Molecular Mechanisms Underlying Drug BF-170 Induces Emergence Of Hematopoietic Stem And Progenitor Cells

During vertebrate development,hematopoietic stem and progenitor cells(HSPCs)differentiate from the hematopoietic endothelium on the ventral wall of the dorsal aorta,and their formation and function are controlled by interactions between various signaling pathways.Zebrafish has proved to be a simple chemogenetic system and have been used to identify various pathways regulating organogenesis,including hematopoiesis.In this study,we discovered the hematopoietic-promoting drug BF-170 through a high-throughput screening system of primary cultured cells of zebrafish blastocyst embryos.BF-170 now acts as an imaging probe for Alzheimer’s disease Tau protein in vivo and indicator in plant secondary xylem cells.However,its effects on vertebrate hematopoietic development are poorly understood.Therefore,we explored the molecular regulatory mechanism and potential theory of BF-170-induced HSPCs generation on the basis of zebrafish embryos.1.Zebrafish primary cultured blastomere cells system in vitro screened out the drug BF-170 for inducing hematopoietic stem and progenitor cellsWe established a method for chemical screening of new pro-hematopoietic molecules using primary cultured blastomere cells of Tg(cmyb:GFP)and Tg(mpx:GFP)fluorescent transgenic embryos,respectively.Fluorescently labeled target cells driven by specific gene promoters were cultured in 384-well plates,about 3000 small molecule compounds were used to treat the cultured cells,and the expression of GFP was analyzed by an automatic imaging system,thereby indicating the expression of the genes cmyb and mpx.97 hits were found to inhibit cmyb-driven GFP intensity in primary cultured cells,25 hits to promote cmyb-driven GFP expression,and 9 hits also simultaneously increased mpx-driven GFP.Further Whole mount in situ hybridization(WISH)in zebrafish embryos showed that 12 of the 25 hits increased cmyb expression in the intermediate cell mass,promoting primary hematopoiesis in the embryos.In addition,8 drugs that increase the fluorescence of cmybGFP positive cells(7 hits can simultaneously increase the mpx-driven GFP fluorescence intensity)screened by the primary cell in vitro culture system were added to the mouse embryoid body induction differentiation experiment.Cell-direcet q RT-PCR was used to detect the expression of c-Myb,Mpx,Gata1,Lmo2,Runx1 and Scl hematopoietic genes,and it was found that 8 drugs could significantly enhance the expression of at least three hematopoietic genes in mouse embryonic stem cells.Among them,the representative drug BF-170 can conservatively induce the expression of all the above hematopoietic genes in zebrafish primary cultured blastomere cells and mouse embryonic stem cells,and the expression of hematopoietic-related gene cmyb,lmo2 can also be significantly increased in zebrafish embryo in the intermediate cell mass.Furthermore,applying BF-170 to radiation recovery experiments,we found that it can induce transplantable cells including HSPCs in vitro,and successfully transplanted into the eyes,thymus and head kidneys of adult fish.In conclusion,the zebrafish primary cultured blastomere cells in vitro screening system is an efficient hits screening system,which can effectively screen out the drug BF-170,which is conserved in zebrafish and mammals to promote hematopoiesis.2.The drug BF-170 promoted the development of hematopoietic stem and progenitor cellsWISH results showed that BF-170 could promote the expression of primary hematopoietic genes lmo2/scl,HSPCs marker cmyb and endothelial cell marker flk1 in zebrafish during the process of primary hematopoiesis.In subsequent definitive hematopoiesis,WISH,Cell-direcet q RT-PCR and confocal microscopy revealed that BF-170 upregulated the expression of the HSPCs marker gene cmyb/runx1,endothelial cell marker fli1/flk1 and arterial marker ephrin B2 a at 28 hpf,and BF-170 promoted the proliferation of HSPCs;then BF-170 increased HSPCs and angiogenesis until 48 hpf.However,focusing on the effect of BF-170 on hematopoietic differentiation,WISH results confirmed that BF-170 inhibited the differentiation of erythroid and lymphoid cells,and slightly promoting the expression of myeloid cells.In conclusion,BF-170 induced the generation of HSPCs.3.The molecular regulation mechanism of the drug BF-170-induced hematopoietic stem and progenitor cellsThis study firstly found that BF-170 can accelerate the blood flow rate in embryos,and the expression of klf2 a is up-regulated.Cilia can transmit changes in blood flow velocity to the Notch signaling pathway to regulate HSPCs generation.This study found that BF-170 could increase the expression of fish embryo cilia genes pkd2,kif3 a,ift88,and fsd1,and BF-170 rescued the reduction of HSPCs in fsd1 Morpholinos;Meanwhile,BF-170 upregulated the expression of Notch signaling pathway receptors,ligands,and target genes,and rescued the defect in HSPCs in embryos treated with Notch signaling pathway inhibitor DAPT;induced the downstream NO signal of Notch signaling pathway,and then regulated the occurrence of HSPCs.In conclusion,this study showed that BF-170 accelerates blood flow,transmits it to the Notch signaling pathway through primary cilia induction,and finally enhances NO signaling to regulate the generation of HSPCs.In this paper,through a high-throughput chemical screening method,the efficiency of identifying drugs that promote hematopoiesis in vitro was improved,and the positive regulation of the drug BF-170 on HSPCs was innovatively discovered.And we revealed the signaling regulatory network that BF-170 induces HSPCs generation in zebrafish embryos.This study not only provided a new screening method,but also improved the theoretical basis for the molecular regulation mechanism for the development of hematopoietic stem and progenitor cells.