Interactive Fly, Drosophila

Hox genes and digit development

Several 5' members of the Hoxd cluster are expressed in nested posterior-distal domains of the limb bud suggesting a role in regulating the anteroposterior pattern of skeletal elements. While loss-of-function mutants have demonstrated a regulatory role for these genes in the developing limb, extensive functional overlaps between various different Hox genes have hampered elucidation of the roles played by individual members. In particular, the function of Hoxd-12 in the limb remains obscure. Using a gain-of-function approach, it has been found that Hoxd-12 misexpression in transgenic mice produces apparent transformations of anterior digits to posterior morphology and digit duplications, while associated tibial hemimelia (shortening) and other changes indicate that formation/growth of certain skeletal elements is selectively inhibited. If the digital arch represents an anterior bending of the main limb axis, then the results are all reconcilable with a model in which Hoxd-12 promotes formation of postaxial chondrogenic condensations branching from this main axis (including the anteriormost digit) and selectively antagonizes formation of 'true' preaxial condensations that branch from this main axis (such as the tibia). Hoxd-12 misexpression can also induce ectopic Sonic hedgehog (Shh) expression, resulting in mirror-image polydactyly in the limb. Misexpression of Hoxd-12 in other lateral plate derivatives (sternum, pelvis) likewise phenocopies several luxoid/luxate class mouse mutants that all share ectopic Shh signaling. This suggests that feedback activation of Shh expression may be a major function of Hoxd-12. Hoxd-12 can bind to and transactivate the Shh promoter in vitro. Expression of either exogenous Hoxd-11 or Hoxd-12 in cultured limb bud cells, together with FGF, induces expression of the endogenous Shh gene. Together these results suggest that certain 5' Hoxd genes directly amplify the posterior Shh polarizing signal in a reinforcing positive feedback loop during limb bud outgrowth (Knezevic, 1997).

The formation of skeletal elements from proximal to distal (hip to toe) proceeds by progressive branching and segmentation of chondrogenic condensations to produce the more distal elements (e.g., the femur branches distally to produce tibia and fibula). Comparisons of the branching pattern in various tetrapods has genearated a model in which the autopod forms by an anterior bending of the main (metapterygial-like) limb axis: the distal row of carpal/tarsals and the digits all arise as successive postaxial branching events from the continuation of the main limb axis along a curving 'digital arch'. In this view, the digits (including digit 1) and the distal row of tarsals are in fact all 'postaxial' structures whereas the tibia or the radius are true preaxial branches. The effects of Hoxd-12 misexpression in the limb are compatible with a model in which Hoxd-12 promotes formation of postaxial condensations branching from the main limb axis, while selectively inhibiting formation or growth of preaxial condensations (Knezevic, 1997 and citations).

Hox genes, located at one end of the HoxD cluster, are essential for the development of the extremities of limbs; that is, the digits. This 'collinear' correspondence is accompanied by a gradual decrease in the transcriptional efficiency of the genes. To decipher the underlying regulatory mechanisms, and thus to understand better how digits develop, a series of deletions and duplications was engineered in vivo. HoxD genes were found to compete for a remote enhancer that recognizes the locus in a polar fashion, with a preference for the 5' extremity. Modifications in either the number or topography of Hoxd loci induced regulatory reallocations affecting both the number and morphology of digits. These results demonstrate why genes located at the extremity of the cluster are expressed at the distal end of the limbs, following a gradual reduction in transcriptional efficiency, and thus highlight the mechanistic nature of collinearity in limbs (Kmita, 2002).

Enhancer preference for the extremity of the cluster may result from different mechanisms. Preferential 5' transcriptional activation may reflect a distance effect, with the gene located at the most 5' position being closest to the enhancer. A distance effect could be produced either by a stochastic process, or through a DNA scanning mechanism, guiding the enhancer towards the closest extremity of the cluster. Alternatively, a sequence-specific mechanism may help to establish a preferential interaction. The results favor the second alternative: (1) duplications introducing additional DNA of the same length but at different loci had distinct effects on the transcription of the gene located at a more 3' position; (2) RXII (a DNA fragment that displays sequence conservation with the chicken genome) is required along with the Hoxd13 locus to implement the position-dependent, preferential activation. Removal of both RXII and the Hoxd13 locus abrogates quantitative collinearity (Kmita, 2002).

