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Cardiac forces regulate zebrafish heart valve delamination by modulating Nfat signaling

['Renee Wei-Yan Chow', 'Institut De Génétique Et De Biologie Moléculaire Et Cellulaire', 'Igbmc', 'Centre National Dela Recherche Scientifique', 'Institut National De La Santé Et De La Recherche Médicale', 'Université De Strasbourg', 'Illkirch', 'Hajime Fukui', 'Department Of Cell Biology', 'National Cerebral']

Date: 2022-02 

In the clinic, most cases of congenital heart valve defects are thought to arise through errors that occur after the endothelial–mesenchymal transition (EndoMT) stage of valve development. Although mechanical forces caused by heartbeat are essential modulators of cardiovascular development, their role in these later developmental events is poorly understood. To address this question, we used the zebrafish superior atrioventricular valve (AV) as a model. We found that cellularized cushions of the superior atrioventricular canal (AVC) morph into valve leaflets via mesenchymal–endothelial transition (MEndoT) and tissue sheet delamination. Defects in delamination result in thickened, hyperplastic valves, and reduced heart function. Mechanical, chemical, and genetic perturbation of cardiac forces showed that mechanical stimuli are important regulators of valve delamination. Mechanistically, we show that forces modulate Nfatc activity to control delamination. Together, our results establish the cellular and molecular signature of cardiac valve delamination in vivo and demonstrate the continuous regulatory role of mechanical forces and blood flow during valve formation.

Funding: This project has received funding from the ERC under the European Union’s Horizon 2020 research and innovation program: GA N°682938, by the ANR grant ANR-SNF 310030E-164245 and by the grant ANR-10-LABX-0030-INRT, a French State fund managed by the Agence Nationale de la Recherche under the frame program Investissements d'Avenir labeled ANR-10-IDEX-0002-02. RC was supported by the foundation Lefoullon Delalande (2019). HF was supported by the University of Strasbourg (USIAS-2017-097), the Takeda Medical Research Foundation, the Uehara Memorial Foundation, the Cell Science Research Foundation, and JSPS KAKENHI. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here, we focus on the second step of zebrafish valve development and show that superior AV valve leaflets are first formed via delamination, where the tissue bilayer splits. By analyzing various EndoMT markers, we show that the extent of EndoMT is carefully controlled and that most abluminal cells of the bilayer undergo mesenchymal–endothelial transition (MEndoT) during delamination. By examining gata1 mutants, where red blood cell formation is inhibited and wall shear stresses are low, we find that interfering with mechanical forces can cause defects in valve delamination that lead to hyperplastic and thickened superior AV valves. Finally, we show that the nuclear factor of activated T cells (Nfat) signaling pathway plays a critical role in the flow response during delamination stages. We propose a model whereby flow-dependent Nfat signaling in luminal endocardial cells of the atrioventricular canal (AVC) is required to inhibit twist1b expression in abluminal valve cells, thereby allowing these abluminal cells to undergo MEndoT and transform AV cellularized endocardial cushions into free-moving valve leaflets.

The zebrafish is a valuable model to study the role of cardiac forces on valve development due to their optical accessibility and their ability to survive even with severe heart defects [ 19 ]. The current model of zebrafish atrioventricular valve (AV) formation postulates that ventricular and atrioventricular endocardial cells first undergo a partial EndoMT and migrate collectively into the CJ to form a bilayered structure [ 20 – 22 ]. The cellularized cushions then transform into free-moving valve leaflets via cellular rearrangement and elongation. Finally, the abluminal endocardial-derived valve interstitial cells (VICs) are joined by cells derived from the neural crest during valve maturation [ 23 ].

Heart valve development is initiated by the endothelial–mesenchymal transition (EndoMT) of a subset of endocardial cells, which migrate into the cardiac jelly (CJ) and subsequently proliferate [ 6 ]. Post-EndoMT, cellularized endocardial cushions remodel to form valve leaflets via various processes, including delamination, excavation, and elongation [ 7 ]. Cellular and molecular mechanisms underlying valve EndoMT and early endocardial cushion morphogenesis have been extensively studied [ 8 – 10 ], and the importance of mechanical forces in regulating EndoMT stages of valve development is becoming ever more appreciated [ 11 – 16 ]. By contrast, mechanisms underlying post-EndoMT valve morphogenesis remain poorly understood, and, except for a few studies [ 17 ], the role of cardiac forces in post-EndoMT processes remains unexplored. This is despite the fact that genetically modified mice with EndoMT defects rarely survive till birth, suggesting that most congenital valvuloseptal defects likely arise from errors that occur post-EndoMT [ 18 ].

