Limb Initiation Across Tetrapods

Abstract

The earliest stage in limb development is limb initiation, where the limb progenitors are specified, and limb proliferation and outgrowth begin. While some aspects of limb initiation have been well characterised, such as the genes which control the timing of location of limb initiation in the mouse, questions remain. In this work, I seek to answer two questions in order to further our knowledge of limb initiation. Firstly, I aim to understand how limb initiation can be delayed so that it no longer takes place within the embryo. Secondly, I aim to understand the functional redundancy of two genes potentially involved in limb progenitor specification. In my first project, I investigated the mode of limb initiation in X. laevis, a metamorphosing frog, to understand how the limb can be initiated post-embryogenesis. I found that the limb progenitors appeared to be specified at a similar time to amniotes, remaining quiescent until just prior to metamorphosis. I found that, unlike amniotes, the limb bud in X. laevis appears to form through convergent migration of Tbx4+/Pitx1+ cells. This more closely resembles limb initiation in zebrafish and may have implications for the evolutionary history of limb initiation. In my second project, I sought to determine whether Evi1 was functionally redundant with Prdm16 in specifying limb progenitors in the mouse. I found that two isoforms of Evi1 were able to substitute for Prdm16 in direct reprogramming assays, but that one isoform, Evi1-Δ324, acted in a dominant negative fashion. My results as to whether Evi1 compensates for loss of Prdm16 in vivo were inconclusive, however, there is some evidence that the levels of expression of these two genes work to determine the size of the limb in the embryo.