青鳉foxl2基因敲除突变体的构建与表型分析
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摘要
【目的】建立青鳉(Oryziaslatipes)foxl2基因突变体材料,为阐释foxl2基因功能提供良好的动物模型和研究基础。【方法】利用CRISPR/Cas9技术敲除青鳉foxl2基因,建立突变体材料。观察foxl2基因突变体的第二性征、性腺等形态特征;组织学分析性腺结构与性腺中细胞类型;用定量PCR检测性腺发育相关基因的表达情况。【结果】通过CRISPR/Cas9技术成功敲除青鳉foxl2基因,建立foxl2基因缺失4个碱基的青鳉突变体。从第二性征与性腺表型上,foxl2-/-突变体均表现为生理雄性;组织切片分析发现,foxl2-/-突变体的性腺结构与野生型卵巢和野生型精巢均有显著差异,存在退化的核仁外周卵母细胞与精子,提示foxl2-/-的性腺在组织结构上类似精巢;定量PCR结果显示,foxl2-/-突变体中雄性性腺发育关键基因sox9b、gsdf、amh等的表达量升高,雌性性腺发育关键基因cyp19a1a等的表达量下降。【结论】通过CRISPR/Cas9技术成功建立青鳉foxl2基因缺失的突变体,foxl2-/-突变体有遗传雌性、生理雄性的表型特征,foxl2对维持青鳉性腺功能有重要作用。
【Objective】Disruption of foxl2 gene in medaka, Oryzias latipes, was generated using CRISPR/Cas9. It provided a new animal model for investigating the function of foxl2 gene in gonadal development. 【Methods】Using CRISPR/Cas9 technique to knock out foxl2 gene, mutant material was generated in Medaka. The morphological characteristics of the mutant material such as secondary sexual characteristics and gonads were observed. Histological analysis was carried out on gonad tissue sections to observe gonad structure, germ cells and somatic cell types of the mutant materials. To comprensively analyze the effects of foxl2 gene on gonadal function, the changes of gene expression level on gonadal development related genes in mutants were detected using qPCR.【Results】The foxl2 gene was successfully disrupted by CRISPR/Cas9 technology, and foxl2 mutants had been generated in this study.According to the phenotype, foxl2-/-mutant is male. The tissue analysis showed that the gonads of foxl2-/-mutant were different with both ovaries of the wild type females and testis of the wild type males. There were degenerated perinucleolar oocytes and spermatids in gonad of mutants. Quantitative real time PCR analysis of the expression of gonadal differentiation and developmental related genes revealed that the loss of foxl2 gene resulted in upregulating the expression of sox9 b, gsdf, and amh, in contrast,downregulating the expression of cyp19 a1 a. 【Conclusion】Disruption of foxl2 gene has been generated in Medaka. Our results revealed the characteristics of foxl2-/-mutants is male in physiology, but female in hereditary. It indicates that foxl2 gene is essential for maintaining gonadal function in Medaka.
【Objective】Disruption of foxl2 gene in medaka, Oryzias latipes, was generated using CRISPR/Cas9. It provided a new animal model for investigating the function of foxl2 gene in gonadal development. 【Methods】Using CRISPR/Cas9 technique to knock out foxl2 gene, mutant material was generated in Medaka. The morphological characteristics of the mutant material such as secondary sexual characteristics and gonads were observed. Histological analysis was carried out on gonad tissue sections to observe gonad structure, germ cells and somatic cell types of the mutant materials. To comprensively analyze the effects of foxl2 gene on gonadal function, the changes of gene expression level on gonadal development related genes in mutants were detected using qPCR.【Results】The foxl2 gene was successfully disrupted by CRISPR/Cas9 technology, and foxl2 mutants had been generated in this study.According to the phenotype, foxl2-/-mutant is male. The tissue analysis showed that the gonads of foxl2-/-mutant were different with both ovaries of the wild type females and testis of the wild type males. There were degenerated perinucleolar oocytes and spermatids in gonad of mutants. Quantitative real time PCR analysis of the expression of gonadal differentiation and developmental related genes revealed that the loss of foxl2 gene resulted in upregulating the expression of sox9 b, gsdf, and amh, in contrast,downregulating the expression of cyp19 a1 a. 【Conclusion】Disruption of foxl2 gene has been generated in Medaka. Our results revealed the characteristics of foxl2-/-mutants is male in physiology, but female in hereditary. It indicates that foxl2 gene is essential for maintaining gonadal function in Medaka.
