AGGF1Is Required In The Differentiation Of Both Hematopoietic And Endothelial Lineages During Zebrafish Embryogenesis

The differentiation of hematopoietic and endothelial lineages plays critical roles in the development of embryogenesis. The abnormal development of hematopoietic and endothelial cells will cause severe defects in development. During embryongenesis, hematopoietic and endothelial lineages develop from a shared progenitor, the hemangioblast. Hemangioblast are developed from the mesoderm in the early vertebrate embryo and can differentiate into both hematopoietic and endothelial cell lineages. Subsequently, the endothelial cell lineages will give rise to vascular endothelial cells, including artery endothelial cells and vein endothelial cells, which form arteries, veins and lymphatic vessels. On the other hand, the hematopoietic line will generate blood cells, including erythroid cells, myeloid cells and lymphoid cells. The generation of both hematopoietic and endothelial lineages is under a complicated and precise control from molecular signal pathways, and failure control of the regulatory network will cause severe defects in development or diseases. To my knowledge, the molecular mechanisms underlying the formation of both hematopoietic and endothelial lineages from hemangioblasts remain largely unknown. Identification of new key regulators and signal pathways in the differentiation of hemangioblasts will help understanding of molecular mechanisms underlying each process, enrich the regulatory network, and provide appropriate molecular targets for clinical treatments of related human diseases.Klippel-Trenaunay Syndrome (KTS) is a congenital vascular disease characterized by malformations of capillary, venous, and lymphatic vessels. Although KTS has been studied for hundreds of years, the etiology is largely unknown. An angiogenic factor with G-Patch and FHA domain1(AGGF1) is a new angiogenic factor that was cloned from by studying a patient with KTS. Similar to VEGFA, purified wild-type AGGF1protein promoted strong angiogenesis in a chick chorioallantoic membrane (CAM) assay. AGGF1binds to the surface of endothelial cells and triggers endothelial cell proliferation. SiRNAs against AGGF1reduced AGGF1expression and inhibited capillary tube formation by HMVECs and HUVECs that were plated onto matrigel. Our recent study has established that AGGF1plays an important role in the specification of vein differentiation and angiogenesis by the AKT signaling pathway in zebrafish embryogenesis. However, it remains to be determined whether AGGF1is involved in important processes upsteam of specification venous endothelial cells and differentiation of hematopoisis.In this study, I have demonstrated new roles of AGGF1in differentiation of both endothelial and hematopoietic lineages in zebrafish embryogenesis. Downregulation of AGGF1expression by specific morpholinos resulted in a decreased number of red blood cells, granulocytes, macrophages and lymphoid cells. Knockdown of AGGF1impaired the initiation of definitive hematopoiesis by reducing the expression of runxl and c-myb in the ventral wall of the dorsal aorta (DA), which suggests that AGGF1plays an important role in the specification of hematopoietic stem cells. Since AGGF1affects the development of both haematopoietic and endothelial lineages in zebrafish embryos, I next tested whether AGGF1affected the differentiation of the precursors of haematopoietic and endothelial lineages, hemangioblasts. Downregulated expression of hemangioblast markers such as flil, scl and lmo2was clearly observed from AGGF1knockdown embryos at the8somits stage, which indicates that A GGF1is required for development of hemangioblasts. The scl and flil-vpl6mRNA rescue experiments indicated that the abnormal reduction of the number of both haematopoietic and endothelial cells is due to the downregulated number of hemangioblasts, and AGGF1may act upstream of scl and flil in the zebrafish hemangioblast development. Capped AGGF1mRNA could not rescue differentiation of the hemangioblasts in cloche mutant zebrafish embryos, and LYCAT mRNA did not rescue the hemangioblasts morphants caused by AGGF1MO, which indicated that AGGF1is in parallel to cloche in hemangioblast differentiation.The study indicates that AGGF1may act upstream of scl and flil to regulate the differentiation of both hematopoietic and endothelial lineages from hemangioblasts in zebrafish embryogenesis. The study will help to advance our understanding of differentiation of hemangioblasts by identifying the detailed regulatory network in both hematopoietic and endothelial lineages, and provide important new insight into the pathogenesis of KTS.