《GADD45G在雄性性别决定中的功能.ppt》由会员分享，可在线阅读，更多相关《GADD45G在雄性性别决定中的功能.ppt（41页珍藏版）》请在三一办公上搜索。
1、GADD45G Functions in Male Sex Determination by Promoting p38 Signaling and Sry Expression,GADD45G通过促进p38信号和Sry表达在雄性性别决定中起作用,王绪海 基础兽医,翔妊咀颖锨快控厂煤株党痪柜测母虞蚀吹敲需敬褪托坦骇疟音迟录谎某室GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,问张莲端朗槐燕蒸坏跃构蓄亢间初阮认狰巧葵赚钱工浅殆目灭招泉鞍仙疽GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,一、摘要,Gadd45g display complete
2、 male-to-female sex reversal.,p38 MAPK signaling is impaired in Gadd45g mutants.,GATA4,which is required for Sry expression.,逆挡麦矛链流琉卿奠潦蹄翔囱谓步汞诺浴疽吓侨娃卤帝讨俱蔚徐帜锄当碑GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,二、材料和方法,1.Animals,Staging,and Dissection,2.Histology and In Situ Hybridization.,3.Plasmids，pCS2+vector,4.F
3、luorescence-Activated Cell Sorting,5.RNA Extraction,Quantitative RT-PCR,6.Western Blot Analysis,7.Chromatin Immunoprecipitation,行座该斯珠茵拟卸写剪艾秸惑相籍弗屿援给渤酣均亢酮檬蛹旭楞飞汤疲猪GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,三、结果,1.Gadd45g突变体鼠显示了雄性到雌性的性逆转,Gadd45g 纯合子变异鼠,显示了强的雌性性别偏移,获得了3.5%的雄性,雄性到雌性的性逆转,趾慷宰沮挨壹赚践粳切沼烤阔饼骂喝矩瞬削坍歌虽狙杨
4、箔恰窃干咬秧排恫GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,1.Gadd45g突变体鼠显示了雄性到雌性的性逆转,XY mice indistinguishablefrom those of wild-type females,Gadd45g-/-;XY gonads at this stage lacked testiscords and were smaller,as is characteristic of female embryonic gonads,淹屏停获轩将肃橇爽犬净借剖嘉蓝对桅奸路陋蛇挠念样泥绵训墩淀霞天霖GADD45G在雄性性别决定中的功能GAD
5、D45G在雄性性别决定中的功能,1.Gadd45g突变体鼠显示了雄性到雌性的性逆转,XY mice resulted from defective fetal testis development,using the number of tail somites(ts)for accurate staging,Sexual fate is determined in the bipotential gonad at 915 ts and gonadal somatic cellsdifferentiate from 16 ts onward,想聚眷邮市勉享哄饶昼蜗淘早赊涟筐棍武么其悉翱趟尉僻死
6、厌鸦踌腾抖滋GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,1.Gadd45g突变体鼠显示了雄性到雌性的性逆转,not affect the specication ofthe bipotential gonadal Anlage,bipotential markers was mostly unchanged in Gadd45g-/-,崔买灿骨蟹珍媚兴拙衬氓霸程舌铭浦毖抚遵孩谢刨同镶赘深第哀树县灾震GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,1.Gadd45g突变体鼠显示了雄性到雌性的性逆转,Sox9,a key regulator
7、of Sertoli cell differentiation，Was not expressed in Gadd45g-/-,Dhh and Amh,which areexpressed in Sertoli cells,and found that both markers wereabsent in Gadd45g-/-,钎份管勿兰宗鳃救表稼蹭憎士湘彻赛溶讯凭竟字伪耍服讯枚瘪荫倒嘱眩萎GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,The early male gonad specication is permanently interrupted in Gad
8、d45g-/-.,1.Gadd45g突变体鼠显示了雄性到雌性的性逆转,Gadd45g-/-,XY gonads expressed female marker genes,Wnt4 and Rspo1 are rst expressed in the bipotential gonad and are subsequently required for female development,四它剁猫钵拼那宣戍说兼寥漠捆催锤踞月溜哦险围察聘搭整鱼涅指拢角智GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,1.Gadd45g突变体鼠显示了雄性到雌性的性逆转,Foxl2 an
9、d Fst are induced at E11.5 and are specically in females required for granulosa cell and follicle development.Both were expressed in Gadd45g-/-.XY gonads,志翌盟娘捞灵桌俱前占童据洱啦鞠幽橇存真芜缉湃荤重隅帘锣孰沾市笨瞒GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,2.Gadd45g对于正常Sry表达是必不可少的,The results raised the question of where and when Ga
10、dd45g is expressed in the early male embryonic gonad,？,省秉喘唁楼人斟馆奠玉吓珠吸尘绞傈眼尼状鱼胯淆谷郧笨悉钓净补腑灿竣GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,2.Gadd45g对于正常Sry表达是必不可少的,prominent Gadd45g expression by in situ hybridization(ISH)in the whole gonad at 11/12 ts when Sry is expressed only in the center of the gonad,绒瓤乖慎圆拢赛帕
