The Role Of Dennd1a,the Gene Of Polycystic Ovary Syndrome,in The Development Of Mouse Embryogenesis

Chapter I The role of Denndla,the gene of polycystic ovary syndrome,in the development of mouse embryogenesisBackgroundPolycystic ovary syndrome(PCOS)is a complex endocrine disorder in women of reproductive age,with a prevalence worldwide of 6-8%.The diagnosis of this disorder includes hyperandrogenemia,chronic anovulation and/or polycystic ovaries.The PCOS is thought to be caused by a combination of genetic and environmental factors.Genome wide association studies conducted in Chinese patients with PCOS found association between single nucleotide polymorphisms in the Denndlagene and PCOS.This association has also been replicated in studies of European patients or patients of European ancestry,suggesting a potentially functional role for Denndla in the pathogenesis of PCOS.It was found that Denndla protein was located in the cytoplasm and nuclei of ovarian theca cells,and its level was increased in the theca cells of PCOS patients.Moreover,knock-down of Dennd1a isoform in isolated PCOS theca cell cultures reduced the androgen biosynthesis,indicating that Denndla plays a key role in the hyperandrogenemia associated with PCOS.14 However,to date,little is known about how the Dennd1a gene function in developmental and reproductive processes.ObjectiveTo investigate the role of Denndla gene in the embryogenesis of mouse.MethodsIn the current study,the mice strains of the dennd1a gene knocked out were used and the disruption of Denndla efficiency was detected.Morphological characteristics,and the histology of the embryo were used to determine the date of embryo death and the difference between the homozygotes embryos and the control group.Immunohistochemical method was used to detect the effects of Denndla deficiency on the proliferation and apoptosis of brain and liver by PHH3 labeled proliferating cells and cleaved caspase-3 labeled apoptotic cells.Furthermore,in situ hybridization was used to detect the effects of Dennd1 a deficiency on other signaling pathways.HNF4?,SOX9,Pecaml and CD45 were used to label the homozygous liver to detect the effect of Denndla deficiency on liver development and hematopoietic function.In addition,the deficiency of Denndla also affected the cell proliferation and apoptosis in each tissue,and the development of primitive germ cells.These findings revealed that Dennd1a is crucial for mouse embryogenesis,suggesting that the Dennd1a-mediated endocytosis pathway may be involved in the regulation of the development of various embryonic organ systems.In addition,the effect of the loss of Dennd1a on the development of primary gonads was further detected.The development process of primary gonads in different stages was marked by AP staining based on the characteristics of high expression of alkaline phosphatase(AP)in primary germ cells.Real-time quantitative RT-PCR was used to detect SOHLH2(a marker of germ cell differentiation)in embryonic 13.5 homozygous female gonads for control analysis and the correlation between Dennd la gene deletion and gonad development was explored.Results1.Homozygous mutation of Denndla leads to fetal lethality.Control and homozygous embryos at the embryonic stage from embryonic 9.5 to 14.5 days were collected.Morphological changes between the two groups were compared.It was found that homozygous embryos had enlarged cerebral ventricles and dimmed liver color compared with the control group.At E14.5,the majority of Dennd1a-/-embryos do not have a heart beat.These results suggest that the disruption of Dennd1a defected multiple organs development,including the brain and liver.These results suggest that Dennd1a is essential for mouse embryogenesisIn situ hybridization analysis showed that Denndla mRNA was widely expressed in E9.5 embryos,and was strongly expressed in the forebrain,gill arch and limb bud.In E10.5,the expression pattern of Denndla in embryos was similar,but the real-time quantitative PCR results showed that the expression level of Denndla mRNA in E10.5 was higher.Our findings suggest that Dennd1a may play a role in morphogenesis of multiple embryonic organs.2.Loss of Denndla causes defects in brain developmentHistological staining of mice at E11.5 and E12.5 stages showed that compared with Dennd1a+/-embryos,the ventricles of Dennd1a-/-embryos became larger and the brain wall became thinner.The positive rate of PHH3 in the dorsal lobe of the E11.5 brain was examined.In the Dennd1a+/-brain,about 45.4%of the neuroepithelial cells were positive for staining,while in the Dennd1a-/-brain,about 23.2%were positive for staining.At the same time,in the brain of Denndla-/-embryos,the expression of c-myc and cyclinD1,which mediating cell cycle,were decreased.Decrease of active-?