Revealing the Trajectory and Critical Roles of Retinoic Acid in Ameloblast Differentiation

About This Grant

PROJECT SUMMARY/ABSTRACT Dental enamel is formed by ameloblasts that are derived from dental epithelium. How dental epithelial stem cells (DESCs) commit to ameloblast lineage remains elusive. A potential critical factor that drives the commitment of DESCs to the ameloblast lineage is retinoic acid (RA). The complex system that drives ameloblast lineage commitment and the abundance of molecules that determine the RA signaling system require us to examine the transcriptomics of developing teeth at single-cell and spatial levels, followed by biological validations. Our preliminary data showed the diversifications of DESCs and the continuous differentiation of ameloblasts in a mouse incisor scRNAseq dataset. We sorted the differentiating ameloblasts into novel clusters in both mouse and human incisor scRNAseq datasets and replicated these findings using RNAscope. Our findings also suggest that RA signaling is inhibited in pre-secretory stage ameloblasts but activated when ameloblasts transition into the secretory stage. We identified potential RA response elements (RARE) in genes that are critical for secretory stage enamel formation. Therefore, we hypothesize that the sequential ameloblast differentiation is specified by critical genes through the retinoic acid (RA) signaling. By integrating scRNA-seq data analyses with molecular validations in developing teeth, we can 1) elucidate the differentiation trajectory of ameloblasts and 2) reveal critical roles of RA signaling in ameloblast differentiation. Aim 1. Determine the ameloblast differentiation trajectory. We will integrate the scRNAseq data obtained from 14 studies in different tooth types from mice, rats, and humans. We will conduct cluster analysis within tooth types and species to identify each phase of the continuous differentiation paths predicted by trajectory inference. We will compare the ameloblast differentiation trajectory across tooth types and species, with an emphasis on molecules relevant to RA degradation and signaling activation. To validate the trajectory defined by bioinformatic analyses, we will perform RNAscope HiPlex assay on mouse developing teeth using identified genes. Aim 2. Determine the critical roles of RA signaling at the onset of secretory stage enamel formation. First, we will conduct spatial transcriptomics and consequent bioinformatic analyses to map spatial distributions of molecules involved in RA synthesis, signaling activation, and metabolism, together with potential target genes of the RA signaling, in mouse enamel organ epithelium and adjacent dental mesenchyme. Second, we will perform the CUT&Tag sequencing to identify RAREs in RA signaling targets across the genome in mouse secretory ameloblasts. We will validate these findings by analyzing spatial transcriptomic and existing scRNAseq data. The completion of this project will allow us to identify critical factors in ameloblast differentiation. These insights will shed light on the mechanisms of tooth morphogenesis and congenital tooth disorders. This project will provide essential information to the development of bioengineering strategies for tooth regeneration.