<i>Keratin 15</i> Regulates Cell Proliferation in Outer Enamel Epithelium

The coordination of dental cells is essential for tooth development. Various dental epithelial cell types are involved in tooth development, and each cell type plays a distinct role; however, the functional role of the outer enamel epithelium (OEE) remains unclear. We performed single-cell RNA-sequence (scRNA-seq) analysis of postnatal day (P) 7 incisors and embryonic day (E) 14 P1 molars of the mouse tooth germ to reveal the gene expression profile and role of OEE during tooth development. We identified <i>keratin 15</i> (<i>Krt15</i>) as a specific marker gene of OEE in the dental epithelium. Characterization of dental epithelial clusters using scRNA-seq suggested that <i>Krt15</i>-negative cycling inner enamel epithelial (IEE) cells give rise to <i>Krt15</i>-positive OEE cells, whereas the proliferative activity of dental epithelial cells decreases toward the development of OEE cells. We performed ex vivo organ cultures of the tooth germ to examine the effects of <i>Krt15</i> knockdown on tooth development. Depletion of <i>Krt15</i> in the tooth germ resulted in ectopic expression of Ki67 in OEE cells, leading to the development of an abnormal dental epithelial structure. We used the dental epithelial cell line CLDE to assess the molecular mechanisms regulated by <i>Krt15</i>. Krt15-depleted CLDE cells showed abnormal cellular morphology and dysregulated gene expression of cytokeratin family members. Furthermore, <i>Krt15</i> knockdown in CLDE cells upregulated the expression of cell proliferation marker genes, such as <i>Mki67</i>. Furthermore, <i>Krt15</i>-depleted CLDE cells exhibited activation of the p38 MAP kinase (MAPK) pathway and high proliferative activity. This suggested that Krt15 may control tooth germ size, inhibits p38 activation, and may act as a suppressor of dental epithelial cell proliferation. These findings provide new insights into the role of OEE in tooth development and contribute to a better understanding of the mechanisms underlying tooth morphogenesis.