-catenin protein expression showed that the loss of Denndla down-regulated the Wnt signaling pathway,a classical signaling pathway that affects mouse brain development.Nestin and microtubule-binding protein 2(MAP2)were used to detect the changes in the expression levels of neurons of E11.5 brain precursor cells.Western blot results showed that the expression levels of NESTIN and MAP2 were low in the brain of Denndla-/-mice.The results showed that fewer neural precursor cells and differentiated neurons in homozygous mutant mice were associated with Dennd1a destruction.These results suggest that abnormal cell proliferation and differentiation may lead to neural defects in the embryonic brain of Dennd1a-/-mice.3.Denndla deficiency impairs embryonic liver developmentIn addition to neurological defects,Denndla-/-embryos also showed abnormal liver development.At E12.5 and E14.5,the size of Denndla-/-embryonic liver was significantly reduced compared to the control group.During the embryo,the liver forms in the foregut endoderm and cells proliferate and migrate to the surrounding septum.To determine whether hepatocyte proliferation and survival were affected by Dennd1a deficiency,PHH3 and Cleaved Caspase 3 immunostaining were performed in cross-sectional E12.5 embryos.Based on PHH3 staining,the percentage of dividing cells in Denndla-/-embryonic liver compared with Dennd1a+/-embryonic liver was 8.9±1.0%vs 12.6±1.4%;P<0.05 indicated that the destruction of Denndla resulted in decreased proliferation of liver cells.On the other hand,Denndla deficiency did not affect apoptosis.In the liver,Cleaved Caspase 3 positive cells are difficult to detect in the liver of E12.5 Denndla-/-or Denndla+/-embryos.During mouse embryonic liver formation,Denndla regulates liver cell proliferation,but is not necessary for liver cell survival.During liver formation,the hepatic endoderm differentiates into hepatocytes or bile duct cells.Hepatocytes are the main parenchymal cell type in the liver.Hepatocyte expression of transcription factor HNF4? is an indicator of regulating the fate of early hepatocytes.Immunohistochemical analysis showed that HNF4? was expressed in hepatocytes of E12.5 Denndla-/-or control fetal livers.Real-time quantitative PCR further revealed that the mRNA expression level of HNF4? was significantly decreased in the liver of E12.5 Dennd1a-/-.It was suggested that the loss of Denndla affected the differentiation of hepatocytes.Next,the effect of Dennd1a deficiency on the differentiation of bile duct cells was examined.Biliary duct cells differentiate first to express SOX9,a transcription factor that controls the development of bile duct cells.Sox9+cells were found confined to the perihepatic bile ducts.However,in Denndla-/-embryonic liver,SOX9 expression is dispersed,not confined to the peri-bile duct region,and is accompanied by an elevated SOX9 transcription level.These results suggest that lack of Denndla can promote the differentiation of hepatogenic cells into intrahepatic bile duct cells.By Ell,the liver of most embryos expressed CD45,a ubiquitically expressed hematopoietic factor.From E12 days to birth,the fetal liver was the main organ for hematopoiesis.The endothelial cells expressed Pecam1,known as a vascular endothelial cell marker.Real-time quantitative PCR results showed that the mRNA level of CD45 was decreased in the E12.5 Dennd1a-/-liver,while increased in Pecam1,suggested that Denndla deficiency also affects liver hematopoiesis and blood vessel formation,which may lead to decreased liver size and fetal hematopoietic damage,ultimately leading to the death of Dennd1a mutant embryos.4.Denndla mutation disturbs the development of primordial germ cellsIn mice,primordial-germ cells(PGCs)appear in the posterior amniotic fold of the extraberminal mesoderm and are characterized by hyperalkaline phosphatase(AP)activity.Between E8.5 and E13.5,the primordial germ cells proliferate and migrate to the reproductive crest through the hindgut endoderm and mesentery.Around E9,the primordial germ cells in the wild-type embryo migrate through the hindgut endoderm and mesentery,while the dominant PGCs have moved much further away.In contrast,the migration of primordial germ cells in Denndla-/-embryos was significantly delayed,with few primordial germ cells reaching the corresponding somatic boundaries.In E10.5,wildtype embryonic primitive germ cells reach to the gonads primordium.At E11.5,primitive germ cells continue to proliferate in the gonads,however,in the corresponding period,mutant embryos found less primitive germ cells in the gonads primordium,after the gonads gonad primordium thickening and determined by the formation of testicular or ovarian sex gene,in female mice,Spermatogenic and oocytic bHLH transcription factor 2(SOHLH2)is expressed at about E13.5 and