Nfatc1 coordinates valve endocardial cell lineage development required for heart valve formation

Abstract

Rationale: Formation of heart valves requires early endocardial to mesenchymal transformation (EMT) to generate valve mesenchyme and subsequent endocardial cell proliferation to elongate valve leaflets. Nfatc1 (nuclear factor of activated T cells, cytoplasmic 1) is highly expressed in valve endocardial cells and is required for normal valve formation, but its role in the fate of valve endocardial cells during valve development is unknown.

Objective: Our aim was to investigate the function of Nfatc1 in cell-fate decision making by valve endocardial cells during EMT and early valve elongation.

Methods and results: Nfatc1 transcription enhancer was used to generate a novel valve endocardial cell-specific Cre mouse line for fate-mapping analyses of valve endocardial cells. The results demonstrate that a subpopulation of valve endocardial cells marked by the Nfatc1 enhancer do not undergo EMT. Instead, these cells remain within the endocardium as a proliferative population to support valve leaflet extension. In contrast, loss of Nfatc1 function leads to enhanced EMT and decreased proliferation of valve endocardium and mesenchyme. The results of blastocyst complementation assays show that Nfatc1 inhibits EMT in a cell-autonomous manner. We further reveal by gene expression studies that Nfatc1 suppresses transcription of Snail1 and Snail2, the key transcriptional factors for initiation of EMT.

Conclusions: These results show that Nfatc1 regulates the cell-fate decision making of valve endocardial cells during valve development and coordinates EMT and valve elongation by allocating endocardial cells to the 2 morphological events essential for valve development.

Figure 1. Fate mapping of Nfatc1^h valve endocardial cells shows they do not undergo EMT

A, Schematic of Nfatc1^h cell fate mapping in Nfatc1^enCre and R26^fslz mice. X-gal staining of Nfatc1^enCre;R26^fslz heart sections shows that Nfatc1^h lineages (arrows) contribute to the endocardium but not the mesenchyme during EMT at E10.5 and E11.5 (B–D) and early valve elongation at E12.5 (E and F). HSP68 indicates heat shock protein 68; AV, atrioventricular.

Figure 2. Fate mapping of endocardial cells in mature valves shows that they do not contribute to valve mesenchyme

A and B, Whole-mount X-gal staining of E14.5 Nfatc1^enCre;R26^fslz heart shows Nfatc1^h cell lineage restricted to 4 heart valves. C–F, Sectional views indicate that cells of the Nfatc1^h lineage only contribute to the endocardium of mature valves; they do not become valve mesenchymal cells. Ao indicates aorta; Pa, pulmonary artery; AV, aortic valve; PV, pulmonary valve; MV, mitral valve; TV, tricuspid valve; LV, left ventricle; RV, right ventricle; and IVS, interventricular septum.

Figure 3. Fate mapping of endocardial cells shows that Nfatc1 deletion results in abnormal OFT morphogenesis

A, Schematic of endocardial cell fate mapping in Tie2^Cre;R26^fslz;Nfatc1^+/+ or Tie2^Cre;R26^fslz;Nfatc1^−/− embryos using Nfatc1^+/+ and Nfatc1^−/− embryos. B and D, E10.5 and E11.5 Nfatc1^+/+ heart sections stained with X-gal show that Tie2^Cre-marked endocardium-derived mesenchymal cells locate at the pOFT, and LacZ-negative neural crest–derived mesenchyme locate at the dOFT. The 2 populations form a tissue boundary at the OFT curvature (B; line). C and E, E10.5 and E11.5 Nfatc1^−/− heart sections show increased endocardium-derived mesenchyme that extends into the dOFT (line). F and G, Graphs show the lengths of dOFT and pOFT and indicate a relative shorting of the pOFT from E10.5 to E11.5 in Nfatc1^+/+ but not in Nfatc1^−/− embryos. n=5 paired embryos analyzed for each time point. A indicates atrium; AS, aortic sac; and V, ventricle.

Figure 4. In vitro collagen gel assays show that Nfatc1 inhibits EMT

A, Schematic shows the landmark to dissect pOFT cushions from E10.5 Wnt1^Cre;R26^fslz embryos without contamination of invasive neural crest (blue staining) from the dOFT. B, Quantification analysis showed an increase in the number of transformed cells in Nfatc1^−/− pOFT (n=11) or AVC (n=13) explants compared with Nfatc1^+/+ pOFT (n=20) or AVC (n=29) explants (P=0.005). Student t test; bar=SD. C–F, Photomicrographs show that Nfatc1^+/+ endocardial cells migrate away from E10.5 OFT (C, D) or E9.5 AVC (E, F) explants but do not invade the gel as readily as Nfatc1^−/− endocardial cells (arrowheads).

