gadd45g在雄性性别决定中的功能

GADD45G Functions in Male Sex Determination by Promoting p38 Signaling and Sry ExpressionGADD45G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 基础兽医实验方法四 .讨论三 .结果二 .摘要一 .设想五 .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.Animals, Staging, and Dissection2. Histology and In Situ Hybridization.3. Plasmids， pCS2+ vector4.Fluorescence-Activated Cell Sorting5. RNA Extraction, Quantitative RT-PCR6. Western Blot Analysis7. 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 indistinguishablefrom those of wild-type femalesGadd45g-/-;XY gonads at this stage lacked testiscords and were smaller, as is characteristic of female embryonic gonads1. Gadd45g突变体鼠显示了雄性到雌性的性逆转XY mice resulted from defective fetal testis developmentusing the number of tail somites (ts) for accurate stagingSexual fate is determined in the bipotential gonad at 915 ts and gonadal somatic cellsdifferentiate from 16 ts onward1. Gadd45g突变体鼠显示了雄性到雌性的性逆转not affect the specication ofthe bipotential gonadal Anlagebipotential markers was mostly unchanged 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 Sertolicell differentiation ， Was not expressed in Gadd45g-/-Dhh and Amh, which areexpressed in Sertoli cells, and found that both markers wereabsent in Gadd45g-/-The early male gonad specication is permanently interrupted 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 genesWnt4 and Rspo1 are rst expressedin the bipotential gonad and are subsequently required for female development1. 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 required for granulosa cell and follicle development. Both were expressed in Gadd45g-/-.XY gonads2. 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 gonad2. 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 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 embryonicgonad reproduced the described kinetics.peaking at 17/18 ts and followed by rapid downregulation2. 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 reportedThe expression of Gadd45g before Sry suggests thatGADD45G might play a role in regulating Sry.the normal onset of Sry expressionwas delayed by approximately 2 ts in heterozygotes and 4 ts in homozygous mutants2. Gadd45g对于正常 Sry表达是必不可少的Sry expression in Gadd45g-/-;XY mice wasreduced to 25% of wild-type level during the criticaltime window between 11 ts and 15 ts.qPCR analysis , Sox9 is never expressed above basal levels in Gadd45g-/-;XY miceSox9 was strongly induced at 15 ts in XY wild-type embryos, whereas this induction was delayed in heterozygotes2. 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 expression2. Gadd45g对于正常 Sry表达是必不可少的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 antibodies；could not detect the protein by immunouorescence3. Gadd45g与 Sry和 Sox9共表达mRNA colocalization ;double-uorescence ISH; coexpression with Sry;72% of Gadd45g+ cells also coexpressed SryGadd45g+ cells were observed throughout the genital ridge at 13/14 tsthe germ cell marker Oct4 ，never coexpressed with Gadd45g in the same cells Sox9 was coexpressed in 25%of Gadd45g+ cells at 15/16 tsThe results indicate that Gadd45gis coexpressed with Sry and Sox9 in somatic cellsreporter 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 embryosdetected 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 SF1expression was high in the GFP+ populationLow in the GFP+ population at 8 ts, and absent in the GFP negative populationThe GFP negative cells most likely represent mesonephric and primordial germ cells3. Gadd45g与 Sry和 Sox9共表达3. Gadd45g与 Sry和 Sox9共表达18 ts， Gadd45g, Sry, and Sox9 expression; in the GFP+ populationcontained pre-Sertoli cells; undetectable in GFP+ and GFP- cellsOct4 was expressed only in the GFP-and GFP+, but not the GFP+(1)Gadd45g-expressing 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 promotersSry promoter is demethylatedat the time of Sry induction；demethylation is required forSry reporter gene expression in vitroSry 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 re