Amelogenin phosphorylation affects key regulatory genes in the enamel organ

Amelogenin (AMELX), the predominant enamel matrix protein (EMP), has a single phosphorylation site at Serine 16 (S16), which enhances its capacity to stabilize amorphous calcium phosphate and inhibit crystallization in vitro. To investigate the role of AMELX phosphorylation in vivo, we generated a knock-in (KI) mouse model, AmelxS16A, in which S16 is substituted with Alanine that prevents AMELX phosphorylation. KI enamel is hypoplastic, lacks the enamel rods, and features multiple ectopic calcifications. Notably, KI ameloblasts lack Tomes’ processes and develop progressive cell pathologies, suggesting that AMELX phosphorylation affects ameloblast biology. To gain a comprehensive view on the effects of AMELX phosphorylation on the biology of the enamel organ (EO) cells, we performed single-cell RNA sequencing on incisal EOs from WT and KI mice. Using this approach, we found that 624 genes were differentially expressed between WT and KI in total EO cell populations. In particular, genes encoding secretory stage EMPs were upregulated in KI mice, while genes encoding maturation-stage EMPs were downregulated. Ingenuity Pathway Analysis revealed that the pathways associated with cell morphology, growth, and proliferation are most affected in KI cells. Ten distinct cell populations of the EO were identified using cluster analysis. Additionally, our analyses showed that AMELX phosphorylation affects the expression of several genes involved in the regulation of odontogenesis. Furthermore, differentially expressed genes were concentrated in the secretory ameloblast (SA) cluster, suggesting that the SAs were most affected by the lack of AMELX phosphorylation. Specifically, there was a substantial 5.1-fold downregulation of Shh, while Wnt5a was upregulated 8.1-fold in KI SAs compared to WT. This dysregulation of regulatory genes may contribute to the observed ameloblast abnormalities in KI. In summary, our transcriptomics analysis reveals that AMELX phosphorylation not only affects numerous extracellular processes during amelogenesis but also influences multiple cellular pathways and processes within the EO.