To investigate the impact of activin receptor type-1 (ACVR1) on odontoblastic differentiation and dentin formation based on single-cell RNA sequencing.

A correlation analysis of ACVR1 with odontogenesis-related transcription factors was performed using single-cell data from healthy wild-type adult mouse incisors obtained from the Gene Expression Omnibus database (GSE146123). This experiment was approved by the Institutional Animal Care and Use Committee of the affiliated institution. Conditional knockout mice with ACVR1 deleted in the dental mesenchyme were generated using the Cre-LoxP system (experimental group: Osterix-Cre; ACVR1^fx/−, control group: Osterix-Cre; ACVR1^fx/+). Dental pulp tissue was extracted from the incisors of 3-week-old mice to prepare single-cell suspensions for 10 × Genomics single-cell transcriptome sequencing. Quality control, normalization, dimensionality reduction, and Harmony batch effect correction were performed using the Seurat pipeline, followed by UMAP dimensionality reduction visualization and cell subpopulation identification. Monocle was used for pseudotime analysis to infer cell differentiation trajectories, and changes in transcription factors positively correlated with ACVR1 were analyzed at the single-cell data level. Immunofluorescence staining was employed to validate the localization and expression changes of the key transcription factor Osterix and the odontoblast-specific protein DSPP.

Sp7/Osterix is widely expressed in the incisor mesenchyme of mice (GSE146123). Using Osterix-Cre, ACVR1 was effectively knocked out in the mesenchyme. Gene correlation analysis between ACVR1 and key odontogenic genes showed positive correlations between ACVR1 and genes such as Msx1, Msx2, and Sp7. Single-cell atlases of incisor dental pulp tissue from “Osterix-Cre; ACVR1^fx/−” and “Osterix-Cre; ACVR1^fx/+” mice were then constructed. The most significant increase in the proportion of pre-odontoblasts was in the experimental group, along with downregulated expression of key genes related to odontoblastic differentiation and polarization in this subpopulation. Differential gene enrichment analysis across all mesenchymal subpopulations indicated downregulated expression of most genes associated with dentin formation. Finally, as observed through single-cell differential gene analysis, pseudotime expression analysis, and immunofluorescence validation, the expression of Sp7 and DSPP were significantly downregulated in the experimental group.

The ACVR1 gene may promote odontoblastic differentiation and subsequently affect dentin formation through transcription factors such as Sp7, Msx1, and Msx2.