n = 3. arm (B) and 3′ arm (C) of Heparin WT ((A) and neural crest specific (B) mouse line were obtained and sectioned, figures show AoV, PV, MV and TV region of RFP+ cell contribution from each lineage. AoV: aortic valve; PV: pulmonary valve; MV: mitral valve; TV: tricuspid valve. Scale Bar = 200m.(TIF) pgen.1007977.s007.tif (1.9M) GUID:?203D47DE-ED51-4A89-83BC-5BB2D381DB61 S7 Fig: Neonatal heart stained with endothelial marker VE-cad and DAPI. Scale bar: 200m.(XLSX) pgen.1007977.s008.xlsx (46K) GUID:?C559B277-D62E-4BE4-8658-0FD6B9E941CE S8 Fig: VGLL4 negatively regulates VEC and VIC proliferation of arterial valves at adult stage. (A) EdU staining shows proliferating cells (green) in arterial Heparin valves of 8-week-old and mice. POSTN staining marks VICs (red). (B,C) Quantitative results of EdU+ cells of VECs and VICs in each individual leaflet of AoV (B) or PV (C). (D) Quantitation of DAPI number per leaflet. (E) Quantitation of DAPI per mm2 valve area. (F,G) Immunostaining of ECM marker ColIII (F) and Versican (G). VEC: valve endothelial cell; VIC: valve intersitital cell; ECM: extracelluar matrix. n = 3. White arrow indicated proliferating cells. Scale bar = 100m. ***P<0.005.(TIF) pgen.1007977.s009.tif (2.6M) GUID:?F54AB7C6-9910-4CE2-A338-3B9A7E8493F1 S9 Fig: Immunostaining of YAP and VE-cad on E15.5 aortic valve. Scale bar:100m.(TIF) pgen.1007977.s010.tif (947K) GUID:?B3339ED5-2073-40D8-B4EA-C03D8B190EE1 S10 Fig: Sections from E15.5 (A) and E17.5 (B) Vgll4-GFP mice were performed immunostaining of endothelial marker VE-cad in red and GFP in green. Mitral valve (MV), tricuspid valve (TV). Scale Bar = 100m.(TIF) pgen.1007977.s011.tif (1.6M) GUID:?19DC3AF4-66A8-4063-922F-8121135346A9 S1 Table: Echocardiographic parameters of Vgll4-/- and Vgl4+/-, Vgll4+/+ control mice. (XLSX) Heparin pgen.1007977.s012.xlsx (44K) GUID:?128913A6-4B2F-4F5B-A1A2-8C7FB3D41005 S2 Table: Echocardiographic parameters of and control mice. (XLSX) pgen.1007977.s013.xlsx (44K) GUID:?66C1F32E-613E-44FF-B4BC-61AF25896D62 S3 Table: Echocardiographic parameters of mice. (XLSX) pgen.1007977.s014.xlsx (63K) GUID:?8CA7CB72-4E60-408A-8ED7-A486475A4FD1 S4 Table: Primer list. (PDF) Cd14 pgen.1007977.s015.pdf (65K) GUID:?5715CEC6-27EC-47A4-99C8-40B93E6B9A7D S5 Table: Antibody list. (PDF) pgen.1007977.s016.pdf (53K) GUID:?D17CA396-ED7E-4827-8453-DA1AEB98F6A5 Data Availability StatementAll relevant data are within the manuscript and its Supporting Information files. Abstract Heart valve disease is usually a major clinical problem worldwide. Cardiac valve development and homeostasis need to be precisely controlled. Hippo signaling is essential for organ development and tissue homeostasis, while its role in valve formation and morphology maintenance remains unknown. VGLL4 is usually a transcription cofactor in vertebrates and we found it was mainly expressed in valve interstitial cells at the post-EMT stage and was maintained till the adult stage. Tissue specific knockout of VGLL4 in different cell lineages revealed that only loss of VGLL4 in endothelial cell lineage led to valve malformation with expanded expression of YAP targets. We further semi-knockout YAP in VGLL4 ablated hearts, and found hyper proliferation of arterial valve interstitial cells was significantly constrained. These findings suggest that VGLL4 is usually important for valve development and manipulation of Hippo components would be a potential therapy for preventing the progression of congenital valve disease. Author summary VGLL4, a new member of the Hippo pathway, is usually intensively investigated in inhibition of tumor progression via competing with YAP to bind TEADs, but its role in cardiovascular field remains unclear. Here we generated VGLL4 knockout mouse line and VGLL4-eGFP reporter mouse line. VGLL4-eGFP reporter mouse line showed VGLL4 was mainly expressed in valve interstitial cells from post-EMT stage to adult stage. Genetic loss of function and lineage tracing data exhibited only endothelial loss of VGLL4 led to.