Therefore, although the refinement of 5' Hoxd gene expression in digits relies on promoter competition, as for the ß-globin locus, the mechanisms underlying this competition are probably different in the two systems: in the ß-globin locus, the relative distance to the locus control region impacts on preferential activation. Yet the efficiency of competition can vary in a gene-specific manner. Competition between Hoxd promoters for the activity of the digit enhancer is not primarily related to gene identity and mostly --although not entirely -- depends on position, owing to the existence of regulatory sequences that assist the enhancer to find the right target with a high probability (Kmita, 2002).

These experiments provide a mechanistic explanation as to how spatial and quantitative collinearity are implemented during limb development. (1) A group of genes at the extremity of the cluster respond to a global digit enhancer sequence located upstream. The enhancer contacts this locus as well as other potentially unrelated loci located nearby. Once the interaction is initiated, a sequence-specific process might refine this contact by directing and strengthening the interaction towards the 5' extremity of the cluster. This mechanism would underlie the correspondence between the extremity of the complex and the distal parts of the limbs. (2) Through the same sequence-specific mechanism, the enhancer would display a strong preference for the promoter located at the most 5' position of this subset of genes. As a consequence, it would gradually lose transcriptional efficiency with genes located further apart, thus accounting for both 'quantitative' and 'reverse' collinearity (the reason why Hoxd13 is expressed more widely than Hoxd12, which conflicts with a strict view of collinearity). This could be due to some neighboring effect -- the enhancer is able to activate promoters located nearby concomitantly with its interaction with the Hoxd13 locus -- or it could be a result of a more stochastic process, such as a flip-flop mechanism, where the enhancer is directed towards these neighbor promoters with a reduced probability (Kmita, 2002).

In the light of these results, one may wonder whether the biological relevance of this complex mechanism could not be to secure a high level of Hoxd13 in distal limbs, due to the importance of this gene in making hands and feet. In this model, quantitative collinearity may have evolved as a by-product of this mechanism, and may reflect the looseness of the process. Analyses of more configurations produced by means of a meiotic recombination strategy will clarify this issue (Kmita, 2002).

Mice carrying transgenes targeted at various locations upstream of the HoxD cluster display abnormal digits, with alterations resembling those obtained with loss of functions of Hoxd genes. Because the HoxD cluster remains entirely untouched by the insertional events, whether these phenotypes were induced by regulatory modifications at a distance was investigated. These targeted relocations behaved as hypomorphic alleles of the distantly located gene Hoxd13 and show that posterior Hoxd genes located in cis with the integration site are down-regulated. Genetic analyses suggests that this down-regulation results from the titration of the activity of a remote located enhancer sequence. These results indicate that the transcriptional efficiency of Hoxd genes in digits can be modulated by the presence of other, unrelated, promoters, within the regulatory landscape of this enhancer. Modifications in these latter transcription units may thus impact upon digit morphology, through misregulation of Hoxd genes, thus illustrating the 'buffering effect' that such a global regulatory element can exert upon a short genomic interval. The observations described in this article suggest that the down-regulation of Hoxd13 to Hoxd10 in digits is due to the presence of competing promoters, located between the target genes and the enhancer, that could titrate out the activity of this latter regulatory element. Therefore, it is concluded that, in the presence of foreign transcription units, the enhancer interacts with these transgenes, at the expense of genuine target promoters located further away (Monge, 2003).

