1.1. E13.5 ErbB2 null mice show subtle alterations in afferent projections
We started our investigation of the effects of ErbB2 abrogation at E13.5 embryos by injecting lipophilic tracers into the brainstem second order auditory (cochlear) and vestibular nuclei. Such injections resulted in labeled fibers extending to all six sensory epithelia of the ear in wild type and ErbB2 null mice (). Closer examination of the vestibular projection showed a somewhat less precise targeting as more fibers seemed to diverge away from the vestibular epithelia. Comparison of the cochlear projection indicated two differences: (1) spiral ganglia were not found close to the basal turn of the organ of Corti in ErbB2 null mice, and (2) spiraling fibers, present in wild type littermates, were absent in ErbB2 mutants (). Interestingly, the cochlear projecting (spiral) neurons were in the center of the cochlear modiolus and not near the organ of Corti.
Fig. 1 The inner ear projections of E13.5 (A, B) and E14.5 (C–F) wild type (B, D, F) and ErbB2 null mice (A, C, E) are shown. Afferent projections were transganglionically labeled after injection of NeuroVue red into the cochlear/vestibular nuclei of (more ...)
1.2. Afferent and efferent fibers show considerable alterations in ErbB2 null mice at E14.5
From this stage onward, we could label selectively the afferent fibers to the cochlea and vestibular sensory epithelia with injections into the cochlea and vestibular nuclei (labeled in red in all figures). Simultaneous injections into the medial vestibular nucleus near the internal facial genu labeled efferent fibers to the cochlea and vestibular epithelia as well as vestibular afferents with dye (labeled green in ). These injections revealed the following differences between ErbB2 null mutants and wild type littermates ().
Wild type littermates showed only afferent and efferent fibers in the modiolus and an assembly of spiral ganglion neurons in a spiral ganglion that paralleled the organ of Corti (). Only short radial fibers emanated from the spiral ganglion to the organ of Corti (arrow in ). In contrast, spiral ganglion cells were not found in the position of the spiral ganglion in erbB2 null mice but apparently filled the much enlarged center of the modiolus (). Long radial fibers emanated from these sensory neurons located in the modiolus. Despite the altered position of the spiral neurons and the increased length of radial fibers in ErbB2 null mice, numerous afferent fibers extended to the organ of Corti ().
Analysis of efferent fibers () also showed differences. Wild type efferent fibers extended along the spiral ganglion cells and started to form the intraganglionic spiral bundle within which single efferent fibers spiral from the base of the organ of Corti toward the apex (). In contrast, no intraganglionic spiral bundle was found in ErbB2 null mice (). Instead efferent fibers extended along and in parallel to the elongated afferent radial fibers from the modiolus to the organ of Corti ().
1.3. ErbB2 null mice show overshooting fibers at E16.5
E16.5 ErbB2 null mice showed an abundance of fibers to the cochlea and beyond (). Indeed, ErbB2 null mice had more fibers reaching the level of the organ of Corti than 2 day older wild type animals in which we found only a limited growth of fibers to the outer hair cells (). Focusing through the confocal stacks of images at different focal planes showed that the vast majority of fibers did not actually enter the organ of Corti but stayed below the basilar membrane. These fibers could be traced to the lateral wall were they coalesced into large bundles that spiraled toward the apex (LW in ). These data show that afferent fiber growth is more profound to the endorgans in ErbB2 null mice than in littermates (). However, the normal pattern of innervation is disrupted and fibers are extending outside the organ of Corti.
Fig. 2 Immunocytochemistry for acetylated tubulin shows differences in fiber organization of wild type (A) and ErbB2 null embryos (B). In older wild type embryos, radial fibers (RF) extend to the organ of Corti (OC) with a few fibers projecting beyond inner (more ...)
1.4. Defects of afferent and efferent innervation persist in ErbB2 null mice at E18.5
All six sensory epithelia were found to be innervated by both afferent and efferent nerves at E18.5 in wild type and ErbB2 null mice. Although these epithelia were innervated, the pattern of innervation was altered, with the cochlea epithelia having the most pronounced alteration in the pattern of innervation. As already observed at E14. 5, the spiral ganglion was no longer present in its usual position (), but resided instead in an expanded central part of the modiolus in ErbB2 null mice (). As a consequence of the unusual position of these neurons, much elongated radial fibers extended from the modiolus to the organ of Corti in ErbB2 null mice () compared to short radial fibers from the prominent spiral ganglion to the organ of Corti in wild type mice (). Closer examination at a higher magnification showed that the radial fibers in the ErbB2 null mice were less organized as demonstrated by several fibers crossing between radial bundles (). Overall, the ErbB2 null mutants showed an apparent reduction of radial fiber density to the organ of Corti (). These data suggest that in ErbB2 null mice the spiral ganglion neurons have migrated beyond their normal location of the spiral ganglion near the osseous spiral lamina and are now in the center of the modiolus.
