At E12.5, the first HCs can be distinguished, and a single cilium emerges from the center of the cell surface. The cilium is surrounded by elongated microvilli [42, 43], and it lengthens to form the kinocilium to subsequently establish subcellular planar polarity as the cilium migrates to one side of the apical cell surface.

Polarization of the stereociliary bundle is cell intrinsic and occurs independently from mechanisms directing planar polarity at the level of PCP or tissue polarity. As described below, auditory and vestibular HCs in vangl2 knockout [44] and frizzled 3/6 double knockout mice [45] are incorrectly oriented relative to the neighboring cells, although their mutant HCs still form polarized stereociliary. Fewer mutations only affect subcellular planar polarity, while a conditional knockout mouse disrupting the function of IFT88/Polaris is an essential factor for intraflagellar transport and cilium formation [46]; protocadherin 15 mutants [47] have been reported to disrupt subcellular planar polarity construction.

Some of the molecular signals that direct formation of the polarized bundles are beginning to be uncovered. The function of P21-activated kinase PAK asymmetrically localized in the vicinity of the kinocilium [48] is further regulated by the small GTPase Rac1 which is itself a well-characterized regulator of actin dynamics [49]. GTPase Rac1 is, in fact, involved in certain functions of P21-activateed kinase PAK. The Rac1-PAK signaling pathway is also dependent upon the function of Kif3a. The Rac1-PAK signaling pathway is also dependent upon the function of Kif3a [50], a component of the Kinesin II motor complex that is necessary for plus-end microtubule trafficking and anterograde intraflagellar transport [51]. However, Kinesin II, whose identity is still unknown, is believed not to be the motor factor that translocates the kinocilium.

Many essential molecules regulating this level of planar polarity have been identified through genetic screening in Drosophila, including the core PCP proteins Frizzled, Dishevelled, Van Gogh, Prickle, Diego, and Flamingo. The subcellular distributions of core PCP proteins are likely conserved between the mouse and Drosophila at the line of polarity reversal (LPR). Core PCP proteins are asymmetrically localized in the epidermal cells of developing Drosophila wings with Frizzled and Dishevelled enriched at the distal cell boundary near the site of hair formation and Van Gogh and Prickle enriched at the proximal side. Flamingo is present at both cell boundaries. In the developing mouse utricle the core PCP proteins Frizzled6 (Fz6) and Prickle-like 2 (Pk2) are located on opposite sides of HCs and supporting cells: a pattern similar to the relative distribution of Frizzled and Prickle in Drosophila, suggesting that other PCP proteins are similarly distributed. The remaining core PCP proteins in mice are asymmetrically localized at the cell boundaries; however, proximal versus distal distributions have not been established. Pk2 is enriched at the same side of vestibular HCs, regardless of the stereociliary bundle orientation or cellular position relative to the LPR [52–56].

Each vestibular afferent neuron has its cell body in the cochleovestibular (CV) ganglion and sends a peripheral axon toward the vestibular sense organs and a central axon to the vestibular nuclei in the brainstem. The CV ganglion consists of two cell clusters extending in a rostral–caudal direction in the internal auditory canal. It consists of a superior and inferior cell group related to the superior and inferior divisions of the vestibular nerve trunk, respectively. The superior division supplies the cristae of the superior and lateral canals, the macula of the utricle, and the anterosuperior part of the macula of the saccule. The inferior division supplies the crista of the posterior canal and the main macular portion of the saccule. The inferior division supplies the crista of the posterior canal and the main portion of the macula of the saccule. Medial to the vestibular ganglion, the nerve fibers of both divisions merge to form a single trunk, which enters the brain stem [57].

As one of the cranial peripheral nerves, the CV nerve is composed of both neurons and glia [58]. These two cell types arise developmentally from distinct sources, viz., the glial cells derived from neural crest cell (NCC) progenitors [59–61], while the neurons originate almost exclusively from the otic placode [59, 62, 63].

Apart from projecting central axons to the hindbrain, CV neurons also project peripheral neuritis to sensory targets developing within the otic epithelium [64]. The glial cells of the CV nerve are derived from NCCs that emigrate from the hindbrain at the level of rhombomere 4 (R4).

CV neurons extend peripheral neuritis to the developing vestibular sensory epithelium as early as E11.5 and extend central axons to the hindbrain as early as E12.5 [64, 65] and release some important factors related this neural development. In mice, disturbance of ERBB2 (a receptor that mediates neuron–glia interactions via the ligand Neuregulin1 [66]) compromises development of the CV nerve. Loss of ErbB2 results in altered migration of CV neuronal cell bodies, abnormal targeting of CV peripheral neurites, and reduced number of CV neurons [67]. Molecularly blocking Semaphorin/Neuropilin signaling in chicks disrupts NCC migratory pathways and impairs the inward movement of epibranchial placodal neurons [68]. In certain studies, ablation of NCC migration by physical or molecular methods in chicks results in reduced numbers of neuroblasts migrating from the epibranchial ganglia and abnormal projection of central axons [69–71].

The maculae contain two types of HCs that are distinguished from each other by the synaptic structure formed with the afferent neurons. Type I HCs receive large calyx nerve endings that surround the cell body, while type II HCs make bouton-like contacts with nerve endings. The two HC classes are innervated by three types of afferent neurons. The calyx-only class of neurons contacts clusters of type I HCs in the striola region. Dimorphic neurons contact HCs throughout the sensory epithelia, forming calyxes with type I HCs and boutons with type II HCs. Bouton-only afferents only contact type II HCs located outside the striola. Each class of afferent neurons may contact multiple HCs, and the dimorphic and bouton-only neurons contact groups of HCs located on either side of the LPR. Despite the range of bundle orientations present in the utricle, afferent neurons only contact HCs with similar stereociliary bundle orientations. Although developmental mechanisms coordinating neuronal innervation and stereociliary bundle orientation are not well known, there is a single cell type that morphologically resembles the mature type II HCs prior to the obvious distinction of the two HC types. From the later appearance of morphologically distinct type I HCs during regeneration in the avian utricle, it has been suggested that type I HCs differentiate from the type II HCs in a serial progression of development. However, earlier studies of spatiotemporal patterns of HC birth have suggested that HCs in regions where type I HCs predominate in the mature sensory epithelium are born first, while hair cells in those regions where type II HCs predominate are born later, indicating that types I and II cells may be birth-specific.