Abstract：
UNASSIGNED: 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.
UNASSIGNED: 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 (MYH6)-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 NF2 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.
UNASSIGNED: Transcriptional regulation and trajectory inference from NF2-null cells reveal the loss of cardiomyocyte identity and the acquisition of nonmesodermal identity. Sustained elevation of early mesoderm lineage repressor SOX2 and upregulation of late anticardiac regulators CDX2 and MSX1 in NF2 knockout cells reflect a necessary role for NF2 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)-LATS (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-dependent formation of the AMOT-NF2-YAP scaffold complex at the adherens junction is required for early cardiomyocyte lineage differentiation.
UNASSIGNED: 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.
UNASSIGNED: 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 (MYH6)-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 NF2 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.
UNASSIGNED: Transcriptional regulation and trajectory inference from NF2-null cells reveal the loss of cardiomyocyte identity and the acquisition of nonmesodermal identity. Sustained elevation of early mesoderm lineage repressor SOX2 and upregulation of late anticardiac regulators CDX2 and MSX1 in NF2 knockout cells reflect a necessary role for NF2 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)-LATS (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-dependent formation of the AMOT-NF2-YAP scaffold complex at the adherens junction is required for early cardiomyocyte lineage differentiation.
UNASSIGNED: 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.
摘要：
■心肌细胞分化涉及通过BMP(骨形态发生蛋白)/Wnt信号通路的调节逐步清除抑制因子和命运限制调节因子。然而,特定发育信号通路的机制以及如何消除监管障碍仍不清楚.
我们使用肌球蛋白重链6（MYH6）-GFP（绿色荧光蛋白）报告系统进行了全基因组CRISPR筛选，以揭示人胚胎干细胞中心肌细胞规格的必需调节因子。在对25个候选物进行独立的二级sgRNA验证后，我们鉴定了NF2（神经纤维蛋白2），膜蛋白-ezrin-radixin样（MERLIN）肿瘤抑制因子，作为早期心肌细胞谱系规范的上游驱动因素。使用CRISPR-Cas9产生独立的单克隆NF2敲除,并且通过跨分化时间点的大量RNA测序和蛋白质表达分析来推断细胞状态。通过使用体外二维微图案化的gapstruoid模型评估末端谱系分化，三系分化，和心肌细胞分化。使用定点诱变评估了NF2与其相互作用伴侣的蛋白质相互作用和翻译后修饰，共免疫沉淀，和邻近连接测定。
■来自NF2无效细胞的转录调节和轨迹推断揭示了心肌细胞身份的丧失和非中胚层身份的获得。NF2敲除细胞中早期中胚层谱系抑制因子SOX2的持续升高和晚期抗心调节因子CDX2和MSX1的上调反映了NF2在消除调节路障中的必要作用。此外，我们发现NF2和AMOT（血管生成素）在中内胚层形成过程中与YAP（Yes相关蛋白）协同结合，从而防止YAP激活，独立于经典MST（哺乳动物无菌20样丝氨酸-苏氨酸蛋白激酶）-LATS（大肿瘤抑制因子丝氨酸-苏氨酸蛋白激酶）信号传导。机械上,通过野生型和NF2丝氨酸-518磷酸根拯救心肌细胞谱系的身份，但不是NF2FERM（ezrin-radixin-meosin同源蛋白）域蓝盒突变体，证明在粘附连接处AMOT-NF2-YAP支架复合物的关键FERM结构域依赖性形成是早期心肌细胞谱系分化所必需的。
■这些结果通过隔离YAP的抑制作用并缓解心肌细胞途径的调节障碍，为NF2在早期上皮-间质转化过程中的重要作用提供了机械见解。
我们使用肌球蛋白重链6（MYH6）-GFP（绿色荧光蛋白）报告系统进行了全基因组CRISPR筛选，以揭示人胚胎干细胞中心肌细胞规格的必需调节因子。在对25个候选物进行独立的二级sgRNA验证后，我们鉴定了NF2（神经纤维蛋白2），膜蛋白-ezrin-radixin样（MERLIN）肿瘤抑制因子，作为早期心肌细胞谱系规范的上游驱动因素。使用CRISPR-Cas9产生独立的单克隆NF2敲除,并且通过跨分化时间点的大量RNA测序和蛋白质表达分析来推断细胞状态。通过使用体外二维微图案化的gapstruoid模型评估末端谱系分化，三系分化，和心肌细胞分化。使用定点诱变评估了NF2与其相互作用伴侣的蛋白质相互作用和翻译后修饰，共免疫沉淀，和邻近连接测定。
■来自NF2无效细胞的转录调节和轨迹推断揭示了心肌细胞身份的丧失和非中胚层身份的获得。NF2敲除细胞中早期中胚层谱系抑制因子SOX2的持续升高和晚期抗心调节因子CDX2和MSX1的上调反映了NF2在消除调节路障中的必要作用。此外，我们发现NF2和AMOT（血管生成素）在中内胚层形成过程中与YAP（Yes相关蛋白）协同结合，从而防止YAP激活，独立于经典MST（哺乳动物无菌20样丝氨酸-苏氨酸蛋白激酶）-LATS（大肿瘤抑制因子丝氨酸-苏氨酸蛋白激酶）信号传导。机械上,通过野生型和NF2丝氨酸-518磷酸根拯救心肌细胞谱系的身份，但不是NF2FERM（ezrin-radixin-meosin同源蛋白）域蓝盒突变体，证明在粘附连接处AMOT-NF2-YAP支架复合物的关键FERM结构域依赖性形成是早期心肌细胞谱系分化所必需的。
■这些结果通过隔离YAP的抑制作用并缓解心肌细胞途径的调节障碍，为NF2在早期上皮-间质转化过程中的重要作用提供了机械见解。
