Stem cell specification and niche formation in developing incisor depend on actomyosin forces

The precise timing of stem cell specification and niche formation during murine incisor development is poorly understood, and it is unclear whether these processes occur simultaneously or in a sequential manner. Functional dental epithelial stem cells are marked by the expression of Sox2, a transcription factor that is broadly expressed in the dental epithelium at the dentition onset and restricted to stem cells in fully developed incisor. Using genetic lineage tracing in Sox2CreERT2/+; R26RmT/mG and Sox2CreERT2/+; R26RtdT/+embryos along with a single-cell RNA sequencing at different stages of incisor development, we investigated the timing of the stem cell specification and its temporal relationship with niche formation. Our results reveal the presence of a Sox2-expressing stem cell-like population prior to formation of the functional niche. These cells localize to the leading edge of the advancing incisor epithelium where they are maintained in an undifferentiated state. Our data demonstrate presence of actomyosin network and a generation of a contractile tension which helps confine Sox2+ stem cells to the leading edge. This mechanical confinement likely plays an important role in maintaining their stemness until the niche is functionally and structurally established. Partial or complete disruption of the actomyosin network disables the clustering of Sox2-expressing cells, potentially triggering their premature differentiation, and ultimately leads to impaired formation of the functional stem cell niche and abnormal growth of the incisor. First, we build the cerebral ischemia model of the male c57 mice, divided into a sham surgery group (Sham) and a stroke group (MCAO). After model establishment, we validated the model using TTC staining, TUNEL staining, and immunofluorescence staining. We assessed the extent of nerve damage in mice after MCAO using the Longa neurological deficit score. We used methylated RNA immunoprecipitation sequencing (MeRIP-Seq) to identify m5C peaks in the mouse transcriptome (lncRNA, circRNA, mRNA), and simultaneously constructed RNA libraries through RNA-Seq to study the expression profile. We analyzed the differentially methylated lncRNA, circRNA, and mRNA, and studied their potential biological functions through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The final validation was performed using RT-PCR