UCSF

Valvular heart disease is a major source of morbidity and mortality with an anticipated increase in prevalence secondary to an aging population and increase in survivorship for patients with congenital disease. Living tissue engineered heart valves are a promising regenerative medicine-based therapeutic objective, but require further developmental studies to become a clinical reality. While many of the molecular regulators of endocardial cushion formation via endocardial-to-mesenchymal transition (EMT) have been identified, much remains to be understood about post-EMT valvulogenesis. In this study, we have interrogated the later stages of valvulogenesis to understand the molecular mechanisms of valve formation and how these mechanisms are disrupted in the context of disease. Leveraging a combination of single-cell RNA and chromatin accessibility sequencing in the developing mouse heart, we identified a novel, rare valvular endocardial subpopulation with a unique transcriptional profile comprised of highly specific developmental signaling pathway genes. These cells are first detectable after valve primordia formation at embryonic day 12.5 and are spatially localized at the leading edge of the developing leaflets. Temporally-restricted ablation of this rare subpopulation results in perinatal lethality and dysplastic valve development, characterized by thickened, immature leaflets associated with valvular stenosis and regurgitation. These dysplastic features are consistent with the features of several congenital valvulopathies including Ebstein’s anomaly, and pulmonary or aortic valve stenosis. Lineage tracing analysis revealed that these cells are neural crest-derived, providing the first evidence of a neural crest-to-endocardial transition. Neural Crest-specific deletion of Pthlh, a marker gene for this rare subpopulation, similarly results in perinatal lethality and dysplastic valve development, implicating a previously undescribed neuroendocrine peptide hormone signaling pathway in the regulation of valvulogenesis. Loss of neural crest-derived Pthlh signaling was associated with a cell autonomous down-regulation of several marker genes and a disruption in valvular myocardial BMP signaling, a previously described pathway required for valvular growth and remodeling. Single-cell RNA sequencing analysis of a human fetal heart with hypoplastic left heart syndrome and critical aortic stenosis demonstrated a depletion of this cell population in the diseased aortic valve relative to the other three healthy valves, suggesting these cells may be required for human valvular development. This study establishes that this rare, highly secretory neural crest-derived endocardial subpopulation and the Pthlh signaling pathway are critical to valve morphogenesis and provide new avenues for investigation into the pathogenesis of human congenital heart defects.