引文
[1]KIYOSHI N P D,MINORU T P D,HIROYUKI T P D.Medaka[M].Japan:Springer,2011.
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[4]KUROKAWA H,SAITO D,NAKAMURA S,et al.Germ cells are essential for sexual dimorphism in the medaka gonad[J].Proceedings of the National Academy of Sciences of the United States of America,2007,104(43):16958-16963.
[5]CARLSSON P,MAHLAPUU M.Forkhead transcription factors:key players in development and metabolism[J].Developmental Biology,2002,250(1):1-23.
[6]SCHMIDT D,OVITT C E,ANLAG K,et al.The murine winged-helix transcription factor Foxl2 is required for granulosa cell differentiation and ovary maintenance[J].Development,2004,131(4):933-942.
[7]PRUEITT R L,ZINN A R.A fork in the road to fertility[J].Nature Genetics,2001,27(2):132-134.
[8]CRISPONI L,DEIANA M,LOI A,et al.The putative forkhead transcription factor FOXL2 is mutated in blepharophimosis/ptosis/epicanthus inversus syndrome[J].Nature Genetics,2001,27(2):159-166.
[9]BOULANGER L,PANNETIER M,GALL L,et al.FOXL2 is a female sex-determining gene in the goat[J].Current Biology,2014,24(4):404.
[10]PAILHOUX E,VIGIER B,CHAFFAUX S,et al.A11.7-kb deletion triggers intersexuality and polledness in goats[J].Nature Genetics,2001,163(1):73-92.
[11]UDA M,OTTOLENGHI C,CRISPONI L,et al.Foxl2disruption causes mouse ovarian failure by pervasive blockage of follicle development[J].Human Molecular Genetics,2004,13(11):1171-1181.
[12]OTTOLENGHI C,OMARI S,GARCIAORTIZ J E,et al.Foxl2 is required for commitment to ovary differentiation[J].Human Molecular Genetics,2005,14(14):2053.
[13]GARCIAORTIZ J E,PELOSI E,OMARI S,et al.Foxl2functions in sex determination and histogenesis throughout mouse ovary development[J].Bmc Developmental Biology,2009,9(1):36.
[14]COCQUET J,PAILHOUX E,JAUBERT F,et al.Evolution and expression of FOXL2[J].Journal of Medical Genetics,2002,39(12):916-921.
[15]COCQUET J,DE BAERE E,GAREIL M,et al.Structure,evolution and expression of the FOXL2transcription unit[J].Cytogenetic and Genome Research,2003,101(3/4):206-211.
[16]NAKAMOTO M,MATSUDA M,WANG D S,et al.Molecular cloning and analysis of gonadal expression of Foxl2 in the medaka,Oryzias latipes[J].Biochemical&Biophysical Research Communications,2006,344(1):353-361.
[17]KOBAYASHI T,CHIBA A,SATO T,et al.Estrogen alters gonadal soma-derived factor(gsdf)/Foxl2expression levels in the testes associated with testis-ova differentiation in adult medaka,Oryzias latipes[J].Aquatic Toxicology,2017,191:209.
[18]URNOV F D,REBAR E J,HOLMES M C,et al.Genome editing with engineered zinc finger nucleases[J].Nature Reviews Genetics,2010,11(9):636-646.
[19]CHRISTIAN M,CERMAK T,DOYLE E L,et al.Targeting DNA Double-Strand Breaks with TALEffector Nucleases[J].Genetics,2010,186(2):757-761.
[20]GAJ T,GERSBACH C A,III C F B.ZFN,TALEN and CRISPR/Cas-based methods for genome engineering[J].Trends in Biotechnology,2013,31(7):397-405.
[21]WANG T,WEI J J,SABATINI D M,et al.Genetic Screens in Human Cells Using the CRISPR-Cas9System[J].Science,2013,343(6166):80-84.
