Neural crest–derived SEMA3C activates endothelial NRP1 for cardiac outflow tract septation
Alice Plein, … , Peter J. Scambler, Christiana Ruhrberg
Alice Plein, … , Peter J. Scambler, Christiana Ruhrberg
Published June 8, 2015
Citation Information: J Clin Invest. 2015;125(7):2661-2676. https://doi.org/10.1172/JCI79668.
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Research Article Vascular biology

Neural crest–derived SEMA3C activates endothelial NRP1 for cardiac outflow tract septation

Abstract

In mammals, the outflow tract (OFT) of the developing heart septates into the base of the pulmonary artery and aorta to guide deoxygenated right ventricular blood into the lungs and oxygenated left ventricular blood into the systemic circulation. Accordingly, defective OFT septation is a life-threatening condition that can occur in both syndromic and nonsyndromic congenital heart disease. Even though studies of genetic mouse models have previously revealed a requirement for VEGF-A, the class 3 semaphorin SEMA3C, and their shared receptor neuropilin 1 (NRP1) in OFT development, the precise mechanism by which these proteins orchestrate OFT septation is not yet understood. Here, we have analyzed a complementary set of ligand-specific and tissue-specific mouse mutants to show that neural crest–derived SEMA3C activates NRP1 in the OFT endothelium. Explant assays combined with gene-expression studies and lineage tracing further demonstrated that this signaling pathway promotes an endothelial-to-mesenchymal transition that supplies cells to the endocardial cushions and repositions cardiac neural crest cells (NCCs) within the OFT, 2 processes that are essential for septal bridge formation. These findings elucidate a mechanism by which NCCs cooperate with endothelial cells in the developing OFT to enable the postnatal separation of the pulmonary and systemic circulation.

Authors

Alice Plein, Amélie Calmont, Alessandro Fantin, Laura Denti, Naomi A. Anderson, Peter J. Scambler, Christiana Ruhrberg

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Figure 9

SEMA3C signals via NRP1 to induce endoMT of OFT endothelium.

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SEMA3C signals via NRP1 to induce endoMT of OFT endothelium. OFT section...
OFT sections from E10.5 Wnt1-Cre RosaYfp (A) and Tie2-Cre RosaYfp mice at E10.5 and E12.5 (B and C) were immunolabeled for YFP and PECAM. Arrows indicate YFP-positive cells within the endocardial cushions; arrowheads indicate YFP-positive OFT endothelium. Curved arrows highlight YFP-positive cells that have undergone endoMT; the open curved arrow shows a lack of PECAM expression in these cells. The wavy arrow labels PECAM-positive capillaries. (D and E) E10.5 OFT explants of the indicated genotypes were labeled for F-actin and YFP (D) or PECAM (E) and counterstained with DAPI after 72 hours of culture. Curved arrows indicate YFP-positive outgrowth cells, demonstrating their origin by endoMT, while open curved arrows show that outgrowth cells lack PECAM after endoMT. (F and G) E10.5 Wnt1-Cre Sema3cfl/fl (n = 6) (F) and Nrp1-null (n = 5) (G) OFTs with their WT littermates (n = 14 and n = 4, respectively) were labeled for F-actin and DAPI after 72 hours of culture and the number of F-actin–positive emigrated cells quantitated. (H and I) E10.5 WT and Nrp1–/– OFTs (n = 3 each) were cultured for 72 hours in 1% FBS with or without 400 ng/ml SEMA3C and labeled for F-actin and DAPI. The number of F-actin–positive emigrated cells was quantitated. Mean ± SD. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001, 2-tailed, unpaired Student’s t test. Scale bars: 100 μm (A and B); 200 μm (C–I).