PROJECT SUMMARY/ABSTRACT Our dentition plays important daily roles in speech, mastication, and determination of facial shape and expression. The tooth root is essential for the proper function of the dentition because it anchors the tooth within the maxilla or mandible. In addition, the root helps transmit and balance occlusal forces through periodontal ligaments and serves as a passageway for the neurovascular bundle that supplies blood and innervation to our teeth. To date, we have limited understanding of the regulatory mechanisms of root development and patterning. Understanding the signaling mechanism that regulates the fate of progenitors during tooth root development and patterning, and how signaling pathway disruption can lead to craniofacial malformations, will advance stem cell-mediated tooth root regeneration. Importantly, we have identified Gli1+ progenitors that are crucial for the development and patterning of roots. These progenitors are heterogenous and may have different functions. To gain a comprehensive understanding of all progenitor cells and their roles in tooth root development, we developed a spatiotemporal single-cell atlas of neural crest lineage diversification and cellular function during this process. Building on this work and preliminary data, we have designed studies to test the hypotheses that (i) FGF signaling from the trigeminal nerve directly controls progenitors during root development; (ii) Piezo1 and Piezo2 function downstream of FGF signaling to control the fate of progenitors; and (iii) Kdm6b and Ezh2 antagonistically regulate Trp53 expression downstream of FGF signaling to regulate root elongation and patterning. We propose the following three specific aims to test our hypotheses. Specific Aim 1: To investigate how FGF signaling from the trigeminal nerve controls the fate of progenitors during root development. We will also investigate how FGF- regulated downstream target genes exert their functional specificity in regulating root development. Specific Aim 2: To investigate the functional significance of Piezo1 and Piezo2 as FGF signaling mediators in regulating the fate of progenitors during root development. We will elucidate the molecular mechanism(s) by which signals from the dental mesenchyme control root development. Specific Aim 3: To investigate how Kdm6b controls the fate of progenitors during root development. We will also investigate the antagonistic interaction between Kdm6b and Ezh2 in regulating Trp53 to control root patterning and uncover the regulatory mechanism by which Trp53 controls progenitors in root development.