The mammalian HoxD cluster lies between two topologically associating domains (TADs) matching distinct enhancer-rich regulatory landscapes. During limb development, the telomeric TAD controls the early transcription of Hoxd genes in forearm cells, whereas the centromeric TAD subsequently regulates more posterior Hoxd genes in digit cells. Therefore, the TAD boundary prevents the terminal Hoxd13 gene from responding to forearm enhancers, thereby allowing proper limb patterning. To assess the nature and function of this CTCF-rich DNA region in embryos, we compared chromatin interaction profiles between proximal and distal limb bud cells isolated from mutant stocks where various parts of this boundary region were removed. The resulting progressive release in boundary effect triggered inter-TAD contacts, favored by the activity of the newly accessed enhancers. However, the boundary was highly resilient, and only a 400-kb deletion, including the whole-gene cluster, was eventually able to merge the neighboring TADs into a single structure. In this unified TAD, both proximal and distal limb enhancers nevertheless continued to work independently over a targeted transgenic reporter construct. We propose that the whole HoxD cluster is a dynamic TAD border and that the exact boundary position varies depending on both the transcriptional status and the developmental context.

Chromatin condensation plays an important role in the regulation of gene expression. Recently, it was shown that the transcriptional activation of Hoxd genes during vertebrate digit development involves modifications in 3D interactions within and around the HoxD gene cluster. This reorganization follows a global transition from one set of regulatory contacts to another, between two topologically associating domains (TADs) located on either side of the HoxD locus. Here, we use 3D DNA FISH to assess the spatial organization of chromatin at and around the HoxD gene cluster and report that although the two TADs are tightly associated, they appear as spatially distinct units. We measured the relative position of genes within the cluster and found that they segregate over long distances, suggesting that a physical elongation of the HoxD cluster can occur. We analyzed this possibility by super-resolution imaging (STORM) and found that tissues with distinct transcriptional activity exhibit differing degrees of elongation. We also observed that the morphological change of the HoxD cluster in developing digits is associated with its position at the boundary between the two TADs. Such variations in the fine-scale architecture of the gene cluster suggest causal links among its spatial configuration, transcriptional activation, and the flanking chromatin context. DNA FISH | super-resolution microscopy | limb development | topologically associating domains | HoxD

A spontaneous semidominant mutation (Ironside, Irn) was isolated in mice, leading to severe hindlimb paralysis following multiple deletions in cis at the HoxD locus. To understand its cellular and molecular etiology, we embarked on a comparative analysis using systematic HoxD cluster deletions, produced via targeted meiotic recombination (TAMERE). Different lines of mice were classified according to the severity of their paralyses, and subsequent analyses revealed that multiple causative factors were involved, alone or in combination, in the occurrence of this pathology. Among them are the loss of Hoxd10 function, the sum of remaining Hoxd gene activity, and the ectopic gain of function of the neighboring gene Evx2, all contributing to the mispositioning, the absence, or misidentification of specific lumbo-sacral pools of motoneurons, nerve root homeosis, and hindlimb innervation defects. These results highlight the importance of a systematic approach when studying such clustered gene families, and give insights into the function and regulation of Hox and Evx2 genes during early spinal cord development.[Keywords: Homeosis; peroneal nerve; chromosome engineering; motoneuron] Supplemental material is available at http://www.genesdev.org.

Vertebrate Hox genes encode transcription factors operating during the development of multiple organs and structures. However, the evolutionary mechanism underlying this remarkable pleiotropy remains to be fully understood. Here, we show that Hoxd8 and Hoxd9, two genes of the HoxD complex, are transcribed during mammary bud (MB) development. However, unlike in other developmental contexts, their coexpression does not rely on the same regulatory mechanism. Hoxd8 is regulated by the combined activity of closely located sequences and the most distant telomeric gene desert. On the other hand, Hoxd9 is controlled by an enhancer-rich region that is also located within the telomeric gene desert but has no impact on Hoxd8 transcription, thus constituting an exception to the global regulatory logic systematically observed at this locus. The latter DNA region is also involved in Hoxd gene regulation in other contexts and strongly interacts with Hoxd9 in all tissues analyzed thus far, indicating that its regulatory activity was already operational before the appearance of mammary glands. Within this DNA region and neighboring a strong limb enhancer, we identified a short sequence conserved in therian mammals and capable of enhancer activity in the MBs. We propose that Hoxd gene regulation in embryonic MBs evolved by hijacking a preexisting regulatory landscape that was already at work before the emergence of mammals in structures such as the limbs or the intestinal tract.enhancers | TAD | mammalian development | mammary gland

Within land vertebrate species, snakes display extreme variations in their body plan, characterized by the absence of limbs and an elongated morphology. Such a particular interpretation of the basic vertebrate body architecture has often been associated with changes in the function or regulation of Hox genes. Here, we use an interspecies comparative approach to investigate different regulatory aspects at the snake HoxD locus. We report that, unlike in other vertebrates, snake mesoderm-specific enhancers are mostly located within the HoxD cluster itself rather than outside. In addition, despite both the absence of limbs and an altered Hoxd gene regulation in external genitalia, the limb-associated bimodal HoxD chromatin structure is maintained at the snake locus. Finally, we show that snake and mouse orthologous enhancer sequences can display distinct expression specificities. These results show that vertebrate morphological evolution likely involved extensive reorganisation at Hox loci, yet within a generally conserved regulatory framework.DOI: http://dx.doi.org/10.7554/eLife.16087.001