Analysis Of Genes Specifically Expressed In Odontoblast And Ameloblast Of Mice Using Single Cell RNA-Seq

Dentine and enamel are two important components of the tooth,which are secreted by odontoblasts and ameloblasts respectively.During the development of the human tooth,dental mesenchymal cells and epithelial cells differentiate into odontoblasts and ameloblasts respectively.These differentiation processes are regulated by multiple gene signaling pathways,the most important of which are transcription factors,which determine the specific differentiation fate of the cells.Therefore,it is very important to analyze the cell-cluster-specific transcription factors during the differentiation of odontoblasts and ameloblasts.It can provide a theoretical basis for dental repair and regeneration.During the differentiation of odontoblasts and ameloblasts,they are closely attached to dentin and enamel,respectively.The traditional transcriptome sequencing method（BulkRNA-Seq）cannot precisely analyze the gene expression of odontoblasts and ameloblasts.As a result,The study of specific transcription factors in the differentiation process of odontoblasts and ameloblasts only stays in the analysis of gene expression patterns by in situ hybridization or immunofluorescence.Single-cellRNA sequencing（scRNA-seq）is a powerful tool to clarify the heterogeneity of developing cell types.Recent scRNA-seq studies in tooth development have mainly focused on the early stages of development.However,there has been no report yet that covers the perspective of transcriptome profiles in tooth during differentiation.In this study,we performed scRNA-seq analysis using P0 OC-cre;R26R^mTmGmice to identify the specific gene expression profile of cell groups,especially found genes specifically expressed in odontoblasts and ameloblasts.Firstly,We mate cell-specific Cre mice with R26R^mTmGmice（GFP reporter mice）to specifically color the related cell lineages with green fluorescence,enabling cell lineage tracing.This allows us to accurately label odontoblasts and distinguish them from other cell types including ameloblasts.Since the formation of dentin is closely related to bone in a number of ways,including composition and formation,we mate three osteoblast-specific Cre mice（OC-Cre,Dmp1-Cre,Col1-Cre）with R26R^mTmGmice.We received P0 OC-Cre;R26R^mTmG,Col1-Cre;R26R^mTmG,Dmp1-Cre;R26R^mTmGmice for GFP antibody immunofluorescence localization.It’s found that only OC-cre;R26R^mTmGcan mark the differentiated odontoblasts.In order to verify the specificity of odontoblasts lineage labeled with OC-Cre;R26R^mTmGmice,we perform the immunofluorescent staining of GFP in the E13.5,E15.5 and P0 OC-cre;R26R^mTmGmice.The results showed that OC-Cre lineage cells did not express Cre enzyme activity in mesenchymal at the early stage of tooth development,but only specifically expressed in the differentiation stage of odontoblasts.Through the above series of experiments,we have determined that OC-cre;R26R^mTmGmice can specifically label the odontoblasts lineage with GFP so that the expression of GFP can be used to trace the odontoblast cluster in scRNA-seq.Secondly,we dissected molars of P0 OC-cre;R26R^mTmGmice littermates and dissociated dental cells into single cells by Trypsin digestion.Single cells were immediately loaded to the 10X Genomics platform,and scRNA-seq was performed.We obtained 8923 single-cell transcriptome data with 1702 mean genes per cell which reached the standard of scRNA-seq data analysis.Unbiased clustering classified them into nine cell clusters.We combined the expression of cell marker genes in the t-SNE plot with GO enrichment to identify the cell populations.We successfully identified Stratum Intermedium cluster,erythrocyte cluster,ameloblast cluster,extracellular matrix cluster,endothelial cluster,leukocyte cluster,B cell cluster,neural cluster and an undefined cluster（Cluster 4）.Meanwhile,to trace the odontoblast lineage,we extracted the EGFP cell population（560 cells）from the single-cell gene expression matrix and performed cell clustering analyses.Unbiased clustering identified osteoblast cluster,odontoblast cluster and an undefined cluster（Cluster 1）.In this part of the study,we have successfully obtained odontoblast