Dissecting the genetic underpinnings of heterochrony in limb bud initiation

Harvard Medical School, Boston MA

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Abstract

ABSTRACT The overarching goal of this project is to understand how timing is controlled during embryonic development and to identify the molecular changes that led to changes in timing, or heterochrony, through evolution. In particular, we will focus on the control of the time when the limb buds start to form in the embryo. The relative timing of initiation of the fore and hind limb buds differs dramatically between birds (2-hour differential in chicks) and mammals (18-hour difference in mice). In simplest terms, the hind limb forms at a relatively later time in mice. We will attempt to understand this heterochrony on two levels: Aim 1 will address the cis-regulatory landscape regulating timing of gene activity driving limb bud initiation. Tbx4 is a transcription factor required for limb bud initiation, whose timing of expression closely matches the time at which the hind limb starts to form in both chick and mouse. Using regulation of Tbx4 as a starting point, putative enhancers have been identified in preliminary experiments that appear to drive proper temporal expression of this gene in mouse and chick. Enhancer swap experiments between these species will validate the contribution of these putative activating enhancers to timing of Tbx4 expression in the forming limbs. In parallel, we will map potential inhibitory cis-regulatory sequences at the Tbx4 locus using a “silence reporter” assay, and through loss of function experiments in mice. In a complementary set of experiments, Aim 2 will focus on identifying transcription factors mediating the relative delay in hindlimb initiation in mice. A list of candidate factors will be assembled from a comparative analysis of scRNA-Seq data on cells taken from mouse and chick fore and hind limb domains, just prior to and just after limb bud initiation. Candidates will be tested functionally through electroporation in developing chick limb field. cRel, a candidate identified in preliminary studies, will be further tested functionally in the mouse. As noted above, the timing of Tbx4 expression correlates well with the timing of hind limb bud initiation in both chick and mouse. Moreover, preliminary data suggest that at least part of the timing of this gene’s activity is established through regulation by the NFKB family member cRel. Interestingly, NFKB activity is regulated by oxygen levels in other settings, and our preliminary data indicates that this is the case for cRel in the limb bud. Accordingly, Aim 3 will dissect the regulatory relationship between oxygen levels and Tbx4 expression in the limb bud. Together, the proposed studies will break new ground towards understanding the important, but under- studied problem of timing during development, and the timing of limb bud initiation in particular.