Genome-wide target analysis of Shh-activated transcription network in limb bud

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Our long term goal is to unravel the steps linking early patterns of gene regulation and expression with the ultimate realization of structure to serve as a paradigm for how signaling networks orchestrate the formation of a complex tissue. To accomplish this, we are developing several combined genetic and genomic, and proteomic approaches to study transcription factors and regulatory cascades operating during limb development with the ultimate aim of elucidating the regulatory hierarchy between early induction of antero-posterior (AP) pattern and the morphogenesis of distinct digits (with different numbers, lengths and shapes of phalanges). Learning how transcription factors orchestrate growth and morphogenesis during normal development will advance our understanding of how to treat genetic diseases and cancers that arise when such regulatory components are either mutated or expressed abnormally. How do Hoxd genes instruct features of digit identity (such as numbers of joints, shape, size) and what is the relation between Hoxd and Gli3 targets? Learning how this 3-dimensional structure forms will be generally relevant for understanding how organogenesis is achieved. A few 5Hoxd in vivo targets have been reported recently (Shh enhancer, Hand2 promoter), albeit not well-characterized. Defining time windows for 5Hoxd functions will be important in choice of limb stages for ChIP analysis. Our genetic results examining effects of removing Hoxd gene function at different times indicates that these genes regulate digit morphology and joint formation at relatively late stages of digit development, when precursor rays for digits have begun to appear. We have developed several antibodies for ChIP, and we have successfully engineered an epitope-tagged Hoxd13 conditional transgene allele for ChIP with anti-tag in collaboration with Steve Vokes (U. Texas, Austin). This inducible transgene will also facilitate biological validation of targets, along with a conditonal knock-out allele that we already have in the lab. Results will be correlated together with anticipated results from Dr. Vokes lab, who is also analyzing Gli3 targets, and who has made an epitope-tagged Gli3 expressing mouse line available to us. Identifying Hoxd and Gli3 targets will provide insight into co-regulated genes and Gli3-Hoxd roles as well as illuminating late effectors of Hoxd genes in limb morphogenesis. The transcriptional network regulated by Hoxd and Gli3 in the limb will also be analyzed in relation to Shh-pathway targets that form two distinct classes, requiring either transient or sustained signaling for their stable activation. In this manner, we hope to uncover the regulatory cascade leading to formation of defined digit morphologies with distinct numbers of segments and joints. Gli and Hox genes are also aberrantly co-expressed in some cancers and may contribute to their pathogenesis, and these studies will also shed light on their possible roles in these contexts.