Temporal and spatial coordination of multiple cell fate decisions is essential for proper organogenesis. Here, we define gene interactions that transform the neurogenic epithelium of the developing inner ear into specialized mechanosensory receptors. By Cre-loxP fate mapping, we show that vestibular sensory hair cells derive from a previously neurogenic region of the inner ear. The related bHLH genes Ngn1 (Neurog1) and Math1 (Atoh1) are required, respectively, for neural and sensory epithelial development in this system. Our analysis of mouse mutants indicates that a mutual antagonism between Ngn1 and Math1 regulates the transition from neurogenesis to sensory cell production during ear development. Furthermore, we provide evidence that the transition to sensory cell production involves distinct autoregulatory behaviors of Ngn1 (negative) and Math1 (positive). We propose that Ngn1, as well as promoting neurogenesis, maintains an uncommitted progenitor cell population through Notch-mediated lateral inhibition, and Math1 irreversibly commits these progenitors to a hair-cell fate.

Inner ear sensory organs and VIIIth cranial ganglion neurons of the auditory/vestibular pathway derive from an ectodermal placode that invaginates to form an otocyst. We show that in the mouse otocyst epithelium, Tbx1 suppresses neurogenin 1-mediated neural fate determination and is required for induction or proper patterning of gene expression related to sensory organ morphogenesis (Otx1 and Bmp4, respectively). Tbx1 loss-of-function causes dysregulation of neural competence in otocyst regions linked to the formation of either mechanosensory or structural sensory organ epithelia. Subsequently, VIIIth ganglion rudiment form is duplicated posteriorly, while the inner ear is hypoplastic and shows neither a vestibular apparatus nor a coiled cochlear duct. We propose that Tbx1acts in the manner of a selector gene to control neural and sensory organ fate specification in the otocyst.

SUMMARY Peripheral axons from auditory spiral ganglion neurons (SGNs) form an elaborate series of radially and spirally oriented projections that interpret complex aspects of the auditory environment. However, the developmental processes that shape these axon tracts are largely unknown. Radial bundles are comprised of dense SGN fascicles that project through otic mesenchyme to form synapses within the cochlea. Here, we show that radial bundle fasciculation and synapse formation are disrupted when Pou3f4 (DFNX2) is deleted from otic mesenchyme. Further, we demonstrate that Pou3f4 binds to and directly regulates expression of Epha4, that Epha4−/− mice present similar SGN defects, and that exogenous EphA4 promotes SGN fasciculation in the absence of Pou3f4. Finally, Efnb2 deletion in SGNs leads to similar fasciculation defects, suggesting that ephrin-B2/EphA4 interactions are critical during this process. These results indicate a model whereby Pou3f4 in the otic mesenchyme establishes an Eph/ephrin-mediated fasciculation signal that promotes inner radial bundle formation.

Vertebrate hearing and balance are based in complex asymmetries of inner ear structure. Here, we identify retinoic acid (RA) as an extrinsic signal that acts directly on the ear rudiment to affect its compartmentalization along the anterior-posterior axis. A rostrocaudal wave of RA activity, generated by tissues surrounding the nascent ear, induces distinct responses from anterior and posterior halves of the inner ear rudiment. Prolonged response to RA by posterior otic tissue correlates with Tbx1 transcription and formation of mostly nonsensory inner ear structures. By contrast, anterior otic tissue displays only a brief response to RA and forms neuronal elements and most sensory structures of the inner ear.axial specification | developmental compartments | morphogen N ormal hearing and balance require that discrete patches of mechanosensory hair cells, each with a distinct function, be precisely positioned within the asymmetric membranous labyrinth of the inner ear (Fig. 1A). Five vestibular sensory patches are present in all vertebrate inner ears: the three cristae (anterior, lateral, and posterior) that detect angular head movements and two maculae (utricle and saccule) that detect linear acceleration. The specialized organ for detecting sound in chickens and mammals is the basilar papilla and organ of Corti, respectively.The entire membranous labyrinth and its innervating neurons are derived from an ectodermal thickening adjacent to the hindbrain known as the otic placode. As the placode deepens to form a cup and then pinches off to form the otocyst, some cells of the otic epithelium delaminate to form neuroblasts of the cochleovestibular ganglion (CVG). Inner ear sensory organs, and the neurons that innervate them, are thought to arise from a neural-sensory competent domain (NSD), most of which is located in the anterior region of the otic cup (1). By contrast, posterior otic epithelium forms nonsensory tissues and only one sensory organ, the posterior crista. This basic organization of functional elements in the ear is thought to be governed by signals emanating from adjacent tissues (2, 3); however, molecular mechanisms that establish the initial anterior-posterior (A-P) asymmetry of the ear primordium are poorly defined. Here, we show that a rostrocaudal wave of retinoic acid activity provides signals to the ear rudiment and establishes structural asymmetries required for normal hearing and balance. ResultsEctoderm Adjacent to the Otic Cup Confers A-P Polarity to the Otocyst. A clear manifestation of A-P asymmetry in developing amniote ears is the anterior expression of transcripts associated with cochleovestibular ganglion neurogenesis. We performed tissue transplantations in ovo to identify source(s) of signals that specify the otic A-P axis in the chicken. Transplantations were carried out at the otic cup stage (11-15 somite stages), before the otic A-P axis is specified (4). As expected, reversing the A-P orientation of the otic cup alone resulted in a high occurrence of otocysts with the axial plan...