Fig. 3 These E18.5 images show the projection of afferents in dissected and whole mounted cochlea after injection of lipophilic tracers into the cochlear/vestibular nuclei of the brainstem (red) and of efferents after injection into the olivo-cochlear bundle (more ...)
Fig. 4 These images illustrate in detail the differences in afferent (red, A, B) and efferent (green, C, D) fiber organization in ErbB2 null mice (A, C) compared to wild type littermates (B, D) in the areas indicated by dotted lines in . The spiral ganglion (more ...)
In the inner ear of the wild type E18.5 embryos, the efferent fibers turn into the intraganglionic spiral bundle (IGSB) inside the spiral ganglion (Figs. and ). The IGSB gives rise to the densely spaced, short radial fiber bundles (). In the ErbB2
null embryos, fibers project as disorganized radial bundles of efferent fibers directly to the organ of Corti without formation of an IGSB (Figs. and ). This disorganization may be due to the abnormal position of the spiral ganglion which is normally present closer to the organ of Corti (). Additionally, fewer and more disorganized efferent fibers can be labeled in the ErbB2 null mutant embryos compared to the control fibers (Figs. and ). Previous studies demonstrated that the efferent fibers rely on the afferent fibers for guidance to the organ of Corti (Farinas et al., 2001
; Tessarollo et al., 2004
). Therefore, it appears that the improper migration of the spiral ganglion and aberrant afferent projections results in disorganization in the efferent fibers () comparable to the afferent fibers.
1.5. PLP-eGFP distribution at E18.5
Afferent fibers interact with surrounding Schwann cells to regulate myelin formation and certain aspects of pathfinding (Corfas et al., 2004
; Michailov et al., 2004
; Morris et al., 1999
). Afferent fiber bundles to the sensory epithelia are surrounded by Schwann cells, which start to form myelin sheaths in the mouse ear as early as E18.5 (). Previous work using PLP
in situ hybridization suggested that the adult Schwann cells of the ear are unique in that they do not express PLP except for the ganglion close to the proximal VIIIth nerve root (Knipper et al., 1998
). We used a recently available PLP
-eGFP expressing mouse line (Mallon et al., 2002
) in which eGFP is expressed under the control of the mouse PLP
promoter and contain an additional mRNA stability region that is found in the 3′ upstream region of the endogenous mouse PLP
gene. In these PLP-eGFP
transgenic mice, all Schwann cells of the ear were strongly PLP-eGFP positive, except for a small population of cells near the VIIIth nerve root, which were negative (). All sensory epithelia contained eGFP positive cells () in wild type embryos. Specifically, in the cochlea, only supporting cells that surrounded the inner and outer hair cells were positive for eGFP. However, some of these supporting cells were not positive after fixation and a patchy distribution with various intensities of label or even apparent absence at E18.5 was observed (). Lipophilic tracers were used to label the nerve fibers (). These data demonstrated that Schwann cells were found around all vestibular and spiral ganglion neurons as well as along all nerve fibers (). Schwann cell process accompanied nerve fibers to the habenula perforate, the entering holes into the organ of Corti (, inserts). In the organ of Corti, fibers extended between eGFP positive supporting cells surrounding inner and outer hair cells (). These data demonstrated that Schwann cells and eGFP positive supporting cells of the sensory epithelia surround the afferent and efferent fibers at E18.5 or earlier. Such cells can provide a cellular basis for afferent and efferent fibers to grow along and be supported by trophic factors in addition to those known to be released from hair cells (Farinas et al., 2001
Fig. 5 Wild type embryos that express eGFP transgene under the control of the PLP promoter were injected with NeuroVue red dye to label afferent vestibular (A–C) and spiral (D–F) sensory neurons. Panels C and F are overlays of panels A, B and (more ...)
1.6. Schwann cells are absent in ErbB2 null mice
In mice null for ErbB2
, Schwann cells are absent in the peripheral nervous system. This absence of Schwann cells suggests an important role for these receptors in Schwann cell development (Morris et al., 1999
) in addition to maturation (Corfas et al., 2004
; Lyons et al., 2005
; Michailov et al., 2004
) but not in adults (Atanasoski et al., 2006
). These results are consistent with our data as we could not detect PLP-eGFP
cells along the peripheral nerves of the inner ear of ErbB2
null embryos. The only eGFP positive cells identified were scattered in the center of the spiral modiolus () and the supporting cells of the sensory epithelia. Nevertheless, nerve fibers reached all sensory epithelia and clearly reached the hair cells in both the cochlea and the vestibular epithelia, albeit in a disorganized manner ().
Fig. 6 Pattern of afferent innervation (NeuroVue red) and distribution of PLP-eGFP (green) is compared in wild type (B, D) and ErbB2 null mice (A, C) and central projections are shown as revealed by dye injections into the posterior canal crista (red, E, F) (more ...)
