or more codes (possibly a combinatorial and posterior prevalence code) that not only specifies the identity of the complete segment but also provides information to the cells on their individual identities and therefore cell type. This is supported by the fact that the three null mutations created to date have resulted in defects affecting different cell types. In addition, the 1acZ expression detected with the Hox-3. I deletion mutants formed a gradient of expression along sections of the spinal cord suggesting that there can be different levels of Hex gene expression within a segment. The elucidation of how Hox genes determine segment and or cell identities via a possible code awaits analysis of expression patterns at the single cell level. Both the combinatorial and posterior prevalence Hox code theories predict that deletion of a given Hox gene would result in a transformed phenotype in the hindbrain, a prediction which deletion of the Hox-1.5 and -1.6 genes has shown not to be true. One possible explanation for this could be that paralogue group members are able to partially compensate for the missing Hox gene resulting in a defective rather than a transformed phenotype. Alternatively, it is possible that deletion of the more 3’ genes in a cluster do not result in a transformed phenotype. In Drosophila deletion of the labial (lab), proboscipedia (pb) and Defomzrd (Dfd) genes, all located at the 3’ end of the HOM-C cluster, does not result in a transformed embryonic phenotype (Merrill, Turner and Kaufman, 1987, 1989; Pultz et al, 1988). This may reflect a complex mechanism by which development of the head region of the embryo is regulated. The fact that deletion of the more posterior Hox-3.1 gene results in a transformed phenotype supports this view. The generation of loss-of-function mutants for genes more 5’ in the Hox-1 cluster and for genes from the other clusters may resolve some of these questions. From the analysis of the dominant gain-of-function and loss-of-function mutant embryos generated so far it is evident that disruption of a single Hox gene produces mutant phenotypes consistent with a role for Hox genes in specifjiing the A-P axis, at least in the hindbrain and paraxial mesoderm. However, it is not known if the mutant phenotypes are a result of indirect disruption of other Hox genes or are a result of a direct effect on downstream target genes. To understand how these mutant phenotypes relate to Hox gene function in specifying axial coordinates it is necessary to understand how Hox gene expression affects individual cells within the embryo, i.e. in relation to the biology of the cell.

F&IW 2. The combinatorial and posterior prevalence Hox codes in the specification of segment identity. Both the combinatorial and posterior prevalence codes can be used to interpret the phenotypes of gain-of-function and lossof-function mutants in the mouse. In the diagrams labelled (a) to (c), anterior is at the top. (a) In the normal phenotype, three Hex genes (A, B and C in the diagram) specie three positions, 1,2 and 3. With the combinatorial Hox code position is determined by the combination of genes expressed, while with the posterior prevalence code, only the most posterior gene expressed at a particular level determines position. (b) In a gain-of-function mutant the domain of a Hax gene (How-B in the diagram) is artificially extended towards the anterior. Both the combinatorial and posterior prevalence codes predict that the anterior segment would adopt the positional identity of one more posterior (posterior transformation). (c) In a loss-of-function mutant a Hex gene (Hw-B in the diagram) has been deleted. Both theories predict that the segment which would normally adopt position 2 in the diagram will undergo an anterior transformation, that is adopt the identity ofposition 1. Using the posterior prevalence code, the segment that would normally develop at position 3 would be unaffected as the expression ofHox-C in the diagram would not be disrupted by the absence of Hex-B. The combinatorial Hox code, however, predicts that deletion ofHox-B would disrupt the Hox code in more posterior segments (position 3 in the diagram).

