Transcriptome analysis identifies EDA1 variants disrupt FOSB-mediated regulation of odontogenic epithelial cell behaviors during dental germ development

Here, we systematically investigated the effects of EDA1 variants on apoptosis, migration, and adhesion in ameloblast-like LS8 cells and identified downstream effectors of the EDA-NF-κB pathway through RNA sequencing (RNA-seq) combined with <i>in vivo</i> and <i>in vitro</i> validation. LS8 cells were transiently transfected with four constructs: wild-type (Wt) EDA1, nonsyndromic tooth agenesis-associated EDA1 variant (EDA1-A259E), X-linked hypohidrotic ectodermal dysplasia-associated EDA1 variant (EDA1-H252L), or empty vector control (pCMV-C-FLAG). We used flow cytometry, wound-healing assay, and cell counting kit 8 assay to assess cell apoptosis, migration, and adhesion, respectively. High-throughput RNA-seq was used to identify differentially expressed genes, which were subsequently validated through quantitative polymerase chain reaction and immunoblotting. Spatiotemporal <i>Fosb</i> expression patterns were comparatively analyzed in Wt and <i>Tabby</i> mouse tooth germs through <i>in situ</i> hybridization by using RNAscope. Wt EDA1 transiently enhances cellular migratory capacity, a function compromised in the pathogenic EDA1 variants. The EDA-NF-κB pathway operates through FOSB-mediated transcriptional regulation, as evidenced by coordinated suppression of <i>Fosb</i> expression at transcriptional and translational levels in the mutant models. Therefore, FOSB is a candidate downstream effector in EDA1-mediated odontogenesis. These results provide mechanistic insights into ectodermal dysplasia pathogenesis.