Mutant mice reveal the molecular and cellular basis for specific sensory connections to inner ear epithelia and primary nuclei of the brain

Abstract

We review the in vivo evidence for afferent fiber guidance to the inner ear sensory epithelia and the central nuclei of termination. Specifically, we highlight our current molecular understanding for the role of hair cells and sensory epithelia in guiding afferents, how disruption of certain signals can alter fiber pathways, even in the presence of normal hair cells, and what role neurotrophins play in fiber guidance of sensory neurons to hair cells. The data suggest that the neurotrophin BDNF is the most important molecule known for inner ear afferent fiber guidance to hair cells in vivo. This suggestion is based on experiments on Ntf3 transgenic mice expressing BDNF under Ntf3 promoter that show deviations of fiber growth in the ear to areas that express BDNF but have no hair cells. However, fiber growth can occur in the absence of BDNF as demonstrated by double mutants for BDNF and Bax. We directly tested the significance of hair cells or sensory epithelia for fiber guidance in mutants that lose hair cells (Pou4f3) or do not form a posterior crista (Fgf10). While these data emphasize the role played by BDNF, normally released from hair cells, there is some limited capacity for directed growth even in the absence of hair cells, BDNF, or sensory epithelia. This directed growth may rely on semaphorins or other matrix proteins because targeted ablation of the sema3 docking site on the sema receptor Npn1 results in targeting errors of fibers even in the presence of hair cells and BDNF. Overall, our data support the notion that targeting of the afferent processes in the ear is molecularly distinct from targeting processes in the central nuclei. This conclusion is derived from data that show no recognizable central projection deviation, even if fibers are massively rerouted in the periphery, as in Ntf3(tgBDNF) mice in which vestibular fibers project to the cochlea.

Fig. 1

The Pou domain factor Pou4f3 (Brn3c) is required for hair cell differentiation and maintenance of hair cells past birth. Only few basal turn hair cells can be identified in neonatal animals using Myosin VII immunocytochemistry (a,b). Despite this absence of differentiated hair cells, afferent fibers are targeted to both vestibular and cochlear sensory epithelia (c,d). Such fibers project in a crudely topographical fashion to the cochlear nuclei as revealed by the injection of different fluorescent tracers into the apex and base of the cochlea, respectively (e,f). These data establish that an overall fairly normal peripheral and a crude central projection can develop in the absence of functional hair cells. AC, anterior crista; HC, horizontal crista; HCs, hair cells; IHCs, inner hair cells; OHCs, outer hair cells; PC, posterior crista; VCN, ventral cochlear nucleus. Modified after (Xiang et al., 2003).

Fig. 2

Fgf10 null mutant mice do not develop semicircular canals and have no posterior canal crista (a,b). Such animals do not show a projection of afferent or efferent fibers to the posterior region of the ear either in late embryonic stages. However, at early embryonic stages posterior crista sensory neurons apparently form and project toward the area of the posterior crista almost like in wildtype or heterorzygotic littermates (c,d). Within two days such projections are lost, presumably because of lack of neurotrophic support. These data support the idea that some pathfinding properties of vestibular afferents reside outside sensory epithelia. AC, anterior cristae; HC, horizontal cristae; PCS, posterior crista sensory neurons; PC, posterior crista; S, saccule. Modified after (Pauley et al., 2003).

Fig. 3

Mice with a targeted replacement of the Sema3a docking site at the neuropilin1 receptor (Npn-1^Sema- knock-in mice) show distinct defects in the inner ear innervation. Specifically, in these mutants, fibers may not stop near hair cells or sensory epithelia but continue to grow until they reach the skin above the ear (a,b). Injections of a lipophilic tracer into the skin above the ear will label the trigeminal system but also fibers that show side-branches to the vestibular endorgans as they pass through the ear (c). In addition, these fibers can be traced to vestibular ganglion cells (d) and can be shown to project centrally like vestibular fibers into the vestibular nuclei rather than like trigeminal fibers. These data suggest that Sema3a is at least one of the stop signals at or near sensory epithelia that directs fibers to hair cells. The data also show that at least during embryonic development specification of central projections of vestibular neurons does not depend on being connected to hair cells. AC, anterior cristae; HC, horizontal crista; MesV, nucleus mesencephalicus V; MVN, medial vestibular nucleus; PC, posterior crista; S, saccule; SVN, superior vestibular nucleus; U, utricle. Modified after (Gu et al., 2003).

