To Explore The Role Of Spliceosome Factor Sart3 In Regulating Hematopoietic Stem/progenitor Cell Development Using Zebrafish

Hematopoietic stem and progenitor cells(HSPCs)possess the potential for self-renew and the capacity,throughout life,to differentiate into all blood cell lineages.Hematopoiesis is tightly regulated by multidimensional pathways,with dysregulation resulting in anemia,leukemia,and other hematological diseases.So,expanding understanding of the mechanisms that regulate HSPC development would be of great significance for shedding light on the pathogenesis of blood-related diseases and developing potential therapeutic treatments for hematopoiesis related disorders.Zebrafish has become an ideal model animal for studying human-related diseases and development because of its high similarity and conservation of genome to mammals.In this study,we used zebrafish to explore the novel regulatory mechanism of HSPCs development.Based on the large-scale forward genetic screening previously performed by the ENU mutagenesis,we obtained a series of zebrafish mutants with hematopoietic defects.Among them,smu471 mutant has defect in definitive hematopoiesis.In this study,we further found that the HSPCs of the smu471 mutant could arise normally in the ventral wall of the dorsal aorta(VDA)region,but the development in the caudal hematopoietic tissue(CHT)region was defective,which was almost disappeared at 4 dpf.To identify the mutant gene of smu471 mutant,we performed positional cloning experiment and identified the causative gene of the smu471 mutant was sart3.A single base substitution occurred in exon 6,which encoded a stop codon.Both zebrafish sart3 gene and human SART3 could effectively rescue the reduced HSPCs in smu471 mutant.Furtherly,using CRISPR/Cas9 to knock out sart3 could mimic the phenotype of smu471 mutant hematopoietic defects.These results suggest that sart3 is indeed the causative gene of smu471 mutant.Next,we explored the regulatory mechanism of sart3 regulating HSPC development.In terms of cytology,we found that both decreased cell proliferation and increased apoptosis mediated the decrease of HSPCs in sart3 mutants.In the molecular mechanism,we performed RNA-seq sequencing on the sart3 mutant,and analyzed the differential genes by the KEGG enriching signaling pathway,and found that the spliceosome pathway and the p53 signaling pathway were significantly enriched.Since the activation of the p53 pathway is closely related to cell cycle arrest and apoptosis,it indicates that the sart3 mutation activates p53,which mediates the inhibition of proliferation and increased apoptosis of HSPCs.To confirm this hypothesis,we injected p53 MO into zebrafish embryos,the results showed that p53 knockdown efficiently rescued the reduced HSPCs in sart3 mutants,which demonstrates that the sart3 gene regulates HSPC development through p53 signaling pathway in zebrafish.Given that sart3 as a splicing factor could promote the recycling of U4 and U6 and the assembly of spliceosome,we speculated the activation of p53 related to the splicing disorder.Through the combined analysis of differentially expressed and abnormally spliced genes,we found that the mdm4 gene in the p53 signaling pathway was enriched.According to previous studies,as a p53 inhibitor,MDM4 could bind to the transactivation domain of p53 and inhibit the transcription of downstream target genes.At the same time,MDM4 can also cooperate with MDM2 to promote the ubiquitination degradation of p53 protein.Whether the abnormal splicing of mdm4 activates p53 and leads to the decrease of HSPCs in the sart3 mutant? By expressing normal mdm4 into sart3 mutants,we found that overexpressing mdm4 could effectively rescue the HSPCs defect in sart3 mutants,indicating that abnormal splicing of mdm4 by sart3 mutation activates p53 and mediates the reduction of HSPCs.Our data indicate that sart3 is required for HSPC proliferation and maintenance to ensure the correct splicing and expression of mdm4 in order to properly inhibit the p53 pathway to prevent eventual hematopoietic failure.This study discovers the regulatory role of the Sart3-Mdm4-p53 pathway in the development of HSPCs,expands our understanding of the regulatory mechanisms that regulate HSPC development,and sheds light on the potential therapeutic treatments for related blood diseases.