Hox genes and embryonic development

Hox Genes and Embryonic Development BRUCE A. MORGAN Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Building 149, 13th Street, Charlestown, Massachusetts 02129

(Key words: hox gene, embryonic development, retroviral vectors, vertebrate limb) 1997 Poultry Science 76:96–104

only for the imposition of unique fate on those segments. In general, the order of genes in the chromosome is reflected in the relative position of their domains of expression along the anterior-posterior (A/ P) axis of the embryo. Most genes are expressed in several contiguous segments and the anterior extent of expression of a given gene frequently overlaps the posterior domain of the adjacent gene on the chromosome. As a result, most regions express more than one member of the hox complex, particularly in the posterior part of the embryo. However, under normal circumstances, the gene with a more posterior extent of expression is sufficient to specify fate in a segment in which it is co-expressed with a gene with a more anterior domain of expression. This phenomenon, referred to as phenotypic suppression or posterior prevalence, means that the phenotypic consequence of ectopic expression in anterior regions of the embryo is conversion of those regions to a more posterior fate (Gonzales-Reyes and Morata, 1990; Duboule, 1991). Conversely, loss of function mutations result in anterior conversions of segmental identity.

HOX GENES SPECIFY SEGMENT IDENTITY IN THE FRUIT FLY The hox genes were identified as a set of genetic loci in the fruit fly whose mutation led to homeotic conversion of structures found in one segment to those normally found in another segment. These genes were cast as master regulators of developmental programs— switches that initiated unique developmental cascades. This interpretation of the genetic analysis of these loci was supported by the discovery that these complexes encoded a group of transcription factors that were clearly related to one another (McGuinness et al., 1984; Scott and Weiner, 1984). In particular, the helix turn helix domain shared by all of the proteins encoded by these genes is highly conserved. This domain mediates the sequence-specific DNA binding of these transcription factors and was christened the homeobox. Multiple genes containing this motif are found in all phyla and are referred to as homeobox genes. Many have important developmental roles and mutations in that some lead to homeotic changes in the organism. However, I will restrict this discussion to the hox genes, those members of the originally (Scott, 1992) identified Homeotic complexes in the fly and their orthologues in other species. The striking feature of the Drosophila hox genes is their ability to specify the characteristic fate of given segments (reviewed in Manak and Scott, 1994). They are not required for the formation of segments per se, but

HOX GENES IN VERTEBRATES The hox genes are found in all animal genomes examined to date, and it has been proposed that they are a defining character for the zootype (Slack et al., 1993). The observation that they are expressed in overlapping domains along the A/P axis of the mouse embryo led to speculation that they played a similar role in the specification of the regional differences in mammalian embryos as well. To consider how much of the information gleaned from the function of hox genes in

Received for publication August 13, 1995. Accepted for publication August 20, 1996.

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mouse is well-suited to the study of their early function, that same function limits the utility of mutational analysis in the investigation of later functions. The use of retroviral vectors to alter gene expression in the chick embryo has emerged as an effective way to address these later functions. This paper reviews that approach and its application to the study of the hox genes in the formation of the vertebrate limb.

ABSTRACT The hox genes specify regional differences along the anterior-posterior (A/P) axis of the vertebrate embryo. This function appears to reflect an ancestral role of the hox gene complex and is conserved across phyla. During the evolution of vertebrates, this gene complex has been recruited to perform other functions as well, many of which occur later in development. Although mutational analysis in the

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homeodomains between members of a paralogue group, do they have distinct function within the cell, or do differences in their role in embryogenesis arise only by differences in their patterns of expression? These questions must be asked separately in the context of each of the roles of the hox cluster in embryogenesis. Below we consider them in the context of an early role, the specification of the primary axis, and a later role, mediation of the morphogenesis of the limb.

