Fig. 1

Notch pathway activation is required for ameloblast maturation in co-culture with odontoblasts. a TopPath pathway analysis of single-cell sequencing data from fetal tissues, identifying key signaling pathways involved in odontoblast (OB) to ameloblast (AM) communication. The Notch pathway (10.6%) is highlighted as a major contributor to AM maturation, alongside other pathways such as FGF, WNT, EGF, and BMP. b Schematic model depicting Notch signaling interactions between OBs and AMs, showing ligand-receptor interactions focusing on incoming signal to eAM/sAM (red arrows). The width of the arrows is proportional to the number of interactions. Black arrow indicates other possible interaction not considered in the analysis of TopPath. c Heatmap representation of Notch ligands (DLL1, DLL4) and Notch receptors (NOTCH1, NOTCH2, NOTCH3, NOTCH4), illustrating ligand expression in OBs and receptor expression in AMs, supporting OB-to-AM signaling. d Differentiation and co-culture model for generating induced ameloblasts (iAMs) from human iPSCs. Induced odontoblasts (iOBs) are co-cultured with induced early ameloblast (ieAM) organoids to promote their maturation into secretory ameloblasts (isAMs), with and without DAPT-mediated Notch inhibition. e 3D confocal images of co-cultured iAM organoids showing ENAM (yellow) and DSPP (red) expression, confirming ameloblast maturation in co-culture. Merged images include DAPI (nuclei, blue) and Phalloidin (cytoskeleton, purple). f Schematic representation summarizing the observed expression patterns of DSPP (red) and ENAM (yellow) in differentiated OB-like and AM-like cells within the co-culture. g Comparative immunofluorescence staining of ENAM in Control (top) vs. DAPT-treated (bottom) conditions, revealing reduced ENAM expression and altered cellular morphology upon Notch inhibition. Scale bar = 100 μm. h Quantification of ENAM fluorescence intensity, normalized to control, showing a significant reduction in ENAM expression in the DAPT-treated group (^****P < 0.000 1)