Heart valves are structures critical for ensuring unidirectional blood flow, and heart valve disease is a significant cause of illness and death worldwide. Given the intimate relationship between cardiac forces and cardiovascular development [ 1 – 5 ], a better understanding of how mechanical forces regulate heart valve morphogenesis can yield valuable insights into the origins of heart valve disease.

Results

Abluminal hinge cells give rise to VICs Abluminal cells of the bilayer have been proposed to become future VICs [23,26]. We thus wondered if the few abluminal cells at the hinge of newly formed superior AV valve leaflets can give rise to the much larger population of endocardial-derived VICs seen at 98 hpf and 144 hpf. To address this, we performed photoconversion analysis [27], where we photoconverted valve progenitors at 48 hpf and imaged the beating heart at 80 hpf when most valves have gained leaflet morphology. As expected, we found that the endocardial cells leading the initial collective migration into the CJ form abluminal cells of the hinge, while cells that enter the CJ behind the leading cells form the AVC wall and the inner layer of the valve leaflet (S3A–S3A”’ Fig). We then repeated our photoconversion experiments but stopped hearts at 98 hpf (S3B Fig and 144 hpf (Fig 2A–2A””) observe the origin of VICs. We find that luminal cells of the valve leaflet remain luminal at later stages, suggesting that the few abluminal hinge cells proliferate at the hinge and their progeny migrates toward the distal part of the valve and give rise to all endocardial-derived VICs (S3C Fig, Fig 2B). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 2. VICs at 144 hpf are derived from abluminal hinge cells at 80 hpf. (A–A””) Tg(fli1a:gal4ff;UAS:Kaede) embryonic hearts stopped using BDM and photoconverted at 48 to 50 hpf (left column). Embryos were then returned to normal media and allowed to grow normally until 144 hpf, when the hearts were stopped using BDM again and imaged (middle and right columns). (A) Embryos where the ventricle and the cell at the ventricular side of the AVC is photoconverted. Photoconverted cells can be seen between the 2 luminal layers of the valve at 144 hpf. (Ventricular cells appear to have degraded photoconverted Kaede.) (A’) Embryos where the ventricle is photoconverted. Photoconverted cells can be seen at the luminal side of the valve base at 144 hpf. (Ventricular cells appear to have degraded photoconverted Kaede.) (A”) Embryo where one cell migrating in is photoconverted. Photoconverted cells can be seen between the 2 luminal layers of the valve at 144 hpf. (A”’) Embryos where the deepest cell inside the CJ and the atrial edge of the AVC are photoconverted. At 144 hpf, photoconverted cells can be seen between the 2 luminal layers of the valve, presumably derived from the cell that was deepest inside the CJ at 48 to 50 hpf. Luminal photoconverted cells can also be seen at the tip of the valve, presumably derived from the atrial side of the AVC at 48 to 50 hpf. (A””) Embryos where the atrial side of the AVC is photoconverted. Photoconverted cells can be found at the ventricular luminal layer of the valve leaflet. Asterisks label abluminal cells. Scale bar left column: 50 μm. Scale bar middle and right columns: 20 μm. (B) Model for zebrafish valve leaflet formation, cells are colored to indicate their position and fate over time. At 50 hpf, red cells represent ventricular endocardial cells. Yellow cells represent endocardial cells at the ventricular edge of the AVC. Green cells represent the remaining endocardial cells in the AVC. Blue cells represent atrial endocardial cells in the AVC. In subsequent stages, color schemes are kept to show the position and fate of cells over time. Cells derived from cells at 50 hpf due to cell proliferation are colored the same color as their mothers. Arrows in the top drawing indicate cell movements. Gray box marks period at which delamination can occur (65 to 80 hpf). Delamination itself takes place within 1 hour. At, atrium; AVC, atrioventricular canal; BDM, 2,3-butanedione monoxime; CJ, cardiac jelly; hpf, hours postfertilization; V, ventricle; VIC, valve interstitial cell. https://doi.org/10.1371/journal.pbio.3001505.g002

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