The Function And Mechanism Of CG5844 In The Germline Stem Cell Nich Of Drosophila Testes

Objective: Germline stem cells(GSCs)are a type of adult stem cells(ASCs).Their selfrenewal and differentiation potential are strictly regulated by the stem cell niche and multiple pathways.Drosophila testis germline stem cells provide an ideal model for exploring the regulatory mechanisms of stem cell self-renewal and differentiation.In previous studies,a new GSC regulatory gene CG5844 was screened in Drosophila testis.However,the underlying mechanistic links between CG5844 and the stem cell niche remain largely undetermined.This study mainly studies and discusses the function and mechanism of CG5844 gene in germline stem cell niche of Drosophila testis.Methods:(1)Through UAS / Gal4-mediated RNAi pathway,two types of Gal4s(Nos-Gal4 and Tj-Gal4),which are mainly expressed in the stem cell niche,cause the loss of CG5844 function.The phenotype of CG5844 in the Drosophila testis stem cell niche was detected and analyzed by immunofluorescent staining of the labeled protein.(2)Small interfering RNA(si RNA)was used to silence CG5844 expression in Drosophila S2 cells,cell proliferation was detected by Phospho-Histone H3(PH3)immunofluorescence staining,TUNEL immunofluorescence staining and flow cytometry to detect apoptosis,and CCK-8 assay to determine whether si RNA affects S2 cell growth to explore the function of CG5844 in vitro.(3)S2 cells were transfected with CG5844 recombinant plasmid to overexpress them in S2 cells.The effects of CG5844 overexpression on the proliferation and apoptosis of Drosophila S2 cells were also detected using PH3 and TUNEL immunofluorescence staining,flow cytometry,and CCK-8 assay.(4)CG5844 rescue experiments were performed to further confirm the phenotype.(5)Liquid chromatography tandem mass spectrometry(LC-MS / MS)was used to identify CG5844 binding proteins and perform bioinformatics analysis.(6)Quantitative real-time PCR was used to detect the relationship between the binding protein highly related to CG5844 and itself.(7)Through UAS / Gal4-mediated RNAi pathway,cell-specific knockdown of the candidate gene ribosomal protein L6(Rp L6)selected in the mass spectrometry results,and analysis of whether Rp L6 has a similar phenotype as the CG5844 by immunofluorescence,so as to further explore the regulation of Drosophila testicular reproduction by CG5844 regulation mechanism of stem cell microenvironment.Results:(1)Confocal microscopy imaging results showed that there were specific defects at the tip of the Drosophila testis.Knocking down CG5844 in early germ cells using Nos-Gal4 caused tiny testis and complete absence of germ cells.However,CG5844 knockdown mediated by Tj-Gal4 led to normal cystic cell dysfunction and accumulation of undifferentiated germ cells,eventually developed into testis tumors.(2)Knockdown of CG5844 in Drosophila S2 cells resulted in inhibition of cell growth.(3)CG5844 overexpression significantly inhibited the cell growth of Drosophila S2 cells.(4)The cell proliferation phenotype induced by silencing the CG5844 gene can be rescued by overexpressing CG5844 in S2 cells,but the apoptotic phenotype cannot be rescued.(5)Both ribosomes and ribonucleoprotein complexes are CG5844 binding proteins.The enrichment analysis revealed that CG5844 participated in the ribosome signaling pathway with high confidence.(6)The results of quantitative real-time PCR showed that CG5844 regulated the expression of spliceosome and ribosome subunits.(7)Rp L6 RNAi completely mimics the self-renewal and differentiation-related phenotypes caused by CG5844 RNAi in Drosophila testis GSCs.Conclusions: The in vivo experiments of Drosophila show that the CG5844 gene is necessary for the formation and differentiation of normal germ cells and cyst cells.The lack of CG5844 will lead to self-renewal and differentiation defects of germline stem cells.In vitro experiments showed that CG5844 is involved in the regulation of Drosophila S2 cell proliferation and apoptotic homeostasis.In addition,CG5844 is highly related to the ribosome complex,and CG5844 can regulate the expression of spliceosome and ribosome subunits.Through the same in vivo experiments in Drosophila,it was found that the ribosomal subunit Rp L6 RNAi completely mimics the self-renewal and differentiation-related phenotypes caused by CG5844 RNAi in Drosophila testis GSCs.It is suggested that CG5844 gene may regulate the self-renewal and differentiation of Drosophila testicular germ stem cells by interacting with Rp L6.This study helps to clarify the fate regulation mechanism of germline stem cells,and provides new ideas for elucidating the formation of germ cell tumors.