HOX GENES SPECIFY REGIONAL DIFFERENCES ALONG THE A/P AXIS Spatial colinearity is observed in the mouse. The hox genes are also expressed in overlapping domains along the A/P axis of the embryo whose order reflects the position of the genes along the chromosome (reviewed in Kessel and Gruss, 1990). In general, the anterior limit of expression is similar among paralogues, although the pattern of expression within a region may vary between paralogues and a precise registration between the domains of expression is not observed. The patterns of expression tend to confirm the proposal that an ancestral function of the hox genes was to specify transitions in the body plan. As shown in Figure 1, the last common ancestor of flies and mice appears to have had five hox genes. These underwent duplication in the two lineages independently. Paralogue Groups 6, 7, and 8 and the Ant, Ubx, and Abd-A genes all arose from a common precursor. It is unclear from sequence comparisons whether paralogue Group 5 and Scr arose from this precursor or the common precursor of Dfd and the paralogue Group 4. Paralogue Groups 9 through 13 and Abd-B all share a common precursor. It is intriguing that the borders of expression of these groups in the mouse correspond to major transitions in the body plan (Figure 2). The Group 4 and 5 paralogues are expressed in the cervical region, expression of the Group 6 paralogues mark the transition to the thorax, and members of paralogue Group 9 are expressed at the beginning of the lumbar region.

FIGURE 1. Evolution of the hox clusters. Evolution of the hox cluster is depicted based on sequence comparisons between the Drosophila hox genes and their orthologues in the primitive chordate Amphioxus as well as the hox clusters in birds and mammals. The last common ancestor of flies and men appears to have had five hox genes. The homologues of these genes are shown in the diverging lineages by the corresponding colors. The 3 and 5 paralogue groups have not been unambiguously assigned to a grouping. Their sequences suggest that they are derived from either the pb or Dfd class or the Dfd and Ant class, respectively. Amphioxus has a single hox gene cluster in which paralogues 1 through 10 have been identified. The more 5′ region of this cluster has not been examined and may contain additional genes. This cluster underwent serial duplication to generate the four clusters found in teleosts. Pictured below are the mouse hox gene clusters. All of these genes have been identified in the chicken and no additional members of the cluster have been reported to date (Nelson et al., 1996). FIGURE 2. Ancestral groupings reflected in expression domains. Shown at center is a schematic representation of the vertebral column of the mouse (below) and chick (above). The cervical (blue), thoracic (red), lumbar (yellow), sacral (purple), and caudal (light blue) vertebrae are shown. The expression domains of the hoxc cluster at equivalent stages in development are diagrammed for both species (after Burke et al., 1995). Note that the genes derived from the red ancestral gene are expressed in the thoracic region of both species, whereas genes derived from the blue and yellow ancestral genes exhibit anterior limits of expression which demarcate the cervical and lumbar regions respectively. Reflecting the different relative body plans, the anterior limits of expression of the hoxc genes are transported towards the caudal end of the somitic scaffold in the chick. The relative limits of expression for successive paralogues is also expanded or compressed to reflect the relative size of each region of the body plan.

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flies can be extrapolated to the experimentally less accessible vertebrates, we need to consider the role of the hox cluster in the last common ancestor of flies and humans, and the way that role has been refined independently since that time. Comparative analysis suggests that a primitive cluster of approximately five genes was present in the early stages of animal evolution (Schubert et al., 1993; and Figure 1). Homologues of genes in the two clusters found in flies are found in single cluster in other insects and the nematode Caenorhabditis elegans (Beeman et al., 1989, Kenyon and Wang, 1991). The cephalochordate Amphioxus has a single hox cluster as well, but gene duplications have increased the number of genes in the cluster to at least 10 and the ancestral chordate cluster may have contained a total of 13 genes (GarciaFernandez and Holland, 1994). In vertebrates, the entire cluster has undergone serial duplication to generate four clusters designated a through d. The corresponding genes in different clusters are referred to as paralogues and each gene is identified by its cluster letter and paralogue number (Scott, 1992; and Figure 1). Paralogues are more similar to each other than they are to adjacent genes within a cluster. They may differ by as little as a few amino acids in the homeodomain. Adjacent genes share considerable homology as well, but show more divergence. The expansion of the hox clusters during a time when they were apparently playing a fundamental role in specification of the primary axis raises a number of questions. 1) Assuming each gene in the ancestral cluster had a distinct role in specifying regional differences along the A/P axis, do all the genes derived from each ancestral gene retain this function, and is that function distinct from that of genes derived from another hox ancestor? 2) Within the group derived from a common ancestor, how much divergence in function has arisen and are novel roles superimposed on some basic function, or modifications of that basic function? This question can be asked at two levels, between paralogue groups, and within paralogue groups. Given the great similarity in

