{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,7]],"date-time":"2026-09-07T11:22:59Z","timestamp":1788780179215,"version":"build-2803163510"},"reference-count":71,"publisher":"Ovid Technologies (Wolters Kluwer Health)","issue":"25","content-domain":{"domain":["www.ahajournals.org"],"crossmark-restriction":true},"short-container-title":["Circulation"],"published-print":{"date-parts":[[2024,6,18]]},"abstract":"<jats:sec>\n                    <jats:title>BACKGROUND:<\/jats:title>\n                    <jats:p>Cardiomyocyte differentiation involves a stepwise clearance of repressors and fate-restricting regulators through the modulation of BMP (bone morphogenic protein)\/Wnt-signaling pathways. However, the mechanisms and how regulatory roadblocks are removed with specific developmental signaling pathways remain unclear.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>METHODS:<\/jats:title>\n                    <jats:p>\n                      We conducted a genome-wide CRISPR screen to uncover essential regulators of cardiomyocyte specification in human embryonic stem cells using a myosin heavy chain 6 (\n                      <jats:italic toggle=\"yes\">MYH6<\/jats:italic>\n                      )-GFP (green fluorescence protein) reporter system. After an independent secondary single guide ribonucleic acid validation of 25 candidates, we identified NF2 (neurofibromin 2), a moesin-ezrin-radixin like (MERLIN) tumor suppressor, as an upstream driver of early cardiomyocyte lineage specification. Independent monoclonal\n                      <jats:italic toggle=\"yes\">NF2<\/jats:italic>\n                      knockouts were generated using CRISPR-Cas9, and cell states were inferred through bulk RNA sequencing and protein expression analysis across differentiation time points. Terminal lineage differentiation was assessed by using an in vitro 2-dimensional-micropatterned gastruloid model, trilineage differentiation, and cardiomyocyte differentiation. Protein interaction and post-translation modification of NF2 with its interacting partners were assessed using site-directed mutagenesis, coimmunoprecipitation, and proximity ligation assays.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>RESULTS:<\/jats:title>\n                    <jats:p>\n                      Transcriptional regulation and trajectory inference from\n                      <jats:italic toggle=\"yes\">NF2<\/jats:italic>\n                      -null cells reveal the loss of cardiomyocyte identity and the acquisition of nonmesodermal identity. Sustained elevation of early mesoderm lineage repressor\n                      <jats:italic toggle=\"yes\">SOX2<\/jats:italic>\n                      and upregulation of late anticardiac regulators\n                      <jats:italic toggle=\"yes\">CDX2<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">MSX1<\/jats:italic>\n                      in\n                      <jats:italic toggle=\"yes\">NF2<\/jats:italic>\n                      knockout cells reflect a necessary role for\n                      <jats:italic toggle=\"yes\">NF2<\/jats:italic>\n                      in removing regulatory roadblocks. Furthermore, we found that NF2 and AMOT (angiomotin) cooperatively bind to YAP (yes-associated protein) during mesendoderm formation, thereby preventing YAP activation, independent of canonical MST (mammalian sterile 20-like serine-threonine protein kinase)\u2013LATS (large tumor suppressor serine-threonine protein kinase) signaling. Mechanistically, cardiomyocyte lineage identity was rescued by wild-type and NF2 serine-518 phosphomutants, but not NF2 FERM (ezrin-radixin-meosin homology protein) domain blue-box mutants, demonstrating that the critical FERM domain\u2013dependent formation of the AMOT-NF2-YAP scaffold complex at the adherens junction is required for early cardiomyocyte lineage differentiation.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>CONCLUSIONS:<\/jats:title>\n                    <jats:p>These results provide mechanistic insight into the essential role of NF2 during early epithelial-mesenchymal transition by sequestering the repressive effect of YAP and relieving regulatory roadblocks en route to cardiomyocytes.<\/jats:p>\n                  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