Cardiac outflow morphogenesis depends on effects of retinoic acid signaling on multiple cell lineages

Abstract

Background: Retinoic acid (RA), the bioactive derivative of vitamin A, is essential for vertebrate heart development. Both excess and reduced RA signaling lead to cardiovascular malformations affecting the outflow tract (OFT). To address the cellular mechanisms underlying the effects of RA signaling during OFT morphogenesis, we used transient maternal RA supplementation to rescue the early lethality resulting from inactivation of the murine retinaldehyde dehydrogenase 2 (Raldh2) gene. Results: By embryonic day 13.5, all rescued Raldh2-/- hearts exhibit severe, reproducible OFT septation defects, although wild-type and Raldh2+/- littermates have normal hearts. Cardiac neural crest cells (cNCC) were present in OFT cushions of Raldh2-/- mutant embryos but ectopically located in the periphery of the endocardial cushions, rather than immediately underlying the endocardium. Excess mesenchyme was generated by Raldh2-/- mutant endocardium, which displaced cNCC derivatives from their subendocardial, medial position. Conclusions: RA signaling affects not only cNCC numbers but also their position relative to endocardial mesenchyme during the septation process. Our study shows that inappropriate coordination between the different cell types of the OFT perturbs its morphogenesis and leads to a severe congenital heart defect, persistent truncus arteriosus. Developmental Dynamics 245:388-401, 2016. © 2015 Wiley Periodicals, Inc. Key Findings: A reproducible murine model of retinoic acid deficiency leads to persistent truncus arteriosus through incomplete fusion of the endocardial cushions, responsible for septation of the outflow tract into the aorta and pulmonary arteries. The mesenchymal component of outflow tract endocardial cushions has two sources: endocardial cells that have detached into the cushions, and neural crest cells that have migrated from the pharynx into the heart, along the endocardium itself. Developing hearts deficient in retinoic acid are missing semaphorin 3C-expressing myocardial progenitors from the base of the pulmonary trunk. They also have fewer, disorganized neural crest cells but more mesenchyme of endocardial origin in the unfused outflow cushions. Retinoic acid signaling thus acts within the same timeframe on cardiac neural crest cell orientation and positioning, myocardial specification, and the endocardial to mesenchymal transition, to complete conotruncal septation into the great arteries of the heart.