Functional Study Of Phf6 During Early Hematopoiesis In Zebrafish

Hematopoiesis consists of primitive hematopoiesis and definitive hematopoiesis,which occurs under conditions of order and complexity,and the dysfunction of these processes can lead to various blood diseases.Zebrafish are small in size and fast in reproduction.They are very similar to humans in embryonic morphogenesis.Some important regulatory factors and signaling pathways are also very conserved,which is very suitable for the study of hematopoietic development.Mutations in phf6 are associated with the development of multiple blood diseases.Phf6 contains two PHD domains in its structure,and mutations sometimes occur in this domain.Although existing studies have shown that mutations in phf6 are associated with the development of blood diseases,the specific function and molecular mechanism of how the gene affects the occurrence of blood diseases are not clear.In order to study the function of phf6 during hematopoiesis,we initially detected the spatiotemporal expression of phf6 by in situ hybridization,and then further investigated its function during early hematopoiesis using phf6 knockdown and knockout.First,phf6 splicing morpholino and phf6 atg morpholino were injected into zebrafish single-cell embryos to reduce the expression of phf6.The expression of specific markers in different hematopoiesis was detected to research the effects of phf6 knockdown during early hematopoiesis by in situ hybridization,O-Dianisidine staining,neutral red staining,qPCR and et al.The results showed that the phenotypes after injection of these two morpholino were consistent,that is,the primitive hematopoiesis was not affected,while during the definitive hematopoiesis,the expression of erythroid hemoglobin was decreased,the number of myeloid neutrophils was decreased but the number of macrophages was increased.Further,we obtained phf6 mutants using the CRISPR/Cas9 system to study the function of phf6 during early hematopoiesis.F0 generations have been obtained.Finally,based on our preliminary real-time quantitative detection of phf6-related factors,we found that the expression of Notch1 a and runx1 was decreased after injection of phf6 morpholino.Previous studies have shown that runx1 is involved in the differentiation of myeloid cells,so we hypothesized that phf6 probably play a critical role mediated by Notch-runx1 pathway in the proliferation and differentiation of myeloid cells.