An ancestral regulatory mechanism underlies Hoxd gene expression in both developing genitals and digits

Coordinated expression of HoxD cluster genes is crucial for the emergence and patterning of both developing digits and external genitalia. As a shared evolutionary origin has been proposed for these secondary structures, which may have accompanied the transition from an aquatic towards a terrestrial life, we investigated whether the regulatory circuitries would also share similarities, indicating a common phylogenetic origin. We show that the transcription of posterior Hoxd genes in the distal part of these different appendages depends, in both cases, upon enhancer sequences located centromeric to the cluster, in a gene poor region, previously described as forming a regulatory archipelago in digit cells. Analysis of the epigenetic status and spatial conformation of the HoxD cluster and its centromeric neighborhood has identified several putative regulatory elements that interact with Hoxd genes via chromatin looping in the developing genital tubercle. In vivo, targeted deletions within the centromeric gene desert further revealed the requirement of these sequences for Hoxd genes transcription in this embryonic structure. We used lentivirus-mediated transgenesis to evaluate the enhancer activity of the identified regulatory elements. While some sequences were able to drive a reporter gene in both digits and genitalia, others were specific for either one of these two structures. We identified Cdx2 transcription factor as a putative interactor of the genitalspecific enhancers. These results suggest a common ancestral regulatory strategy for digits and genitals, yet with specific adaptations in each developmental context. This work should help understand the regulatory mechanism underlying Hoxd gene expression in the genital tubercle and digits, as well as the phylogenetic histories of such global regulations.