CG5033基因在果蝇睾丸中调控生殖干细胞的自我更新和分化
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摘要
目的:探讨CG5033基因在果蝇睾丸中对生殖干细胞自我更新和分化过程的影响。方法:采用UAS/Gal4系统驱动果蝇睾丸生殖干细胞(nos-Gal4)、分化的精原细胞(bam-Gal4)和包囊细胞(tj-Gal4)中UAS-CG5033 RNAi的表达。在F1代果蝇中挑选单只nos>CG5033 RNAi、bam>CG5033 RNAi及tj>CG5033 RNAi基因型成年雄蝇,与3只野生型处女蝇杂交,观察是否可获得后代果蝇;通过光学显微镜和免疫荧光染色方法观察野生型和CG5033 RNAi雄性果蝇睾丸中Vasa、眼缺陷蛋白(eyes absent,Eya)、DE-cad、锌指结构域1蛋白(Zn finger homeodomain 1,Zfh1)和1B1的表达模式。结果:与野生型果蝇组比较,nos>CG5033 RNAi组和tj>CG5033 RNAi组果蝇呈雄性不育,而bam>CG5033 RNAi果蝇组生育率未受影响。光学显微镜下,与野生型果蝇组比较,用nos-Gal4驱动UAS-CG5033 RNAi雄性果蝇的睾丸明显变小。免疫荧光染色结果表明,与野生型果蝇组比较,用nos-Gal4驱动UAS-CG5033 RNAi的雄性果蝇睾丸中的生殖细胞消失,包囊细胞代偿性增多,而用tj-Gal4驱动UAS-CG5033 RNAi的果蝇睾丸中出现生殖细胞肿瘤;bam>CG5033 RNAi睾丸未见明显异常。结论:CG5033基因编码的蛋白在果蝇睾丸生殖干细胞中影响细胞的自我更新能力,并可通过体细胞干细胞影响生殖干细胞的分化过程,最终导致生殖细胞肿瘤的形成。
Objective: To explore the regulatory effect of self-renewal and differentiation of germline stem cells for CG5033 gene in the Drosophila testis. Methods: This study uses the UAS/Gal4 system to drive the expression of UAS-CG5033 RNAi in germline stem cells( nos-Gal4),differentiated spermatogonial cells( bam-Gal4) and cyst cells( tj-Gal4). Single adult F1 male fly with genotype of nos > CG5033 RNAi,bam > CG5033 RNAi or tj > CG5033 RNAi was hybridized with three wildtype( WT) virgin flies to observe whether they could obtain offspring flies. To understand the function of CG5033 gene in the stem cell niche,the expression patterns of Vasa,eyes absent( Eya),DE-cad,Zn finger homeodomain 1( Zfh1) and 1 B1 were observed by light microscopy and immunofluorescence staining in WT and CG5033 RNAi testes. Results: Nos > CG5033 RNAi and tj > CG5033 RNAi flies were male infertile when compared with WT flies,while bam > CG5033 RNAi flies do not affect male fertility. Nos-Gal4 driven UASCG5033 RNAi testes were significantly smaller than that of the normal group. Immunofluorescence staining showed that nos-Gal4 driven UAS-CG5033 RNAi testes led to germ cell lost and compensatory increase of cyst cells when compared with the normal group. However,tj-Gal4 driven UAS-CG5033 RNAitestes resulted in the formation of testicular germ cell tumor; there was no observable abnormality in bam> CG5033 RNAi testes. Conclusion: CG5033 encoded protein can influence the self-renewal ability of germline stem cells in the Drosophila testis,and CG5033 can affect the differentiation process of germline stem cells by somatic stem cells,eventually leading to the formation of germ cell tumor.
Objective: To explore the regulatory effect of self-renewal and differentiation of germline stem cells for CG5033 gene in the Drosophila testis. Methods: This study uses the UAS/Gal4 system to drive the expression of UAS-CG5033 RNAi in germline stem cells( nos-Gal4),differentiated spermatogonial cells( bam-Gal4) and cyst cells( tj-Gal4). Single adult F1 male fly with genotype of nos > CG5033 RNAi,bam > CG5033 RNAi or tj > CG5033 RNAi was hybridized with three wildtype( WT) virgin flies to observe whether they could obtain offspring flies. To understand the function of CG5033 gene in the stem cell niche,the expression patterns of Vasa,eyes absent( Eya),DE-cad,Zn finger homeodomain 1( Zfh1) and 1 B1 were observed by light microscopy and immunofluorescence staining in WT and CG5033 RNAi testes. Results: Nos > CG5033 RNAi and tj > CG5033 RNAi flies were male infertile when compared with WT flies,while bam > CG5033 RNAi flies do not affect male fertility. Nos-Gal4 driven UASCG5033 RNAi testes were significantly smaller than that of the normal group. Immunofluorescence staining showed that nos-Gal4 driven UAS-CG5033 RNAi testes led to germ cell lost and compensatory increase of cyst cells when compared with the normal group. However,tj-Gal4 driven UAS-CG5033 RNAitestes resulted in the formation of testicular germ cell tumor; there was no observable abnormality in bam> CG5033 RNAi testes. Conclusion: CG5033 encoded protein can influence the self-renewal ability of germline stem cells in the Drosophila testis,and CG5033 can affect the differentiation process of germline stem cells by somatic stem cells,eventually leading to the formation of germ cell tumor.