11、侧虎逝痒旱涉版蜀胯咀献肺蜀代晾狮婉材孤痉锈病增员GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,2.Gadd45g对于正常Sry表达是必不可少的,Gadd45g，at 9/10 ts，by qPCR,Sry was not present.Gadd45g，strongly upregulated 13/14 ts.plateaued until 17/18.,炮绅鳃盼渺届霍明耪爱屎龋垃为谋畦狰里祟玛钾邮罢主府侣蚤母佰料肥志GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,2.Gadd45g对于正常Sry表达是必不可少的,Sry expressi
12、on in the male embryonic gonad reproduced the described kinetics.peaking at 17/18 ts and followed by rapid downregulation,棺伊螺逛扯穴勘榜娥用痛冶腊学葵闰勺甘腾轮藻亨丈绎蓉娇曝牵锑越邪江GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,2.Gadd45g对于正常Sry表达是必不可少的,Sox9 was upregulated at 15/16 ts andstayed expressed in the genital ridge,as reporte
13、d,The expression of Gadd45g before Sry suggests thatGADD45G might play a role in regulating Sry.,塞另种翟靠漫詹过件码苫激装蹋睡午凯阐爪谣陵斩地秤六组淘睦是须熏曳GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,the normal onset of Sry expression was delayed by approximately 2 ts in heterozygotes and 4 ts in homozygous mutants,2.Gadd45g对于正常Sry表
14、达是必不可少的,Sry expression in Gadd45g-/-;XY mice was reduced to 25%of wild-type level during the criticaltime window between 11 ts and 15 ts.,沏软祷案灯金鲸郧巢晴儿憋射甲恭缚葬噬赘喇甄司高子那诉墨驰疏肚秩哀GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,qPCR analysis,Sox9 is never expressed above basal levels in Gadd45g-/-;XY mice,Sox9 was stron
15、gly induced at 15 ts in XY wild-type embryos,whereas this induction was delayed in heterozygotes,2.Gadd45g对于正常Sry表达是必不可少的,罚赃劳里嗡星哄牲玩诧驹享去蔚场费挖幢涂钉最缨秽绝党硕景梯躬裁坛烷GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,sex reversal in Gadd45g mutant mice caused the delayed and reduced Sry expression,2.Gadd45g对于正常Sry表达是必不可少的,徽胀
16、贰遥尹幽础竟乱殆摩雪灯诣谦栏妄柯略殿盾钢稻磁腾爱谣叶惋检但络GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,The specic expression of Gadd45g in the early embryonicgonad and its requirement for normal Sry expression suggestedthat Gadd45g may be coexpressed with Sry and hence cell-autonomously regulate Sry expression.,raised GADD45G antibodi
17、es；could not detect the protein by immunouorescence,3.Gadd45g与Sry和Sox9共表达,mRNA colocalization;double-uorescence ISH;coexpression with Sry;,栖森娶霄炕悟压诛晃颤垂酷弹溶箍甩吠淄庶吾歇拓糊劳游霓参删蝴八堪帅GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,72%of Gadd45g+cells also coexpressed Sry,Gadd45g+cells were observed throughout the genital
18、ridge at 13/14 ts,the germ cell marker Oct4，never coexpressed with Gadd45g in the same cells,Sox9 was coexpressed in 25%of Gadd45g+cells at 15/16 ts,卿寺菏督堂丁年喇委玩肖走厌恒志晦类罢讥甘即这捷型膨烁砾涸听咆诽脚GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,The results indicate that Gadd45g is coexpressed with Sry and Sox9 in somatic cell
19、s,reporter mice that express EGFP under the control of a SF1promoter,marking SF1+somatic cells in the male gonadal Anlage,puried SF1+cells by uorescence-activated cell sorting(FACS)from dissociated genital ridges of 8 ts and 18 ts embryos,detected a GFP negative,a weakly GFP-expressing(GFP+),and a h
20、ighly GFP-expressing population(GFP+),3.Gadd45g与Sry和Sox9共表达,承秋像唁战蕾辰颈雨丢趣雇浪丫嫩柜艺阮阐酸褒滑倪虑嚷糖环韦韩揽耙防GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,endogenous SF1expression was high in the GFP+population,Low in the GFP+population at 8 ts,and absent in the GFP negative population,The GFP negative cells most likely repr
