Reshuffling genomic landscapes to study the regulatory evolution of Hox gene clusters

The emergence of Vertebrata was accompanied by two rounds of whole-genome duplications. This enabled paralogous genes to acquire novel functions with high evolutionary potential, a process suggested to occur mostly by changes in gene regulation, rather than in protein sequences. In the case of Hox gene clusters, such duplications favored the appearance of distinct global regulations. To assess the impact of such “regulatory evolution” upon neo-functionalization, we developed PANTHERE (PAN-genomic Translocation for Heterologous Enhancer RE-shuffling) to bring the entire megabase-scale HoxD regulatory landscape in front of the HoxC gene cluster via a targeted translocation in vivo. At this chimeric locus, Hoxc genes could both interpret this foreign regulation and functionally substitute for their Hoxd counterparts. Our results emphasize the importance of evolving regulatory modules rather than their target genes in the process of neo-functionalization and offer a genetic tool to study the complexity of the vertebrate regulatory genome

Ulnaless (Ul), a regulatory mutation inducing both loss-of-function and gain-of-function of posterior Hoxd genes

Ulnaless (Ul), an X-ray-induced dominant mutation in mice, severely disrupts development of forearms and forelegs. The mutation maps on chromosome 2, tightly linked to the HoxD complex, a cluster of regulatory genes required for proper morphogenesis. In particular, 5'-located (posterior) Hoxd genes are involved in limb development and combined mutations within these genes result in severe alterations in appendicular skeleton. We have used several engineered alleles of the HoxD complex to genetically assess the potential linkage between these two loci. We present evidence indicating that Ulnaless is allelic to Hoxd genes. Important modifications in the expression patterns of the posterior Hoxd-12 and Hoxd-13 genes at the Ul locus suggest that Ul is a regulatory mutation that interferes with a control mechanism shared by multiple genes to coordinate Hoxd function during limb morphogenesis

Genetic analysis of a Hoxd-12 regulatory element reveals global versus local modes of controls in the HoxD complex

Vertebrate Hoxd genes are essential determinants of limb morphogenesis. In order to understand the genetic control of their complex expression patterns, we have used a combined approach involving interspecies sequence alignments in parallel with transgenic analyses, followed by in vivo mutagenesis. Here, we report on the identification of a regulatory element that is located in the vicinity of the Hoxd-12 gene. While this element is well conserved in tetrapods, little sequence similarity was scored when compared to the cognate fish DNA. The regulatory potential of this region XI (RXI) was first assayed in the context of a Hoxd-12/lacZ reporter transgene and shown to direct reporter gene expression in posterior limb buds. A deletion of this region was generated by targeted mutagenesis in ES cells and introduced into mice. Analyses of animals homozygous for the HoxDRXI mutant allele revealed the function of this region in controlling Hoxd-12 expression in the presumptive posterior zeugopod where it genetically interacts with Hoxa-11. Downregulation of Hoxd-12 expression was also detected in the trunk suggesting that RXI may mediate a rather general function in the activation of Hoxd-12. These results support a model whereby global as well as local regulatory influences are necessary to build up the complex expression patterns of Hoxd genes during limb development