引文
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[16]Lim JG,Fuller MT.Somatic cell lineage is required for differentiation and not maintenance of germline stem cells in Drosophila testes[J].Proc Natl Acad Sci USA,2012,109(45):18477-18481.
[17]Yu J,Wu H,Wen Y,et al.Identification of seven genes essential for male fertility through a genome-wide association study of non-obstructive azoospermia and RNA interference-mediated large-scale functional screening in Drosophila[J].Hum Mol Genet,2015,24(5):1493-1503.
[18]Demontis F,Perrimon N.Integration of Insulin receptor/Foxo signaling and d Myc activity during muscle growth regulates body size in Drosophila[J].Development,2009,136(6):983-993.
[19]Chen D,Wu C,Zhao S,et al.Three RNA binding proteins form a complex to promote differentiation of germline stem cell lineage in Drosophila[J].PLo S Genet,2014,10(11):e1004797.
[20]Insco ML,Bailey AS,Kim J,et al.A self-limiting switch based on translational control regulates the transition from proliferation to differentiation in an adult stem cell lineage[J].Cell Stem Cell,2012,11(5):689-700.
[21]Li Y,Maines JZ,Tastan OY,et al.Mei-P26 regulates the maintenance of ovarian germline stem cells by promoting BMP signaling[J].Development,2012,139(9):1547-1556.
[2]Fuller MT,Spradling AC.Male and female Drosophila germline stem cells:two versions of immortality[J].Science,2007,316(5823):402-404.
[3]Spradling A,Fuller MT,Braun RE,et al.Germline stem cells[J].Cold Spring Harb Perspect Biol,2011,3(11):a002642.
[4]Kiger AA,Jones DL,Schulz C,et al.Stem cell self-renewal specified by JAK-STAT activation in response to a support cell cue[J].Science,2001,294(5551):2542-2545.
[5]Tulina N,Matunis E.Control of stem cell self-renewal in Drosophila spermatogenesis by JAK-STAT signaling[J].Science,2001,294(5551):2546-2549.
[6]Kawase E,Wong MD,Ding BC,et al.Gbb/Bmp signaling is essential for maintaining germline stem cells and for repressing bam transcription in the Drosophila testis[J].Development,2004,131(6):1365-1375.
[7]Shivdasani AA,Ingham PW.Regulation of stem cell maintenance and transit amplifying cell proliferation by TGF-βsignaling in Drosophila spermatogenesis[J].Curr Biol,2003,13(23):2065-2072.
[8]Bunt SM,Hime GR.Ectopic activation of Dpp signalling in the male Drosophila germline inhibits germ cell differentiation[J].Genesis,2004,39(2):84-93.
[9]Yu J,Lan X,Chen X,et al.Protein synthesis and degradation are essential to regulate germline stem cell homeostasis in Drosophila testes[J].Development,2016,143(16):2930-2945.
[10]de Cuevas M,Matunis EL.The stem cell niche:lessons from the Drosophila testis[J].Development,2011,138(14):2861-2869.
[11]Joti P,Ghosh-Roy A,Ray K.Dynein light chain 1 functions in somatic cyst cells regulate spermatogonial divisions in Drosophila[J].Sci Rep,2011,1:173.
[12]Davies EL,Fuller MT.Regulation of self-renewal and differentiation in adult stem cell lineages:lessons from the Drosophila male germ line[J].Cold Spring Harb Symp Quant Biol,2008,73:137-145.
[13]White-Cooper H.Tissue,cell type and stage-specific ectopic gene expression and RNAi induction in the Drosophila testis[J].Spermatogenesis,2012,2(1):11-22.
[14]Singh SR,Zhen W,Zheng Z,et al.The Drosophila homolog of the human tumor suppressor gene BHD interacts with the JAK-STAT and Dpp signaling pathways in regulating male germline stem cell maintenance[J].Oncogene,2006,25(44):5933-5941.
[15]Liu M,Lim TM,Cai Y.The Drosophila female germline stem cell lineage acts to spatially restrict DPP function within the niche[J].Sci Signal,2010,3(132):ra57.
[16]Lim JG,Fuller MT.Somatic cell lineage is required for differentiation and not maintenance of germline stem cells in Drosophila testes[J].Proc Natl Acad Sci USA,2012,109(45):18477-18481.
[17]Yu J,Wu H,Wen Y,et al.Identification of seven genes essential for male fertility through a genome-wide association study of non-obstructive azoospermia and RNA interference-mediated large-scale functional screening in Drosophila[J].Hum Mol Genet,2015,24(5):1493-1503.
[18]Demontis F,Perrimon N.Integration of Insulin receptor/Foxo signaling and d Myc activity during muscle growth regulates body size in Drosophila[J].Development,2009,136(6):983-993.
[19]Chen D,Wu C,Zhao S,et al.Three RNA binding proteins form a complex to promote differentiation of germline stem cell lineage in Drosophila[J].PLo S Genet,2014,10(11):e1004797.
[20]Insco ML,Bailey AS,Kim J,et al.A self-limiting switch based on translational control regulates the transition from proliferation to differentiation in an adult stem cell lineage[J].Cell Stem Cell,2012,11(5):689-700.
[21]Li Y,Maines JZ,Tastan OY,et al.Mei-P26 regulates the maintenance of ovarian germline stem cells by promoting BMP signaling[J].Development,2012,139(9):1547-1556.