Project Summary/Abstract The overall dental morphology of an adult vertebrate is set in motion by the initiation of tooth fields at specific regions in the body plan during early development. Thereafter, primary teeth form sequentially, expanding each tooth field as new teeth are added at the tooth field margin. Basic research into the genes and signaling pathways underlying these processes will reveal which cell types and genetic signatures are associated with tooth field initiation and expansion, thus informing future attempts to create and successfully implant live teeth. By determining which cell types are capable of undergoing transformation to a dental fate, new avenues for the creation of lab-made tooth organs will be revealed. Additionally, identifying the genetic signatures associated with tooth field expansion and arrest will provide information about the greater context under which dental arcade expansion is facilitated. The present study will use a newly developed set of stable transgenic stickleback fish and zebrafish to identify the cell types, transcript profiles, and signaling events that underlie the processes of tooth field initiation, expansion, and arrest. These model fishes present a unique opportunity to understand tooth field morphogenesis, because tooth field position and size can be manipulated using genetic tools. In response to Eda overexpression, both species form ectopic tooth fields in highly consistent locations in or on the head, while also expanding endogenous tooth fields. By contrast, Dkk2 overexpression in stickleback reduces tooth field size by inhibiting tooth field expansion. Aim 1 seeks to understand the gene expression dynamics of the switch to a tooth organ fate by assessing the fine spatial expression patterns of genes encoding Tumor Necrosis Factor Receptors (TNFRs), candidate receptors that may confer the response to Eda, and performing single-cell RNA sequencing on tooth-competent regions microdissected from Eda overexpressing and WT sticklebacks. Aim 2 of this project will longitudinally observe the process of ectopic tooth formation by repeatedly imaging fish as they grow ectopic facial teeth, utilizing lineage tracing techniques, reporter gene expression, and vital dye labeling to understand which cells contribute to ectopic teeth. Aim 3 will elucidate which developmental pathways are associated with tooth field size and tooth row number by comparing the gene expression profiles of the dissected tooth field margins derived from expanded (Eda overexpression), arrested (Dkk2 overexpression), and normal stickleback tooth fields. Overall, these experiments will yield information on how teeth can be specified in development (Aims 1 and 2), and which gene expression responses are concomitant with favorable or unfavorable conditions for tooth field expansion (Aim 3). The first portion of this work will occur at UC-Berkeley (K99 phase), facilitated by Dr. Square’s advisory committee, institution, and curre...