regulates oocyte differentiation.According to AP staining,the gonads of E13.5 female Dennd1a-/-mice were retained,which were slightly longer and narrower than those of the control group.Real-time quantitative PCR results showed that the deficiency of Denndla significantly reduced the expression of SOHLH2 in embryonic ovaries.These results suggest that lack of Dennd1a may interfere with proliferation,migration and differentiation of PGCs.ConclusionIn this study,for the first time,we studied the deletion of Denndla caused the death of embryonic mice at about E14.5,and clarified that the mutation affected the development of brain,liver and gonad,respectively,and disrupted the proliferation,differentiation and apoptosis of the corresponding tissue cells,further confirmed that Denndla gene is essential in embryonic development.At the same time,it was found that Dennd1a gene affected the proliferation,differentiation and migration of primary germ cells.The role of Dennd1a in the development of primary germ cells remains to be further explored.Chapter ? The mechanism of DENND1A regulating the development of embryonic germ cellsBackgroundDenndla functions as a guanine nucleotide exchange factor(GEF)for the small GTPase Rab35,is essential for mouse embryogenesis.The destruction of dennd1a can damage the migration and differentiation of embryonic embryonic cells.In this study,we further clarified the role of denndla in the occurrence and reduction of fetal ovarian germ cells.Dennd1a is mainly expressed in the fetal ovarian cells.Dennd1a's deletion desrupted the expression of the mRNA of the sohlh2 in the E13.5 dennd la-mutants ovaries.Objective:The purpose of our study was to discover the effects of DENND1A on the meiosis and differentiation of germ cells and explore the effects of the molecular mechanisms mediated by DENND1A on the differentiation and meiosis of germ cells through the embryonic ovaries.Methods:The mRNA expressions of SOHH2,FIGLA,STRA8 and REC8 in the ovary of E13.5 DENND1A-/-mutant were detected by real-time quantitative PCR in DENND1A knockout mouse model.The expression of SOHLH2,FIGLA,STRA8 and REC8 was further verified by in vitro culture of female gonads of E12.5 with DENND1A shRNA adenovirus infected or not.DENND1A was required for oocyte differentiation and meiosis initiation in somatic cells.The gonads were infected with adenovirus of DENND1A shRNA,and the possible effects of altered Wnt5a and retinoic acid synthesis in somatic cells on oocyte differentiation and meiosis were examined at the transcriptional and translation levels.Results1.Expression of Denndla is gradually increased in the fetal ovary,with a higher level in the somatic cells than in the germ cells.Dennd1a protein was located on both the hindgut mesentery and genital ridge of E11.5 mouse embryos,suggesting Dennd1a may play a role when PGCs are migrating through the mesentery and colonized in the genital ridge.In fetal ovaries,the levels of Denndla mRNA were gradually increased on E12.5,E13.5,and E15.5,during early stages of the oocyte differentiation and meiosis.Whereas in the postnatal 1-week,3-week,or 6-week ovaries,the expression of Denndla mRNA and protein was not significantly changed.Using the stagespecific embryonic antigen-1(SSEA1)as the fetal germ cell marker,immunofluorescence results showed that Denndla was mainly expressed in the somatic cells(SC)in fetal gonad at E11.5 and E13.5,and barely co-localized with SSEA1.The same expression pattern was found in E14.5 and E17.5 fetal ovaries using Stella and DDX4 as germ cell markers,respectively.The fetal germ cells were then isolated from the somatic cells of E11.5 and E1 3.5 female gonads by fluorescence activated cell sorting.The mRNA expression of Denndla was higher in somatic cells than in fetal germ cells.These findings suggest that Denndla is mainly expressed in the somatic cells of the fetal ovary,and Dennd la-mediated endocytic pathway in the somatic cells may be involved in regulation of cell signaling that is crucial for the development of fetal germ cells.2.Dennd1a Deficiency impairs initiation of oogenesis and meiosis in the fetal ovary.To assess the oocyte differentiation and initiation of meiosis during embryonic development,we examined mRNA expression of Sohlh2,Figla(Factor in the germline ?),Stra8,and Rec8 in the fetal ovaries at E12.5,E13.5,and E15.5.As transcription factors in the germ cells or oocytes,Sohlh2 mRNA level was elevated at E13.5 and then declined at E15.5,while mRNA expression of Figla was activated at E13.5 and dramatically promoted at E15.5.The transcription of Stra8 and Rec8,two genes essential for oocyte meiosis,were both activated circa E13.5.However,using Denndla gene knockout mice,we discovered that in the ovaries of homozygous Dennd1a-/-mutants at E13.5,mRNA expression