An essential role for Notch in neural crest during cardiovascular development and smooth muscle differentiation

Abstract

The cardiac outflow tract develops as a result of a complex interplay among several cell types, including cardiac neural crest cells, endothelial cells, and cardiomyocytes. In both humans and mice, mutations in components of the Notch signaling pathway result in congenital heart disease characterized by cardiac outflow tract defects. However, the specific cell types in which Notch functions during cardiovascular development remain to be defined. In addition, in vitro studies have provided conflicting data regarding the ability of Notch to promote or inhibit smooth muscle differentiation, while the physiological role for Notch in smooth muscle formation during development remains unclear. In this study, we generated mice in which Notch signaling was specifically inactivated in derivatives of the neural crest. These mice exhibited cardiovascular anomalies, including aortic arch patterning defects, pulmonary artery stenosis, and ventricular septal defects. We show that Notch plays a critical, cell-autonomous role in the differentiation of cardiac neural crest precursors into smooth muscle cells both in vitro and in vivo, and we identify specific Notch targets in neural crest that are implicated in this process. These results provide a molecular and cellular framework for understanding the role of Notch signaling in the etiology of congenital heart disease.

Figure 1. Multiple Notch receptors and ligands are expressed in the developing cardiac outflow tract.

In situ hybridization analysis of cross-sections through the outflow tract and aortic arch arteries of E12.5 embryos is shown. (A) Notch1 is expressed by endothelial cells lining the aortic arch arteries (AAA) and by endocardial cushion tissue of the developing aortic valve (AV). (B) Notch2 is expressed broadly throughout the pharyngeal mesenchyme and notably by the neural crest–derived cells surrounding the aortic arch arteries. (C) Notch3 is expressed by neural crest–derived cells surrounding the aortic arch arteries. (D) Notch4 is expressed by endothelial cells of the aortic arch arteries. (E) Jagged1 is expressed by neural crest–derived cells surrounding the aortic arch arteries. (F) Jagged2 is expressed broadly in the pharyngeal region, including cells surrounding the aortic arch arteries. (G) Delta-like1 is expressed by endocardium but is absent from the aortic arch arteries. (H) Delta-like4 is expressed by endothelium of the aortic arch arteries. Scale bars: 100 μm.

Figure 2. Neural crest–specific expression of DNMAML results in cardiac outflow tract defects.

(A and B) Lateral view of the left side of hearts removed from E17.5 control (A) and Pax3^Cre/+ DNMAML (B) embryos. Compared with the control pulmonary artery, the mutant pulmonary artery and ductus arteriosus are significantly narrowed (arrowheads in A and B). (C and D) Hematoxylin and eosin stained cross-sections through the hearts of E17.5 control (C) or Pax3^Cre/+ DNMAML (D) embryos demonstrate a membranous ventricular septal defect in the mutant (arrow in D). (E–J) Photographs and drawings depicting the aortic arch phenotypes in several E17.5 embryos. A control embryo (E) shows the normal branching pattern of the great vessels. (F) Pax3^Cre/+ DNMAML mutant showing an abnormal arch structure similar to the human defect known as persistent fifth arch. In addition, the ductus arteriosus is absent, and there is a retroesophageal right subclavian artery. (G) Pax3^Cre/+ DNMAML mutant showing a right-sided aortic arch and isolated left subclavian artery arising from the pulmonary artery (arrowhead). (H) Pax3^Cre/+ DNMAML mutant with an atretic ductus arteriosus (arrow) and isolated right subclavian artery (arrowhead). (I) Wnt1-Cre DNMAML mutant showing duplication of the left common carotid artery (arrows). (J) Wnt1-Cre DNMAML mutant with an absent ductus arteriosus. The asterisk indicates ventricular septal defects that were observed upon sectioning the hearts. rv, right ventricle; lv, left ventricle; ao, aorta; da, ductus arteriosus; rsa, right subclavian artery; rca, right common carotid artery; lca, left common carotid artery; lsa, left subclavian artery. Scale bars: 200 μm.

Figure 3. DNMAML-GFP is activated specifically in neural crest and somites by Pax3-Cre and does not affect neural crest cell number or migration.