[22]BASSETT A R,TIBBIT C,PONTING C P,et al.Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system[J].Cell Reports,2013,4(1):220-228.
[23]JAO L E,WENTE S R,CHEN W.Efficient multiplex biallelic zebrafish genome editing using a CRISPRnuclease system[J].Proc Natl Acad Sci USA,2013,110(34):13904-13909.
[24]LI Z Z G,HE L,CHEN S.Novel gene transfer into the fertilized eggs of gold fish(Carassius auratus,L.1758)[J].Journal of Applied Ichthyology,1985,1(1):31-34.
[25]WANG T,HONG Y.Direct gene disruption by TALENs in medaka embryos[J].Gene,2014,543(1):28-33.
[26]LUO D,LIU Y,CHEN J,et al.Direct production of XYDMY-sex reversal female medaka(Oryzias latipes)by embryo microinjection of TALENs[J].Scientific Reports,2015,5:14057.
[27]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCRand the 2(-Delta Delta C(T))Method[J].Methods,2001,25(4):402-408.
[28]WANG D S,KOBAYASHI T,ZHOU L Y,et al.Foxl2up-regulates aromatase gene transcription in a female-specific manner by binding to the promoter as well as interacting with ad4 binding protein/steroidogenic factor 1[J].Molecular Endocrinology,2007,21(3):712.
[29]LI M H,ZHOU L Y,WANG D S,et al.Antagonistic roles of dmrt1 and foxl2 in sex differentiation via estrogen production in tilapia as demonstrated by TALENs[J].Endocrinology,2013,154(12):4814-4825.
[30]ZHANG X,LI M,MA H,et al.Mutation of foxl2 or cyp19a1a results in female to male sex reversal in XXNile tilapia[J].Endocrinology,2017,158(8):2634.
[31]YANG Y J,WANG Y,LI Z,et al.Sequential,divergent and cooperative requirements of Foxl2a and Foxl2b in ovary development and maintenance of zebrafish[J].Genetics,2017,205(4):1551.
[32]IJIRI S,KANEKO H,KOBAYASHI T,et al.Sexual dimorphic expression of genes in gonads during early differentiation of a teleost fish,the Nile tilapia Oreochromis niloticus[J].Biology of Reproduction,2008,78(2):333-41.
[33]NAKAMOTO M,MURAMATSU S,YOSHIDA S,et al.Gonadal sex differentiation and expression of Sox9a2,Dmrt1,and Foxl2,in Oryzias luzonensis[J].Genesis,2010,47(5):289-299.
[34]WANG D,KOBAYASHI T,ZHOU L,et al.Molecular cloning and gene expression of Foxl2 in the Nile tilapia,Oreochromis niloticus[J].Biochemical&Biophysical Research Communications,2004,320(1):83-89.
[35]KOBAYASHI Y,HORIGUCHI R,NOZU R,et al.Expression and localization of forkhead transcriptional factor 2(Foxl2)in the gonads of protogynous wrasse,Halichoeres trimaculatus[J].Biology of Sex Differences,2010,1(1):3.
[36]HU Q,GUO W,GAO Y,et al.Molecular cloning and analysis of gonadal expression of Foxl2 in the rice-field eel Monopterus albus[J].Sci Rep,2014,4(4):6884.
[37]PASCAL D S B,BONNEAUD N,BOIZET B,et al.Direct interaction of SRY-related protein SOX9 and steroidogenic factor 1 regulates transcription of the human anti-Müllerian hormone gene[J].Molecular and Cellular Biology,1998,18(11):6653-6665.
[38]DA SILVA S M,HACKER A,HARLEY V,et al.Sox9expression during gonadal development implies a conserved role for the gene in testis differentiation in mammals and birds[J].Nature Genetics,1996,14(1):62-68.
[39]VIZZIANO D,RANDUINEAU G,BARON D,et al.Characterization of early molecular sex differentiation in rainbow trout,Oncorhynchus mykiss[J].Developmental Dynamics,2007,236(8):2198.
[40]CHIANG E F,PAI C I,WYATT M,et al.Two sox9genes on duplicated zebrafish chromosomes:expression of similar transcription activators in distinct sites[J].Developmental Biology,2001,231(1):149.