Sonic hedgehog (Shh) signaling regulates both digit number and identity, but how different distinct digit types (identities) are specified remains unclear. Shh regulates digit formation largely by preventing cleavage of the Gli3 transcription factor to a repressor form that shuts off expression of Shh target genes. The functionally redundant 5'Hoxd genes regulate digit pattern downstream of Shh and Gli3, through as yet unknown targets. Enforced expression of any of several 5'Hoxd genes causes polydactyly of different distinct digit types with posterior transformations in a Gli3(+) background, whereas, in Gli3 null limbs, polydactylous digits are all similar, short and dysmorphic, even though endogenous 5'Hoxd genes are broadly misexpressed. Hoxd12 interacts genetically and physically with Gli3, and can convert the Gli3 repressor into an activator of Shh target genes. Several 5'Hoxd genes, expressed differentially across the limb bud, interact physically with Gli3. It is proposed that a varying [Gli3]:[total Hoxd] ratio across the limb bud leads to differential activation of Gli3 target genes and contributes to the regulation of digit pattern. The resulting altered balance between 'effective' Gli3 activating and repressing functions may also serve to extend the Shh activity gradient spatially or temporally (Chen, 2004).

The results show a genetic interaction between a 5'Hoxd member and Gli3 in regulating digit formation. Biochemical and transfection analyses further indicate that the 5'Hoxd class protein interacts physically with Gli3 via the homeodomain, and can convert the truncated Gli3 repressor form into an activator of its target promoters. This suggests a model in which Gli3-responsive target promoter activity would depend, at least in part, on the ratio of Gli3 to total Hoxd protein expression at a given site. This model is consistent with the known functional overlap and additive effects of 5'Hoxd genes, as cumulative recruitment of Hoxd proteins to bound Gli3 repressor protein would modify the overall effect on Gli3 target promoters. Rather than a combinatorial Hox code, a quantitative Hox-activity gradient, determined by the total Hox protein relative to Gli3 protein at a particular site, would modify 'net' Gli3 function to regulate expression levels of Gli3 target promoters differentially, and thereby potentially activate downstream Shh pathway targets indirectly. The genetic evidence presented in this study suggests that Gli3-Hoxd interaction pertains mainly to the regulation of digit morphogenesis. This is not unexpected for an interaction with Gli3 shared among several posterior Hox proteins, given that some of the 5'Hoxd members normally only regulate digits physiologically (e.g., Hoxd13). In fact, the long bone shortening observed may represent a distinct dominant-negative effect independent of Gli3. Gli3-Hox interactions may represent a recent evolutionary acquisition that, together with the distal recruitment of 5'Hox genes, enables the development of the distal autopod with its multiple digits. Since the distal autopod is probably a neomorphic structure of tetrapod vertebrates, it is not surprising that an interaction between the homologous Drosophila Ci and AbdB proteins has not been described (Chen, 2004).

The genetic mechanisms that regulate the complex morphogenesis of generating cartilage elements in correct positions with precise shapes during organogenesis, fundamental issues in developmental biology, are still not well understood. By focusing on the developing mouse limb, importance was confirmed of transcription factors encoded by the Sall gene family in proper limb morphogenesis, and it was further shown that they have overlapping activities in regulating regional morphogenesis in the autopod (the distal elements of a limb that will give rise to the wrist and the fingers in the forelimb, and the ankle and toes in the hindlimb). Sall1/Sall3 double null mutants exhibit a loss of digit1 as well as a loss or fusion of digit2 and digit3, metacarpals and carpals in the autopod. Sall activity affects different pathways, including the Shh signaling pathway, as well as the Hox network. Shh signaling in the mesenchyme is partially impaired in the Sall mutant limbs. Additionally, the data suggest an antagonism between Sall1-Sall3 and Hoxa13-Hoxd13. Expression of Epha3 and Epha4 is downregulated in the Sall1/Sall3 double null mutants, and, conversely, is upregulated in Hoxa13 and Hoxd13 mutants. Moreover, the expression of Sall1 and Sall3 is upregulated in Hoxa13 and Hoxd13 mutants. Furthermore, by using DNA-binding assays, it was shown that Sall and Hox compete for a target sequence in the Epha4 upstream region. In conjunction with the Shh pathway, the antagonistic interaction between Hoxa13-Hoxd13 and Sall1-Sall3 in the developing limb may contribute to the fine-tuning of local Hox activity that leads to proper morphogenesis of each cartilage element of the vertebrate autopod (Kawakami, 2009).

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