During the previous funding period, we found that Tbx1, encoding a T-box containing transcription factor, the gene for velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS) on 22q11.2, is required in the otic vesicle (OV) and periotic mesenchyme (POM), for inner ear development. Mutations in Brn4 (Pou3f4), an X-linked POU domain containing transcription factor, causes DFN3. Patients with DFN3 have hearing loss and mice with inactivating mutations are deaf. Tbx1 and Brn4 are co-localized to the POM and genetically interact for cochlear outgrowth. Inactivation of Tbx1 in the mouse using a pan-mesodermal Cre driver, T-Cre, leaves the vestibular system intact but results in failed cochlear outgrowth. In Specific Aim 1, we propose to dissect the signaling pathways downstream of Tbx1 and Brn4 in the POM needed to for inner ear development using conditional loss and gain of function alleles of Tbx1. Upstream factors regulating expression of Tbx1 in the OV have not been identified. Preliminary data suggests that the Wnt/B-catenin signaling pathway may act upstream of Tbx1. In Specific Aim 2, we will determine the role of Wnt/B-catenin signaling in inner ear morphogenesis via Tbx1 using conditional B-catenin loss and gain-of-function mouse mutants. We will also take an unbiased approach in finding the OV enhancer using transient mouse transgenesis methods. Inactivation of Tbx1 in the otic vesicle results in a missing cochlea and vestibular system as well as a duplicated cochleovestibular ganglion rudiment, suggesting that it acts as a selector gene for inner ear morphogenesis. In Specific Aim 3, we will focus on the pathways downstream of Tbx1 to further understand its roles in restricting neurogenesis and promoting cell proliferation in the otic epithelium. This competitive renewal seeks to reveal the molecular genetic pathway of Tbx1 in inner ear development. This program will enable us to understand the molecular pathogenesis of VCFS/DGS and DFN3, two human genetic syndromes. Being able to inactivate or overexpress genes in mammalian model organisms will make it possible to learn about the genetic pathways upstream and downstream of Tbx1 and downstream of Brn4. In addition, Tbx1 and Brn4 may be required for modulating retinoic acid levels. Too much or too little retinoic acid causes birth defects including inner ear anomalies. The genes we identify may serve as genetic modifiers to alter the severity in patients with VCFS/DGS and DFN3 and may contribute to more commonly occurring sporadic birth defects in the general population. In addition, understanding cross talk between these genes and the retinoic acid and other morphogen pathways we study will shed light on gene-environment interactions, which will lead to novel therapeutics in the future. The goal of this project is to understand the molecular basis of inner ear defects in patients with congenital malformation disorders.