Bmp signaling repressesVegfato promote outflow tract cushion development

Abstract

SUMMARYCongenital heart disease (CHD) is a devastating anomaly that affects ~1% of live births. Defects of the outflow tract (OFT) make up a large percentage of human CHD. We investigated Bmp signaling in mouse OFT development by conditionally deleting both Bmp4 and Bmp7 in the second heart field (SHF). SHF Bmp4/7 deficiency resulted in defective epithelial to mesenchymal transition (EMT) and reduced cardiac neural crest ingress, with resultant persistent truncus arteriosus. Using a candidate gene approach, we found that Vegfa was upregulated in the Bmp4/7 mutant hearts. To determine if Vegfa is a downstream Bmp effector during EMT, we examined whether Vegfa is transcriptionally regulated by the Bmp receptor-regulated Smad. Our findings indicate that Smad directly binds to Vegfa chromatin and represses Vegfa transcriptional activity. We also found that Vegfa is a direct target for the miR-17-92 cluster, which is also regulated by Bmp signaling in the SHF. Deletion of miR-17-92 reveals similar phenotypes to Bmp4/7 SHF deletion. To directly address the function of Vegfa repression in Bmp-mediated EMT, we performed ex vivo explant cultures from Bmp4/7 and miR-17-92 mutant hearts. EMT was defective in explants from the Bmp4/7 double conditional knockout (dCKO; Mef2c-Cre;Bmp4/7 f/f ) and miR-17-92 null. By antagonizing Vegfa activity in explants, EMT was rescued in Bmp4/7 dCKO and miR-17-92 null culture. Moreover, overexpression of miR-17-92 partially suppressed the EMT defect in Bmp4/7 mutant embryos. Our study reveals that Vegfa levels in the OFT are tightly controlled by Smad-and microRNA-dependent pathways to modulate OFT development.

Thyroid follicle development requires Smad1/Smad5- and endothelial-dependent basement membrane assembly

“…Although endothelial cell density in epithelial-specific Smad1/5 dKO was comparable to that of control thyroid glands, expression of endothelial cell identity markers (Cdh5, Pcdh12 and Tie1) and endothelial-enriched basement membrane proteins (type IV collagen α1, α2 and laminin α3) was impaired, consistent with ongoing reciprocal paracrine communications between epithelial and endothelial cells (Hick et al, 2013). In this report, we further found that BMP-Smad signaling in thyroid epithelial cells controls the expression of Vegfa, in agreement with reports in other cell types (Bai et al, 2013;Deckers et al, 2002;Shao et al, 2009;Shimizu et al, 2012). Of note, this regulation in zebrafish could be either positive (for Smad1) or negative (for Smad5) (He and Chen, 2005).…”

“…4B), but expression of Vegfr2, Pecam1 and endothelial cell-specific molecule 1 (Esm1) was normal. Vegfa controls endothelial cell development and BMP-Smad1/5 signaling is known to control Vegfa expression in several cell types (Bai et al, 2013;Deckers et al, 2002;Shao et al, 2009;Shimizu et al, 2012). We measured Vegfa in developing Smad1/5 dKO thyroids and found that it was reduced starting at E14.5; this reduction reached statistical significance starting at E16.5 (Fig.…”

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

“…Instead, Wnt1-Cre-targeted cardiac NCCs contributed to the semilunar valve leaflets and septal bridge ( Figure 3A). In the Tie2-Cre lineage-traced OFT, many individual YFP-positive cells were present in the endocardial cushions ( Figure 3A), consistent with the previously described contribution of endoMT-derived cells to the endocardial cushions (17)(18)(19). Tie2-Cre lineage-traced cells also contributed to the semilunar valves ( Figure 3A).…”

“…To show that endoMT was reduced in the OFT of mice lacking NRP1 in vivo, we investigated the expression of the transcription factor Slug, a well-established effector of endoMT (42,43). We first performed reverse-transcription PCR (RT-PCR) analysis signaling lacking NCC-derived SEMA3C display disorganized endocardial cushions and lack proximal OFT septation, 2 processes known to rely on endoMT (17)(18)(19). We therefore investigated whether cardiac NCC immigration and the onset of endoMT correlate during OFT development.…”

Akt1 signaling coordinates BMP signaling and β-catenin activity to regulate second heart field progenitor development

“…This Cre line has been extensively utilized to delete genes for investigation of the SHF development in mice (Ai et al, 2007;Bai et al, 2013;Engleka et al, 2012;Xie et al, 2012). Using Mef2c-Cre, Pten was deleted in the SHF.…”