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/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. Limb Initiation: How does Tbx5 regulate limb outgrowth?: Tbx5 is critical for both heart and forelimb development and human mutations (Holt-Oram syndrome) lead to serious defects in both organ systems. Identifying the Tbx5-target repertoire will illuminate how limb outgrowth is controlled and provide new insights on how context-dependent activity of the same factor regulates very different developmental programs. We are developing ChIP assays in embryos for genome-wide direct identification of target promoters in vivo for transcriptional regulators of limb development and unravel the regulatory networks operating during pattern formation and morphogenesis. Tbx5 is an excellent prototype for this analysis: 1) It is highly expressed in early forelimb bud (also heart);Tbx5 mutations cause cardiac and limb abnormalities in humans (Holt-Oram disease) and similar defects in mice. 2) A direct Tbx5 target, Fgf10, has been identified, which will aid in validation and troubleshooting;nevertheless most targets are unknown. We have generated and validated high affinity polyclonal antibodies against Tbx5 that efficiently and specifically enrich the Fgf10 promoter by ChIP, optimized the approach, and have analyzed pilot genome-wide promoter microarrays to identify about 200 targets, some of which are novel. We have now extended this analysis to genome-wide tiling arrays and identified several novel, unexpected targets. These targets are currently being validated both in vitro, and in vivo, using a conditional Floxed Tbx5 gene mutant allele crossed with a tamoxifen inducible Cre-deleter line to remove Tbx5 function from limb buds at different stages. Digit morphogenesis: 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?: We plan to extend ChIP analysis to several other developmental regulators important in digit specification and patterning. We have generated polyclonal antibodies for Hoxd12 and Gli3 to identify direct binding targets for Hoxd and Gli3 proteins and elucidate the role of Hoxd-Gli3 interaction in gene regulation. A few 5Hoxd in vivo targets have been reported recently (Shh enhancer, Hand2 promoter), albeit not well-characterized. Defining time windows for 5Hoxd functions (III) will be important in choice of limb stages for ChIP analysis. We have developed several antibodies for ChIP, and we are also engineering an epitope-tagged Hoxd13 conditional transgene allele for ChIP with anti-tag in collaboration with Steve Vokes (U. Texas, Austin. The 5'Hoxd-flox allele will facilitate biological validation of targets. Results will be correlated with anticipated results from Dr. Vokes lab, who is also analyzing Gli3 targets. Identifying 5'Hoxd and Gli3 targets will provide insight into co-regulated genes and Gli3-Hoxd roles as well as illuminating late effectors of 5Hoxd genes in limb morphogenesis. Gli and Hox genes are also aberrantly expressed in some cancers and may contribute to their pathogenesis, and these studies will shed light on their possible roles in these contexts.
