Molecular Mechanisms Of Mouse M(?)llerian Duct Development

Background:Before sexual differentiation,an embryo possesses two sets of reproductive tract progenitors,a pair of mesonephric ducts(Wolffian duct,WD)and a pair of paramesonephric ducts(Mullerian duct,MD).The Mullerian duct is the precursor the female reproductive tract(FRT),including the oviduct,uterus,cervix and upper vagina.Dysplasia of different domains of the Mullerian duct can lead to various degrees of Mullerian anomalies,while the mechanism of Mullerian anomalies remains to be elucidated.From a "homogenous" tube to the fallopian tube,uterus,cervix,and vagina,there must be up-regulated or down-regulated genes and key signaling pathways that regulate differentiation of Mullerian duct.Identifying the heterogeneity of Mullerian mesenchyme,the differential expression genes of heterogenous clusters and crucial signaling pathway will help to uncover the mechanism of Mullerian anomalies.Single-cell RNA sequencing technology has become a powerful tool for the comprehensive analysis of cell heterogeneity because of its ability to sequence the transcriptome of a single cell.Objectives:1.To identify the heterogeneity of the Mullerian mesenchyme of female mice;2.To uncover the key genes and signaling pathways that regulate the differentiation of Mullerian mesenchyme into fallopian tubes and uterus.Methods:We mated Amhr2-Cre mice with mT/mG mice and obtained the mT/mG;Amhr2-Cre mice,for which the Mullerian mesenchyme cells were labeled with Green Fluorescent Protein(GFP).Mesonephro tissues(without gonads)were then harvested on Embryonic day 14.5(E14.5),E15.5,E16.5,E18.5 and postnatal day 0(PO).Then mesonephro tissues were dissociated into single-cell suspension and 10 X genomics single-cell RNA sequencing was performed.Cell ranger software was used to group the cells based on the level of gene expression.The differentially expressed genes of subgroups were obtained based on the results of cell clustering.A gene set enrichment analysis based on geneontology(GO)terms was conducted to characterize various gene sets in the analysis.Monocle software was used to carry out pseudotime analysis on the green fluorescent protein(GFP)positive cell groups,to reconstruct the developmental trajectory.Beam analysis was used to identify genes whose expression was significantly associated with the pseudotime.Results:1.After removal of endothelial clusters,immune cell clusters,erythroblasts clusters,and double cells,single-cell RNA sequencing identified 15 clusters of mice developing mesonephros.2.Mullerian mesenchyme contains two heterogenous cells,which are HoxalO+Hoxall+and Hoxa7+Hoxc8+respectively.The heterogeneity could be detected as early as E14.5.3.Hoxa7,Hoxc8,Celf2,Itm2a,Nr2f1 and Vstm2b specifically express in Mullerian mesenchyme that would differentiate into fallopian tube;Hoxa10 Hoxa11?Pkdcc and Fnl exclusively express in Miillerian mesenchyme that would differentiate into uterus.These genes are expected to be new marker genes for the Mullerian duct,neonatal fallopian tube and uterus.4.The Mullerian mesenchyme in mice originates from Amhr2+coelomic epithelial cells.There are two branches of differentiation of the coelom epithelium.One is Hoxa7+Hoxc8+Mullerian mesenchyme which eventually differentiates into the mesenchyme of fallopian tube;The other is Hoxa10+Hoxall+Mullerian mesenchyme that eventually differentiates into the mesenchyme of uterus.5.As the coelomic epithelial cells differentiate into the oviduct,the expression of Hoxa7,Hoxc8 and Celf2 increase.While the expression of Hoxa10,Hoxall and Aldh1a2,an enzyme synthesizing RA,decrease.No significant changes in the expression of family members of BMP,anti-BMP or Wnts signaling were found.6.As the coelomic epithelial cells differentiate into the uterus,the expression levels of Hoxa10,Hoxall and Aldh1a2,an enzyme synthesizing RA,increase.While the expression of Hoxa 7,Hoxc8 and Celf2 decrease.Also,no significant changes in the expression of family members of BMP,anti-BMP or Wnts signaling were found.Conclusions:1.The Mullerian mesenchyme in mice originates from Amhr2+coelomic epithelial cells.Mullerian mesenchyme contains two clusters of heterogenous cells,Hoxa10+Hoxa11+cluster and Hoxa7+Hoxc8+ cluster,which will differentiate into uterine and fallopian tube mesenchyme at P0,respectively.2.Hoxa 7,Hoxc8 and Celf2 regulate the differentiation of coelomic epithelia into the Mullerian mesenchyme that will develop into fallopian tube;Hoxa10 and Hoxa11 regulate the differentiation of coelomic epithelia into the Mullerian mesenchyme that will develop into uterus;RA may initiate the differentiation of coelomic epithelia into Mullerian mesenchyme that will develop into uterus.Background and objective:Before sexual differentiation,an embryo possesses two sets of reproductive tract progenitors,a pair of mesonephric ducts(Wolffian duct,WD)and a pair of paramesonephric ducts(Mullerian duct,MD).The Mullerian duct is the precursor the female reproductive tract(FRT),including the oviduct,uterus,cervix and upper vagina.Hence,any abnormality in the development of Mullerian duct may lead to congenital malformations of FRT.But mechanisms regulating these processes are poorly understood.The Wilms' tumor 1(Wt1)gene is involved in the regulation of development of multiple organs.Wt1 participates in the regression of Mullerian duct in male mice.While it is unclear what role it plays in the development of FRT.Anti-Mullerian hormone type 2 receptor(Amhr2)specifically expressed in the mesenchyme of Mullerian duct and Amhr2-Cre mice are always used to conditional knock-out of target genes to study the development and regression of Mullerian duct.Given its important role in embryonic development and the overlapped expression pattern of Wt1 and Amhr2 transcripts in the developing Mullerian duct,we hypothesized that conditional knock-out of Wtl in Mullerian mesenchyme by Amhr2-Cre might result in severe defect or even aplasia of Mullerian duct.Here we examined the role of Wtl in postnatal Mullerian duct differentiation.Methods:Wt1 was ablation within the Mullerian duct mesenchyme using Amhr2-Cre.Mice with the genotype of Wt1-/flox;Amhr2-Cre was referred to as mutants.Hematoxylin-eosin staining was used to study the morphology of female reproductive tract.Results:1)Wt1 and Amhr2 showed overlapping expression patterns in the developing mesonephros.Both genes were expressed in the coelomic epithelium and around the Mullerian duct.2)Female reproductive tract abnormalities caused by ablation of Wt1 in Mullerian duct mesenchyme using Amhr2-Cre and the phenotypic varied a lot,from normal gross appearance to distal vaginal atresia.3)Ablation of Wtl in Mullerian duct mesenchyme by Amhr2-Cre resulted in myometrial hypoplasia and reduced uterine glands of female adult mice.Moreover,with the aggravation of the vaginal phenotype,the phenotype of uterine hypoplasia is more obvious.4)Ablation of Wtl in Mullerian duct mesenchyme by Amhr2-Cre resulted in aberrant epithelial cell proliferation and stratification in the basal cell layers and led to failure of keratinized differentiation of vaginal epithelium.Conclusions:1)Ablation of Wt1 by Amhr2-Cre didn't impede the formation of Mullerian duct but resulted in abnormal differentiation of Mullerian duct.Wt1 is required for proper development of female reproductive tract.2)Wt1 in Mullerian duct mesenchyme regulates vaginal epithelial cell differentiation.Ablation of Wt1 by Amhr2-Cre resulted in aberrant proliferation and stratification in the basal cell layers and led to failure of keratinized differentiation of vaginal epithelium.