<i>Foxf</i> Genes Integrate <i>Tbx5</i> and Hedgehog Pathways in the Second Heart Field for Cardiac Septation

<div><p>The Second Heart Field (SHF) has been implicated in several forms of congenital heart disease (CHD), including atrioventricular septal defects (AVSDs). Identifying the SHF gene regulatory networks required for atrioventricular septation is therefore an essential goal for understanding the molecular basis of AVSDs. We defined a SHF Hedgehog-dependent gene regulatory network using whole genome transcriptional profiling and GLI-chromatin interaction studies. The Forkhead box transcription factors <i>Foxf1a</i> and <i>Foxf2</i> were identified as SHF Hedgehog targets. Compound haploinsufficiency for <i>Foxf1a</i> and <i>Foxf2</i> caused atrioventricular septal defects, demonstrating the biological relevance of this regulatory network. We identified a <i>Foxf1a</i> cis-regulatory element that bound the Hedgehog transcriptional regulators GLI1 and GLI3 and the T-box transcription factor TBX5 <i>in vivo</i>. GLI1 and TBX5 synergistically activated transcription from this cis-regulatory element <i>in vitro</i>. This enhancer drove reproducible expression <i>in vivo</i> in the posterior SHF, the only region where <i>Gli1</i> and <i>Tbx5</i> expression overlaps. Our findings implicate <i>Foxf</i> genes in atrioventricular septation, describe the molecular underpinnings of the genetic interaction between Hedgehog signaling and <i>Tbx5</i>, and establish a molecular model for the selection of the SHF gene regulatory network for cardiac septation.</p>