Single Cell Transcriptomes Of Mouse Bladder Urothelium Uncover Novel Cell Type Markers And Urothelial Differentiation Characteristics

BackgroudThe bladder urothelium consists of a layer of basal cells,intermediate cells,and a luminal layer of fully differentiated superficial cells that serves as a crucial barrier between the blood and urine.Normal bladder urothelial function is critical in maintaining the homeostasis of bladder.Many bladder diseases are related to bladder urothelial biology and function.Common technique to study the bladder urothelium is using cell culture preparations and enzyme digested or surgically isolated tissue.However,it seems difficult,or even impossible,to study cell-type specific characteristics and functions of bladder urothelial cells by these traditional methods.The heterogeneity and hierarchy of bladder urothelial cells have not been well revealed.Single-cell RNA sequencing is an advanced technology that can greatly facilitates the studies on cell function under normal physiological activities as well as during disease processes.By utilizing this powerful tool,the cellular hierarchy and developmental trajectory of multiple organs have been discovered.Although previous studies have mapped all major mouse organs,high-throughput single-cell RNA sequencing of bladder urothelial cells have not been reported so far.PurposeThe present study aims to investigate the unsupervised classification and function of mouse bladder urothelial cells precisely by single-cell RNA sequencing and learn more about the relationship between bladder urothelial cells and bladder diseases.Method1.Single Cell RNA Sequencing10-week-old Female C57BL/6J mice were sacrificed by cervical vertebra dislocation and bladder urothelium was removed.Single-cell RNA-seq libraries were prepared with Chromium Single cell 3’Reagent v3 Kits according to the manufacturer’s protocol.Using the Cell Ranger software to produce a matrix of gene counts versus cells.We removed low quality cells and likely multiplet captures.Principal component analysis(PCA)was performed to reduce the dimensionality on the log transformed gene-barcode matrices of top variable genes.Cells were clustered based on a graph-based clustering approach.2.Pseudotime Analysis and RNA velocity analysisWe determined the developmental pseudotime with the Monocle2 package and the RNA velocity analysis with the velocyto package.We used SCENIC for the simultaneous reconstruction of gene regulatory networks(GRNs)and the identification of regulons.Cell-cell communication networks were identified and visualized by Cell Chat package.3.Cell Identification and Functional VerificationThe expression of Plxna4+and ASPM+ cells was identified by immunofluorescence.The cell functions were verified by cytological experiment.Result1.Single Cell Profiling and Unbiased Clustering of Mouse Bladder Urothelial CellsSingle bladder urothelial cell suspension was obtained from the mouse bladder urothelium using trypsin digestion.The percent of alive cell is more than 90%conformed by trypan blue staining and Calcein-AM/PI staining.Using stringent quality controls,18917 bladder urothelial cells were further analyzed.2.Classification of Urothelial Cells Based on Cell-Type Specific Marker GenesUnsupervised clustering analysis identified 8 distinct cell clusters.We performed differential gene expression analysis to further investigate the identity of each cell cluster.Based on the specific expression of these known cell type-specific markers,Cluster 0,Cluster 5 and Cluster 6 were identified as Basal-like cells,which was named as Basal cell 1(Bc 1),Basal cell 2(Bc 2)and Basal cell 3(Bc 3).Cluster 1 and Cluster 2 were defined as Intermediate cell 1(Ic 1)and Intermediate cell 2(Ic 2).Cluster 3 and Cluster 4 were defined as superficial cell 1 and superficial cell 2(Sc 1 and Sc 2).Cluster 7 was named as Plxna4+urothelial cell according to the most specific gene--Plxna4.3.Developmental Trajectory and RNA Velocity Analysis Revealed the Developmental Lineages of Mouse Bladder Urothelial CellsThe utilized reverse graph embedding to generate a trajectory plot and perform cell trajectory analysis by using pseudotime analysis.Bc 3 is the starting point of the trajectory,and followed by Bc 1 and Bc 2.The cluster of Ic 2,Sc 1 and Sc 2 were the end of the