Partial requirement of endothelin receptor B in spiral ganglion neurons for postnatal development of hearing

Impairments of endothelin receptor B (Ednrb/EDNRB) cause the development of Waardenburg-Shah syndrome with congenital hearing loss, hypopigmentation, and megacolon disease in mice and humans. Hearing loss in Waardenburg-Shah syndrome has been thought to be caused by an Ednrb-mediated congenital defect of melanocytes in the stria vascularis (SV) of inner ears. Here we show that Ednrb expressed in spiral ganglion neurons (SGNs) in inner ears is required for postnatal development of hearing in mice. Ednrb protein was expressed in SGNs from WT mice on postnatal day 19 (P19), whereas it was undetectable in SGNs from WT mice on P3. Correspondingly, Ednrb homozygously deleted mice (Ednrb(-/-) mice) with congenital hearing loss showed degeneration of SGNs on P19 but not on P3. The congenital hearing loss involving neurodegeneration of SGNs as well as megacolon disease in Ednrb(-/-) mice were markedly improved by introducing an Ednrb transgene under control of the dopamine β-hydroxylase promoter (Ednrb(-/-);DBH-Ednrb mice) on P19. Neither defects of melanocytes nor hypopigmentation in the SV and skin in Ednrb(-/-) mice was rescued in the Ednrb(-/-);DBH-Ednrb mice. Thus, the results of this study indicate a novel role of Ednrb expressed in SGNs distinct from that in melanocytes in the SV contributing partially to postnatal hearing development.

Endothelin receptor B (Ednrb/EDNRB) belongs to the G-protein-coupled receptor family, mediating pleiotropic actions of endothelins (5,6). ET-1, ET-2 and ET-3 are ligands for Ednrb with equal affinity (6,7). Impairments of Ednrb/EDNRB have been shown to cause embryonic defects of melanocytes and enteric ganglion neurons derived from the neural crest, resulting in hypopigmentation, megacolon disease and congenital hearing loss. In rodent models, piebald-lethal rats in which Ednrb is spontaneously mutated (8) and Ednrb homozygous knock-out [Ednrb(-/-)] mice (9) have been shown to have typical WS-IV phenotypes. Thus, these previous studies indicate that Ednrb is one of the key regulatory molecules for embryonic development of melanocytes and peripheral neurons including neurons in the enteric nervous system.
The inner ears contain the organ of Corti and stria vascularis (SV). The organ of Corti, which contains two kinds of sensory cells (inner hair cells and outer hair cells), is responsible for mechanotransduction, by which sound impulses are converted into neural impulses. Auditory information from the sensory cells is transmitted to spiral ganglion neurons (SGNs) as the primary sensory carrier for the auditory system, followed by eventual transmission to the auditory cortex (10,11). Impairments of SGNs have been shown to cause hearing loss (12). Our recent study has also shown that c-Ret-mediated degeneration of SGNs directly causes severe congenital hearing loss (13). The SV consists of marginal cells, melanocytes (also known as intermediate cells) and basal cells and has been shown to maintain high levels of potassium ion for endocochlear potential (EP) (14,15). Melanocytes in the inner ear are specifically located in the SV and these defects lead to impaired EP levels resulting in hearing loss (16). Thus, disturbance of these constituent cells in inner ears has been shown to cause hearing losses (10).
Dopamine beta-hydroxylase (DBH) is an enzyme that converts the neurotransmitter dopamine to noradrenaline. DBH has been using as a specific marker of noradrenergic/adrenergic neurons since noradrenaline converted by DBH is secreted as a neurotransmitter from noradrenergic/adrenergic neurons. DBH promoter has been used as a valuable tool to allow a target gene to be expressed in peripheral neurons derived from the neural crest in vivo (17). A previous study showed that aganglionic megacolon disease in Ednrb homozygously deleted mice [Ednrb(-/-)-mice] was recovered by the introduction of an Ednrb transgene driven by the human DBH promoter [Ednrb(-/-);DBH-Ednrb-mice] (18). However, there is no information about hearing levels in Ednrb(-/-);DBH-Ednrb-mice. Previous studies have shown that endogenous DBH is expressed in SGNs of inner ears (19), while neither endogenous DBH nor a transgene driven by the DBH promoter are expressed in melanocytes or their precursors (17,20). Thus, these results of previous studies suggest that the DBH promoter enables Ednrb protein to be specifically expressed in SGNs.