(A and B) E10.5 Pax3^Cre/+ Z/EG control (A) and Pax3^Cre/+ DNMAML mutant (B) embryos demonstrating expression of DNMAML-GFP in pharyngeal arches (arrows) and somites (arrowheads). (C and D) Immunostaining for GFP with Hoechst nuclear counterstain on frontal sections through the pharyngeal arches of E10.5 Pax3^Cre/+ DNMAML embryos. (C) Low-magnification view showing GFP-positive cells investing the third, fourth, and sixth aortic arch arteries. (D) Higher magnification showing GFP expression specifically in the neural crest–derived mesenchyme of the pharyngeal arch (nc), but not in pharyngeal epithelium (ep) or endothelial cells (ec). (E and F) Immunostaining for GFP on frontal sections through the conotruncus of E11.5 embryos, showing an equivalent number of GFP-positive cells in the conotruncal cushions (arrows) of control (E) and mutant (F) embryos. (G–J) Immunostaining for GFP and α-SMA on frontal sections through the aortic arch arteries of E11.5 embryos. (G and H) Low-magnification view showing equivalent numbers of GFP-positive cells in the pharyngeal region surrounding the 6 major aortic arch arteries (arrows) in control (G) and mutant (H) embryos. (I and J) High-magnification views of the left sixth aortic arch arteries shown in G and H. (K–N) In situ hybridizations for the neural crest cell marker PlexinA2 on frontal sections through the conotruncus (K and L) and the aortic arch arteries (M and N) of E11.5 embryos, showing equivalent expression in control (K and M) and mutant (L and N) embryos. (O and P) GFP expression in the mature aortic arch of control Pax3^Cre/+R26R^GFP (O) and mutant Pax3^Cre/+ DNMAML (P) mice. Scale bars: 100 μm (C, E–H, and K–N), 20 μm (D).

Figure 4. Notch target genes are expressed in the developing smooth muscle layer of the aortic arch arteries and are suppressed by DNMAML.

(A–H) Frontal sections through the aortic arch arteries of E11.5 control (A–D) and Pax3^Cre/+ DNMAML (E–H) embryos showing the aortic arch arteries (arrows) and pharyngeal epithelium (ep). Adjacent sections were used for in situ hybridizations for the Notch target genes HRT1, HRT2, and HRT3 (A–C and E–G) and immunostaining for α-SMA (D and H). (A–D) In control embryos, HRT1, HRT2, and HRT3 are expressed in the cells surrounding the aortic arch arteries, consistent with expression in the developing smooth muscle layer. (E–H) Pax3^Cre/+ DNMAML embryo shows a loss of HRT1, HRT2, and HRT3 expression surrounding the aortic arch arteries, while HRT1 and HRT3 are maintained in the pharyngeal epithelium (ep), and a thin layer of HRT2-positive cells persists in the endothelium of the aortic arch arteries. (I) Semiquantitative RT-PCR from primary smooth muscle cells derived from the aortic arches of late-gestation control and Pax3^Cre/+ DNMAML embryos. Cells were stimulated with immobilized control-Fc (ctrl) or Jagged1-Fc (Jag1). Jagged1 stimulation induces HRT1, HRT2, and HRT3 expression in control cells (lane 2) but not in DNMAML-expressing cells (lane 4). In contrast, expression of the Mef2 target genes histidine-rich calcium-binding protein (HRC) and Mef2c is unchanged in cells expressing DNMAML compared with controls. Scale bars: 100 μm.

Figure 5. Neural crest–specific inhibition of Notch results in loss of smooth muscle markers in the developing aortic arch arteries.

(A, B, E, and F) Lateral views of the aortic arch arteries as visualized by whole mount staining for β-galactosidase activity in E11.5 SM22αLacZ/+ embryos. Control embryo shows strong staining in all 3 major aortic arch arteries on both the right (A) and left (B). In contrast, a Pax3^Cre/+ DNMAML embryo shows absence of β-galactosidase activity in the right sixth aortic arch artery (E) and diminished β-galactosidase activity in the left sixth aortic arch artery (F). (C, D, G, and H) Immunohistochemistry for α-SMA on frontal sections through the developing aortic arch arteries of E12.5 embryos. At low magnification, the smooth muscle layer surrounding all aortic arch arteries is robust and uniform in control embryos (C). However, a Pax3^Cre/+ DNMAML embryo shows diminished α-SMA staining in the sixth aortic arch arteries but normal staining in the other vessels (G). (D and H) High-magnification views of the left sixth aortic arch artery shown in C and G. Compared with control (D), the mutant artery (H) shows diminished α-SMA staining and a general disruption in the architecture of the smooth muscle layer. Scale bars: 100 μm (C and G), 20 μm (D and H).

Figure 6. Inhibition of Notch activity blocks the differentiation of neural crest precursors into smooth muscle ex vivo.

(A–C) Neural tube explants from E8.5 control (A) and Pax3^Cre/+ DNMAML (B and C) embryos, immunostained for α-SMA (green) and GFP (red). The majority of cells in the control explant express α-SMA, as do the DNMAML-GFP–negative cells in the Pax3^Cre/+ DNMAML explants (B and C). Conversely, most of the DNMAML-GFP–positive cells in B and C are α-SMA negative. (D–F) Wild-type neural tube explants treated with the γ-secretase inhibitor DAPT, immunostained for α-SMA (green) and SM22α (red). Compared with DMSO-treated controls (D), explants treated with 1 μM DAPT (E) or 5 μM DAPT (F) show a decrease in the number of cells expressing smooth muscle markers. (G) Quantification of neural tube explant assays from Pax3^Cre/+R26R^GFP (control) and Pax3^Cre/+ DNMAML embryos. (H) Quantification of assays on neural tube explants treated with the γ-secretase inhibitor DAPT. Error bars indicate 1 SD. P values, indicated by brackets, were determined by Student’s t test. Scale bars: 100 μm.