Specifies Hemangioblasts at the Top of Regulatory Hierarchy via and mTOR-S6K-Emp2-ERK Signaling.

Background: 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.

Methods: CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (type II CRISPR RNA-guided endonuclease) editing was used to develop and knockout zebra fish. Whole-mount in situ hybridization and transgenic Tg(-EGFP [enhanced green fluorescent protein]), Tg(-EGFP), Tg(-DsRed [discosoma sp. red fluorescent protein]), Tg(-EGFP), Tg(-EGFP), and Tg(;-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.

Results: Knockout of impaired specification of hemangioblasts and HSPCs, hematopoiesis, and vascular development in zebra fish. Expression of /-the presumed earliest marker for hemangioblast specification-was significantly reduced in embryos and increased by overexpression of in embryos. Overexpression of rescued the impaired specification of hemangioblasts and HSPCs and development of hematopoiesis and intersegmental vessels in embryos, placing upstream of in hemangioblast specification. To identify the underlying molecular mechanism, we identified as a key downstream gene. Similar to , 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 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 knockdown.

Conclusions: These results indicate that acts at the top of 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.