Previous studies demonstrated that impairments of Ednrb/EDNRB cause hypopigmentation and megacolon disease due to defects of melanocytes and peripheral neurons such as enteric ganglion neurons, respectively (4-6, 8, 9). There has been only one report showing that Ednrb-mediated hearing loss involved a congenital defect of melanocytes in the SV (9). However, there was no information in that report about the role of Ednrb in SGNs, which serve as peripheral neurons in inner ears for the auditory system, although it was shown in the present study that Ednrb protein is expressed in SGNs. The results of the present study indicate a novel etiology for Ednrb-mediated hearing loss in mice that can involve postnatal degeneration of SGNs besides congenital defects of melanocytes in the SV.

EXPERIMENTAL PROCEDURES
Mice---Ednrb(-/-)-mice (5) and Ednrb(-/-);DBH-Ednrb-mice (18) were previously reported. All experiments were authorized by the Institutional Animal Care and Use Committee in Chubu University (approval number: 2110017) and the Institutional Recombinant DNA Experiment Committee in Chubu University (approval number: 06-01) and followed the Japanese Government Regulations for Animal Experiments.
Measurement of hearing---Tone-burst-evoked auditory brainstem response (ABR) measurements (AD Instruments Pty. Ltd.) were performed as described previously (13,21). Tone burst stimuli were measured 5 dB-stepwise from 0 decibel sound pressure level (dB SPL) to 70 or 90 dB SPL. The threshold was obtained by identifying the lowest level of the I wave of ABR recognized. Data are presented as means ± SE.
Morphological analysis with a light microscope---After perfusion fixation by Bouin's solution, cochleae from postnatal day 19 (P19) mice were immersed in the same solution for 1 week. H&E staining and immunohistochemical analyses with anti-Ednrb (1:2000; Chemicon) and anti-Kir4.1 (1:500; Santa Cruz Biotechnology, Inc.) antibodies were performed with paraffin sections. The VECTASTAIN ABC kit (Vector) and Envision kit/HRP (diaminobenzidine; DAB) (DAKO) were used in the immunohistochemical analyses with a hematoxylin counterstain. In the case of anti-Kir4.1, the Vector VIP substrate kit for peroxydase (Vector) was used with counterstained Methyl Green. LacZ staining of dopachrome tautomerase (Dct)-LacZ melanocytes was performed as described previously (13). In brief, after fixation with PBS containing 0.25% glutaraldehyde, the inner ears were stained with 0.04% X-gal by intracochlear perfusion. The samples were post-fixed with 4% paraformaldehyde (PFA) and decalcified with EDTA and then paraffin sections were prepared. Estimation of cell density of SGNs with H&E staining basically followed the previous method (13,(22)(23)(24). In brief, the area of Rosenthal's canal in 3 sections from each mouse was measured with the software program WinROOF (version 6.2, Mitani Corp., Fukui, Japan) as previously reported (13,24). Cell density of SGNs from 3 mice for each mouse strain was calculated by dividing the cell number of SGNs in the measured Rosenthal's canal by the area of the section examined. A total of 100-150 SGNs in 3 sections from each mouse were examined. Percentage of positive signals histochemically detected by antibodies or LacZ staining was estimated with WinROOF (version 6.2) as previously reported (13,24). Briefly, the number of positive SGNs was divided by the total number of SGNs. A total of 100-150 SGNs in 5 sections from each mouse were examined. In the case of SV, positive signals in the measured SV were divided by the area of the section measured. In order to compare the positive levels among mouse strains, the normalized positive signals in Ednrb(-/-)-mice and Ednrb(-/-);DBH-Ednrb-mice were divided by the normalized positive signals in WT-mice. Rosenthal's canal or SV from 3 or 4 mice for each mouse strain was measured for each estimation.
