The role of Hox in pisiform and calcaneus ossification and the nature of the zeugopod/autopod boundary

The wrist and ankle, or mesopodium, form at the boundary between the zeugopod and autopod and are composed of short nodular bones that typically lack growth plates. Hoxa11 and Hoxa13 are expressed in mutually exclusive proximal-distal domains that demarcate the zeugopod/autopod boundary. Similarly, Hoxd genes are deployed in two distinct phases during limb development. The early phase corresponds to proximal segments including the zeugopod and a late phase occurs in the digits. This arrangement produces a gap of low Hoxd expression that generally corresponds to the mesopodium. In contrast to the other bones of the wrist and ankle, the mammalian pisiform and calcaneus form true growth plates. We show that these bones develop within the Hoxa11 and Hoxd11 expression domains. We also observe that the pisiform growth plate becomes disorganized with Hoxa11 or Hoxd11 loss-of-function indicating a direct role for Hox11 in its development. Hoxa13 loss-of-function has minimal effect on the pisiform and proximal calcaneus as these bones still form secondary centers and undergo longitudinal growth. Consideration of the phenotypes resulting from hypodactyly (Hd) and synpolydactyly homolog (Spdh) mutations, which result from altered HOXA13 and HOXD13 proteins respectively, confirms that Hox13 plays a limited role in the development of the pisiform and calcaneus and suggests that they lie within the early phase of Hox expression. Therefore, with respect to patterns of ossification and gene expression, these bones share much more in common with the zeugopod than the autopod. Such an interpretation fits with the timing of autopod origins during tetrapod evolution. This work is supported by the NSF (BCS-1540418) and a Hill Fellowship, Department Anthropology, Pennsylvania State University.