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Examination of hox gene expression in the chick confirms the significance of these expression patterns (Figure 2). The chicken and the mouse have different numbers of somites devoted to the major regions of the vertebral axis. The long neck of the chick incorporates 15

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cervical somites supported by a short thorax composed from 7 somites. In contrast, the mouse has only 7 cervical somites and 13 thoracic somites. To specify these transitions in morphology, expression of the hox genes must be transposed along the somitic scaffold.

FIGURE 3. Retrovirus mediated gene transfer in the chick. The RCAS replication competent retroviral vectors will accept up to 2.4 kb of novel coding sequence in the viral genome. These vectors retain the viral sequences necessary for virus production and will spread from cell to cell in the infected embryo. By choosing the time and position at which the virus is injected, different domains of infection, and therefore altered gene expression, can be achieved (Morgan and Fekete, 1996). To study hox gene function, the protocol shown in A was employed. A cluster of five injections were made in the lateral plate mesenchyme of a Stage 10 embryo that will give rise to the right leg bud 1 d later. By 3 d of incubation, the virus has spread throughout the limb but is largely restricted to the limb (B). The extent of infection can be assessed by whole mount in situ hybridization using a probe to the inserted sequences (C,D).

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MUTATIONAL ANALYSIS IN THE MOUSE The inferences based on expression data have been corroborated by mutational analysis in the mouse. Mutations in the 6 and 5 paralogues confirm a role for these genes in specification of the cervico/thoracic transition (Rancourt et al., 1995). In a similar fashion, ectopic expression of a 6 paralogue in the cervical region results in the formation of ribs anterior to the normal thorax (Jegalian and De Robertis, 1992). High level expression of an 8 paralogue in the lumbar region can also result in the formation of ribs in this region (Pollock et al., 1992, 1995). While demonstrating the thoracic character induced by the paralogue 8 genes, this experiment also shows that posterior prevalence represents a relative efficiency in competition with more anterior genes that can be overcome by high levels of expression. This mutational analysis has also suggested that successive paralogue groups specify progressively more posterior regions of the body. Within a region, paralogues cooperate in the formation of structures, the phenotype associated with the mutation of a single paralogue is less severe than that associated with the removal of several paralogues (Condie and Capecchi, 1994; Kostic and Capecchi, 1994; Davis et al., 1995; Horan et al., 1995). Overexpression studies suggest that paralogues have overlapping but partially distinct functions (Pollock, et al., 1995).

OTHER ROLES OF THE HOX GENES The hox genes are also expressed in the “secondary axes” of the embryo, the limbs and developing genitalia, as well as in many other developing tissues (Dolle et al.,

