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During vertebrate limb development, gene members of the HoxD cluster are transcribed in two subsequent waves, following a collinear strategy. Early on, genes located in the center of the cluster are transcriptionally activated and pattern a "central" part of the limbs, such as the forearm and part of the arm. Subsequently, a partially overlapping group of genes, located at one extremity of the cluster, are activated in a distal territory, which will expand and organize the distal pieces of our limbs: the hands. Although enhancer sequences controlling the latter phase have been characterized and mapped in a gene desert centromeric to the gene cluster, the location of the early enhancers, as well as the mechanism underlying the transition from the early to the late phases of transcription, remained elusive.
To localize enhancer sequences, we screened a series of conserved sequences using a transgenic lentivector-based approach in mice. We also analyzed various histone modifications by chromatin immunoprecipitation, as well as the interaction profiles by multiplex circular chromosome conformation capture sequencing (4C-seq) on microdissected wild-type and mutant limb buds. The regulatory switch was monitored using 4C-seq, and mutant configurations were produced using embryonic stem cell targeting, targeted meiotic recombination, and sequential targeted recombination strategies. In situ hybridizations and reverse transcription quantitative real-time fluorescence polymerase chain reaction analyses were used as readouts for gene transcription.
We show that the early phase of transcription requires enhancers located in the telomeric gene desert. Therefore, the early and late phases of Hoxd gene transcription in limb buds are controlled by two opposite deserts flanking the cluster on either side and corresponding to two adjacent topological domains. The transition between early and late regulation involves a functional and conformational switch between these domains, as reflected by a subset of genes mapping centrally into the cluster, which initially interact with the telomeric domain and subsequently shift to establish new contacts with the opposite side. This polarization of the cluster between the two domains ensures a proper collinear distribution of HOX products in both proximal and distal limb structures.
The intriguing collinear correspondence between Hoxd gene topology and the patterning of proximal versus distal limb structures relies on the sequential implementation of two regulatory landscapes flanking the gene cluster. Genes located around the boundary between these two topological domains will swing from one to the other, along with the switch in regulation. The existence of independent regulation allows for cellular offset to occur between the two expression domains, where a reduced HOX protein dose is present, and which will develop into the wrist. Therefore, the mechanism patterning vertebrate proximal and distal limb pieces also contains the intrinsic capacity to build the necessary articulation in between—an adaptive value presumably explaining the selection of this complex regulatory system in tetrapods.
Collinearity Cracked in Tetrapod Limbs
During limb development, the time a nd place of Hox transcription are fixed by respective gene position within the gene cluster. Andrey et al. (p. 1234167; see the Perspective by Rodrigues and Tabin) found that this enigmatic property results from the opposite and successive actions of two large regulatory landscapes located on either side of the mouse Hox locus. In the early phase, one of these topological domains regulates transcription in the proximal limb until a switch occurs toward the other topological domain, which takes over the regulation in the distally developing digits. As a side effect of this antagonistic regulatory strategy, cells in-between have lessened Hox transcription, which generates the wrist.
Hox genes are major determinants of the animal body plan, where they organize structures along both the trunk and appendicular axes. During mouse limb development, Hoxd genes are transcribed in two waves: early on, when the arm and forearm are specified, and later, when digits form. The transition between early and late regulations involves a functional switch between two opposite topological domains. This switch is reflected by a subset of Hoxd genes mapping centrally into the cluster, which initially interact with the telomeric domain and subsequently swing toward the centromeric domain, where they establish new contacts. This transition between independent regulatory landscapes illustrates both the modularity of the limbs and the distinct evolutionary histories of its various pieces. It also allows the formation of an intermediate area of low HOX proteins content, which develops into the wrist, the transition between our arms and our hands. This regulatory strategy accounts for collinear Hox gene regulation in land vertebrate appendages.