Functional Study Of NR4A3 During The Process Of Hematopoiesis Development And Differentiation

Hematopoietic process is regulated by complex and sophisticated molecular signaling pathway,any deficiency of key molecules can lead to severe developmental defects.So far,molecular mechanisms involving hematopoietic development is not very clear.Hematopoietic development in vertebrate is highly conserved,and the developmental pattern of zebrafish hematopoiesis is highly similar to that of mice.Using zebrafish as a model to study hematopoietic mechanisms has many unique advantages.Compared to other nuclear receptor families,members in the orphan nuclear receptor family share many similarities in their structure domains,but their endogenous ligand is unknown.Nr4a3?NOR-1?belongs to the orphan nuclear receptor family Nur77 and is expressed in various tissues such as skeletal muscle,adipose tissue,heart,kidney,liver,brain and T lymphocytes.Double deletion of Nr4a1 and Nr4a3 led to rapid development of acute myeloid leukemia?AML?in mice,Nr4a1 and Nr4a3 were identified as tumor suppressors in acute myeloid leukemia?AML?.Studies have shown that reduced gene dosage of Nr4a1 and Nr4a3 in mice(Nr4a1^+/-/Nr4a3^-/-or Nr4a1^-/-/Nr4a3^+/-),of which below a critical threshold leads to a chronic myeloid malignancy that recapitulates the pathologic symptoms of mixed myelodysplastic/myeloproliferative neoplasms?MDS/MPNs?.However,the role of Nr4a3 in hematopoietic development and molecular mechanisms involving is not clear.In this study,we firstly injected Nr4a3 morpholino antisense RNA?Nr4a3-MO?into one cell zebrafish embryos after fertilization,which can disturb the mRNA splicing to lead to the down-regulation of Nr4a3 expression,we discovered that the obvious decrease in erythrocytes and increase in myeloid neutrophils.To further study the function of Nr4a3 during the process of hematopoiesis development and differentiation,we knock out gene Nr4a3 using Crispr-cas9 genome editing technique in zebrafish,and homozygous lines of frameshift mutations in Nr4a3 gene were obtained.Then we did in situ hybridization,erythrocyte fast blue staining and sudan black staining of myeloid neutrophils,and we found that Nr4a3 knockout had no significant effect on the differentiation and development of erythroid and lymphoid cells;However,the results of in situ hybridization of hematopoietic stem cells?HSCs?and Sudan black staining confirmed the significant increase in hematopoietic stem cells?HSCs?and in myeloid neutrophils.Based on the results of Nr4a3 in mice and our results in zebrafish,we hypothesized that Nr4a3,probably acting as a direct target gene for Notch-runx1,can affect the homeostasis of hematopoietic stem cells,the proliferation,differentiation and apoptosis of myeloid cells.