Integration of Single-Cell RNA- and CAGE-seq Reveals Tooth-Enriched Genes

Abstract

Organ development is dictated by the regulation of genes preferentially expressed in tissues or cell types. Gene expression profiling and identification of specific genes in organs can provide insights into organogenesis. Therefore, genome-wide analysis is a powerful tool for clarifying the mechanisms of development during organogenesis as well as tooth development. Single-cell RNA sequencing (scRNA-seq) is a suitable tool for unraveling the gene expression profile of dental cells. Using scRNA-seq, we can obtain a large pool of information on gene expression; however, identification of functional genes, which are key molecules for tooth development, via this approach remains challenging. In the present study, we performed cap analysis of gene expression sequence (CAGE-seq) using mouse tooth germ to identify the genes preferentially expressed in teeth. The CAGE-seq counts short reads at the 5′-end of transcripts; therefore, this method can quantify the amount of transcripts without bias related to the transcript length. We hypothesized that this CAGE data set would be of great help for further understanding a gene expression profile through scRNA-seq. We aimed to identify the important genes involved in tooth development via bioinformatics analyses, using a combination of scRNA-seq and CAGE-seq. We obtained the scRNA-seq data set of 12,212 cells from postnatal day 1 mouse molars and the CAGE-seq data set from postnatal day 1 molars. scRNA-seq analysis revealed the spatiotemporal expression of cell type–specific genes, and CAGE-seq helped determine whether these genes are preferentially expressed in tooth or ubiquitously. Furthermore, we identified candidate genes as novel tooth-enriched and dental cell type–specific markers. Our results show that the integration of scRNA-seq and CAGE-seq highlights the genes important for tooth development among numerous gene expression profiles. These findings should contribute to resolving the mechanism of tooth development and establishing the basis for tooth regeneration in the future.

Establishment of inclusive single-cell transcriptome atlases from mouse and human tooth as powerful resource for dental research

“…With the aim of establishing an inclusive sc atlas of postnatal mouse teeth, we set out to integrate the at present nine publicly available scRNA-seq datasets ( Figure 1A ; Table 1 ) ( Sharir et al, 2019 ; Takahashi et al, 2019 ; Chen et al, 2020 ; Chiba et al, 2020 ; Krivanek et al, 2020 ; Wen et al, 2020 ; Chiba et al, 2021 ; Nagata et al, 2021 ; Zhao et al, 2021 ). After rigorous quality control (i.e., removal of low-quality cells and doublets), 37,948 cells were retained for further analysis, with the datasets contributing between 1,645 and 11,714 cells to the total pool, and with 24,290 and 13,658 cells obtained from molars and incisors, respectively ( Supplementary Figures S1A,B , Table 2 ).…”

“…In recent years, sc transcriptomic interrogations have been applied to mouse and human tooth. Regarding mouse, single-cell RNA-sequencing (scRNA-seq) datasets were generated from the constantly (re-)growing incisor and the more static, human-resembling molar at different timepoints, either of whole tooth or specific tissue components ( Sharir et al, 2019 ; Takahashi et al, 2019 ; Chen et al, 2020 ; Chiba et al, 2020 , 2021 ; Krivanek et al, 2020 ; Wen et al, 2020 ; Nagata et al, 2021 ; Zhao et al, 2021 ). Human sc transcriptomic data were predominantly generated from dental pulp and periodontal tissues of molars in both healthy and diseased states ( Krivanek et al, 2020 ; Pagella et al, 2021b ; Shi et al, 2021 ; Yin et al, 2021 ; Hemeryck et al, 2022 ; Lin et al, 2022 ; Opasawatchai et al, 2022 ).…”

Diverse stem cells for periodontal tissue formation and regeneration

“…Further characterization of mesenchymal progenitors in the PDL is required to define the function of these stem cells in both mice and humans. Together with mouse genetic approaches, scRNA‐seq analysis has been extensively applied in recent years to reveal the cellular heterogeneity of dental tissues including the periodontium in both mice and humans at a single‐cell level (Chiba et al, 2020; Chiba et al, 2021; Jones et al, 2019; Krivanek et al, 2020; Nagata et al, 2021; Pagella, de Vargas Roditi, Stadlinger, Moor, & Mitsiadis, 2021; Takada et al, 2022; Takahashi et al, 2019; J. Zhao et al, 2021). Moreover, spatial transcriptomic approaches are currently developed to compensate for the disadvantage of conventional RNA‐sequencing approaches that do not allow to retain positional information of each individual cells (Rodriques et al, 2019; Ståhl et al, 2016; Vickovic et al, 2022).…”

Single‐cell RNA sequencing in oral science: Current awareness and perspectives

“…However, not all marker genes discovered by scRNA‐seq were functional and critical genes in tooth histogenesis. The cap analysis of gene expression sequence (CAGE‐seq) can identify the preferentially expressed genes in a particular organ without transcript length bias, thus helping determine if the genes identified by scRNA‐seq are preferentially expressed in tooth 28 . Combined scRNA‐seq with CAGE‐seq, Cldn10 was found to be a novel SI marker, 29 as well as FXYD domain‐containing ion transport regulator 4 (Fxyd4) and Dspp to be unrecognized ameloblast markers 28 .…”

“… 28 Combined scRNA‐seq with CAGE‐seq, Cldn10 was found to be a novel SI marker, 29 as well as FXYD domain‐containing ion transport regulator 4 (Fxyd4) and Dspp to be unrecognized ameloblast markers. 28 The mouse incisor serves as a major model to study tooth development. The in‐depth exploration of the model by scRNA‐seq updates our knowledge of the development and maintenance of tooth.…”