Using Zebrafish To Evaluate The Hematopoietic Inhibition Risk Of Famcilovir And Study The Role Of Smu1539 Mutant In Hematopoiesis

Hematopoiesis refers to the process of the production and maturation of various blood cells in the hematopoietic system.The whole process is very complex and is precisely regulated by various transcription factors and signaling pathways.Although there are thousands of reports about hematopoiesis,the knowledge on the whole regulation network of hematopoiesis is still little.By the forward genetic screening of hematopoiesis,unknown genes regulating hematopoiesis or unknown functions of known genes in hematopoiesis can be found.In addition to the factors of the body itself,external substances also have an impact on hematopoiesis.At present,we still have unknown region about effects on the clinical application of drugs,including hematopoiesis.By using zebrafish,we can study the role of drugs in hematopoiesis and may find new clinical applications of drugs.In the first part of this paper,we explored the effect of famciclovir on hematopoiesis in zebrafish.By usingTg(cd41:eGFP)transgenic zebrafish,a specific marker of thrombocytes,to screen drugs affecting hematopoietic cells,we found that famciclovir could reduce the GFP+ signal,which means thrombocytes decreased.Famciclovir(FCV)is an oral prodrug of penciclovir(PCV),a guanosine analog antiviral drug commonly used for the treatment of virus infection.FCV will be rapidly converted to PCV in vivo,and then PCV will be stpwise phosphorylated to a bioactivated form,PCV triphosphate,mediated by viral thymidine kinase and following by host cellular kinase.Therefore,we wanted to explore how famciclovir affects hematopoiesis and what is the mechanism of action,and this may provide a new understanding of its clinical application.Through the identification of phenotype by whole-mount in situ hybridization(WISH)and antibody staining,we found that the number of HSPCs in CHT was reduced,accompanied by the defect on all lineages of the definitive hematopoiesis in famiclovir-treated embryos.By TUNEL assay,AnnexinV assay,cell cycle analysis and BrdU proliferation assay,we found that the decrease of HSPCs in CHT of the famciclovir-treated embryos was due to increased apoptosis and decreased proliferation.On the other hand,FCV treatment could effectively relieve myeloid malignancies in the c-mybhyper MDS-like fsh model,and played a role not only in the embryonic stage but also in adult zebrafsh.In summary,famciclovir induces the HSPCs defect in CHT,leading to abnormalities in all blood lineages,and appropriate FCV treatment may be benefcial for the treatment of MDS.In the second part of this paper,we carried out gene mapping and function study of the hematopoietic deficient mutant smu1539 in zebrafish.Through the large-scale forward genetic screening based on ENU mutagenesis carried out by our laboratory before,we got a lot of hematopoietic deficient mutants in zebrafish,including smu1539 mutant,which shows the lack of βe1-globin expression in definitive hematopoiesis.Therefore,we wanted to explore that the mutated gene of mutant smu1539 and its role in hematopoiesis.We located the mutation site between two markers chr17-1001 and chr17-1009 on chromosome 17 by gene mapping.Based on whole genome sequencing and CDS sequencing of some genes,we found some base changes that can cause amino acid changes in edaradd,si:ch21-125o16.4,exo1 and ttc27 genes.In particular,the base change of ttc27 can cause premature termination of protein translation.Through smu1539 mutant phenotyping by WISH,we found that the primitive hematopoiesis of smu1539 mutant was not affected,but definitive hematopoiesis was abnormal.The emergence of HSPCs of smu1539 mutant in VDA region was not affected,but their maintenance in CHT region was abnormal.In addition,we found the proliferation of HSPCs in CHT did not change but the apoptotic cells in CHT were increased by BrdU assay,acridine orange staining and RNA-seq,suggesting that the decrease of HSPCs in CHT region in smu1539 mutant may be due to the increased apoptosis.In summary,the mutated gene of smu1539 mutant locates in chromosome 17,and may affect the maintenance of HSPCs in CHT by promoting the apoptosis of HSPCs,and plays an essential role in hematopoiesis.