周运迪,吴星星,赵海萍,罗大极

• 1:武汉大学基础医学院
• 2:中国科学院水生生物研究所淡水生态与生物技术国家重点实验室

摘要(Abstract)：

【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。

关键词(KeyWords)： 青鳉;foxl2;基因敲除;CRISPR/Cas9;性腺发育

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(31472263)

作者(Author): 周运迪,吴星星,赵海萍,罗大极

参考文献(References)：

• [1]KIYOSHI N P D,MINORU T P D,HIROYUKI T P D.Medaka[M].Japan:Springer,2011.
• [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.