Murine Jagged1/Notch signaling in the second heart field orchestrates Fgf8 expression and tissue-tissue interactions during outflow tract development

Abstract

Notch signaling is vital for proper cardiovascular development and function in both humans and animal models. Indeed, mutations in either JAGGED or NOTCH cause congenital heart disease in humans and NOTCH mutations are associated with adult valvular disease. Notch typically functions to mediate developmental interactions between adjacent tissues. Here we show that either absence of the Notch ligand Jagged1 or inhibition of Notch signaling in second heart field tissues results in murine aortic arch artery and cardiac anomalies. In mid-gestation, these mutants displayed decreased Fgf8 and Bmp4 expression. Notch inhibition within the second heart field affected the development of neighboring tissues. For example, faulty migration of cardiac neural crest cells and defective endothelial-mesenchymal transition within the outflow tract endocardial cushions were observed. Furthermore, exogenous Fgf8 was sufficient to rescue the defect in endothelial-mesenchymal transition in explant assays of endocardial cushions following Notch inhibition within second heart field derivatives. These data support a model that relates second heart field, neural crest, and endocardial cushion development and suggests that perturbed Notch-Jagged signaling within second heart field progenitors accounts for some forms of congenital and adult cardiac disease.

Figure 1. Cardiovascular defects in Notch second heart field mutants.

E17.5–P0 control (A), Islet1^Cre/+;DNMAML (B and C), and Mef2c-AHF-Cre;DNMAML (D–F) mutants. (A) Arrows indicate pulmonary artery and aorta. (B) A single OFT vessel (arrow) and retroesophageal right subclavian artery. (C) Double outlet right ventricle (arrows) with a right aortic arch and left ductus arteriosus producing a vascular ring. (D) Cervical right aortic arch (arrow). (E) Interruption of the aortic arch, type B (IAA-B, arrow), and retroesophageal right subclavian artery. (F) Double aortic arch (arrows). (G–P) H&E sections of E17.5–E18.5 hearts. (G) Control with separate aorta and pulmonary artery. (H) Islet1^Cre/+;DNMAML mutant with single OFT arising from right ventricle. (I) Islet1^Cre/+;DNMAML mutant with retroesophageal right subclavian artery dorsal to the trachea. (J and K) Sections from a Mef2c-AHF-Cre+;DNMAML mutant showing a vascular ring. A right-sided circumflex arch (J) runs posterior to the trachea and esophagus en route to a left-sided descending aorta and left ductus arteriosus (K). (L) Control with normal tricuspid valve (arrow). (M and N) Islet1^Cre/+;DNMAML with tricuspid atresia (arrow, M), right ventricular hypoplasia, and atrial septal defect (arrow in N). (O and P) Islet1^Cre/+;DNMAML (O) and Mef2c-AHF-Cre+;DNMAML (P) hearts showed ventricular septal defects (arrows). ao, aorta; da, ductus arteriosus; e, esophagus; IAA-B, interruption of the aortic arch, type B; la, left atrium; lca, left carotid artery; lsa, left subclavian artery; lv, left ventricle; pa, pulmonary artery; pv, pulmonic valve; ra, right atrium; rca, right carotid artery; rersa, retroesophageal right subclavian artery; rsa, right subclavian artery; rv, right ventricle; ta, truncus arteriosus; tr, trachea; tv, tricuspid valve. Original magnification: ×30 (A–F). Scale bars: 250 μm (G–K), 500 μm (L–P).

Figure 2. Notch inhibition in the second heart field results in abnormal patterning of the pharyngeal arches and impaired cardiac neural crest cell migration.

(A–D) PECAM immunostaining of coronal sections through E10.5 embryos to visualize the endothelium of the aortic arch arteries (numbered). Note the hypoplasia of the fourth pharyngeal arch and narrowing of the fourth and sixth aortic arch arteries in the mutant (B). (C and D) Higher-magnification views of the fourth aortic arch artery in control (C) and Islet1^Cre/+;DNMAML (D) embryos show severe hypoplasia in the mutant. (E–H) In situ hybridization for the neural crest markers Sema3C (E and F) and PlexinA2 (G and H) of coronal sections through the OFT of E11.5 control (E and G) and mutant (F and H) embryos. Dotted lines in G highlight neural crest cells within the outflow endocardial cushions expressing PlexinA2. Control genotype was Islet1^+/+;DNMAML. Scale bars: 50 μm (A–D), 100 μm (E–H).

