Similarities and differences in the regulation of HoxD genes during chick and mouse limb development

In all tetrapods examined thus far, the development and patterning of limbs require the activation of gene members of the HoxD cluster. In mammals, they are regulated by a complex bimodal process that controls first the proximal patterning and then the distal structure. During the shift from the former to the latter regulation, this bimodal regulatory mechanism allows the production of a domain with low Hoxd gene expression, at which both telomeric (T-DOM) and centromeric regulatory domains (C-DOM) are silent. These cells generate the future wrist and ankle articulations. We analyzed the implementation of this regulatory mechanism in chicken, i.e., in an animal for which large morphological differences exist between fore- and hindlimbs. We report that although this bimodal regulation is globally conserved between the mouse and the chick, some important modifications evolved at least between these two model systems, in particular regarding the activity of specific enhancers, the width of the TAD boundary separating the two regulations, and the comparison between the forelimb versus hindlimb regulatory controls. At least one aspect of these regulations seems to be more conserved between chick and bats than with mouse, which may relate to the extent to which forelimbs and hindlimbs of these various animals differ in their morphologies.
A comparison of Hox gene regulation during the development of limbs in birds and mammals reveals that whereas the characteristic bimodal regulatory system, based on large chromatin domains, is largely conserved between these morphologically distinct structures, some differences are revealed in the way this is implemented in various vertebrates.
Author summary The shapes of limbs vary greatly among tetrapod species, even between the forelimbs and hindlimbs of the same animal. Hox genes regulate the proper growth and patterning of tetrapod limbs. In order to evaluate whether variations in the complex regulation of a cluster of Hox genes—the Hoxd genes—during limb development contribute to the differences in limb shape, we compared their transcriptional control during limb bud development in the forelimbs and hindlimbs of mouse and chicken embryos. We found that the regulatory mechanism underlying Hoxd gene expression is highly conserved, but some clear differences exist. For instance, we observed a variation in the topologically associating domain (TAD; a self-interacting genomic region) boundary interval between the mouse and the chick, as well as differences in the activity of a conserved enhancer element situated within the telomeric regulatory domain. In contrast to the mouse, the chicken enhancer has a stronger activity in the forelimb buds than in the hindlimb buds, which is correlated with the striking differences in the mRNA levels of the genes. We conclude that differences in both the timing and duration of TAD activities and in the width of their boundary may parallel the important decrease in Hoxd gene transcription in chick hindlimb buds versus forelimb buds. These differences may also account for the slightly distinct regulatory strategies implemented by mammals and birds at this locus.