1989, 1991; Yokouchi et al., 1991). Many of these structures arose relatively late in evolution, and the hox genes may have been recruited to employ the same functions performed in the primary axis, or may have acquired entirely new functions. The analysis of the role of the hox genes in specification of the primary axis of vertebrate embryos is well suited to analysis in the mouse because of the applicability of gene disruption approaches to assay function. However, gene disruption has disadvantages for studying the later roles of a gene that is reused sequentially during development. For instance, the effect of a hox gene knockout on formation of the limb can reflect an alteration in the specification of the region along the primary axis that will give rise to the limb as well as a direct role in the formation of the appendage. Tissue-specific knockouts may prove informative in this regard, but they have not yet been applied to this question. The alternative approach of ectopic expression is often the most useful for gaining insights into later gene function. The chick embryo is well suited to the ectopic expression approach because the embryo is generally accessible to microinjection and convenient retroviral vectors for the chick have been developed (e.g., Hughes and Kosik, 1984, reviewed in Morgan and Fekete, 1996). Recombinant retroviruses can be used to ectopically express a gene at the time and place of choice (Figure 3 and Morgan and Fekete, 1996). This ectopic approach is proving to be a powerful technique for the study of gene function and has made it possible to combine the advantages of the chick embryo for experimental embryology with the powers of molecular genetic manipulation (e.g., Morgan et al., 1992; Riddle et al., 1993; Laufer et al., 1994; Yang and Niswander, 1994; Roberts et al., 1995). The hox genes represent a special challenge for the ectopic expression technique in that they are expressed in large contiguous domains early in development. Furthermore, they act within the cell in which they are produced, so that all the cells in a field must be infected to achieve a concerted response. However, by using replication competent vectors to take advantage of the spread of virus from cell to cell, domains of altered gene expression of comparable size to normal expression domains may be generated, and this approach has been used to analyze the role of the hox genes in limb morphogenesis.

HOX GENE EXPRESSION IN THE LIMB Members of all four clusters of hox genes are expressed in the chick limb. These expression patterns are described in detail elsewhere and the expression patterns of representative members of the hoxd and hoxa clusters are diagrammed in Figure 4 (IzpisuaBelmonte et al., 1991; Nohno et al., 1991; Yokouchi et al., 1991; Nelson et al., 1996). The role of these proteins was tested by altering their expression patterns following the

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This transposition is observed when the expression of the hox genes is examined in a stage 25 chick embryo (Burke et al., 1995). The expression of paralogue Groups 5 and 6 flank the cervical thoracic transition and are shifted eight somites toward the posterior end of the chick embryo relative to the mouse. In a similar fashion, members of the paralogue Group 9 are expressed at the transition from the thorax to the lumbar region in both species. Hence, relative to the mouse there is a posterior shift along the axis as well as a change in the relative position of the expression domains of the 6 and 9 paralogue groups (six somite interval vs nine somite interval). This correlation between the domains of expression of these groups of genes suggests that hox genes were functionally distinct in the five gene ancestral cluster and that this function is reflected in the descendants of those genes in the vertebrate lineage. Analysis of hox gene expression in the arthropod lineage yields similar conclusions (Averof and Akam, 1995). This finding would suggest that in specifying vertebral morphologies the hox genes are in part expressing a more basic function in specifying regional distinctions in the body plan that preceded the appearance of vertebrae in evolution.

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affected (Figure 5). Although this includes the bones affected by hoxd-11, it also includes several bones that were not. This observation also holds true for paralogues. Ectopic expression of hoxa-11 throughout the limb did not give the phenotypes observed when hoxd11 was ectopically expressed. Hence, paralogues have distinct activities in the limb as well.

