Semaphorin-5B Controls Spiral Ganglion Neuron Branch Refinement during Development.

During nervous system development, axons often undergo elaborate changes in branching patterns before circuits have achieved their mature patterns of innervation. In the auditory system, type I spiral ganglion neurons (SGNs) project their peripheral axons into the cochlear epithelium and then undergo a process of branch refinement before forming synapses with sensory hair cells. Here, we report that Semaphorin-5B (Sema5B) acts as an important mediator of this process. During cochlear development in mouse, immature hair cells express Sema5B, whereas the SGNs express both PlexinA1 and PlexinA3, which are known Sema5B receptors. In these studies, genetic sparse labeling and three-dimensional reconstruction techniques were leveraged to determine the morphologies of individual type I SGNs after manipulations of Sema5B signaling. Treating cultured mouse cochleae with Sema5B-Fc (to activate Plexin-As) led to type I SGNs with less numerous, but longer terminal branches. Conversely, cochleae from Sema5b knock-out mice showed type I SGNs with more numerous, but shorter terminal branches. In addition, conditional loss of Plxna1 in SGNs (using Bhlhb5Cre) led to increased type I SGN branching, suggesting that PlexinA1 normally responds to Sema5B in this process. In these studies, mice of either sex were used. The data presented here suggest that Sema5B-PlexinA1 signaling limits SGN terminal branch numbers without causing axonal repulsion, which is a role that distinguishes Sema5B from other Semaphorins in cochlear development.SIGNIFICANCE STATEMENT The sensorineural components of the cochlea include hair cells, which respond mechanically to sound waves, and afferent spiral ganglion neurons (SGNs), which respond to glutamate released by hair cells and transmit auditory information into the CNS. An important component of synapse formation in the cochlea is a process of SGN "debranching" whereby SGNs lose extraneous branches before developing unramified bouton endings that contact the hair cells. In this work, we have found that the transmembrane ligand Semaphorin-5B and its receptor PlexinA1 regulate the debranching process. The results in this report provide new knowledge regarding the molecular control of cochlear afferent innervation.

Recent advances in the development and function of type II spiral ganglion neurons in the mammalian inner ear.

In hearing, mechanically sensitive hair cells (HCs) in the cochlea release glutamate onto spiral ganglion neurons (SGNs) to relay auditory information to the central nervous system (CNS). There are two main SGN subtypes, which differ in morphology, number, synaptic targets, innervation patterns and firing properties. About 90-95% of SGNs are the type I SGNs, which make a single bouton connection with inner hair cells (IHCs) and have been well described in the canonical auditory pathway for sound detection. However, less attention has been given to the type II SGNs, which exclusively innervate outer hair cells (OHCs). In this review, we emphasize recent advances in the molecular mechanisms that control how type II SGNs develop and form connections with OHCs, and exciting new insights into the function of type II SGNs.

Neuropilin-2/Semaphorin-3F-mediated repulsion promotes inner hair cell innervation by spiral ganglion neurons.

Auditory function is dependent on the formation of specific innervation patterns between mechanosensory hair cells (HCs) and afferent spiral ganglion neurons (SGNs). In particular, type I SGNs must precisely connect with inner HCs (IHCs) while avoiding connections with nearby outer HCs (OHCs). The factors that mediate these patterning events are largely unknown. Using sparse-labeling and time-lapse imaging, we visualized for the first time the behaviors of developing SGNs including active retraction of processes from OHCs, suggesting that some type I SGNs contact OHCs before forming synapses with IHCs. In addition, we demonstrate that expression of Semaphorin-3F in the OHC region inhibits type I SGN process extension by activating Neuropilin-2 receptors expressed on SGNs. These results suggest a model in which cochlear innervation patterns by type I SGNs are determined, at least in part, through a Semaphorin-3F-mediated inhibitory signal that impedes processes from extending beyond the IHC region.

Expression and Misexpression of the miR-183 Family in the Developing Hearing Organ of the Chicken.

The miR-183 family consists of 3 related microRNAs (miR-183, miR-96, miR-182) that are required to complete maturation of primary sensory cells in the mammalian inner ear. Because the level of these microRNAs is not uniform across hair cell subtypes in the murine cochlea, the question arises as to whether hair cell phenotypes are influenced by microRNA expression levels. To address this, we used the chicken embryo to study expression and misexpression of this gene family. By in situ hybridization, expression of all 3 microRNAs is robust in immature hair cells of both auditory and vestibular organs and is present in the statoacoustic ganglion. The auditory organ, called the basilar papilla, shows a weak radial gradient (highest on the neural side) in prosensory cells near the base on embryonic day 7. About nine days later, the basilar papilla also displays a longitudinal gradient (highest in apical hair cells) for the 3 microRNAs. Tol2-mediated gene delivery was used to ask whether cell phenotypes are malleable when the prosensory epithelium was forced to overexpress the miR-183 family. The expression plasmid included EGFP as a reporter located upstream of an intron carrying the microRNA genes. The vectors were electroporated into the otic cup/vesicle, resulting in strong co-expression of EGFP and the miR-183 family that persisted for at least 2 weeks. This manipulation did not generate ectopic hair cells in non-sensory territories of the cochlear duct, although within the basilar papilla, hair cells were over-represented relative to supporting cells. There was no evidence for a change in hair cell phenotypes, such as short-to-tall, or basal-to-apical hair cell features. Therefore, while increasing expression of the miR-183 family was sufficient to influence cell lineage decisions, it did not redirect the differentiation of hair cells towards alternative radial or longitudinal phenotypes.

The genetics of hair cell development and regeneration.

Sensory hair cells are exquisitely sensitive vertebrate mechanoreceptors that mediate the senses of hearing and balance. Understanding the factors that regulate the development of these cells is important, not only to increase our understanding of ear development and its functional physiology but also to shed light on how these cells may be replaced therapeutically. In this review, we describe the signals and molecular mechanisms that initiate hair cell development in vertebrates, with particular emphasis on the transcription factor Atoh1, which is both necessary and sufficient for hair cell development. We then discuss recent findings on how microRNAs may modulate the formation and maturation of hair cells. Last, we review recent work on how hair cells are regenerated in many vertebrate groups and the factors that conspire to prevent this regeneration in mammals.