trajectory branch 1.Plxna4+urothelial cells appeared as the ends of the trajectory branch 2.To further analyze the genes in terms of the changes in pseudotime analysis,we found Malatl,Ftl1,Tpt1,S100a6 and Ly6d highly expressed at the the end stage of developmental trajectory.Gstml and Tmsb4x were highly expressed at the early stage of developmental trajectory.RNA velocity was analyzed to study the developmental lineage of urothelial cells.The results showed that Ic 1 and Ic 2 cells were involved in the transformation of Sc 1 and Sc 2.Sc 1 and Sc 2 have no obvious transforming relation.4.The Expression Patterns of Transcription Factors in Bladder Urothelial CellsTo systematically characterize the combinatorial patterns,we compared the atlas-wide similarity of regulon activity scores of every regulon pair based on the CSI.Strikingly,regulons were organized into 4 major modules.Module M2 contains 12 regulators were considered to play important role in cell differentiation.The activity of those regulons were higher in Basal-like cell subpopulations but lower in Sc 2 cell subpopulations.The regulon specificity score of Foxa1,Foxq1 and Ikzf2 were higher in the Bc 1 cell subpopulations and Ic 1 cell subpopulations.At the gene expression level,all of the genes were highest in Sc 2 cell subpopulations.5.Cell-Cell Communication AnalysisThe signal intensity of urothelial cell subsets was analyzed by Cellchat.Bc 3 has the strongest outgoing signal intensity,while Bc 1,Bc 3 and Sc 2 have strong incoming signal intensity.Subsequently,the main communication pathways among urothelial cells were predicted.Bc 3 cells could influence other clusters through PTN and ncWNT signaling pathways,while Bc 1 and Bc 2 cells could affect Bc 3 cells through WNT signaling pathways.In addition,FGF,IGF and GRN growth factor signaling pathways were more active in urothelium,and Plxna4+ urothelial cells could affect basal cells through FGF and IGF signals.Sc 2 cells regulate their biological functions through GRN signal.6.Novel ASPM+ Basal Cells Participated in Urothelial RepairCluster 6 specifically expresses the ASPM gene,hence the name ASPM+urothelial basal cells.Immunofluorescence results showed that ASPM+cells were low expressed in urinary tract epithelium of healthy mice.The number of ASPM positive cells in basal layer increased significantly during the repair period of urinary tract infection.The results showed that the loss of ASPM gene affected the ability of Urothelial Repair.7.Novel Plxna4+Cells Participated in Inflammatory Response of UrotheliumCluster 7 was enriched for Plxna4 and named as Plxna4+urothelial cell.GSVA analysis showed Plxna4+cells were closely associated with immunity.Immunofluorescence results showed that a group of superficial cells in the mouse bladder urothelium were positive for Plxna4.Similar results were also obtained in the bladder urothelium of rats and human beings.The results showed that Plxna4 protein could enhance the release of LPS-induced inflammatory cytokines IL6 and TNFa in urothelial cells.Conclusion and Shortcomings of this researchIn this study,we improved a single-cell suspension preparation method of mice bladder urothelial cells for single-cell RNA sequencing.Our study provides the most comprehensive information on the cell types of mouse bladders urothelium that would be fundamental to understanding bladder biology and bladder diseases.We innovatively identified two new subtypes of bladder urothelial cells.A deficiency should be pointed out that we chosed mice bladder urothelial cells as sequencing sample rather than human being because it is difficulty to get normal bladder urothelium from healthy volunteers.We analyzed Plxna4 and ASPM,the novel bladder urothelial cell subtype specificity expressed genes homologous comparison.The results showed that mouse Plxna4 and ASPM genes were highly homologous with human.Gene set variation analysis and cell function experiment of cells suggested that ASPM could affect the proliferation of urothelial cells.And Plxna4+urothelial cells might have the properties of immune cells to participate in the physiological process of immune responses such as bladder infection.These results indicated that Plxna4 and ASPM play an important role in guiding clinical bladder diseases caused by bladder urothelial cells.