INDEPENDENT REGULATION OF HOX EXPRESSION IN THE THREE SEGMENTS OF THE LIMB These ectopic expression experiments have demonstrated that the different hox genes expressed in the limb mediate morphogenesis of the appendage and that their complex patterns of expression are crucial to the normal genesis of the limb. We have employed these genes as markers to study the inductive interactions which organize their expression and direct the patterning of the limb bud. Examination of the normal patterns of expression of the hoxd genes suggests that discrete inductive interactions are responsible for patterning the three segments of the limb along the proximal distal axis; the stylopod (thigh), zeugopod (calf), and autopod (foot) (Figure 4). The recent identification of Sonic hedgehog as one of the signals mediating these inductions (Riddle et al., 1993) has allowed us to test this proposal directly (Nelson et al., 1996). Sonic hedgehog (SHH) is a signaling molecule expressed in the zone of polarizing activity (ZPA) that can induce mirror image duplications in the chick limb when expressed on the anterior margin of the limb bud. These mirror image duplications are presaged by the induction of the hoxd genes in a mirror image pattern in the anterior of the bud; SHH regulates the expression of the hoxd cluster (Riddle et al., 1993). Cells in the distal margin of the limb must receive both the SHH signal derived from the ZPA and fibroblast growth factor (FGF) derived from the apical ectodermal ridge to respond with the organized induction of hox gene expression (Laufer et al., 1994). During the initial stages of limb outgrowth when the stylopod is formed, SHH is not expressed (Laufer et al., 1994). hoxd-9 and -10 are expressed uniformly along the A/P axis of the limb, apparently in response to a signal distributed across the limb ectoderm (Figure 4). hoxd-11 through -13 do not respond to this signal and are not expressed. At Stage 18, SHH is expressed at the posterior margin of the limb bud and hoxd-11 through 13 are sequentially expressed in the posterior mesenchyme of the bud. Stage 18 is the period of development during which the zeugopod is formed. In this segment of the limb, the expression domains of hoxd-11, -12, and -13 are progressively restricted towards the posterior margin of the limb. During the final phase of limb development when the foot is being formed, SHH continues to be expressed at the posterior margin of the limb and hoxd-10 through -13 are expressed. However,

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protocol depicted in Figure 3. hox proteins normally expressed in restricted regions of the limb were expressed throughout the limb during much of its development. To date, this analysis has been restricted to the descendants of the 5′ gene in the ancestral cluster (paralogues 9 through 13), so the question of ancestral groupings has not been addressed. The first test of this approach examined the role of the hoxd-11 gene in limb formation (Morgan et al., 1992). It is normally expressed in the posterior half of the limb bud throughout development. Ectopic expression in the anterior half of the limb has a dramatic consequence that can be separated into sequential activities. The early expression of hoxd-11 in the anterior mesenchyme results in a reduction in the normal posterior bias in limb bud outgrowth and a change in the pattern of condensing skeletal elements such that the anterior digit has an additional phalange and resembles digit 2 (Figure 5). Persistent expression of hoxd-11 in the anterior half of the limb leads to a subsequent inhibition in the maturation and growth of bones in the region where hoxd-11 is not normally expressed (Morgan and Tabin, 1994). The role of the hox genes in the formation of the limb has also been addressed by gene ablation studies in the mouse, although these studies suffer from the possibility of indirect effects of the mutation on the primary axis of the embryo, they are nevertheless consistent with the ectopic expression experiments in the chick. Knockout of the hoxd-11 gene results in reduction of the second phalange of digit 2, as well in the reduction or fusion of a number of other bones in the hand plate and wrist (Davis and Capecchi, 1994; Favier et al., 1995). Deletion of the hoxd-13 gene had inhibitory effects of the formation of a distinct but overlapping set of bones (Dolle et al., 1993). The question of the role of paralogues in the formation of the limb was also examined. Deletion of the hoxa-11 gene results in defects in the forearm and wrist bones (Small and Potter, 1993). Combined deficiency for hoxd-11 and hoxa-11 results in a much more drastic phenotype in which the distal phalanges of digits 2 and 5 are entirely missing, and the hand and wrist, and forearm bones are more severely reduced (Davis et al., 1995). These experiments suggest that the different hox genes expressed in the limb have cooperative roles in its formation; however, the specific effects of deleting different genes could reflect the differential expression of the genes, rather than distinct functions for the encoded proteins (e.g., Hanks et al., 1995). Alternatively, the proteins could have distinct functions, but both functions are required for the normal morphogenesis of the limb. Distinct activities for the different hox proteins in the limb were demonstrated by ectopic expression. Similar to the results obtained with hoxd-11, expression of hoxd-13 throughout the limb resulted in the inhibition of bone growth in specific regions. However, in this case the bones in regions not expressing hoxd-13 were

