Neural crest–derived SEMA3C activates endothelial NRP1 for cardiac outflow tract septation
Alice Plein, Amélie Calmont, Alessandro Fantin, Laura Denti, Naomi A. Anderson, Peter J. Scambler, Christiana Ruhrberg
Alice Plein, Amélie Calmont, Alessandro Fantin, Laura Denti, Naomi A. Anderson, Peter J. Scambler, Christiana Ruhrberg
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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 10

SEMA3C signaling through NRP1 induces endoMT in the OFT.

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SEMA3C signaling through NRP1 induces endoMT in the OFT. (A–C) qRT-PCR o...
(A–C) qRT-PCR of E10.5 OFT was used to evaluate Nrp1, Slug, and Snail expression relative to Gapdh, shown by gel electrophoresis (A) and qRT-PCR (B and C) of Nrp1+/– versus Nrp1–/– (n = 3 each) and Sema3cfl/fl (n = 3) versus Wnt1-Cre Sema3cfl/fl (n = 7). (D) Immunolabeling of OFT sections from E11.25 Tie2-Cre Nrp1fl/– and control OFTs (n = 3 each) for PECAM and SLUG; the single SLUG channel is shown in gray scale. The boxed area is shown at a higher magnification. Arrowheads indicate SLUG-expressing cells within the endothelium; the curved arrow indicates an example of a SLUG-expressing cell adjacent to the endothelium in the control, while the dotted cross indicates an area with few SLUG-positive cells in the mutant. (E) Serial sections of E12.5 OFTs from Tie2-Cre RosaYfp mice on a control Nrp1fl/+ or mutant Nrp1fl/fl background at corresponding distal, medial, and proximal levels, immunolabeled for YFP, SMA, and PECAM. Solid arrowheads indicate endothelium; the solid arrow indicates septal bridge myocardialization; wavy arrows indicate migrating SMA-positive cells; and open arrows indicate absence of septal bridge formation. Dotted lines indicate the axis of septal bridge formation. Boxed areas are shown in higher magnification, with curved arrows highlighting cells that have undergone endoMT, dotted crosses showing areas lacking cells derived by endoMT, and the open arrowhead indicating an ectopic endothelial association with the myocardium. (F) Quantitation of YFP+PECAM– area in Tie2-Cre RosaYfp and Tie2-Cre RosaYfp Nrp1fl/fl E12.5 OFTs, n = 3 each. Mean ± SD. *P ≤ 0.05; ***P ≤ 0.001, 2-tailed, unpaired Student’s t test. Scale bars: 50 μm (D); 100 μm (E).