AGGF1is Required In The Differentiation Of Both Hematopoietic And Endothelial Lineages During Zebrafish Embryogenesis

The differentiation of hematopoietic and endothelial lineages plays critical roles inthe development of embryogenesis. The abnormal development of hematopoietic andendothelial cells will cause severe defects in development. During embryongenesis,hematopoietic and endothelial lineages develop from a shared progenitor, thehemangioblast. Hemangioblast are developed from the mesoderm in the early vertebrateembryo and can diferentiate 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 andlymphatic vessels. On the other hand, the hematopoietic line will generate blood cells,including erythroid cells, myeloid cells and lymphoid cells. The generation of bothhematopoietic and endothelial lineages is under a complicated and precise control frommolecular signal pathways, and failure control of the regulatory network will cause severedefects in development or diseases. To my knowledge, the molecular mechanismsunderlying the formation of both hematopoietic and endothelial lineages fromhemangioblasts remain largely unknown. Identification of new key regulators and signalpathways in the differentiation of hemangioblasts will help understanding of molecularmechanisms underlying each process, enrich the regulatory network, and provideappropriate molecular targets for clinical treatments of related human diseases.Klippel-Trenaunay Syndrome (KTS) is a congenital vascular disease characterized bymalformations of capillary, venous, and lymphatic vessels. Although KTS has beenstudied for hundreds of years, the etiology is largely unknown. An angiogenic factor withG-Patch and FHA domain1(AGGF1) is a new angiogenic factor that was cloned from bystudying a patient with KTS. Similar to VEGFA, purified wild-type AGGF1proteinpromoted strong angiogenesis in a chick chorioallantoic membrane (CAM) assay. AGGF1binds to the surface of endothelial cells and triggers endothelial cell proliferation. SiRNAsagainst AGGF1reduced AGGF1expression and inhibited capillary tube formation byHMVECs and HUVECs that were plated onto matrigel. Our recent study has establishedthat AGGF1plays an important role in the specification of vein differentiation and angiogenesis by the AKT signaling pathway in zebrafish embryogenesis. However, itremains to be determined whether AGGF1is involved in important processes upsteam ofspecification venous endothelial cells and differentiation of hematopoisis.In this study, I have demonstrated new roles of AGGF1in differentiation of bothendothelial and hematopoietic lineages in zebrafish embryogenesis. Downregulation ofAGGF1expression by specific morpholinos resulted in a decreased number of red bloodcells, granulocytes, macrophages and lymphoid cells. Knockdown of AGGF1impaired theinitiation of definitive hematopoiesis by reducing the expression of runx1and c-myb in theventral wall of the dorsal aorta (DA), which suggests that AGGF1plays an important rolein the specification of hematopoietic stem cells. Since AGGF1affects the development ofboth haematopoietic and endothelial lineages in zebrafish embryos, I next tested whetherAGGF1affected the differentiation of the precursors of haematopoietic and endotheliallineages, hemangioblasts. Downregulated expression of hemangioblast markers such asfli1, scl and lmo2was clearly observed from AGGF1knockdown embryos at the8somitsstage, which indicates that AGGF1is required for development of hemangioblasts. The scland fli1-vp16mRNA rescue experiments indicated that the abnormal reduction of thenumber of both haematopoietic and endothelial cells is due to the downregulated numberof hemangioblasts, and AGGF1may act upstream of scl and fli1in the zebrafishhemangioblast development. Capped AGGF1mRNA could not rescue differentiation ofthe hemangioblasts in cloche mutant zebrafish embryos, and LYCAT mRNA did not rescuethe hemangioblasts morphants caused by AGGF1MO, which indicated that AGGF1is inparallel to cloche in hemangioblast differentiation.The study indicates that AGGF1may act upstream of scl and fli1to regulate thedifferentiation of both hematopoietic and endothelial lineages from hemangioblasts inzebrafish embryogenesis. The study will help to advance our understanding ofdifferentiation of hemangioblasts by identifying the detailed regulatory network in bothhematopoietic and endothelial lineages, and provide important new insight into thepathogenesis of KTS.