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FIGURE 4. hox gene expression in the limb. The expression patterns of hoxd-10, -11, and -13 in the developing leg bud at Stages 19, 23, and 26 are shown at left. These patterns are diagrammed at right at the composite of three independently regulated phases of expression. The first phase (blue) is initiated at Stage 16 and results in the expression of hoxd-10 across the anterior-posterior (A/P) axis of the limb at the distal margin. This expression is still apparent at Stage 19 shown at left, when the second phase of hoxd gene expression has initiated at the posterior margin of the limb bud (yellow). An independently regulated phase of expression directs the expression of the hoxd genes in the foot plate (red). In the calf, the domain of expression of hoxd-11 is broader than that of hoxd-13. In the foot, this relative order is reversed, hoxd-13 is expressed across most of the A/P axis whereas hoxd-11 is restricted to the region posterior to digit 2. FIGURE 5. Early and late roles for hox genes in limb morphogenesis. A) Early effects on limb outgrowth. A limb infected with the hoxd-11 virus shows roughly symmetrical outgrowth along the anterior-posterior (A/P) axis. In contrast, the contralateral limb from the same embryo (inverted photographically for comparison) shows the normal posterior bias in growth. B) Chick leg buds are diagrammed. The pattern of expression of hoxd-11, hoxd-13, and hoxa-11 are shown. Bones which show reduced growth after expression of the respective genes throughout the limb bud are shown in red. hoxd-11 affects the growth of the tibia, fibula, and anterior two tarsometatarsals. hoxd-13 reduces the growth of all of these bones as well as the posterior tarsometatarsals but does not affect the phalanges. hoxa-11 expression throughout the limb does not inhibit the growth of bones.

CONCLUSIONS The hox genes have been specifying regional differences along the primary axis of the embryo during much of animal evolution. This ancestral function is reflected in the primary role of each group of genes derived from a different ancestral gene in a different subdivision of the body plan. Within each region, the different genes in each subgroup have developed distinct functions defining the morphological differences within major regions and it is a combination of hox gene activities that

specifies the fate of a region. It appears that different hox genes compete for access to similar target genes to specify morphology, but the identity of those genes and their function remains largely unknown. Examination of the role of the hox genes in limb morphogenesis suggests that some of those target genes regulate the response of undifferentiated mesenchyme to the signals from the apical ectodermal ridge which regulate the growth of the limb. A later role in the growth and maturation of bone may involve similar target genes as well. Continued analysis of hox gene function in the chick embryo will help us to understand the role of the hox genes in modulating developmental subroutines to achieve distinctive morphologies.

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in this segment of the limb, the relative extent of the domains of expression are reversed. hoxd-13 is expressed across the A/P axis of the foot, whereas hoxd12, -11, and -10 are progressively restricted towards the posterior margin. The absence of SHH expression during the first, unpolarized phase of limb development demonstrates that it is not required for the initial expression of hox d genes and formation of the stylopod. Transplantation of the ZPA, or ectopic expression of SHH during the second phase of limb development leads to a complete, albeit inverted duplication of the normal pattern of hoxd gene expression in the calf and foot, so SHH is capable of organizing hoxd gene expression in both of these segments. However, by varying the time at which SHH is added to the anterior margin of the limb, a difference in the regulation of hox d gene expression in these two segments is revealed. If SHH is added while the calf is being formed, hoxd-11 is activated before hoxd-13 and is induced in a broader domain. However, if SHH is added while the foot is being formed, a different effect is observed. hoxd-13 is now activated before hoxd-11 and comes to be expressed in a broader domain. hox d gene expression reveals this change in the response of the cells to the same inductive signal as they progress from forming the calf to forming the foot (Nelson et al., 1996). Because the hox genes mediate the morphogenesis of the limb, their independent regulation in the three segments of the limb has implications for the evolution of this structure. The three segments may be modified independently by changing the strength of these inductive signals to alter the patterns of hox gene expression.

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