[41]NAKAMURA S,AOKI Y,SAITO D,et al.Sox9b/sox9a2‐EGFP transgenic medaka reveals the morphological reorganization of the gonads and a common precursor of both the female and male supporting cells[J].Molecular Reproduction&Development,2008,75(3):472-476.
[42]UHLENHAUT N H,JAKOB S,ANLAG K,et al.Somatic sex reprogramming of adult ovaries to testes by FOXL2 ablation[J].Cell,2009,139(6):0-1142.
[43]MA?LLE Pannetier,STéPHANE Fabre,FRANKBatista,et al.FOXL2 activates P450 aromatase gene transcription:towards a better characterization of the early steps of mammalian ovarian development[J].Journal of Molecular Endocrinology,2006,36(3):399-413.
[44]SHEN Z G,WANG H P.Molecular players involved in temperature-dependent sex determination and sex differentiation in Teleost fish[J].Genetics Selection Evolution,2014,46(1):1-21.
[45]CHANG X,KOBAYASHI T,SENTHILKUMARAN B,et al.Two types of aromatase with different encoding genes,tissue distribution and developmental expression in Nile tilapia(Oreochromis niloticus)[J].General&Comparative Endocrinology,2005,141(2):101-115.
[46]SAWYER S J,GERSTNER K A,CALLARD G V.Real-time PCR analysis of cytochrome P450 aromatase expression in zebrafish:gene specific tissue distribution,sex differences,developmental programming,and estrogen regulation[J].General&Comparative Endocrinology,2006,147(2):108-117.
[47]NAKAMURA S,KUROKAWA H,ASAKAWA S,et al.Two distinct types of theca cells in the medaka gonad:germ cell-dependent maintenance of cyp19a1-expressing theca cells[J].Developmental Dynamics,2009,238(10):2652-2657.
[48]NAKAMOTO M,SHIBATA Y,OHNO K,et al.Ovarian aromatase loss-of-function mutant medaka undergo ovary degeneration and partial female-to-male sex reversal after puberty[J].Molecular&Cellular Endocrinology,2017,460:104-122.
[2]MATSUDA M,NAGAHAMA Y,AI S,et al.DMY is a Y-specific DM-domain gene required for male development in the medaka fish[J].Nature,2002,417(6888):559-563.
[3]OTAKE H,SHINOMIYA A,KAWAGUCHI A,et al.The medaka sex-determining gene DMY acquired a novel temporal expression pattern after duplication of DMRT1[J].Genesis,2010,46(12):719-723.
[4]KUROKAWA H,SAITO D,NAKAMURA S,et al.Germ cells are essential for sexual dimorphism in the medaka gonad[J].Proceedings of the National Academy of Sciences of the United States of America,2007,104(43):16958-16963.
[5]CARLSSON P,MAHLAPUU M.Forkhead transcription factors:key players in development and metabolism[J].Developmental Biology,2002,250(1):1-23.
[6]SCHMIDT D,OVITT C E,ANLAG K,et al.The murine winged-helix transcription factor Foxl2 is required for granulosa cell differentiation and ovary maintenance[J].Development,2004,131(4):933-942.
[7]PRUEITT R L,ZINN A R.A fork in the road to fertility[J].Nature Genetics,2001,27(2):132-134.
[8]CRISPONI L,DEIANA M,LOI A,et al.The putative forkhead transcription factor FOXL2 is mutated in blepharophimosis/ptosis/epicanthus inversus syndrome[J].Nature Genetics,2001,27(2):159-166.
[9]BOULANGER L,PANNETIER M,GALL L,et al.FOXL2 is a female sex-determining gene in the goat[J].Current Biology,2014,24(4):404.
[10]PAILHOUX E,VIGIER B,CHAFFAUX S,et al.A11.7-kb deletion triggers intersexuality and polledness in goats[J].Nature Genetics,2001,163(1):73-92.
[11]UDA M,OTTOLENGHI C,CRISPONI L,et al.Foxl2disruption causes mouse ovarian failure by pervasive blockage of follicle development[J].Human Molecular Genetics,2004,13(11):1171-1181.
