Development and evolution of the vestibular sensory apparatus of the mammalian ear

Abstract

Herein, we will review molecular aspects of vestibular ear development and present them in the context of evolutionary changes and hair cell regeneration. Several genes guide the development of anterior and posterior canals. Although some of these genes are also important for horizontal canal development, this canal strongly depends on a single gene, Otx1. Otx1 also governs the segregation of saccule and utricle. Several genes are essential for otoconia and cupula formation, but protein interactions necessary to form and maintain otoconia or a cupula are not yet understood. Nerve fiber guidance to specific vestibular end-organs is predominantly mediated by diffusible neurotrophic factors that work even in the absence of differentiated hair cells. Neurotrophins, in particular Bdnf, are the most crucial attractive factor released by hair cells. If Bdnf is misexpressed, fibers can be redirected away from hair cells. Hair cell differentiation is mediated by Atoh1. However, Atoh1 may not initiate hair cell precursor formation. Resolving the role of Atoh1 in postmitotic hair cell precursors is crucial for future attempts in hair cell regeneration. Additional analyses are needed before gene therapy can help regenerate hair cells, restore otoconia, and reconnect sensory epithelia to the brain.

Fig. 1

A brief survey of major evolutionary changes in the ear is presented. Hagfish, a jawless vertebrate, have a single torus that contains two canal cristae and a single common macula (A). Mammals such as mice have three canal cristae and two organs for linear acceleration perception with otoconia as well as a cochlea (B). The canal cristae show differences in their organization. Mammals have a crista that sits on a ridge separated by a cruciate eminence (CE) that is covered by a cupula (removed in D to show the hair cell bundles). In contrast, hagfish have no cupula and the sensory hair cells are arranged to form a ring around the largest diameter of ampullary enlargement (C). AC, anterior crista; C, cochlea; CE; cruciate eminence; HC, horizontal crista; MC, macula communis; PC, posterior crista; S, saccule; U, utricle. Bar indicates 100 μm. Modified after [45].

Fig. 2

Morphogenetic defects of three null mutant lines are shown. Fgf10 null mutants lack formation of any canals (A,B) but form a single undifferentiated pouch that carries the reduced and malformed anterior and horizontal cristae. These mutants lack a posterior crista entirely (B). Gata3 null mutants show arrest of ear formation at the level of the otocyst with barely recognizable initiation of sensory epithelia formation (D). Interestingly, the expression of Gata3 as revealed with the β-galactosidase marker shows disorganization suggestive of alterations of expression of Gata3 (C,D). Otx1 null mutants lack formation of the horizontal canal and show a fusion of the utricular and saccular epithelium across the enlarged utriculo-saccular foramen (E,F). The loss of a horizontal canal and fusion of the utriculo-saccular epithelium in the absence of Otx1 suggests that Otx1 expression in the ear was a crucial step in vertebrate ear evolution to segregate the common macula of cyclostomes into two gravistatic organs, the utricle and saccule. AC, anterior crista; HC, horizontal crista; PC, posterior crista; S, saccule; SN, sensory neurons; U, utricle. Bar indicates 100 μm. Modified after [44,68,109].

Fig. 3

The regional differences of otoconia related gene expression in the utricle and structural and protein differences in the utricular otoconia as revealed by immunocytochemistry are shown for a guinea pig. Note the differences in the size of otoconia crystals in striola (S) and extrastriolar regions (see insert). Oc90/95 is expressed in the non-sensory epithelium of the ear (red), otopetrin is expressed throughout the sensory epithelium (yellow); osteopontin is in hair cells (dark blue); otogelin is in supporting cells (cyan); α-tectorin is in supporting cells (orange); β-tectorin is in the striola (green). All proteins are found throughout the otoconia (lilac) except for otogelin (cyan) and β-tectorin (green) which are in the lower layers. No information on concentration differences of proteins within the otoconia matrix are available. Modified after [84].

Fig. 4

The effect of loss of hair cells (B,D) loss of a neurotrophin (C,E,F) or misexpression of a neurotrophin (F) on the pattern of vestibular innervation is shown. Late embryonic loss of hair cells in Pou4f3 null mice (B) and absence of differentiated hair cells in Atoh1 null mice (D) are both compatible with a targeted projection to vestibular sensory epithelia. Note, however, that the projection to the utricle is more profoundly affected (A,B,D). Loss of Bdnf results in complete loss of crista innervation and reduced innervation of the utricle (E). Some projection to cristae organs can be rescued in mutants in which sensory neuron degeneration is blocked in the absence of Bax (C). More fibers can be rescued to the canal cristae organs in Ntf3^tgBDNF misexpressors (F) suggesting that a limited expression of Ntf3 is able to guide fibers to the canal cristae (C) but is not sufficient to rescue survival of the neurons (E). Note that misexpression of Bdnf under Ntf3 promoter control can also guide fibers to areas of the ear that have minor Ntf3 expression but no hair cells (arrow in F). AC, anterior crista; HC, horizontal crista; U, utricle. Bar indicates 100 μm. Modified after [50,61,139,155].

Fig. 5

The distribution of afferent fibers (A) and sensory neurons (B) after injections of two different lipophilic tracers into the cerebellum (red) and the superior vestibular nucleus (green) is shown in this 7 day old mouse. Note that red and green fibers are mixed to the canal cristae but are segregated along the striola in the utricle (u in A). These data imply that hair cell polarity reversal is accompanied by an almost complete segregation of fibers across the area of hair cell polarity reversal. Despite this extraordinary degree of segregation in their target organs, sensory neurons in the superior and inferior vestribular ganglion (SVG, IVG) are almost randomly mixed (B). AC, anterior crista; HC, horizontal crista; U, utricle. Bar indicates 200 μm.

Fig. 6

The formation of Atoh1 and Bdnf expressing cells is shown in the absence of Atoh1, a gene necessary for hair cell differentiation. Atoh1 and Bdnf genes were replaced with the LacZ reporter gene and the distribution of β-galactosidase, the enzyme generated by the LacZ gene, was revealed with a histochemical reaction that generates a blue reaction product. Note that either Atoh1-LacZ or Bdnf-LacZ reveals all hair cells of vestibular epithelia (A,C,E) in mice heterozygotic for these genes. Interestingly, in mice null for Atoh1 and that show no differentiated hair cells, expression of Atoh1-LacZ (B) and Bdnf-LacZ (D) shows a distribution of cells consistent with the formation of sensory epithelia. In addition, those sensory epithelia receive an innervation that is somewhat targeted toward those cells (E,F). Note that Bdnf expression in the utricle depends on the expression of Atoh1 (D). AC, anterior crista; HC, horizontal crista; PC, posterior crista; S, saccule; SN, sensory neurons; U, utricle. Bar indicates 100 μm. Modified after [50].