Fig. 4

The neurotrophins BDNF (a) and Ntf3 (b) are both expressed in late embryonic canal cristae. However, only hair cells are positive for BDNF (a) while Ntf3 is expressed in the stroma of the crista as well as in adjacent dark cells near the crista (b). In wildtype mice afferent fibers are targeted to cristae as well as the utricle and innervate densely hair cells (c). In mutants, in which BDNF has been eliminated, hardly any fiber projects to canal cristae (e). However, breeding BDNF null mice into a Bax null background results in survival of some neurons and growth of some neurites in the absence of BDNF (d,f). These fibers extend toward cristae (d) but innervate predominantly areas of Ntf3 expression with only an occasional fiber extending to hair cells (f). These data suggest that BDNF is not only a major survival factor but also helps direct growth of fibers to hair cells. Nevertheless, the residual ability of fibers to grow toward hair cells suggests that some additional attractive substances may be released from hair cells. AC, anterior crista; HC, horizontal crista; U, utricle. Modified after (Hellard et al., 2004).

Fig. 5

The effect of misexpression of BDNF under the Ntf3 promoter control on vestibular fiber pathfinding is shown without (a) or with a combined elimination of BDNF (b–f). Replacing of Ntf3 with BDNF results in rerouting of saccular and posterior crista afferents even in the presence of normal BDNF expression (a). However, combining the transgenic expression of BDNF under Ntf3 promoter control with absence of BDNF results in more profound rerouting of fibers to the basal turn of the cochlea (b) as well as rescue of fibers to the anterior and horizontal crista (d,e). Despite the fact that no neurotrophin is expressed in hair cells in these animals, fibers preferentially innervate hair cells (e) but also project outside the sensory epithelia to areas of expression of Ntf3 (c–e). These data suggest that attractors other than BDNF must exist in hair cells but also suggest that such attractors can be overridden by BDNF which by itself can attract fibers outside the sensory epithelia. Central projections from the cochlea (f) show fibers not only to the cochlear nuclei but also to vestibular nuclei. These data suggest that the central projection is regulated molecularly distinct from the peripheral projection. AC, anterior crista; DCN, dorsal cochlear nucleus; HC, horizontal crista; MVN, medial vestibular nucleus; PC, posterior crista; U, utricle. Modified after (Tessarollo et al., 2004).

Fig. 6

This schematic illustrates (a) the normal areas of expression of BDNF and Ntf3 in the utricle, hair cells of the anterior canal crista, and cells surrounding the sensory area of the crista, and the normal pattern of innervation of the crista. (b–f) The variations in expression pattern of neurotrophins and resultant patterns of crista innervation are shown. BDNF is required for neuron growth and survival (b) whereas Ntf3 is not (c). When Bax is also knocked out in the BDNF null mutant, fibers survive and project to areas of Ntf3 expression (d). When Ntf3 is replaced with BDNF, fibers innervate the sensory cells as usual, but also project to non-sensory, BDNF-expressing areas, demonstrating a positive attraction of fibers to BDNF even when expressed in an abnormal location (e). Combining the transgenic mouse in which Ntf3 is replaced with BDNF with BDNF null mutant shows that fibers can innervate hair cells that do not express BDNF, suggesting that other hair cell or supporting cell factors are involved in fiber attraction, but are also attracted to BDNF expressing non-sensory patches (f). White areas are areas in which neither BDNF or Ntf3 are expressed. Red represents Ntf3 expression. Blue represents BDNF expression. Purple represents normal overlap of BDNF and Ntf3 expression in hair cells and supporting cells, respectively. Green represents BDNF expression in place of Ntf3 expression and teal represents an overlap of normal BDNF expression in hair cells with BDNF expressed under the Ntf3 promoter in supporting cells. AC, anterioventral crista.