Functional Study Of Thbs1 During Hematopoiesis In Zebrafish

Hematopoietic stem cells play a very important role during early hematopoiesis.It is derived from arterial endothelial cells and has the potential to differentiate into various types of blood cells.The molecular mechanisms that regulate the development and differentiation of hematopoietic stem cells are complex.A slight hurdle can lead to serious blood diseases.Diamond-Blackfan is a rare autosomal dominant disease,most of which are infancy,manifested as abnormal red blood cells and lack of bone marrow erythroid progenitor cells.The expression of thrombospondin-1?TSP-1?were significantly changed in the patient.TSP-1 is an integral part of platelet alpha granules and has been reported to act on vascular endothelial cells to inhibit the formation of new blood vessels and participate in inflammatory reactions.The hematopoietic stem cells originate from vascular endothelial cells and originate from a process known as endothelial-to-hematopoietic transition?EHT?.And it has been reported that inflammatory factors such as NF-?B can regulate the process of hematopoietic stem cells.Therefore,we speculate that TSP-1 can influence the development of hematopoietic stem cells through the EHT process and affect the formation of various types of blood cells.To prove our hypothesis,we first knocked down the expression level of thbs1 in zebrafish using morpholino and detected the expression levels of marker genes during primitive hematopoiesis?gata1,scl?and definitive hematopoiesis?runx1,cmyb?process.We found that the expression levels of both primitive hematopoietic and definitive hematopoietic marker genes were significantly decreased in thbs1^-/-embryos,suggesting that knockdown of thbs1 has an effect on hematopoiesis.Further,we used the CRISPR/Cas9 system to knock out thbs1 and constructed a thbs1^-/-homozygous mutant zebrafish model(thbs1^-/-).Using in situ hybridizatio,sudan black,o-dianisidine,neutral red staining technique,we found the expression of hematopoietic stem cell marker genes runx1and cmyb were significantly decreasedInaddition,we use real-time fluorescence quantification confirmed that EHT was affected during the development of hematopoietic stem cells in thbs1^-/-,demonstrating that thbs1 is involved in the EHT process and is associated with the early development of hematopoietic stem cells.