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HOX GENES AND CELL BIOLOGY:

TARGETS

For normal development to proceed, cells in the early vertebrate embryo must be provided with information which results in cells ‘knowing’their position relative to the A-P axis. This information must then be interpreted to provide cells with the correct identity for those axial coordinates. In Drosophila, where the embryo is progressively subdivided into small units (parasegments), the specification of segment identity has been shown to involve a cascade of transcription factors many of which contain a homeodomain. No such cascade has yet been identified in vertebrates, but as discussed previously it is thought to be the Hox code (either combinatorial or posterior prevalence) that specifies axial coordinates in the mouse embryo. Such a code, by virtue of the fact that Hox gene products are transcription factors, must be a nuclear code. This nuclear code must therefore be translated into an effect on cell biology that enables cells to differentiate according to their physical location. One way to determine how the expression ofHox genes affects cell fate would be to identify the genes that are switched on or off as a result of the information provided by the Hox code. The identification of such target genes would perhaps show how the expression of Hox genes relates to changes in cell biology and lead to an understanding of how positional identity is achieved. As yet the identification of downstream target genes in both invertebrates and vertebrates has proved onerous. Although interactions between homeodomain proteins and Hox genes, including auto regulation, have been demonstrated (for review see Dessain and McGinnis, 1991), very few targets that are not transcription factors have been identified. In vitro analysis of DNA binding sites of homeodomain proteins has shown that they are all capable of binding &elements with a core ATTA motif. The HOX4 homeoproteins have been shown to activate transcription in vivo via ATTA-related binding sites (Zappavigna et al, 1991). In Drosophila, on the basis of in vitro DNA binding, it has been suggested that a homeodomain protein could bind to several thousand sites in the Drosophilagenome (Laughon, Howell and Scott, 1988). Clearly the in vivo binding of homeodomain proteins must be much more specific. That this is the case is demonstrated by the fact that a temporal and spatial pattern of gene expression is obtained in transgenic mice using a reporter gene under the control of a weak promoter and three copies of an ATTA core cis-element (P. L. Coletta, unpublished observation). It is not known how this specificity ofbinding is achieved invivo but it is possible that it involves complex interactions of homeodomain proteins and other transcription factors. In Drosophila the cooperative interaction of homeodomain and zinc-finger proteins has been demonstrated in the activation and repression of transcription (Zuo et al, 1991). The in vitro analysis of homeodomain binding sites has therefore not proved useful in the identification of downstream targets of Hox genes. Indirect evidence of downstream target genes in Drosophilahas come from the study of the inductive interactions between the visceral mesoderm and endoderm in Drosophilamutants. It has been shown that a cascade of gene activity involving five genes is required for the expression of the lab gene, during which the Ultrabithorax (Ubx) and abdominal-A (abd-A) homeo-proteins activate the decapentaplegik(dpp) and wingless (wg) genes respectively (Immergliick, Lawrence and Bienz, 1990). Both dpp and wg encode putative extracellular proteins which are thought to regulate the expression of lab, another homeodomain protein, in the endoderm. In this case therefore, Hox genes are used to activate signalling molecules which regulate the expression of a Hox gene in a different germ layer. These interactions have yet to be confirmed at the level of specific DNA/protein interactions.

Colrttai’tal:

