Hedgehog Signals Regulate The Proliferation Of Cardiac Progenitor Cells In Pharyngeal Arches

Congenital heart disease (CHD) is one of the most common live birth defects. The study of its underlying molecular and genetic mechanisms is crucial to our understanding of the origin of CHD and to the improvement of existing treatments. Previous studies have shown the importance of the Sonic Hedgehog (Shh) signaling pathway, among others, in the embryonic development of heart. Shh from distinct pulmonary and pharyngeal endoderm modulates second heart field (SHF) development and atrial septation. We provided evidence that the pharyngeal ectoderm may be another source of the Hedgehog signal. The conditional knockout of Shh and its paralog, Indian Hedgehog (Ihh), led to hypoplastic hearts missing the right ventricle and part of the outflow tract in embryonic day 9.5 mice. We used mouse embryonic stem cells (mESCs) to study how Hedgehog signaling controls the proliferation of SHF cardiac progenitor cells (CPCs), a potential contributing factor to the hypoplastic heart. By treating SHF CPCs with Hedgehog agonist and antagonist, we observed that Hedgehog activation increased proliferation within 24 hours after treatment while Hedgehog inhibition had the opposite effect. Furthermore, the heart phenotypes observed in Shh mutants are similar to those observed in mice that model Down syndrome (here, “Down syndrome mice). Disruption of Shh signaling in Down syndrome mice resulted in increased frequencies of complete atrioventricular septal defect (AVSD) in which the dorsal mesenchymal protrusion was absent. Further, we examined the interactions between Hedgehog signaling and trisomy in DS mice and identified two genes orthologous to chromosome 21, Ripk4 and Grik1, that were misregulated beyond the expected 50% increase in expression level. In mESC, we found that Grik1 overexpression was correlated with downregulation of Ptch1, a Hedgehog pathway gene, at the cardiomyocyte stage. The activation of Hedgehog signaling suppressed Grik1 transcriptional expression. The knockdown of Hedgehog pathway genes Gli1 and Gli2 resulted in normal the Grik1 level back control level. This indicates a potential regulatory interaction between Hedgehog signaling and trisomy for Grik1