Canonical Wnt/β-catenin signaling has been implicated in multiple developmental events including the regulation of proliferation, cell fate, and differentiation. In the inner ear, Wnt/β-catenin signaling is required from the earliest stages of otic placode specification through the formation of the mature cochlea. Within the avian inner ear, the basilar papilla (BP), many Wnt pathway components are expressed throughout development. Here, using reporter constructs for Wnt/β-catenin signaling, we show that this pathway is active throughout the BP (E6-E14) in both hair cells (HCs) and supporting cells. To characterize the role of Wnt/β-catenin activity in developing HCs, we performed gain- and loss-of-function experiments in vitro and in vivo in the chick BP and zebrafish lateral line systems, respectively. Pharmacological inhibition of Wnt signaling in the BP and lateral line neuromasts during the periods of proliferation and HC differentiation resulted in reduced proliferation and decreased HC formation. Conversely, pharmacological activation of this pathway significantly increased the number of HCs in the lateral line and BP. Results demonstrated that this increase was the result of up-regulated cell proliferation within the Sox2-positive cells of the prosensory domains. Furthermore, Wnt/β-catenin activation resulted in enhanced HC regeneration in the zebrafish lateral line following aminoglycoside-induced HC loss. Combined, our data suggest that Wnt/β-catenin signaling specifies the number of cells within the prosensory domain and subsequently the number of HCs. This ability to induce proliferation suggests that the modulation of Wnt/β-catenin signaling could play an important role in therapeutic HC regeneration.

Background In the cochlea patterning of the organ of Corti is tightly regulated to produce a single row of sound detecting inner hair cells and three rows of outer hair cells, which amplify and refine the signal. The recently identified R-Spondin family of signaling molecules usually act as co-activators of Wnt signaling; it is thought that they regulate turnover of Wnt receptors at the membrane. We sought to test whether R-Spondins function in the developing cochlea. Results Expression analysis of all four members of the R-Spondin family showed that only R-Spondin2 (Rspo2) is expressed in the cochlea during development of the sensory epithelium. Examination of an Rspo2 −/− mouse showed that loss of Rspo2 results in an additional single row of outer hair cells and disruption of peripheral innervation pattern. Addition of Rspo2 recombinant protein to organotypic cochlear cultures resulted in a small but significant decrease in the number of outer hair cells. Conclusions Rspo2 is required to limit the number of outer hair cells to three rows and for optimal arrangement of peripheral nerve fibers. The Rspo2 gain and loss-of-function studies show that in the ear, Rspo2 function is not consistent with its assigned role as a Wnt potentiator.

LIM-domain containing transcription factors (LIM-TFs) are conserved factors important for embryogenesis. The specificity of these factors in transcriptional regulation is conferred by the complexes that they form with other proteins such as LIM-domain-binding (Ldb) proteins and LIM-domain only (LMO) proteins. Unlike LIM-TFs, these proteins do not bind DNA directly. LMO proteins are negative regulators of LIM-TFs and function by competing with LIM-TFs for binding to Ldb's. Although the LIM-TF Lmx1a is expressed in the developing mouse hindbrain, which provides many of the extrinsic signals for inner ear formation, conditional knock-out embryos of both sexes show that the inner ear source of Lmx1a is the major contributor of ear patterning. In addition, we have found that the reciprocal interaction between Lmx1a and Lmo4 (a LMO protein within the inner ear) mediates the formation of both vestibular and auditory structures. Lmo4 negatively regulates Lmx1a to form the three sensory cristae, the anterior semicircular canal, and the shape of the utricle in the vestibule. Furthermore, this negative regulation blocks ectopic sensory formation in the cochlea. In contrast, Lmx1a negatively regulates Lmo4 in mediating epithelial resorption of the canal pouch, which gives rise to the anterior and posterior semicircular canals. We also found that Lmx1a is independently required for the formation of the endolymphatic duct and hair cells in the basal cochlear region.SIGNIFICANCE STATEMENT The mammalian inner ear is a structurally complex organ responsible for detecting sound and maintaining balance. Failure to form the intricate 3D structure of this organ properly during development most likely will result in sensory deficits on some level. Here, we provide genetic evidence that a transcription factor, Lmx1a, interacts with its negative regulator, Lmo4, to pattern various vestibular and auditory components of the mammalian inner ear. Identifying these key molecules that mediate formation of this important sensory organ will be helpful for designing strategies and therapeutics to alleviate hearing loss and balance disorders.