and ameloblast populations as well as their specific gene sets with high expression by scRNA-seq experiments and data analysis.Thirdly,in order to find potential odontoblast transcription factors,we compared specific and highly expressed genes in the odontoblast population with the mouse transcription factor database.We selected three transcription factors（Irx5、Dlx5 and Runx2）that were specifically expressed in odontoblast cluster.We also carried out a literature search on the specific and highly expressed genes in the odontoblast population,and screened 3 genes related to the differentiation of odontoblasts:Wnt6,Notum and Smpd3.We collected P0 OC-cre;R26R^mTmGmouse tooth tissues and performed immunofluorescence colocalization experiments with RUNX2,DLX5,WNT6,NOTUM,IRX5 antibodies and GFP antibodies.Runx2 was localized in odontoblasts.Dlx5,Wnt6and Notum were expressed in odontoblasts and ameloblasts.Irx5 was not expressed in mouse molar tooth germs.We hypothesized that there might be a quality issue with the IRX5 antibody that led to the failure of the immunofluorescence experiment,and that Smpd3 did not purchase commercially available antibodies,we further used RT-PCR to verify whether Irx5 and Smpd3 are odontoblast-specific genes.We collected P0 OC-cre;R26R^mTmGmouse molar tooth germs were separated by flow cytometry to separate green fluorescent cells（ie odontoblasts）and non-green fluorescent cells for RT-PCR.The results showed that Irx5 was microexpressed in odontoblasts and Smpd3 was expressed in odontoblasts.Furthermore,in order to find potential ameloblast transcription factors,we compared specific and highly expressed genes in the ameloblast population with the mouse transcription factor database.We selected three transcription factors（Foxq1,Id4,Irx3,Foxo1）that were specifically expressed in odontoblast cluster.We collected P0 OC-cre;R26R^mTmGmouse tooth tissues and performed immunofluorescence colocalization experiments with ID4,IRX3,FOXO1 antibodies and GFP antibodies.Foxo1 was expressed in odontoblasts and ameloblasts.Id4 and Irx3 were not expressed in mouse molar tooth germs.Recent advancements in scRNA-seq have furthered the understanding of heterogeneous cell compositions in complex tissues through the characterization of different cell types based on gene expression levels.In the data processing protocols of scRNA-seq experiments,cell type identification is a vital step for subsequent analysis.A level of uncertainty is introduced by the fact that one cell type is commonly associated with multiple cell markers and one cell marker can be linked with multiple cell types.In order to eliminate this uncertainty,the cells of interest are usually labeled with fluorescent markers when performing scRNA-seq experiments,so that the expression analysis of fluorescent markers can be used to trace the cell population of interest and increase the accuracy of cell type annotations.In this experiment,we used the OC-cre;R26R^mTmGmice molar tooth germ that can specifically mark the odontoblast lineage with GFP to perform scRNA-seq,and trace the odontoblast population by analyzing the expression of GFP,which can be successful screened genes specifically expressed in odontoblasts.However,since there are no fluorescently labeled mice that can trace the ameloblast population,we speculate that the ameloblast population identified lacks specificity in this experiment,so we have not successfully screened genes specifically expressed in ameloblasts.To sum up,we found that OC-cre;R26R^mTmGmice could specifically label the odontoblast lineage.Further,we used P0 OC-cre;R26R^mTmGmice to perform scRNA-seq and successfully obtained odontoblast and ameloblast clusters.Meanwhile,we identified two transcription factors specifically expressed in odontoblast:Runx2,Irx5;One gene specifically expressed in odontoblast cells:Smpd3;Four genes expressed in both odontoblasts and ameloblasts:Dlx5,Wnt6,Notum and Foxo1.It is suggested that they may play a role in the differentiation of odontoblasts and ameloblasts.The results not only provided a resoure of transcriptome data in dental cells but also provides a theoretical basis for further understanding the differentiation mechanism of odontoblasts and ameloblasts.