Project Summary Using zebrafish as a model organism, experiments will explore mechanisms by which mechonsensory hair cells and neurons of the inner ear are formed and maintained. Fgf and Wnt signaling pathways cooperate to establish hair cells, and at the same time Fgf and Wnt work in opposition during formation of neurons. How Fgf and Wnt are coordinated to regulate these diverse responses is poorly understood. This will be investigated in three specific aims. 1) Study how transcription factors Pax2, Pax5, Sp5a, and Sp5l mediate discrete aspects of Fgf and Wnt signaling. Experiments will examine how mutations in these genes alter the response to Fgf and Wnt. In addition to evaluating loss-of-function mutations, transgenic lines will be used to overexpress these factors. 2) Explore how Pax2 and Pax5 promote hair cell survival. In embryos lacking Pax2 or Pax5, hair cells initially form but are later extruded from the developing inner ear. Experiments will test the idea that Pax2 and Pax5 are required to maintain cell adhesion molecules that normally hold hair cells in place. This will be tested by treating embryos with drugs that stabilize adhesion complexes, and by using transgenic lines to overexpress cell adhesion molecules to see whether hair cell loss is prevented. 3) Previous studies uncovered a completely novel mechanism by which cells in the developing ear undergo a change in metabolism similar to the “Warburg Effect” seen in metastatic tumors. Specifically, ear cells upregulate glycolysis and fermentation (despite abundant oxygen) in order to produce and secrete high levels of lactate. Disruption of lactate production impairs production of hair cells and neurons, in part by weakening the response to Fgf. Proposed experiments will further explore how lactate impacts Fgf signaling, and also test whether lactate affects Wnt signaling. In addition, the function of Foxm1, a transcription factor often responsible for activating the Warburg Effect in tumors, will be tested in the context of inner ear development. Foxm1 also mediates Wnt and Fgf in tumors, hence the effects of mutations in Foxm1 or misexpressing Foxm1 on Fgf and Wnt signaling will be tested. Together, these studies will provide fundamental insights into mechanisms of hair cell and neural development. Because developmental mechanisms are broadly conserved, studying how these genes work in zebrafish could suggest candidates for “gene therapy” to restore hearing in mammals.