Abstract：
Hemangioblasts are mesoderm-derived multipotent stem cells for differentiation of all hematopoietic and endothelial cells in the circulation system. However, the underlying molecular mechanism is poorly understood.
CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (type II CRISPR RNA-guided endonuclease) editing was used to develop aggf1-/- and emp2-/- knockout zebra fish. Whole-mount in situ hybridization and transgenic Tg(gata1-EGFP [enhanced green fluorescent protein]), Tg(mpx-EGFP), Tg(rag2-DsRed [discosoma sp. red fluorescent protein]), Tg(cd41-EGFP), Tg(kdrl-EGFP), and Tg(aggf1-/-;kdrl-EGFP) zebra fish were used to examine specification of hemangioblasts and hematopoietic stem and progenitor cells (HSPCs), hematopoiesis, and vascular development. Quantitative real-time polymerase chain reaction and Western blot analyses were used for expression analysis of genes and proteins.
Knockout of aggf1 impaired specification of hemangioblasts and HSPCs, hematopoiesis, and vascular development in zebra fish. Expression of npas4l/cloche-the presumed earliest marker for hemangioblast specification-was significantly reduced in aggf1-/- embryos and increased by overexpression of aggf1 in embryos. Overexpression of npas4l rescued the impaired specification of hemangioblasts and HSPCs and development of hematopoiesis and intersegmental vessels in aggf1-/- embryos, placing aggf1 upstream of npas4l in hemangioblast specification. To identify the underlying molecular mechanism, we identified emp2 as a key aggf1 downstream gene. Similar to aggf1, emp2 knockout impaired the specification of hemangioblasts and HSPCs, hematopoiesis, and angiogenesis by increasing the phosphorylation of ERK1/2 (extracellular signal-regulated protein kinase 1/2). Mechanistic studies showed that aggf1 knockdown and knockout significantly decreased the phosphorylated levels of mTOR (mammalian target of rapamycin) and p70 S6K (ribosomal protein S6 kinase), resulting in reduced protein synthesis of Emp2 (epithelial membrane protein 2), whereas mTOR activator MHY1485 (4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine) rescued the impaired specification of hemangioblasts and HSPCs and development of hematopoiesis and intersegmental vessels and reduced Emp2 expression induced by aggf1 knockdown.
These results indicate that aggf1 acts at the top of npas4l and becomes the earliest marker during specification of hemangioblasts. Our data identify a novel signaling axis of Aggf1 (angiogenic factor with G-patch and FHA domain 1)-mTOR-S6K-ERK1/2 for specification of hemangioblasts and HSPCs, primitive and definitive hematopoiesis, and vascular development. Our findings provide important insights into specification of hemangioblasts and HSPCs essential for the development of the circulation system.
CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (type II CRISPR RNA-guided endonuclease) editing was used to develop aggf1-/- and emp2-/- knockout zebra fish. Whole-mount in situ hybridization and transgenic Tg(gata1-EGFP [enhanced green fluorescent protein]), Tg(mpx-EGFP), Tg(rag2-DsRed [discosoma sp. red fluorescent protein]), Tg(cd41-EGFP), Tg(kdrl-EGFP), and Tg(aggf1-/-;kdrl-EGFP) zebra fish were used to examine specification of hemangioblasts and hematopoietic stem and progenitor cells (HSPCs), hematopoiesis, and vascular development. Quantitative real-time polymerase chain reaction and Western blot analyses were used for expression analysis of genes and proteins.
Knockout of aggf1 impaired specification of hemangioblasts and HSPCs, hematopoiesis, and vascular development in zebra fish. Expression of npas4l/cloche-the presumed earliest marker for hemangioblast specification-was significantly reduced in aggf1-/- embryos and increased by overexpression of aggf1 in embryos. Overexpression of npas4l rescued the impaired specification of hemangioblasts and HSPCs and development of hematopoiesis and intersegmental vessels in aggf1-/- embryos, placing aggf1 upstream of npas4l in hemangioblast specification. To identify the underlying molecular mechanism, we identified emp2 as a key aggf1 downstream gene. Similar to aggf1, emp2 knockout impaired the specification of hemangioblasts and HSPCs, hematopoiesis, and angiogenesis by increasing the phosphorylation of ERK1/2 (extracellular signal-regulated protein kinase 1/2). Mechanistic studies showed that aggf1 knockdown and knockout significantly decreased the phosphorylated levels of mTOR (mammalian target of rapamycin) and p70 S6K (ribosomal protein S6 kinase), resulting in reduced protein synthesis of Emp2 (epithelial membrane protein 2), whereas mTOR activator MHY1485 (4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine) rescued the impaired specification of hemangioblasts and HSPCs and development of hematopoiesis and intersegmental vessels and reduced Emp2 expression induced by aggf1 knockdown.
