Deciphering The Development Of Human Embryonic Müllerian Ducts By Single-Cell RNA-Sequencing

[Background]In the early stage of mammalian embryonic development,the reproductive tract primordium of both sexes is similar before sex determination and sex differentiation.And after sex determination,the paramesonephric ducts(Müllerian Duct,MD)which develop as female reproductive tract in females,gradually develop into the structure as oviduct,uterus,and cervix,while the counterpart,mesonephric ducts,which develop as male reproductive duct(Wolffian Duct,WD)gradually degenerates into remnants.The Müllerian ducts are bilateral in early human embryo,which are uniform ducts extend cranially to caudally alone with the mesonephroi.In the process of female genital track development,the caudal segments of the bilateral Müllerian ducts gradually merge into one canal in about the 9th week of gestation.In the direction of the anterior-posterior axis,the embryonic Müllerian ducts have different fates to differentiate into the oviduct and the uterus commitment.In mammals such as mice,their uteri are bicornuate.Unlike human uterus,the bilateral Müllerian ducts in mice would not fuse during the development as the uterus.We use the method of single-cell RNA-sequencing to profile the transcriptome of human female embryonic mesonephroi.By this cell-resolution sequencing method and referring to the previous data of the mouse embryonic mesonephros single-cell RNA-sequencing completed by our team,we are about to search for the regulatory mechanism of the heterogeneity among the development and differentiation of the human female embryonic Müllerian ducts,and to find the differential expression genes related to the fusion of the human uterine tube by comparing with the mouse.And this would provide us with the clues of genes and regulatory mechanisms in the development progression of female genital track.[Objective]The purpose of this study is to explore the regulation process of cell fate differentiation in Müllerian duct development,especially Müllerian interstitial development in human female track;and we are going to explore the crucial genes involved in human uterine fusion and the transcription factors or signal path which play an important role in the regulation process of female genital track development.[Methods]This study was approved by the institutional review board(IRB)of Peking Union Medical College Hospital.Embryos by medical abortion were collected from Peking Union Medical College Hospital which is in the 8th to 11th weeks of gestation.And the embryonic mesonephroi were dissected under stereo microscopes.The polymerase chain reaction(PCR)was used to identify the sex of the embryos,and we collected the female embryonic mesonephroi.According to the standard workflow of single-cell transcriptome sequencing,we digest the obtained mesonephric tissue to a single-cell suspension step by step.After validation of the sample status and sex,the samples were sent to 10x Genomics single-cell transcriptome sequencing.And right after obtained the raw sequencing data,we analyzed by the conventional pipeline for single-cell transcriptome data.Firstly,we used the 10x Genomics recommended official software,Cell Ranger,by reading and aligning the reads to generate the expression matrix,then processed dimension reduction and visualization by R studio backpacks,such as Seurat or Monocle.By using these bioinformatic tools,we have mapped the human female mesonephros single-cell transcriptome and the trajectories of Müllerian duct differentiation in different lineage during Mullerian duct-uterine tube development.Afterwards,according to the analysis results,further downstream bioinformatics analysis,such as GSEA(Gene Set Enrichment Analysis),GO(Gene Ontology),and KEGG(Kyoto Encyclopedia of Genes and Genomes)websites or tools were applied to further interpret our results.Differentially expressed genes(DEGs)and high variable genes(HVGs)are discriminated in enrichment and comparison to find the pivotal regulator genes or signaling pathways.[result]1.The single-cell transcriptome map of the embryonic mesonephros was depicted.We clustered all the female mesonephric cells into 15 clusters,and we defined each cluster by their DEGs,which allowed us to focus on these Müllerian epithelium cells and Müllerian stromal cells.2.By pseudotime analysis,we observed that the coelomic mesenchymal progenitors in the early female fetal mesonephroi have different cell fate commitment into two lineages,which could differentiate as both coelomic epithelial and Mullerian stromal precursors.Furthermore,by dividing the interested cell clusters,the Mullerian stromal precursors could also be traced by two developmental trajectories,those are,Müllerian stromal cells to differentiate the oviduct stroma and Müllerian stromal cells to differentiate the uterine stroma.3.Combined with the HVGs that were calculated in the pseudo-time analysis and highly dependent on cell fate determination.We conducted the BEAM analysis to find the HVGs,which are related to Wnt signaling pathway,extracellular matrix(ECM),transmembrane protein and transporter channels that may be involved in the regulation of Müllerian stromal cell development and differentiation process.In the same way,we analyzed the different trajectories between Mullerian stromal precursor cells to Müllerian stromal cells which differentiate the oviduct stroma versus Miillerian stromal cells which differentiate the uterine stroma.We found that,the HOX gene cluster and the RA signaling pathway play a significant role in these two branches of cell fate differentiation.These regulators are consistent with our previous analysis of mouse Mullerian stromal cell differentiation into uterine or oviduct stromal cells,that is,this procedure is conserved between human and mouse.4.By conducting Gene Set Enrichment Analysis(GSEA)between human and mouse embryonic mesonephric transcriptome data on our interested signaling pathways and biological process.We found that in the relatively late stage of our studied window.That is,comparison of the group of in 10th weeks human and the E16.5 mouse,human was positively enriched to mouse in the development of Tgf-β signaling pathway,Notch signaling pathway,Wnt-β-Cantenin signaling pathway,Hedgehog signaling pathway,estrogen response signaling pathway and epithelial-mesenchymal transition(EMT).[Conclusion]The Müllerian duct stroma originates from EMX2+and AMHR2+coelomic mesenchymal progenitors,and the coelomic mesenchymal progenitors have the potential either to diferentiate into Müllerian duct stromal precursors or to maintain the coelomic epithelial state.Both in human and mouse Müllerian duct stromal development,HOX Gene cluster,RA signaling pathway and Wnt signaling pathway are similarly involved conservatively in the regulation of female reproductive tract development.It can be speculated that the occurrence of Mullerian duct development is a multiple coordinate regulated result in anterior and posterior,dorsal and ventral and left and right axis by the signaling pathways and transcription factors which mentioned above temporally and spatially.