Spatiotemporal Profiling of Fat Family Genes Highlights Fat2 As a Novel Marker of Outer Enamel Epithelium and Stratum Intermedium

OBJECTIVES:The Fat gene family, also known as atypical cadherins, contributes to the formation of planar cell polarity, which determines organ shape, size, and polarity. However, their roles in tooth morphogenesis remain unclear. In this study, the aim was to investigate the spatiotemporal expression patterns of Fat1-Fat4 during murine tooth development. METHODS:Quantitative polymerase chain reaction was performed using total RNA from newborn C57BL/6J murine tissues to determine Fat1-Fat4 expression. In situ hybridization was performed to examine the localization of Fat family transcripts during tooth development from embryonic Day 12.5 to birth. The dental epithelial stem cell line, M3H1, was used as an in vitro differentiation model, and small-interfering-RNA-mediated knockdown of Fat2 was performed to assess its function. Public single-cell RNA-sequence (scRNA-seq) datasets were analyzed to confirm cell-type specificity. RESULTS:All Fat genes were expressed in the teeth, with Fat2 having the highest level among tissues examined. Fat1 was expressed in the epithelial and mesenchymal cells, and Fat2 expression was restricted to the outer enamel epithelium (OEE) and stratum intermedia (SI). Fat3 and Fat4 were expressed in the dental mesenchyme. In M3H1 cells, Fat1 and Fat2 had biphasic expression at undifferentiated and differentiated stages. Fat2 knockdown decreased Sox2 and nephronectin expression, and increased Foxj1 expression. scRNA-seq supported Fat2 specificity in the OEE and SI. CONCLUSION:Fat genes have cell-type-specific and developmentally regulated expression during tooth development. Among them, Fat2 emerges as a novel marker of OEE and SI, and it may contribute to epithelial identity and differentiation during odontogenesis.