[12]OTTOLENGHI C,OMARI S,GARCIAORTIZ J E,et al.Foxl2 is required for commitment to ovary differentiation[J].Human Molecular Genetics,2005,14(14):2053.
[13]GARCIAORTIZ J E,PELOSI E,OMARI S,et al.Foxl2functions in sex determination and histogenesis throughout mouse ovary development[J].Bmc Developmental Biology,2009,9(1):36.
[14]COCQUET J,PAILHOUX E,JAUBERT F,et al.Evolution and expression of FOXL2[J].Journal of Medical Genetics,2002,39(12):916-921.
[15]COCQUET J,DE BAERE E,GAREIL M,et al.Structure,evolution and expression of the FOXL2transcription unit[J].Cytogenetic and Genome Research,2003,101(3/4):206-211.
[16]NAKAMOTO M,MATSUDA M,WANG D S,et al.Molecular cloning and analysis of gonadal expression of Foxl2 in the medaka,Oryzias latipes[J].Biochemical&Biophysical Research Communications,2006,344(1):353-361.
[17]KOBAYASHI T,CHIBA A,SATO T,et al.Estrogen alters gonadal soma-derived factor(gsdf)/Foxl2expression levels in the testes associated with testis-ova differentiation in adult medaka,Oryzias latipes[J].Aquatic Toxicology,2017,191:209.
[18]URNOV F D,REBAR E J,HOLMES M C,et al.Genome editing with engineered zinc finger nucleases[J].Nature Reviews Genetics,2010,11(9):636-646.
[19]CHRISTIAN M,CERMAK T,DOYLE E L,et al.Targeting DNA Double-Strand Breaks with TALEffector Nucleases[J].Genetics,2010,186(2):757-761.
[20]GAJ T,GERSBACH C A,III C F B.ZFN,TALEN and CRISPR/Cas-based methods for genome engineering[J].Trends in Biotechnology,2013,31(7):397-405.
[21]WANG T,WEI J J,SABATINI D M,et al.Genetic Screens in Human Cells Using the CRISPR-Cas9System[J].Science,2013,343(6166):80-84.
[22]BASSETT A R,TIBBIT C,PONTING C P,et al.Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system[J].Cell Reports,2013,4(1):220-228.
[23]JAO L E,WENTE S R,CHEN W.Efficient multiplex biallelic zebrafish genome editing using a CRISPRnuclease system[J].Proc Natl Acad Sci USA,2013,110(34):13904-13909.
[24]LI Z Z G,HE L,CHEN S.Novel gene transfer into the fertilized eggs of gold fish(Carassius auratus,L.1758)[J].Journal of Applied Ichthyology,1985,1(1):31-34.
[25]WANG T,HONG Y.Direct gene disruption by TALENs in medaka embryos[J].Gene,2014,543(1):28-33.
[26]LUO D,LIU Y,CHEN J,et al.Direct production of XYDMY-sex reversal female medaka(Oryzias latipes)by embryo microinjection of TALENs[J].Scientific Reports,2015,5:14057.
[27]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCRand the 2(-Delta Delta C(T))Method[J].Methods,2001,25(4):402-408.
[28]WANG D S,KOBAYASHI T,ZHOU L Y,et al.Foxl2up-regulates aromatase gene transcription in a female-specific manner by binding to the promoter as well as interacting with ad4 binding protein/steroidogenic factor 1[J].Molecular Endocrinology,2007,21(3):712.
[29]LI M H,ZHOU L Y,WANG D S,et al.Antagonistic roles of dmrt1 and foxl2 in sex differentiation via estrogen production in tilapia as demonstrated by TALENs[J].Endocrinology,2013,154(12):4814-4825.
[30]ZHANG X,LI M,MA H,et al.Mutation of foxl2 or cyp19a1a results in female to male sex reversal in XXNile tilapia[J].Endocrinology,2017,158(8):2634.
[31]YANG Y J,WANG Y,LI Z,et al.Sequential,divergent and cooperative requirements of Foxl2a and Foxl2b in ovary development and maintenance of zebrafish[J].Genetics,2017,205(4):1551.
