Effect of Setd2 knockout in dental germ mesenchymal cells on early tooth development

To investigate the effect of specific deletion of SET-domain-containing 2 (Setd2) in dental germ mesenchymal cells on early tooth development and provide a research basis for exploring the etiology of dental developmental disorders.

This study was approved by the Animal Care and Ethical Committee of Wuhan University. Wnt1^Cre mice with a C57BL/6J background were crossed with Setd2^flox/flox mice to generate the Setd2 knockout experimental group (Wnt1^Cre; Setd2^flox/flox) and the control group (Setd2^flox/flox). Tail tissues of mouse embryos were collected at embryonic day (E) 13.5, E15.5, and E18.5 for polymerase chain reaction (PCR)-based genotyping, and embryonic heads were collected for paraffin sectioning to observe the development of the first mandibular molar germs. Immunohistochemical (IHC) staining was performed to verify the specific knockout of Setd2 in dental germ mesenchymal cells; hematoxylin and eosin (HE) staining and subrenal culture were used to evaluate the effect of Setd2 knockout in dental germ mesenchymal cells on tooth development; Ki67 staining and a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were employed to evaluate the effects of specific Setd2 knockout on the proliferation and apoptosis of dental germ mesenchymal cells; immunofluorescence (IF) staining was conducted to observe the impact of Setd2 knockout on the trimethylation of lysine 36 on histone 3 (H3K36me3).

PCR-based genotyping showed that the experimental group presented a single band (266 bp) for the Setd2^flox/flox homozygote and a characteristic band for Wnt1^Cre, while the control group only showed a single band (266 bp) for the Setd2^flox/flox homozygote. IHC staining confirmed the successful knockout of Setd2 in dental germ mesenchymal cells of the experimental group; HE staining and subrenal culture demonstrated that specific knockout of Setd2 in the experimental group led to reduced size of tooth germs and increased condensation of mesenchymal cells at E15.5 and E18.5 (E15.5, P < 0.05; E18.5, P < 0.01); Ki67 staining showed no statistically significant effect on the proportion of division of dental germ mesenchymal cells between the experimental and control groups at E13.5 and E15.5 (E13.5, P = 0.694; E15.5, P = 0.503); the TUNEL assay demonstrated increased apoptosis of dental germ mesenchymal cells in the experimental group at both E13.5 and E15.5 (P < 0.001); IF staining revealed the absence of H3K36me3 modification in the dental germ mesenchymal cells of the experimental group across E13.5, E15.5, and E18.5 (P < 0.000 1).

Specific knockout of Setd2 in dental germ mesenchymal cells leads to increased cell apoptosis by impairing H3K36me3 modification, resulting in early developmental defects in mouse teeth characterized by reduced tooth germ volume.