The cochlea is innervated by neurons that relay sound information from hair cells to central auditory targets. A subset of these are the type II spiral ganglion neurons, which have nociceptive features and contribute to feedback circuits providing neuroprotection in extreme noise. Type II neurons make a distinctive 90° turn towards the cochlear base to synapse with 10-15 outer hair cells. We demonstrate that this axon turning event requires planar cell polarity (PCP) signaling and is disrupted in and knockout mice, and that VANGL2 acts non-autonomously from the cochlea to direct turning. Moreover, VANGL2 is asymmetrically distributed at intercellular junctions between cochlear supporting cells, and in a pattern that could allow it to act directly as an axon guidance cue. Together, these data reveal a non-autonomous function for PCP signaling during axon guidance occurring in the tissue that is innervated, rather than the navigating growth cone.

Calcium and integrin-binding protein 2 (CIB2) and CIB3 bind to transmembrane channel-like 1 (TMC1) and TMC2, the pore-forming subunits of the inner-ear mechano-electrical transduction (MET) apparatus. Whether these interactions are functionally relevant across mechanosensory organs and vertebrate species is unclear. Here we show that both CIB2 and CIB3 can form heteromeric complexes with TMC1 and TMC2 and are integral for MET function in mouse cochlea and vestibular end organs as well as in zebrafish inner ear and lateral line. Our AlphaFold 2 models suggest that vertebrate CIB proteins can simultaneously interact with at least two cytoplasmic domains of TMC1 and TMC2 as validated using nuclear magnetic resonance spectroscopy of TMC1 fragments interacting with CIB2 and CIB3. Molecular dynamics simulations of TMC1/2 complexes with CIB2/3 predict that TMCs are structurally stabilized by CIB proteins to form cation channels. Overall, our work demonstrates that intact CIB2/3 and TMC1/2 complexes are integral to hair-cell MET function in vertebrate mechanosensory epithelia.

The vestibular maculae of the inner ear contain sensory receptor hair cells that detect linear acceleration, contribute to equilibrioception, and thereby coordinate posture and ambulatory movements. These hair cells are divided between two groups, separated by a line of polarity reversal (LPR), with oppositely oriented planar-polarized stereociliary bundles that detect motion in opposite directions. The transcription factor EMX2 is known to establish this planar polarized organization by regulating the distribution of the transmembrane receptor GPR156 at the hair cell surface in one group of cells, however those genes regulated by EMX2 in this context were previously not known. We have identified the serine threonine kinase STK32A as a downstream effector negatively regulated by EMX2. Stk32a is expressed in hair cells on one side of the LPR in a pattern complementary to Emx2 due to transcriptional repression. Stk32a is necessary to align the intrinsic polarity of the bundle with the core planar cell polarity (PCP) proteins in EMX2-negative regions, and is sufficient to reorient bundles when ectopically expressed in neighboring EMX2-positive regions. We demonstrate that STK32a reinforces LPR formation by regulating the apical localization of GPR156. These observations support a model in which bundle orientation is determined through separate mechanisms in hair cells on opposite sides of the LPR, with EMX2-mediated repression of Stk32a determining the position of the LPR.

FGF8 signaling plays diverse roles in inner ear development, acting at multiple stages from otic placode induction to cellular differentiation in the organ of Corti. As a secreted morphogen with diverse functions, Fgf8 expression is likely to be spatially restricted and temporally dynamic throughout inner ear development. We evaluated these characteristics using genetic labeling mediated by Fgf8mcm gene-targeted mice and determined that Fgf8 expression is a specific and early marker of Type-I vestibular hair cell identity. Fgf8mcm expression initiates at E11.5 in the future striolar region of the utricle, labeling hair cells following EdU birthdating, and demonstrates that sub-type identity is determined shortly after terminal mitosis. This early fate specification is not apparent using markers or morphological criteria that are not present before birth in the mouse. While analyses of Fgf8 conditional knockout mice did not reveal developmental phenotypes, the restricted pattern of Fgf8 expression suggests that functionally redundant FGF ligands may contribute to vestibular hair cell differentiation and supports a developmental model in which Type-I and Type-II hair cells develop in parallel rather than from an intermediate precursor.