Mesenchymal-epithelial interactions regulated by TBXI are essential for inner ear development

Abstract

Tbx1, a transcription factor of the T-box family, has been implicated in the pathogenesis of Velo-cardio-facial/DiGeorge syndrome (VCFS/DGS) and is expressed in both the developing otic epithelium (OV) and the periotic mesenchyme (POM) during inner ear development. Approximately 10% of VCFS patients, as well as 100% of homozygous null Tbx1 mouse models, display defects in inner ear development and sensorineural hearing loss. The role of Tbx1 in the OV has been well established and thought to play a major developmental role, while little is known about its role in the POM. Through conditional POM ablation of Tbx1 (POM KO) using mesodermal Cre lines (TCre, Mesp1Cre), we have identified an essential role for mesenchymal-epithelial signaling downstream of Tbx1 in inner ear development. Such POM KO embryos display defects in OV-derived structures including the cochlea and semicircular canals. In order to identify possible mediators of this mesenchymal-epithelial signaling, we performed gene profiling at the stages of OV elongation (E11.5) and early auditory and vestibular system development (E12.5). This unbiased approach led to the identification of genes in the retinoic acid (Clu, Adm, Mia), Wnt (Frzb, Wise1), and BMP (Bmper, Bmp14) pathways. Expression changes were validated using microdissected in situ hybridization as well as quantitative real-time PCR. Based on expression changes in regions of the ear phenotypically affected in POM KO embryos as well as the availability of mouse knockout models, the cochlear gene Clusterin (Clu) and vestibular gene BMP-binding endothelial regulator (Bmper) were chosen as candidates for downstream targets of mesenchymal-epithelial signaling regulated by Tbx1. Using such mouse knockout models, we have shown that loss of Bmper in the developing vestibular system leads a severe semicircular canal (SCC) defect, with absence of anterior and posterior SSCs and variable presence of the lateral SSC (25%). Additionally, Tbx1 genetically interacts with Bmper during SSC development, as Bmper-/-;Tbx1+/- embryos display loss of all three SSCs with 100 percent penetrance. This data implicates retinoic acid and Wnt/BMP signaling as the first identified mesenchymal-epithelial signaling pathways during inner ear development and implicates mesenchymal Tbx1 in the regulation of such signaling.