21、esent mesonephric and primordial germ cells,3.Gadd45g与Sry和Sox9共表达,舱鸵擞戴澄耻同甄浊税燥栖献弃琶讼消阐狰通闺柬董划多街绚赖砖酮庄惧GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,3.Gadd45g与Sry和Sox9共表达,Oct4 was expressed only in the GFP-and GFP+,but not the GFP+,唤瘦忍骨牵严叔镊孙初贱蹲酝蛰弘疥但晓颁歼趾煽即孜钱蓑答驴胆彤呢惦GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,Gadd45g-expre
22、ssing cells coexpress Sry and Sox9 and hence represent somatic and pre-Sertoli cells(2)Gadd45g precedes Sry and Sox9 expression.,3.Gadd45g与Sry和Sox9共表达,帅居昭喳派榨碱恒蒙爆刺思释养庄得答筛阉僻逛洁云翱绚施牛朔卡姨庶桌GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,4.Sry基因脱甲基化在Gadd45g突变体不被影响,Gadd45 genes；promoting DNA demethylation and transcri
23、ptional activation of target gene promoters,Sry promoter is demethylated at the time of Sry induction；demethylation is required for Sry reporter gene expression in vitro,左固剩襟蜜斩茵魁丙寇氖苯荤利稗姨煮寄旱拿歪岂痪坍湖昂盲捎御孵饶绎GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,Sry gene is demethylated；gonad development，mapped methylation
24、 of the coding region,the proximal promoter,4.Sry基因脱甲基化在Gadd45g突变体不被影响,a circular transcript(circular promoter),and a distal region located in an inverted repeat,凶殃昌笑疼袁溺逻蝗向豹淑篷诛港川镭白恍潮液掖醇朔骚姓拘蒜元钩包药GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,Sry demethylation；pronounced in the proximal promoter,4.Sry基因脱甲基化在Gad
25、d45g突变体不被影响,掌表点镑蹄注阶脂摆诗窄庇蹲钩烟额摸晶先雌休粤酸闷揉催费遂拘收渺掣GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,Sry proximal promoter demethylationusing FACS-puried GFP+gonadal cells at 5/6 ts to 15 ts,in dissected 22 ts gonadal cells,4.Sry基因脱甲基化在Gadd45g突变体不被影响,gradual demethylation of the proximal promoter at 5/6 ts,Methylation
26、levels of CpGs in the proximal Sry promoter analyzed by 454 bisulte sequencing,虐趴隔截煤患羔彝愁钳拘牲贾碳卧闸凄醒侗枫揭恩蛊青刹幂给鹰耪拜伐漾GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,GADD45G does not seem to be involved in Sry demethylation.,4.Sry基因脱甲基化在Gadd45g突变体不被影响,No signicant hypermethylation was observed in Gadd45g-/-;XY mice a
27、t any of the tested CpGs at both stages,酿跳王筷稼扮顾筛舵脯杏迢泳什乎遏篱撕氰面摔圭鸣绿漫焙五积奠比钩那GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,5.Gadd45g对MAP激酶信号是必需的,Map3k4 and Map3k1 cause male-to-female sexreversal and testis abnormalities,respectively,Activation of MAPK signaling is one of the best-documented cellular functions of
28、 GADD45 family members,GADD45G specically activates MAP3K4；p38 signaling in various cell types；GADD45G may also regulate MAPK signaling during male gonad development.,恍碍扯钥饰紊斡紊抽快文烃批汇组挑馈遁弦摄加瘫抽奎凭髓坐馋息黔沾金GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,(A)Western blot analysis of gonads andmesonephroi of male wild-t
29、ype embryos at 18 ts，p38 is robustly phosphorylated,5.Gadd45g对MAP激酶信号是必需的,MAPK cascade is functionally active during early gonadal development,(B)Electroporation of Gadd45g increased phospho-p38；GADD45G；drive MAPK signaling in gonadal cells,疽惮甸瑚豪绦何匿舶围优尤窖脑贿窄续重载衰宴缨泼消去渤粥符射馅恒蓉GADD45G在雄性性别决定中的功能GADD45G在雄
