Endocardial to myocardial notch-wnt-bmp axis regulates early heart valve development

Abstract

Endocardial to mesenchymal transformation (EMT) is a fundamental cellular process required for heart valve formation. Notch, Wnt and Bmp pathways are known to regulate this process. To further address how these pathways coordinate in the process, we specifically disrupted Notch1 or Jagged1 in the endocardium of mouse embryonic hearts and showed that Jagged1-Notch1 signaling in the endocardium is essential for EMT and early valvular cushion formation. qPCR and RNA in situ hybridization assays reveal that endocardial Jagged1-Notch1 signaling regulates Wnt4 expression in the atrioventricular canal (AVC) endocardium and Bmp2 in the AVC myocardium. Whole embryo cultures treated with Wnt4 or Wnt inhibitory factor 1 (Wif1) show that Bmp2 expression in the AVC myocardium is dependent on Wnt activity; Wnt4 also reinstates Bmp2 expression in the AVC myocardium of endocardial Notch1 null embryos. Furthermore, while both Wnt4 and Bmp2 rescue the defective EMT resulting from Notch inhibition, Wnt4 requires Bmp for its action. These results demonstrate that Jagged1-Notch1 signaling in endocardial cells induces the expression of Wnt4, which subsequently acts as a paracrine factor to upregulate Bmp2 expression in the adjacent AVC myocardium to signal EMT.

Figure 1. Loss of Notch1 in the endocardium is embryonic lethal.

A and B, wholemount X-gal staining of the E10.5 R26^fslz;c1^Cre embryo and yolk sac showing that the Cre recombinase-mediated LacZ expression (blue) was restricted to the heart (A) and not present in the yolk sac (B). C, sections of the X-gal stained embryos showing that LacZ expression was localized in the endocardium (arrowheads) and endocardial-derived cushion mesenchymal cells at the atrioventricular canal (AVC). D–G, Immunofluorescence showing that Notch1 protein is present in the AVC endocardium (D, arrowheads) and the pharyngeal vascular endothelium of N1 ^f/f embryo (F, arrows), but not in the AVC endocardium of N1 ^f/f ;c1 ^Cre embryo (E, arrowheads). Note that Notch1 protein remains in the pharyngeal vascular endothelium of N1 ^f/f ;c1 ^Cre embryo (G, arrows). H and I, Wholemount views showing that E11.5 N1 ^f/f ;c1 ^Cre embryos were runted and had dilated pericardial sac (H) and E12.5, N1 ^f/f ;c1 ^Cre embryos were absorbed (I). J, summarizing the total number of embryos analyzed at different stages, indicating that N1 ^f/f ;c1 ^Cre embryos died between E11.5 and E12.5. The expected number of embryos at different stages is indicated in the parentheses.

Figure 2. Endocardial-specific deletion of Notch1 does not affect early vascular formation.

A–C, wholemount view showing the yolk sac vessels in E9.5 N1^f/f (A) and N1^f/f;c1^Cre (B) embryos; they are not present in the N1^f/f;Tie1^Cre embryos (C). D–I, Pecam1 staining showing mature vessels in the yolk sac and head of N1^f/f (D and G) and N1^f/f;c1^Cre embryos (E and H); they are not formed in the N1^f/f;Tie1^Cre embryos (F and I). J–L, wholemount view showing mature yolk sac vessels in E10.5 N1^f/f (J) and N1^f/f;c1^Cre (K), but not in N1^f/f;Tie1^Cre embryos (L). M–R, Pecam1 staining showing mature vascular networks in the yolk sac and head of E10.5 N1^f/f (M and P) and N1^f/f;c1^Cre (N and Q) but not N1^f/f;Tie1^Cre (O and R) embryos.

Figure 3. Disruption of endocardial Jagged1-Notch1 signaling blocks EMT and formation of endocardial cushions.