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Two in vivo methods for identifying target genes have been successfully employed in Drosophila. Gould et al (1990) used an immunopurification procedure to identify chromatin fragments from embryos that contain binding sites for the Ubx protein. Antibodies to the Ubx homeoprotein were used to detect the Ubx protein bound to chromatin. Using this method two transcripts were identified that are good candidates for direct regulation by Ubx, but as yet the identity of these genes has not been published. Such an approach, although successful in Drosophila, is unlikely to work in species with larger genomes. However, the identification of target genes for homeoproteins in Drosophila may enable the identification of homologous genes in vertebrates assuming that some of the targets for Hox genes, like the Hox genes themselves, are conserved between different phylogenetic groups. A second in vivo approach to identifying target genes in Drosophila utilizes the technology of enhancer traps. Enhancer trapping uses constructs in which the reporter gene 1ncZ is placed under the control of a weak promoter and then randomly integrated into the genome. The expression of the reporter gene is then regulated by elements surrounding the integration site. Thus, the enhancer trap often reflects part or all of the expression pattern of one or more genes. Wagner-Bernholz et al (1991) have used this approach to identify downstream targets of the Antp protein. By comparing the expression patterns of enhancer traps in mutant flies in which Antp is either ectopically expressed or deleted, they have identified a new- gene spalt major (salm) which is thought to be negatively regulated by Antp. Although similar experiments are now technically feasible in the mouse they would be extremely difficult and time consuming. Obviously alternative strategies are required for the identification of downstream targets for Hox genes in the mouse. We have devised such a strategy based on the hypothesis that one class of target genes for homeoproteins must be genes that encode cell surface molecules. This hypothesis is based on the fact that the cell surface is central to almost all developmental processes including cell migration, cell adhesion and cell-cell signalling. It is possible, therefore, that the cell surface is involved in specifying positional identity and that cell surface markers reflect the Hox code of a giv-en cell thereby providing a cellular as opposed to a nuclear code of positional identity. This view is supported by recent results which show that the Hox-2.4 and -2.5 homeoproteins can modulate transcription from the promoter of the N-CAM gene in vivo (Jones et al, 1992a). In addition, it has been shown that the mouse homeobox-containing gene, EZX-1, can activate the cytoactin gene promoter, an extracellular matrix molecule which affects cell shape, division and migration (Jones et al, 1992b). To determine whether or not cell surface changes can be mediated by Hox gene expression we have used a technique capable of detecting changes in cell surface molecules in response to ectopic expression of a given Hox gene. Multiple aqueous two phase partition via thin layer countercurrent distribution (TLCCD) can detect extremely subtle changes in cell surface properties regardless of the type of cell surface molecule involved (Sharpe, 1984). Thus it is not necessary to know the identity of cell surface molecules in order to detect changes in their expression. We have shown that ectopic expression of the Hox-3.3 gene in tissue culture cells results in changes at the cell surface as detected by TLCCD (Shimeld and Sharpe, 1992). The biochemical nature of the surface molecules involved is not yet known and is the next goal of this work. However this is the first experimental evidence to show that Hox gene expression can alter molecules at the cell surface. Using this information it is possible to make predictions as to how a Hox code could specie position via cell surface molecules (Figure 3). As shown in Figure 3 both the combinatorial and posterior prevalence codes can be used together as part of the same mechanism for specifying position. In this interpretation cells

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520 Target Hex gene Actlon genes _-___-____________-_-_---__-________ Hox-A

Lb

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respond to the combinatorial HOXcode by producing specific cell surface molecules. The nature of the surface molecules can then reflect posterior prevalence of each Hex gene specifying a different cell surface molecule. One interesting question that arises from this hypothesis is whether paralogues expressed in the same A-P domains are expressed in the same cells within a given tissue. This is clearly a very important question particularly in relation to the interpretation of the phenotype of deletion mutants In addition to A-P expression domains each Hex locus has a characteristic dorsal-ventral (D-V) expression pattern in the developing nervous system (Gaunt et al, 1990). Thus paralogues have different D-V expression patterns showing that at a given A-P position cells are expressing different paralogues. It remains to be seen how close to reality such ideas are. What can be said with certainty is that homeobox gene products function as transcriptional regulators and as such must have target genes. IfHox genes do play a role in positional specification whether via a code or some other mechanism it is difficult to envisage their immediate target genes being extracellular signalling molecules. From our results on cell surface changes, we suggest that cells are given a positional value by their position specific surface molecules with which they can communicate with neighbouring cells so enforcing their positional identity.

Coletta et al: Hox genes

521 ACKNOWLEDGEMENTS

1Z’e thank The Wellcome Trust for funding (PLC) and Dr IMichael Kessel for critical reading of this manuscript,

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Wagner-Bemholz, J. T., Wilson, C., Gibson, G., &huh, R. & Gehring, W. J. (1991) Identification of target genes of the homeotic gene Antennapedia by enhancer detection. Genes and Development, 5,2467-2480. Zappavigna, V., Renucci, A., Izpisua-Belmonte, J-C., Urier, G., Peschle, C. & Duboule, D. (1991) HOX4 genes encode transcription factors with potential auto- and cross-regulatory capacities. EMB03otmal, 10,41774187. Zuo, P., Stanojevic, D., Co&n, J., Han, K., Levine, M. & Manley, J. L. (1991) Activation and repression of transcription by the gap proteins htwhback and Kriippel in cultured Drosophila cells. Genes and Development, 5, 254-264.