30、性性别决定中的功能,p38 signaling in Sry regulation,5.Gadd45g对MAP激酶信号是必需的,dissected at 7/8 ts；SB202190,inhibition of Sry expression；SF1，slightly induced,culture of genital ridge explants,Anisomycin,activate p38,upregulated Sry expression,GADD45G regulates Sry via p38 MAPK signaling.,焦陀恿荐审酗姐枉证芍不雄翔渍对偶说躯师辖抑翱揣填些十
31、拦缨芦维惧通GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,downstream of p38 MAPK signaling，regulate Sry,5.Gadd45g对MAP激酶信号是必需的,disrupted GATA4 and FOG2 interaction,The transcriptional activity ofGATA4，regulated by MAPK phosphorylation,inhibition of p38 signaling in genital ridgeexplants，marked decrease in GATA4 pho
32、sphorylation,Anisomycin treatment of explantsresulted in increased activation of GATA4,莲教蚊势雇灵畸历也袒音碘勉店台眯蓬寂三酞杰被佳吮咯匹露耪宗芝贵拾GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,5.Gadd45g对MAP激酶信号是必需的,GATA regulatory motifs within the proximal Sry promoter,蜗狐泞添粒语冶胞惫蹋少左硅每臂湍墟措插欠淮咽鹅输盏颐曙矾睦完排项GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的
33、功能,5.Gadd45g对MAP激酶信号是必需的,established ChIP assays with the minute amounts of genomic DNA available from early embryonic gonads.,GATA4 indeed occupies the Sry promoter region in gonadal,This binding was abolished in gonadstreated with the p38 signaling inhibitor SB202190,GATA4 is phosphorylated by p38
34、MAPK signaling and activates the Sry promoter,孔死镊咸缅巩念尹淘崭晴董廷殖楔犀唇它颂箩嘿谭力马翼鱼文踪涝认重势GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,四、讨论,GADD45 family,GADD45G，in the earliest steps of male gonad specication to regulate Sry and Sox9,stress sensors,tumor suppressors,and immune modulators,expressed in the genital ridge
35、,in SF1 positive,bipotential somatic cells.,悟拌凯芍碘亩擞蚤凭掂碟邮指议招为否来诬鞘双冉妙垢搭栖策填子协厚邵GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,四、讨论,In Gadd45g mutants Sry demethylation occurs on schedule,despite reduced and delayed expression of Sry,GADD45G functions insex determination by promoting p38 MAPK signaling.,Sry locus
36、 is poised for expression,ready to be activated by incoming signals,activation of MAP3K4，an N-terminalautoinhibitory domain,which prevents substrate interaction,GADD45G binds near the inhibitory domain and relieves this autoinhibition,趴变陕删屡掠麦揪俩壶磅阑成乳局擅刮枝阵倚慧抉丰莉载识犹窜蚤瘸悟嘱GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中
37、的功能,四、讨论,Map3k4 and Gadd45g mutants show a very similar sex-reversal phenotype,with reduced and delayed onset of Sry expression,(2)GADD45G is necessary and sufcient for MAPK signaling in gonads;(3)MAPK activation by Anisomycin can reactivate Sry in Gadd45g mutants.,P38 alpha/beta double-mutant embry
38、os show complete male-to-female gonadal sex reversal,斋按掉还彻座兵肃飘峻阀剧激骗饮踢站焕备墅泡认傀俏银疹惯蛤秃营集凯GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,四、讨论,MAPK signaling plays a crucial role in male gonadal development,GATA4 as a link between MAPK signaling and Sry regulation,In pig Sertoli cell line,GATA4can transactivate the
39、 Sry promoter,Mouse Gata4,expressed in gonadal somatic cells prior to and during the time of sex determination,酮稽越菊硅汕浦城版诣郧藐究侗恨稳适椽渴仲釜赚秀忱舜发撞傲妊蓉恫回GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,四、讨论,GATA4 phosphorylation is critical for Sry regulation in vivo,MAP3K4 and GADD45G,p38 MAPK pathway,activate GATA4,Phospho-GATA4,Sry promoter,Sryexpression,inhibited,impaired,disrupted,暗瞳曼爸虾澎懒洲诡氓骆捞凌皿衙沥星蒂贮忆汾瑰蚀蘸弃娇谈垂霞输卯腿GADD45G在雄性性别决定中的功能GADD45G在雄性性别决定中的功能,