Figure 5. Blastocyst complementation analysis shows Nfatc1 inhibits EMT in a cell-autonomous manner

A, Diagram shows generation of chimeric embryos by wild-type blastocyst (LacZ-labeled) injection with Nfatc1^−/− ES cells. B, X-gal–stained E9.5 heart section shows Nfatc1^−/− endocardial cells (negative for LacZ) were integrated into the endocardium at AVC and OFT (arrowheads) and invaded the cushions (arrows). C and D, X-gal–stained E10.5 heart sections show transformed Nfatc1^+/+ (positive for LacZ) Nfatc1^−/− (negative for LacZ) endocardial cells at AVC and OFT cushions. More Nfatc1^−/− transformed cells appear in both cushions (★). E, Quantitative analyses demonstrate significant increases in the transformation of Nfatc1^−/− endocardial cells compared with Nfatc1^+/+ endocardial cells (n=8 or 6 chimeric embryos examined at E9.5 or E10.5; Student t test; bar=SD).

Figure 6. Nfatc1 promotes the proliferation of the valve endocardial lineage

A and C, Bromodeoxyuridine (BrdU) antibody–stained E11.5 Nfatc1^+/+ heart sections show BrdU⁺ endocardium-derived mesenchymal cells in pOFT (A; ★) and AVC (C; ★) and Nfatc1^h cells (C; arrowheads). B and D, BrdU antibody–stained E11.5 Nfatc1^−/− heart sections show fewer BrdU⁺ endocardium-derived mesenchymal cells in pOFT (B; ★) and AVC (D; ★). E and F, Quantitative analyses show a significant reduction in BrdU⁺ endocardial (E) and mesenchymal cells (F) of pOFT and AVC in E11.5 Nfatc1^−/− heart. Serial sections from 4 paired E11.5 Nfatc1^+/+ and Nfatc1^−/− embryos were examined. Student t test; bar=SD.

Figure 7. Nfatc1 regulates expression of genes involved in EMT and cell-cell contact

A and B, Photomicrographs of the in vitro endocardial cell differentiation assay using mouse EPCs show that differentiated Nfatc1^h endocardial cells form clusters within vessel-like lumens positive for Pecam1 expression (arrows). C, Quantitative RT-PCR analyses show significantly increased expression of Nfatc1, VE-Cad, or Pecam1 transcripts when EPCs undertake endocardial differentiation. D, Semiquantitative RT-PCR analyses show marked upregulation of Snail1 and Snail2, the transcriptional repressors of VE-Cad and EMT, and downregulation of their target, VE-Cad, in E10.5 Nfatc1^−/− hearts. E and F, Snail2 staining of sections of E10.5 Nfatc1^+/+ (E) and Nfatc1^−/− (F) shows the number of Snail2-expressing cells is increased in the endocardium (F; arrowheads) and cushion mesenchymal cells (F; ★) in Nfatc1^−/− embryos.

Figure 8. Working model for the role of Nfatc1 in semilunar valve development

A, Diagram showing the role of Nfatc1 in defining valve mesenchymal interaction during semilunar valve morphogenesis. In E11.5 Nfatc1^+/+ embryos, apposition of endocardium-derived mesenchyme from EMT at pOFT (blue cells) and cardiac neural crest–derived mesenchyme from migration at dOFT (green cells) establishes a mesenchymal tissue boundary for semilunar valve formation. From E11.5 to E12.5, the valve elongates from the boundary and becomes primitive valve leaflets. In Nfatc1^−/− embryos, this tissue boundary is shifted into the dOFT because of an increased endocardium-derived mesenchyme from augmented EMT in the pOFT and subsequently decreased neural crest–derived mesenchyme in the dOFT. The alteration of heterogeneous mesenchymal tissue populations (and reduced valve endocardial proliferation not shown in the model) disrupts post-EMT valve elongation. B, A simple diagram shows that Nfatc1 transcriptionally regulates EMT and valve endocardial cell proliferation. Nfatc1 maintains an endocardial cell phenotype through suppression of expression of Snail1 and Sanil2, thereby maintaining the VE-Cad expression necessary for a tight cell-cell contact of the valve endocardial cells that prevents them from undergoing EMT. Nfatc1 also positively regulates the proliferation of valve endocardial cells necessary for the growth of valve leaflets mediated by unknown factors.