Foxf Genes Integrate Tbx5 and Hedgehog Pathways in the Second Heart Field for Cardiac Septation

Abstract

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 Foxf1a and Foxf2 were identified as SHF Hedgehog targets. Compound haploinsufficiency for Foxf1a and Foxf2 caused atrioventricular septal defects, demonstrating the biological relevance of this regulatory network. We identified a Foxf1a cis-regulatory element that bound the Hedgehog transcriptional regulators GLI1 and GLI3 and the T-box transcription factor TBX5 in vivo. GLI1 and TBX5 synergistically activated transcription from this cis-regulatory element in vitro. This enhancer drove reproducible expression in vivo in the posterior SHF, the only region where Gli1 and Tbx5 expression overlaps. Our findings implicate Foxf genes in atrioventricular septation, describe the molecular underpinnings of the genetic interaction between Hedgehog signaling and Tbx5, and establish a molecular model for the selection of the SHF gene regulatory network for cardiac septation.

Author Summary

Atrioventricular septal defects (AVSDs) are a common severe class of congenital heart defects. Recent work demonstrates that events in the second heart field (SHF) progenitors, rather than in the heart, drive atrioventricular (AV) septation. Our laboratory has shown that both Hedgehog signaling and the T-box transcription factor, Tbx5, are required in the SHF for AV septation. To understand the molecular underpinnings of the AV septation process we investigated SHF Hedgehog-dependent gene regulatory networks. Transcriptional profiling and chromatin interaction assays identified the Forkhead box transcription factors Foxf1a and Foxf2 as SHF Hedgehog targets. Compound haploinsufficiency for Foxf1a and Foxf2 caused AVSDs in mice, demonstrating the biological relevance of this pathway. We identified a cis-regulatory element at Foxf1a that bound TBX5 and Hedgehog transcriptional regulators GLI1 and GLI3 in-vivo. Furthermore, TBX5 and Gli1 co-activate transcription of the identified cis-regulatory element in-vitro. The enhancer is expressed primarily in the pSHF in-vivo, where Tbx5 and Gli1 expression overlap. Our findings implicate Foxf1a and Foxf2 in AV septation and establish Tbx5 and Hedgehog signaling upstream of Foxf genes in a gene regulatory network for cardiac septation.