of these genes were insufficiently induced.Deletion of Denndla affected the transcription of not only Sohlh2,but Figla,Stra8 and Rec8 as well at this developmental stage.Since homozygous Denndla-/-mutants die circa E14.5,they are not suitable for further determining whether loss of Denndla eventually disrupts the expression of genes associated with oocyte differentiation and meiotic initiation.Therefore,we established an in vitro gonad culture by incubating genital ridges isolated from E12.5 wild-type female embryos.The long and narrow E12.5 gonads in vitro became short and thick after being cultured for 24,48,and 72 h,recapitulating the morphological changes of developing gonads in vivo from E12.5 to E15.5.The mRNA expression of Sohlh2,Figla,Stra8 and Rec8 in wild-type gonads in vitro were activated after being incubated for 48 h,showing a similar expression pattern as that in gonads in vivo at E13.5.To knockdown the expression of Dennd1a,the in vitro E12.5 gonads were infected with adenoviral Denndla shRNA carrying a GFP fluorescent tag for 24,48,72,or 96 h.After 48 h or longer of infection,the expression of Denndla mRNA and protein were decreased to approximately half the wildtype level,and the mRNA expression of Sohlh2,Figla,Stra8 and Rec8 were not activated,as shown in representative samples treated for 72 h.These results suggest that Dennd 1 a deficiency disrupted the proper activation of genes associated with oocyte differentiation and initiation of meiosis in fetal ovaries.2.Production of Wnt5a and RA is reduced in the somatic cells of the fetal ovary lacking Dennd1a.DENND1A-mediated FIGLA,STRA8,and REC8 expression is independent of Wnt5a.The initiation of meiosis in embryonic germ cells requires DENND1A to mediate the production of RA by somatic cells,thereby inducing the expression of germ cells,STRA8 and REC8.In summary,DENND1A may be involved in multiple somatic signaling pathways that are critical to the various processes and stages of oocyte differentiation and meiosis.3.The expression pf Wnt5a and RA in somatic cells mediating the oocyte differentiation and meiotic initiation in the fetal ovary.Treatment with exogenous Wnt5a(350ng/mL)could stimulate the expression of Soh1h2 in Dennd1a knockdown fetal ovaries to the level comparable to that in wild-type control,showing representative samples treated for 48 h.However,exogenous Wnt5a could not rescue the decreased mRNA expression of Figla,Stra8,or Rec8 in the fetal ovaries affected by Dennd1a knockdown(Fig.4B).On the other hand,RA treatment(1uM)stimulated the expression of Stra8 and Rec8 in both wild-type and adenoviral Denndla shRNA infected fetal ovaries,although it had no effects on the expression of Sohlh2 and Figla.The above results suggested that Denndla might play a role in regulating the somatic cells of fetal ovaries to produce Wnt5a and RA,which were required for the oocyte differentiation and meiotic initiation respectively.3.The Denndla mediating the synthesis of wnt5a and RA in somatic cells through the PI3K-Akt and canonical wnt/?-catenin pathwayWe further investigated the signaling target of Dennd1a in somatic cells and its mechanism of controlling fetal oocyte differentiation and meiosis.Dennd1a knockdown led to reduced accumulation of non-phosphorylated ?-catenin and activation of Axin 2,a downstream target of ?-catenin-dependent transcription showing representative samples treated for 72 h.This could be due to decreased phosphorylation of Akt and increased phosphorylation of GSK3?.Downregulated ?-catenin signaling could be the altered upstream regulators for Wnt5a and RA synthesis in the fetal ovary,through decreased expression and phosphorylation of Stat3 and reduced expression of Aldh1a1.Treatment with exogenous Wnt3a(100ng/mL)could stimulate the expression of wnt5a and RA in somatic cells from mesenophros to the level comparable to that in wild-type control.showing representative samples treated for 48 h.However,exogenous LY294002(PI3K inhibitor)could the decreased protein level of wnt5a and RA in the somtic cells.The above results suggested that Denndla might affect the synthesis of wnt5a and RA in somatic cells through the PI3K-Akt and canonical wnt/?-catenin pathway.Conclusions:In this study,we further clarified the role of denndla in the differentiation and meiosis offetal germ cells.Denndla regulating the synthesis of wnt5a and ra in somatic cells through mediating the PI3K-AKT and wnt/?-catenin pathway.The denndla affects the mRNA expression of sohlh2,figla and stra8 rec8,which is related to the differentiation and meiosis.The above results showed that the denndla in somatic cells is a necessary for the development of fetal germ cells,and is critical for the different processes and stages of the differentiation and meiosis of the fetal germ cells.