Figure 3. Jag1 is an essential Notch ligand in the second heart field.

(A and B) In situ hybridizations for Jag1 in control embryos. (A) Jag1 expression in pharyngeal endoderm (arrows, E10.5 embryo). (B) Jag1 expression in pharyngeal mesenchyme (arrow), OFT (arrowheads), and aortic arch arteries (asterisks, E11.5 embryo). (C and D) Jag1 immunostaining through E11.5 control (C) and mutant (D) embryos demonstrated loss of Jag1 expression in pharyngeal mesenchyme (arrow) and OFT (black arrowheads) of the mutant. Expression was maintained in the aortic arch arteries (white arrowheads). (E–G) Photographs and diagrams of hearts from E18.5 control (E) and mutant (F and G) embryos. (E) Arrows indicate pulmonary artery and aorta. (F) Double outlet right ventricle (arrows). (G) Double outlet right ventricle (arrows) and an interrupted aortic arch. (H–K) H&E-stained sections of E18.5 control (H and J) and mutant (I and K) hearts, demonstrating a double outlet right ventricle and a ventricular septal defect (arrow). (L–O) In situ hybridization of E10.5 control (L and N) and mutant (M and O) embryos. Control sections (L and N) demonstrated normal neural crest expression of Sema3C (L) and PlexinA2 (N) in the 2 clusters of cells in the center of the developing OFT cushions (dotted circles in N), while mutants (M and O) demonstrated reduced expression. Original magnification, ×30 (E–G). Scale bars: 250 μm (A, C, D, H, and I), 500 μm (B, J, and K), 100 μm (L–O).

Figure 4. Notch regulates Fgf8 expression in the second heart field.

(A and B) Light microscopy of E9.5 hearts of control (A) and Islet1^Cre/+;DNMAML mutants (B). The mutant OFT (arrow) and right ventricle (arrowhead) were hypoplastic. (C and D) H&E-stained sections of E10.5 OFT cushions in control (C) and Islet1^Cre/+;DNMAML mutant (D). The mutant OFT cushions (arrowheads) were hypocellular (D). (E and F) Quantitative RT-PCR of DAPT-treated cultured pharyngeal explants (E) or tissue directly isolated from E10.5 embryos (F), expressed relative to controls. (E) The data represent an average of 4 independent experiments, with error bars indicating 1 standard deviation. Asterisks indicate gene products that showed statistically significant changes in all 4 experiments (P < 0.02). (F) Fgf8 expression levels in the anterior pharynx and OFT of E10.5 control and mutant embryos. The data represent the average of 3 pools of 3–4 embryos per genotype. (G–J) In situ hybridizations showing Fgf8 expression in the OFT of E9.5 control (G and I) and mutant embryos (H and J). Arrowheads point to developing OFT myocardium. (K and L) In situ hybridization for Bmp4 at E11.5 in control (K) and mutant (L) embryos. (M and N) Immunohistochemistry for phospho-Smad in the OFT at E10.5 of control (M and O) and mutant (N and P) embryos showed robust phospho-Smad expression in the OFT cushions (circled) and endothelium (arrowheads) of controls with decreased expression in mutants. Control genotype was Islet1^+/+;DNMAML. Scale bars: 100 μm (C–H, K–P), 250 μm (A, B, I, and J).

Figure 5. Defective EMT in Islet1^Cre/+;DNMAML mutants is rescued by rFgf8.

(A–D) Representative images of outflow explants taken 48 hours (24 hours after treatment with rFgf8 or vehicle) after plating. Control explants treated without (A) or with (B) rFgf8 underwent EMT and invasion into the collagen gel. (C) Islet1^Cre/+;DNMAML explants displayed deficient EMT. (D) Addition of rFgf8 to mutant explants rescued EMT. (E–H) Similar results were seen at 72 hours. (I–K) Quantitative analysis was performed on samples pooled from 3 independent experiments at 24, 48, and 72 hours. Error bars represent 1 standard deviation. Control genotype was Islet1^+/+;DNMAML. Scale bars: 100 μm.

Figure 6. Model depicting tissue-tissue interactions during OFT development.

The model depicts second heart field myocardium (red), endothelium undergoing EMT (green), and cardiac neural crest (yellow). Jag1/Notch signaling in the second heart field is proposed to stimulate Fgf8, which functions within the second heart field to regulate downstream cascades including Bmp4, which in turn signals to endothelium and neural crest.