A–D, wholemount views showing that at E10.5 the gross morphology was comparable between N1^f/f (A) andN1^f/f;c1^Cre (B) embryos. Similar results were observed between J1^f/f (C) and J1^f/f;c1^Cre (D) embryos. E–L, H&E stained sections through the atrioventricular canal (avc) region of E10.5 embryos showing dense mesenchymal cells (arrows) in the cushion of N1^f/f (E and I) or J1^f/f (G and K) hearts, but fewer mesenchymal cells in the same region of N1^f/f;c1^Cre (F and J) or J1^f/f;c1^Cre (H and L) hearts. M and N, quantitative analysis of the number of mesenchymal cells in the cushion of N1^f/f;c1^Cre (M) or J1^f/f;c1^Cre (N) hearts. *p<0.001. O–Q, in vitro collagen gel EMT assay showing that while ∼80 endocardial cells (arrows) migrated away from each N1^f/f explant (O and Q), fewer (25/explant) cells underwent this process in cultured N1^f/f;c1^Cre explants (P and Q). *p<0.001.

Figure 4. Endocardial Jagged1-Notch1 signaling regulates expression of endocardial Wnt4 and myocardial Bmp2.

A, qPCR analysis of EMT gene expression in the atrioventricular canal (avc) from E10.5 N1^f/f or N1^f/f;c1^Cre hearts. Each cDNA sample was prepared from five avc tissues and three samples of each group were used for qPCR. Gene expression was normalized to Gapdh. *p<0.05; **p<0.01. B–E, RNA in situ hybridization showing endocardial Wnt4 (B, ec, arrowheads) and myocardial Bmp2 expression (D, myo, arrows) in E10.5 N1^f/f hearts. Their expression is dramatically reduced in N1^f/f;c1^Cre hearts (C and E). F, qPCR analysis of EMT gene expression in the AVC cushions of E10.5 J1^f/f or J1^f/f;c1^Cre hearts. G–J, RNA in situ hybridization showing that Wnt4 and Bmp2 expression is downregulated in J1^f/f;c1^Cre hearts. a, atrium and v, ventricle.

Figure 5. Bmp2 expression is regulated by Wnt signaling.

A, qPCR analysis showing that Bmp2 expression was inhibited by Wif1 and induced by Wnt4. E9.5 embryos were cultured in the control media or media with Wnt inhibitor Wif1 or recombinant mouse Wnt4. After 24-hour culture, each RNA sample was prepared from AVC tissues of 5 hearts for each treatment. The data from three independent samples for each group were used for statistical calculation. *p<0.05 and **p<0.01. B–D, RNA in situ hybridization analysis showing Bmp2 expression (indicated by arrows) in cultured wild type embryos under indicated condition; cushion myocardial Bmp2 expression was inhibited by Wif1 (C) and induced by Wnt4 (D). E–G, RNA in situ hybridization showing Bmp2 expression in cultured N1^f/f (E), N1^f/f;c1^Cre embryos (F), and N1^f/f;c1^Cre embryo treated with Wnt4 (G). The data indicated that Bmp2 expression was reduced in the N1^f/f;c1^Cre embryo when compared to the N1^f/f embryo. However, Wnt4 treatment restored its expression in the N1^f/f;c1^Cre embryo (G). a, atrium and v, ventricle.

Figure 6. Wnt4 and Bmp2 act downstream of Notch to regulate EMT.

A, showing in vitro collagen gel EMT assay analysis of AVC explants from E9.5 R26^fsGFP;c1^Cre embryos in which the endocardial cells were labeled by GFP (indicated by arrowheads). B, showing that Notch inhibitor DAPT blocked EMT by endocardial cells. C–E, Bmp2, Wnt2, or Wnt4 treatment rescued EMT defect caused by DAPT. F, showing that Bmp inhibitor Noggin abolished the Wnt4 rescuing. G, showing quantitative analysis of the number of transformed mesenchymal cells under each condition. The number of explants analyzed is indicated in parentheses.

Figure 7. Working model shows Notch-Wnt-Bmp signaling axis that regulates EMT and early valve formation.

A, Schematic showing the cardiac phenotypes found in the endocardial Jagged1 or Notch1 knockout embryos. During E9.5-E10.5, cushion endocardial cells undergo EMT and form endocardial cushions at the atrioventricular canal and outflow tract of the wild-type (WT) embryos. This process is disrupted in the endocardial Jagged1 (J1^f/f;c1^Cre) or Notch1 (N1^f/f;c1^Cre) knockout (KO) embryos, which results in hypocellular endocardial cushions. B, Endocardial Jagged1-mediated Notch1 activation induces expression of Wnt4, which subsequently upregulates expression of Bmp2 in the adjacent myocardium. Myocardial Bmp2 then acts on endocardial cells to promote EMT. This Notch-Wnt-Bmp signaling axis promotes EMT during heart valve development.