[32]IJIRI S,KANEKO H,KOBAYASHI T,et al.Sexual dimorphic expression of genes in gonads during early differentiation of a teleost fish,the Nile tilapia Oreochromis niloticus[J].Biology of Reproduction,2008,78(2):333-41.
[33]NAKAMOTO M,MURAMATSU S,YOSHIDA S,et al.Gonadal sex differentiation and expression of Sox9a2,Dmrt1,and Foxl2,in Oryzias luzonensis[J].Genesis,2010,47(5):289-299.
[34]WANG D,KOBAYASHI T,ZHOU L,et al.Molecular cloning and gene expression of Foxl2 in the Nile tilapia,Oreochromis niloticus[J].Biochemical&Biophysical Research Communications,2004,320(1):83-89.
[35]KOBAYASHI Y,HORIGUCHI R,NOZU R,et al.Expression and localization of forkhead transcriptional factor 2(Foxl2)in the gonads of protogynous wrasse,Halichoeres trimaculatus[J].Biology of Sex Differences,2010,1(1):3.
[36]HU Q,GUO W,GAO Y,et al.Molecular cloning and analysis of gonadal expression of Foxl2 in the rice-field eel Monopterus albus[J].Sci Rep,2014,4(4):6884.
[37]PASCAL D S B,BONNEAUD N,BOIZET B,et al.Direct interaction of SRY-related protein SOX9 and steroidogenic factor 1 regulates transcription of the human anti-Müllerian hormone gene[J].Molecular and Cellular Biology,1998,18(11):6653-6665.
[38]DA SILVA S M,HACKER A,HARLEY V,et al.Sox9expression during gonadal development implies a conserved role for the gene in testis differentiation in mammals and birds[J].Nature Genetics,1996,14(1):62-68.
[39]VIZZIANO D,RANDUINEAU G,BARON D,et al.Characterization of early molecular sex differentiation in rainbow trout,Oncorhynchus mykiss[J].Developmental Dynamics,2007,236(8):2198.
[40]CHIANG E F,PAI C I,WYATT M,et al.Two sox9genes on duplicated zebrafish chromosomes:expression of similar transcription activators in distinct sites[J].Developmental Biology,2001,231(1):149.
[41]NAKAMURA S,AOKI Y,SAITO D,et al.Sox9b/sox9a2‐EGFP transgenic medaka reveals the morphological reorganization of the gonads and a common precursor of both the female and male supporting cells[J].Molecular Reproduction&Development,2008,75(3):472-476.
[42]UHLENHAUT N H,JAKOB S,ANLAG K,et al.Somatic sex reprogramming of adult ovaries to testes by FOXL2 ablation[J].Cell,2009,139(6):0-1142.
[43]MA?LLE Pannetier,STéPHANE Fabre,FRANKBatista,et al.FOXL2 activates P450 aromatase gene transcription:towards a better characterization of the early steps of mammalian ovarian development[J].Journal of Molecular Endocrinology,2006,36(3):399-413.
[44]SHEN Z G,WANG H P.Molecular players involved in temperature-dependent sex determination and sex differentiation in Teleost fish[J].Genetics Selection Evolution,2014,46(1):1-21.
[45]CHANG X,KOBAYASHI T,SENTHILKUMARAN B,et al.Two types of aromatase with different encoding genes,tissue distribution and developmental expression in Nile tilapia(Oreochromis niloticus)[J].General&Comparative Endocrinology,2005,141(2):101-115.
[46]SAWYER S J,GERSTNER K A,CALLARD G V.Real-time PCR analysis of cytochrome P450 aromatase expression in zebrafish:gene specific tissue distribution,sex differences,developmental programming,and estrogen regulation[J].General&Comparative Endocrinology,2006,147(2):108-117.
[47]NAKAMURA S,KUROKAWA H,ASAKAWA S,et al.Two distinct types of theca cells in the medaka gonad:germ cell-dependent maintenance of cyp19a1-expressing theca cells[J].Developmental Dynamics,2009,238(10):2652-2657.
[48]NAKAMOTO M,SHIBATA Y,OHNO K,et al.Ovarian aromatase loss-of-function mutant medaka undergo ovary degeneration and partial female-to-male sex reversal after puberty[J].Molecular&Cellular Endocrinology,2017,460:104-122.