Integration of a Notch-dependent mesenchymal gene program and Bmp2-driven cell invasiveness regulates murine cardiac valve formation

Abstract

Cardiac valve formation is crucial for embryonic and adult heart function. Valve malformations constitute the most common congenital cardiac defect, but little is known about the molecular mechanisms regulating valve formation and homeostasis. Here, we show that endocardial Notch1 and myocardial Bmp2 signal integration establish a valve-forming field between 2 chamber developmental domains. Patterning occurs through the activation of endocardial epithelial-to-mesenchymal transition (EMT) exclusively in prospective valve territories. Mice with constitutive endocardial Notch1 activity ectopically express Hey1 and Heyl. They also display an activated mesenchymal gene program in ventricles and a partial (noninvasive) EMT in vitro that becomes invasive upon BMP2 treatment. Snail1, TGF-β2, or Notch1 inhibition reduces BMP2-induced ventricular transformation and invasion, whereas BMP2 treatment inhibits endothelial Gsk3β, stabilizing Snail1 and promoting invasiveness. Integration of Notch and Bmp2 signals is consistent with Notch1 signaling being attenuated after myocardial Bmp2 deletion. Notch1 activation in myocardium extends Hey1 expression to nonchamber myocardium, represses Bmp2, and impairs EMT. In contrast, Notch deletion abrogates endocardial Hey gene transcription and extends Bmp2 expression to the ventricular endocardium. This embryonic Notch1-Bmp2-Snail1 relationship may be relevant in adult valve disease, in which decreased NOTCH signaling causes valve mesenchyme cell formation, fibrosis, and calcification.

Notch1 regulates progenitor cell proliferation and differentiation during mouse yolk sac hematopoiesis

“…Scale bars: 800 mm Notch regulates yolk sac hematopoietic progenitors I Cortegano et al accompanied by severe cardiac defects that presumably resulted in hemodynamic alterations, causing embryonic death. 33 CD31/PECAM1 was expressed on hematopoietic and endothelial cells in the P-Sp/AGM dorsal aorta and the YS vitelline artery (Supplementary Figure 1a-j), indicating that early endothelial differentiation occurs in these vessels. Nevertheless, the arterial marker Efnb2 was ectopically expressed in venous endothelium ( Supplementary Figure 1k and l) while the venous marker Ephb4 was not expressed (Supplementary Figure 1m and n).…”

“…32 Tie2-Cre;N1ICD embryos that constitutively expressed N1ICD and EGFP in Tie2 þ hematovascular progenitor cells died at E11.0. 33 At E9.5 (Figures 1a-d), transgenic embryos were smaller than wild-type (WT) littermates and the YS was pale and lacked well-formed blood vessels (Figure 1b). Similarly, although the dorsal aorta and the umbilical and vitelline arteries were preserved, the intraembryonic P-Sp/AGM area lacked hemoglobinized cells (Figure 1d).…”

Co-ordinating Notch, BMP, and TGF-β signaling during heart valve development

“…This allows examination of effects in the presence or absence of Notch signalling. Gain of function experiments with constitutive endocardial Notch activation using NICD leads to the up-regulation of a mesenchymal gene program in the ventricular endocardium and ventricular explants have the ability to undergo a non-invasive EMT and interestingly upon addition of BMP2 ventricular explants can undergo a full invasive EMT [51]. This data indicates that Notch signalling plays an important role in endocardial patterning of the AVC and chambers of the heart and that BMP2 has a key role in inducing invasive EMT.…”

“…Additionally, although explants normally require AVC myocardium for EMT to take place, BMP2 induces EMT in mouse AVC explants without AVC myocardium. Moreover, noggin, a BMP antagonist, blocks EMT [50] and addition of BMP2 to ventricular explants (normally unable to undergo EMT) induces EMT [51]. These together indicate that BMP2 is a critical factor required for EMT and initial formation of the cardiac cushions.…”

“…The reduction in TGFβ-Smad phosphorylation was surprising and suggest that BMP signalling allows endocardial cells to be responsive to TGFβ signalling [45]. This endocardial responsiveness could be attributable to ALK2-mediated BMP signalling that leads to activation of Notch signalling resulting in TGFβ2 expression and subsequently induction of Smad2/3 signalling [51]. Similar to endocardial loss of ALK2, deletion of ALK3 in endocardium causes a failure in EMT with a dramatic reduction in mesenchymal cells in the cardiac cushions [60].…”