Genome-wide identification of Hand2 target regions in mouse embryos using dRMCE, a new genetic tool

Limb bud development is a paradigmatic model to study the molecular signals that orchestrate cell growth and behaviour. Anterior-posterior patterning of the limb bud mesenchyme is dependent on the secreted ligand Sonic hedgehog (Shh). Shh expression in the posterior limb bud mesenchyme defines the zone of polarizing activity (ZPA) and controls cell survival and proliferative expansion during limb bud outgrowth. The bHLH transcription factor Hand2 binds to the limb-specific far-upstream Shh enhancer termed ZPA regulatory sequence (ZRS) and is essential for Shh activation. With the exception of the ZRS, no other direct Hand2 target regulatory regions and genes have been identified. Given that Hand2 is also required for development of the heart and neural crest derivatives, determining the genome-wide range of Hand2 target regions in mouse embryos will contribute to the understanding of underlying gene-regulatory networks. We decided to insert an epitope tag into the endogenous Hand2 protein to be able to precisely determine the range of Hand2 target sequences by ChIP-seq analysis. However, as genetic engineering of the Hand2 locus by homologous recombination is very inefficient, we developed dRMCE to re-engineer the Hand2 conditional allele. In doing so, we realized that dRMCE is compatible with thousands of conditional alleles and allows highly efficient custom-modification of the endogenous locus. dRMCE allowed me to rapidly generate a mouse model encoding an epitope tag within the endogenous Hand2 protein, which permits highly sensitive detection and localization of endogenous Hand2 in differentiated ES cells and embryonic tissues. We successfully used this fully functional epitope-tagged Hand2 protein to identify the large range of Hand2 target sequences in mouse embryonic tissues using a ChIP-seq approach. Our results indicate that Hand2 interacts with Gli3 and Tbx regulatory sites in limb buds and binds to a minimal ZRS element associated with human point mutations that cause polydactyly. I show that Hand2 is required for the development of the proximal skeleton of the hindlimb, likely by interacting directly with a Tbx4 enhancer. Furthermore, I describe the Hand2 target range associated with essential regulators of cardiac or craniofacial development. Thus, my approach begins to provide insight into the regulatory gene networks regulated by Hand2 during embryogenesis.