Ribosome Biogenesis Protein Urb2 Regulates Hematopoietic Stem Cells Development Via P53 Pathway In Zebrafish

Ribosomes are essential machines for protein synthesis in cells,and the biogenesis of ribosome is a highly conserved process in different species.Ribosome biogenesis is crucial to cellular adaptation,proliferation,and growth.Disruption of the process will result in many improper genetic programs,thus triggering some specific diseases such as Diamond Blackfan anemia and Dyskeratosis congenita.Ribosome biogenesis is a process that needs many different ribosome biogenesis proteins.If one of these proteins is mutated,the process may be dysfunctioned.Urb2 is an essential protein for ribosome biogenesis,and it locates in the nucleolus.Research in yeast suggests that Urb2 protein is necessary for 60S ribosomal subunit biogenesis,and mutation or depletion of urb2 will lead to the disruption of ribosomal subunits and rRNAs.Urb2 contains a conserved nucleolar 27S pre-rRNA processing domain,implying that this domain may be vital in the regulation of Urb2 in ribosome biogenesis.The deficiency of ribosome biogenesis will lead to the stress responses of ribosomes,resulting in the accumulation and stabilization of P53 protein.P53 pathway plays significant roles in cell cycle,apoptosis and DNA repair.Studies in mouse and zebrafish demonstrate that the disruption of ribosome biogenesis will lead to the activation of P53 pathway,which then affects several tissues or organs development.Those data suggest that P53 pathway can cooperate with ribosome biogenesis,thus regulating the proper developmental process in vivo.Hematopoietic stem cells have the potential to self-renew and differentiate to all blood lineages,validating their important roles in the maintenance and self-renewal of vertebrate blood system.HSCs transplantation is restrictively used to treat hematological diseases for the shortage of sources.For the sake of producing transplantable HSCs,the molecular mechanisms of HSCs development need to be fully understood.Zebrafish is widely used to study hematopoietic process for its particular advantages to other models.The key genes and signaling pathways that regulate hematopoiesis are highly conserved in zebrafish and mammals,the research in zebrafish would provide useful theoretical basis for the hematopoietic studies in mammals.We isolated a zebrafish mutant cq42 through an ENU-induced forward genetic screen,and the mutant showed hematopoietic defects.Genetic mapping results displayed that cq42 carried a non-sense mutation in gene urb2.In addition,we detected urb2 expression pattern using whole mount in situ hybridization?WISH?and fluorescent in situ hybridization,and we found that urb2 showed little expression during hematopoietic stem cells?HSCs?emergence stage.However,urb2 showed strong expression in caudal hematopoietic tissue?CHT?during HSCs expansion stage,implying its important roles in HSCs expansion.Molecular characterization of urb2^cq42mutant suggests that urb2 deficiency notably decreases the population of HSCs in CHT and early T cells in thymus.However,the development of primitive hematopoiesis and vascular in urb2^cq42 mutant seems normal compared to the WT,indicating the specific regulation of urb2 in definitive hematopoiesis.Further analysis shows that compromised cell proliferation and superfluous apoptosis are observed in the CHT of urb2^cq42 mutant,which could account for the hematopoietic defects in CHT and thymus.These results suggest that ribosome biogenesis protein Urb2 regulates HSCs development through P53 pathway in zebrafish.Shh/Vegfa/Notch signaling and Wnt16/Dlc/Dld signaling play significant roles in HSCs budding and specification,but those signalings show normal expression levels in urb2^cq42 mutant.To explore the molecular mechanisms that might underlie the HSCs defects in urb2^cq42 mutant,we examined the expression of those genes implicated in cell proliferation or apoptosis.Previous studies have shown that the mutations of ribosomal proteins will cause the upregulation of P53 pathway,which is implicated in cell cycle and apoptosis.Hence,we assumed that P53 pathway might be regulated by Urb2 to affect HSCs development.The whole mount in situ hybridization results showed that p53 and its response genes?p21,mdm2,and ccng1?were both upregulated in urb2^cq42 mutant.Thus,we speculated that activated P53 pathway may cause the hematopoietic defects in urb2^cq42 mutant.To test the hypothesis,we performed two approaches.Firstly,we generated a double mutant by outcrossing urb2^cq42 heterozygote to p53^M214K homozygous mutant.WISH results revealed that the hematopoietic defects in urb2^cq42 mutant were fully rescued after the additional loss of p53.Secondly,we knocked down p53 by injecting p53 morpholino?MO?.We also checked the rescue efficiency by WISH,the results showed that the injection of p53 MO could equally rescue the HSCs defects in the CHT and thymus of urb2^cq42 mutant.mTOR signaling pathway palys significant roles in cell metabolism and growth,and it can also regulate embryonic development and tissue regeneration.Previous study have demonstrated that Urb1 can act as a downstream regulator of mTORC1 to mediate the zebrafish digestive organs development.We have known that Urb2 is essential to HSCs development in zebrafish,but we don't know if mTORC1 has any relationship with Urb2 in HSCs development.Raptor is an important factor of mTORC1.We injected wild-type embryos with raptor MO and found that it would not affect the budding of HSCs from dorsal aorta.However,the raptor MO embryos displayed hematopoietic defects at 72 hours post-fertilization?hpf?,suggesting that mTOR signaling is involved in zebrafish hematopoiesis.The hematopoietic defects of raptor MO embryos are similar to the hematopoietic phenotype in urb2^cq42 mutant.Hence,we guessed that Urb2 may function as a downstream regulator of mTORC1 to influence the HSCs development.In order to check the relationship between Urb2 and mTOR signaling pathway,we constructed a heatshock transgenic line Tg?Hsp70L:urb2?.Using this transgenic line,we found that overexpression of urb2could rescue the hematopoietic defects in raptor MO embryos,implying that Urb2 can act as a downstream mediator of mTORC1 to regulate zebrafish hematopoiesis.In conclusion,our study demonstrate that Urb2 can regulate HSCs development through P53 pathway,and it also acts as a downstream mediator of mTORC1 to regulate zebrafish hematopoiesis.