Abstract

Abstract: The vertebrate limb has provided a deep understanding of the cellular, genetic, and molecular mechanisms that generate an appendage. The majority of limb experimentation has been in animals where the limb forms early in embryogenesis. Yet, several tetrapods, most notably frogs, form their limbs well after embryonic patterning and differentiation have occurred. Surprisingly, we know very little about the processes that direct limb formation in amphibian tadpoles since most limb research in these animals has focused on regeneration. Here we use both molecular methods and classical transplant experiments to investigate the earliest steps in Xenopus hindlimb initiation. We found that posterior lateral plate mesoderm from the late neurula contributes to the hind limb and that this tissue expresses Pitx1 and Tbx4 in the early tadpole, both key genes involved in limb initiation. Surprisingly, Fgf10 , a major contributor limb formation in amniotes, is not expressed until well after bud formation. These results suggest that, unlike amniotes, Fgf10 is dispensable for bud formation. Single-cell sequencing confirmed these observations and revealed several factors consistent with cell motility and migration in the limb. Histological analyses revealed the limb generating cells are mesenchymal at stage 40 and appear to condense into a bud by stage 46. Together, these data suggest a model that resembles zebrafish fin formation whereby the limb-forming mesenchyme is specified early. However, bud formation occurs several days later via cell migration and condensation. This work presents new insights into how limb development varies across tetrapods.

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