GADD45G在雄性性别决定中的功能.ppt

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,一、摘要,Gadd45g display complete male-to-female sex reversal.,p38 MAPK signaling is impaired in Gadd45g mutants.,GATA4, which is required for Sry expression.,二、材料和方法,1.An

2、imals, Staging, and Dissection,2. Histology and In Situ Hybridization.,3. Plasmids，pCS2+ vector,4.Fluorescence-Activated Cell Sorting,5. RNA Extraction, Quantitative RT-PCR,6. Western Blot Analysis,7. Chromatin Immunoprecipitation,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

3、,雄性到雌性的性逆转,1. Gadd45g突变体鼠显示了雄性到雌性的性逆转,XY mice indistinguishable from those of wild-type females,Gadd45g-/-;XY gonads at this stage lacked testis cords and were smaller, as is characteristic of female embryonic gonads,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

4、,using the number of tail somites (ts) for accurate staging,Sexual fate is determined in the bipotential gonad at 915 ts and gonadal somatic cells differentiate from 16 ts onward,1. Gadd45g突变体鼠显示了雄性到雌性的性逆转,not affect the specication of the bipotential gonadal Anlage,bipotential markers was mostly un

5、changed in Gadd45g-/-,1. Gadd45g突变体鼠显示了雄性到雌性的性逆转,Sox9, a key regulator of Sertoli cell differentiation ，Was not expressed in Gadd45g-/-,Dhh and Amh, which are expressed in Sertoli cells, and found that both markers were absent in Gadd45g-/-,The early male gonad specication is permanently interrupted

6、 in Gadd45g-/-.,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,1. Gadd45g突变体鼠显示了雄性到雌性的性逆转,Foxl2 and Fst are induced at E11.5 and are specically in females re

7、quired for granulosa cell and follicle development. Both were expressed in Gadd45g-/-.XY gonads,2. Gadd45g对于正常Sry表达是必不可少的,The results raised the question of where and when Gadd45g is expressed in the early male embryonic gonad,？,2. Gadd45g对于正常Sry表达是必不可少的,prominent Gadd45g expression by in situ hybri

8、dization (ISH) in the whole gonad at 11/12 ts when Sry is expressed only in the center of the gonad,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.,2. Gadd45g对于正常Sry表达是必不可少的,Sry expression in the male embryoni

9、c gonad reproduced the described kinetics. peaking at 17/18 ts and followed by rapid downregulation,2. Gadd45g对于正常Sry表达是必不可少的,Sox9 was upregulated at 15/16 ts and stayed expressed in the genital ridge, as reported,The expression of Gadd45g before Sry suggests that GADD45G might play a role in regula

10、ting Sry.,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表达是必不可少的,Sry expression in Gadd45g-/-;XY mice was reduced to 25% of wild-type level during the critical time window between 11 ts and 15 ts.,qPCR analysis ,

11、 Sox9 is never expressed above basal levels in Gadd45g-/-;XY mice,Sox9 was strongly 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,sex reversal in Gadd45g mutant mice caused the delayed and reduced Sry expression,2. Gadd45g对于正常

12、Sry表达是必不可少的,The specic expression of Gadd45g in the early embryonic gonad and its requirement for normal Sry expression suggested that Gadd45g may be coexpressed with Sry and hence cell- autonomously regulate Sry expression.,raised GADD45G antibodies； could not detect the protein by immunouorescence

13、,3. Gadd45g与Sry和Sox9共表达,mRNA colocalization ; double-uorescence ISH; coexpression with Sry;,72% of Gadd45g+ cells also coexpressed Sry,Gadd45g+ cells were observed throughout the genital ridge at 13/14 ts,the germ cell marker Oct4 ， never coexpressed with Gadd45g in the same cells,Sox9 was coexpress

14、ed in 25% of Gadd45g+ cells at 15/16 ts,The results indicate that Gadd45g is coexpressed with Sry and Sox9 in somatic cells,reporter mice that express EGFP under the control of a SF1 promoter, marking SF1+ somatic cells in the male gonadal Anlage,puried SF1+ cells by uorescence-activated cell sortin

15、g (FACS) from dissociated genital ridges of 8 ts and 18 ts embryos,detected a GFP negative,a weakly GFP-expressing (GFP+), and a highly GFP-expressing population (GFP+),3. Gadd45g与Sry和Sox9共表达,endogenous SF1 expression was high in the GFP+ population,Low in the GFP+ population at 8 ts, and absent in

16、the GFP negative population,The GFP negative cells most likely represent mesonephric and primordial germ cells,3. Gadd45g与Sry和Sox9共表达,3. Gadd45g与Sry和Sox9共表达,Oct4 was expressed only in the GFP- and GFP+, but not the GFP+,Gadd45g-expressing cells coexpress Sry and Sox9 and hence represent somatic and

17、pre-Sertoli cells (2) Gadd45g precedes Sry and Sox9 expression.,3. Gadd45g与Sry和Sox9共表达,4.Sry基因脱甲基化在Gadd45g突变体不被影响,Gadd45 genes；promoting DNA demethylation and transcriptional activation of target gene promoters,Sry promoter is demethylated at the time of Sry induction； demethylation is required for

18、Sry reporter gene expression in vitro,Sry gene is demethylated ；gonad development ， mapped methylation 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,Sry demethylation ； pronounced in

19、 the proximal promoter,4.Sry基因脱甲基化在Gadd45g突变体不被影响,Sry proximal promoter demethylation using 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 levels of CpGs in the proxima

20、l Sry promoter analyzed by 454 bisulte sequencing,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 at any of the tested CpGs at both stages,5. Gadd45g对MAP激酶信号是必需的,Map3k4 and Map3k1 cause male-to-fe

21、male sex reversal and testis abnormalities, respectively,Activation of MAPK signaling is one of the best-documented cellular functions of GADD45 family members,GADD45G specically activates MAP3K4； p38 signaling in various cell types； GADD45G may also regulate MAPK signaling during male gonad develop

22、ment.,(A) Western blot analysis of gonads and mesonephroi of male wild-type 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 signalin

23、g in gonadal cells,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.,downstream of

24、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 of GATA4 ， regulated by MAPK phosphorylation,inhibition of p38 signaling in genital ridge explants ， marked decrease in GATA4 phosphorylation,Anisomycin treatment of explants res

25、ulted in increased activation of GATA4,5. Gadd45g对MAP激酶信号是必需的,GATA regulatory motifs within the proximal Sry promoter,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,

26、This binding was abolished in gonads treated with the p38 signaling inhibitor SB202190,GATA4 is phosphorylated by p38 MAPK signaling and activates the Sry promoter,四、讨论,GADD45 family,GADD45G， in the earliest steps of male gonad specication to regulate Sry and Sox9,stress sensors, tumor suppressors,

27、and immune modulators,expressed in the genital ridge, in SF1 positive, bipotential somatic cells.,四、讨论,In Gadd45g mutants Sry demethylation occurs on schedule, despite reduced and delayed expression of Sry,GADD45G functions in sex determination by promoting p38 MAPK signaling.,Sry locus is poised for expression, ready to be activated by incoming signals,activation of MAP3K4，an N-terminal autoinhibitory domain, which prevents substrate inter