These results indicate that aggf1 acts at the top of npas4l and becomes the earliest marker during specification of hemangioblasts. Our data identify a novel signaling axis of Aggf1 (angiogenic factor with G-patch and FHA domain 1)-mTOR-S6K-ERK1/2 for specification of hemangioblasts and HSPCs, primitive and definitive hematopoiesis, and vascular development. Our findings provide important insights into specification of hemangioblasts and HSPCs essential for the development of the circulation system.
摘要：
背景：成血管细胞是中胚层衍生的多能干细胞，用于分化循环系统中的所有造血细胞和内皮细胞。然而,潜在的分子机制知之甚少。
方法：CRISPR（成簇规则间隔的短回文重复序列）/Cas9编辑用于开发aggf1-/-和emp2-/-敲除斑马鱼。全量原位杂交和转基因Tg（gata1-EGFP），Tg(mpx-EGFP)，Tg(rag2-DsRed),Tg(cd41-EGFP),Tg(kdrl-EGFP)，和Tg（aggf1-/-;kdrl-EGFP）斑马鱼用于检查血管母细胞和造血干细胞和祖细胞（HSPC）的规格，造血,和血管发育。定量实时聚合酶链反应和Western印迹分析用于基因和蛋白质的表达分析。
结果：敲除aggf1受损的成血管细胞和HSPC的规格，造血,斑马鱼的血管发育。npas4l/cloche-假定是成血管细胞规格的最早标记-在aggf1-/-胚胎中的表达显着降低，而在胚胎中aggf1的过表达则增加。npas4l的过表达挽救了成血管细胞和HSPC的受损规格以及aggf1-/-胚胎中造血和节间血管的发育，将aggf1放在血管母细胞规范中npas4l的上游。为了确定潜在的分子机制，我们确定emp2是一个关键的aggf1下游基因。与aggf1相似，emp2敲除损害了成血管细胞和HSPC的规格，造血,通过增加ERK1/2（细胞外信号调节蛋白激酶1/2）的磷酸化和血管生成。机制研究表明，aggf1敲低和敲除显著降低mTOR（哺乳动物雷帕霉素靶蛋白）和p70S6K（核糖体蛋白S6激酶）的磷酸化水平，导致Emp2（上皮膜蛋白2）的蛋白质合成减少，而mTOR激活剂MHY1485挽救了成血管细胞和HSPCs的受损规格以及造血和节间血管的发育，并降低了aggf1敲低诱导的Emp2表达。
结论：这些结果表明aggf1在npas4l的顶部起作用，并成为成血管细胞特化过程中最早的标记。我们的数据确定了Aggf1（具有G-patch和FHA结构域1的血管生成因子）-mTOR-S6K-ERK1/2的新信号轴，用于规范成血管细胞和HSPC，原始和确定的造血，和血管发育。我们的发现为循环系统发展所必需的成血管细胞和HSPCs的规格提供了重要见解。
方法：CRISPR（成簇规则间隔的短回文重复序列）/Cas9编辑用于开发aggf1-/-和emp2-/-敲除斑马鱼。全量原位杂交和转基因Tg（gata1-EGFP），Tg(mpx-EGFP)，Tg(rag2-DsRed),Tg(cd41-EGFP),Tg(kdrl-EGFP)，和Tg（aggf1-/-;kdrl-EGFP）斑马鱼用于检查血管母细胞和造血干细胞和祖细胞（HSPC）的规格，造血,和血管发育。定量实时聚合酶链反应和Western印迹分析用于基因和蛋白质的表达分析。
结果：敲除aggf1受损的成血管细胞和HSPC的规格，造血,斑马鱼的血管发育。npas4l/cloche-假定是成血管细胞规格的最早标记-在aggf1-/-胚胎中的表达显着降低，而在胚胎中aggf1的过表达则增加。npas4l的过表达挽救了成血管细胞和HSPC的受损规格以及aggf1-/-胚胎中造血和节间血管的发育，将aggf1放在血管母细胞规范中npas4l的上游。为了确定潜在的分子机制，我们确定emp2是一个关键的aggf1下游基因。与aggf1相似，emp2敲除损害了成血管细胞和HSPC的规格，造血,通过增加ERK1/2（细胞外信号调节蛋白激酶1/2）的磷酸化和血管生成。机制研究表明，aggf1敲低和敲除显著降低mTOR（哺乳动物雷帕霉素靶蛋白）和p70S6K（核糖体蛋白S6激酶）的磷酸化水平，导致Emp2（上皮膜蛋白2）的蛋白质合成减少，而mTOR激活剂MHY1485挽救了成血管细胞和HSPCs的受损规格以及造血和节间血管的发育，并降低了aggf1敲低诱导的Emp2表达。
结论：这些结果表明aggf1在npas4l的顶部起作用，并成为成血管细胞特化过程中最早的标记。我们的数据确定了Aggf1（具有G-patch和FHA结构域1的血管生成因子）-mTOR-S6K-ERK1/2的新信号轴，用于规范成血管细胞和HSPC，原始和确定的造血，和血管发育。我们的发现为循环系统发展所必需的成血管细胞和HSPCs的规格提供了重要见解。