Comparison between wild type and ErbB2 null embryos demonstrated fewer fibers in the cochlea and vestibular sensory epithelia of ErbB2 null mice, many of which with aberrant projections (Figs. and ). For example, many fibers did not enter the cochlea but stayed on the scala tympani side of the basilar membrane (, arrow). Other fibers extended beyond the hair cell region of the cochlea and reached the lateral wall which also displayed some PLP-eGFP positive cells (). Overall, fibers did not form a regular lattice of fiber projections to the cochlea in distinct radial bundles but rather showed fibers which crossed paths, particularly in the region adjacent to the organ of Corti () where all fibers enter the cochlea in ErbB2 wild type mice (). In addition, ErbB2 null mice had expression of PLP-eGFP in almost all supporting cells of the cochlear and vestibular region, with particularly high levels of eGFP expression in the border cells of the organ of Corti, leaving very few cells unlabeled ().
Fig. 7 NeuroVue red labels bundled afferent fibers that extend to the base of the organ of Corti in wild type littermates and reach the (unlabeled) inner hair cells next to the border cells and pillar cells (B) in these overlays. Some type II fibers extend in (more ...)
Given the disorganization of afferent fibers in the ear, we wanted to know whether central projections of vestibular and cochlear fibers are at least grossly normal. NeuroVue dye tracing revealed grossly normal projections to cochlear and vestibular nuclei (). However, the labeled fiber density of central projections was markedly reduced compared to wild type animals, proportional to the apparent reduction at the periphery. To analyze further these effects and separate ear-specific form, brain-specific effects will require generation of ear-specific ErbB2 conditional null mice.
We next examined the histology of the organ of Corti to verify that development of the sensory epithelia is grossly normal and that Schwann cells are indeed absent as suggested by the virtually complete lack of PLP-eGFP positive cells along the nerve fibers of ErbB2 null mice (). Epoxy resin sections showed that the spiral ganglion cells were indeed further away from the organ of Corti in the ErbB2 null mice (). Higher magnification of the spiral ganglion showed both neurons and Schwann cells in the E16.5 wild type () but only neurons in the ErbB2 null mice (). Moreover, large empty spaces were present in the spiral ganglion of ErbB2 null mice and many apoptotic cells were distributed throughout the ganglion (). Whether these apoptotic cells are neurons or Schwann cells remains unknown.
Fig. 8 These 1-μm thick epoxy resin sections show the histological differences between E16.5 ErbB2 null mice (A, C, E) and wild type littermates (B, D). ErbB2 null mice and wild type littermates (A, B) have an apparently normal development of the organ (more ...)
1.7. ErbB2 null effects on neurotrophin expression
Previous work has shown that inner ear innervation is critically dependent in the embryo on two neurotrophins, Bdnf
(Fritzsch et al., 2004
). Of these two neurotrophins, Ntf3
is predominantly expressed in E13–18 embryos in supporting cells of the cochlea and some vestibular organs (utricle and saccule; Farinas et al., 2001
). In contrast, Bdnf
is predominantly expressed in hair cells in E13–18 embryos with only a limited expression in supporting cells, particularly near the apex of the cochlea (Fritzsch et al., 2005a
). Recent experiments have shown that over-expression of a dominant negative ErbB4 in juvenile supporting cells can reduce Ntf3
to the extent that spiral neurons die in adult mice whereas Bdnf
is only mildly affected (Stankovic et al., 2004
). In order to determine if Bdnf
expression was altered in the ErbB2
null embryos, the presence of Bdnf
in the ear of wild type and ErbB2
null embryos at E18.5 was determined using RT-PCR analysis. No significant reduction of Bdnf
mRNA was observed, while Ntf3
was significantly reduced to 25% compared to wild type embryos (). Overall, these effects are similar to observations in adult mice overexpressing a dominant-negative ErbB4
(Stankovic et al., 2004
) suggesting that similar molecular changes occurred at the level of the supporting cells of the inner ear sensory epithelia in ErbB2
Fig. 9 RNA extracted from the inner ears of E18.5 ErbB2 null and littermate control embryos was analyzed using real-time RT-PCR to determine the expression of Bdnf and Ntf3. The level of expression of Bdnf (left) and Ntf3 (right) relative to GAPDH indicated (more ...)
In summary, our data demonstrate that ErbB2 null mutants have aberrant projections of afferents, and aberrant migration of spiral neurons; both effects are possibly related to the absence of Schwann cells that may provide a stop and guidance signal. ErbB2 null mice display a reduction and disorganization of both afferent and efferent fibers in the much longer trajectory from the center part of the modiolus to the organ of Corti. In contrast, wild type mice have much shorter and straight trajectories of afferent and efferent fibers coming off the spiral ganglion and IGSB, respectively, that are adjacent and parallel to the organ of Corti. However, despite the absence of Schwann cells, there is an obvious innervation of all sensory epithelia in the ErbB2 null mice.