Integration of a Notch-dependent mesenchymal gene program and Bmp2-driven cell invasiveness regulates murine cardiac valve formation
Luis Luna-Zurita, … , José María Pérez-Pomares, José Luis de la Pompa
Luis Luna-Zurita, … , José María Pérez-Pomares, José Luis de la Pompa
Published September 20, 2010
Citation Information: J Clin Invest. 2010;120(10):3493-3507. https://doi.org/10.1172/JCI42666.
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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.

Authors

Luis Luna-Zurita, Belén Prados, Joaquim Grego-Bessa, Guillermo Luxán, Gonzalo del Monte, Alberto Benguría, Ralf H. Adams, José María Pérez-Pomares, José Luis de la Pompa

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Figure 5

BMP2 treatment rescues EMT in Notch1-expressing myocardium.

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BMP2 treatment rescues EMT in Notch1-expressing myocardium. Heart explan...
Heart explants triple stained as in Figure 2. (A–I) Details of explants. Full lateral views are shown below. (A) WT ventricular explants show an endocardial monolayer. (B and C) WT AVC explants ± BMP2 undergo EMT. (D) In Nkx2.5-Cre;N1ICD ventricular explants, ENCs migrate across the collagen surface, but are not invasive. (E) Nkx2.5-Cre;N1ICD AVC explants produce migratory mesenchymal cells that show reduced invasion, which is rescued by BMP2 (F). (G) In cTnT-Cre;N1ICD ventricular explants, most ENCs grow as a monolayer and are noninvasive. (H) Mesenchymal cells in cTnT-Cre;N1ICD AVC explants migrate but show reduced invasion, which is rescued by BMP2 (I). Arrows, ENCs; arrowheads, invasive mesenchymal cells. (J) TI analysis of ventricular explants. Most ENCs in Nkx2.5-Cre;N1ICD explants (Nkx) migrate over the gel surface (P = 2.2 × 10–7 for 2D TI versus WT explants), whereas few cells in cTnT-Cre;N1ICD explants (abbreviated cTnT) migrate in 2D. 3D TI is very low for both genotypes. (K) TI analysis of AVC explants. BMP2 treatment of WT explants significantly increases 2D and 3D TI (P = 3.7 × 10–2 and 6.1 × 10–3). Nkx2.5-Cre;N1ICD explants (Nkx) show increased 2D TI (P = 3.5 × 10–4) but a markedly reduced 3D TI compared with WT (P = 5.7 × 10–3). BMP2 treatment increases Nkx2.5-Cre;N1ICD 3D TI (P = 6.7 × 10–4) and slightly reduced 2D TI. BMP2 treatment sharply increases the 3D TI of cTnT-Cre;N1ICD explants (cTnT; P = 1.9 × 10–3). m, myocardium. Scale bar: 50 μm. Results are expressed as mean + SD. *P < 0.005; **P < 0.01; ***P < 0.001.