Abstract 541: A Hedgehog-signaling Dependent Gene Regulatory Network Directly Delays Second Heart Field Differentiation to Prevent Congenital Heart Disease

Abstract

The first heart field (FHF) and the second heart field (SHF) comprise the major progenitor pools for the developing vertebrate heart. We investigated the delayed differentiation of the SHF relative to the FHF, a major distinguishing feature of these fields. Single-cell transcriptional profiling of the SHF revealed a differentiation trajectory of SHF progenitors to cardiomyocytes. Hedgehog (Hh) signaling was highly enriched in the progenitor state in signaling pathway analysis, suggesting a possible role in cardiomyocyte differentiation control. Transcriptional profiling of the SHF following removal of active Hh signaling in vivo revealed inappropriate cardiomyocyte gene expression. We observed precocious cardiomyocyte differentiation in the SHF in vivo in Hh mutants, which led to Congenital Heart Disease (CHD). Modeling active Hh signaling in a novel mouse embryonic stem cell (mESC) line through transient expression of the activating Hh transcription factor (TF), GLI1, in cardiac progenitors delayed the onset of cardiomyocyte differentiation. GLI1 directly activated a progenitor-specific gene regulatory network, dominated by repressive TFs, that prevented the acquisition of the cardiomyocyte gene regulatory network. Maintained expression of one GLI1 target TF, FOXF1, repressed the cardiomyocyte differentiation program. FOXF1 binding sites were identified at putative regulatory elements near repressed cardiac genes involved in contraction, electrical impulse propagation and transcriptional regulation. Finally, FOXF1 repressed the activity of these elements in vitro , indicating that FOXF1 can directly repress the activation of genes essential for cardiomyocyte differentiation. Together, these results indicate that a Hh-dependent gene regulatory network including transcriptional repressors directly delays the onset of cardiomyocyte gene expression to delay SHF differentiation. Abrogation of Hh signaling and the resultant premature differentiation of cardiac progenitors provides a molecular mechanism for the ontogeny of some CHD.