Morphological analysis by transmission electron microscopy (TEM)---Preparation of tissues for TEM basically followed the previous method (13,22). In brief, after perfusion fixation with a mixture of 2% PFA and 2% glutaraldehyde in 0.3 M HEPES-buffer (pH 7.4), dissected murine cochleae were immersed in the same fixative solution overnight at 4ºC. The cochleae were then fixed with 2% osmium tetroxide in 0.3 M HEPES-buffer (pH 7.4) at 4ºC for 3 hours. After rinsing off the fixative solution, the cochleae were dehydrated with a graded series of ethanol and embedded in epoxy resin (Quetol 651). Ultrathin sections (t = 70 nm) were observed under an electron microscope at 80 kV (JEOL JEM1200EX, Tokyo, Japan).

Discussion
This study demonstrated that Ednrb(-/-)-mice had severe congenital deafness (ABR threshold > 90 dB SPL) with not only a defect of melanocytes in the SV ( Fig. 1 and 5) but also neurodegeneration of SGNs ( Fig. 2 and 3). These results indicate a novel etiology for Ednrb-mediated hearing loss in Ednrb(-/-)-mice that involves degeneration of SGNs, which serve as peripheral neurons in inner ears, besides defects of melanocytes in the SV.
This study showed neurodegeneration of SGNs resulting in decreased numbers of SGNs in Ednrb(-/-)-mice on P19 (Fig. 2 and 3), while cell density and morphology of SGNs were comparable in Ednrb(-/-)-mice and WT-mice on P3 (Fig. 2). These results suggest that SGNs in Ednrb(-/-)-mice developed normally at least until P3, when the level of Ednrb expression in SGNs from WT-mice was undetectable ( Fig. 2A). However, SGNs from Ednrb(-/-)-mice no longer survived on P19 (Fig. 2 I and J), when the level of Ednrb expression in SGNs from WT-mice was clearly detectable (Fig. 2C). We therefore conclude that a defect of Ednrb expression affects survival of SGNs during hearing development after birth in mice.
Ednrb has been reported to mediate embryonic development of melanocytes (30) and the enteric nervous system (18,26) derived from the neural crest. Our results indicate a novel possibility that Ednrb is essential for postnatal development of SGNs, although the development process of SGNs (e.g., differentiation or migration of precursors) during prenatal and postnatal hearing development has not been completely elucidated (12). Our previous study also showed that impairment of c-Ret causes severe congenital hearing loss with degeneration of SGNs and with intact morphology of hair cells and the SV (13). Since both EDNRB and c-RET cause megacolon disease with congenital intestinal aganglionosis in mice and humans, further study is needed to determine whether megacolon-related molecules such as SOX10 and PAX3 are involved in congenital hearing loss caused by degeneration of SGNs.
The degeneration of SGNs from Ednrb(-/-)-mice did not involve the hallmark of apoptotic signals (Fig. S3). The results of a previous study also showed that neurodegeneration of enteric neurons did not involve apoptotic signals during the developmental stage in mice with deletion of Ednrb (30). On the other hand, our results showed that hair bundles of inner-and outer-hair cells in Ednrb(-/-)-mice, which have already developed congenital hearing loss, were comparable to those in littermate WT-mice (Fig.  S2). Immunohistochemical analysis correspondingly showed that expression of Ednrb protein was nearly undetectable in hair cells from WT-mice (Fig. S1). These results suggest that the congenital hearing loss in Ednrb(-/-)-mice involves postnatal degeneration of SGNs as well as defects of melanocytes in the SV rather than disturbance of hair cells.
Several mouse models for Ednrb-mediated WS-IV have been reported (summarized in Fig.  S7). sl mice, in which exon 1 and intron 1 are spontaneously deleted, and WS-IV mice, in which exons 2 and 3 are spontaneously deleted, have been shown to develop megacolon disease and hearing loss. On the other hand, the hearing level of Ednrb(-/-)-mice with deletion of exon 3, which we analyzed in this study, has not been reported. In humans, although impairments of EDNRB caused by nonsense mutations of exon 3 have been reported to also result in the development of WS with hearing loss, the etiology has not been clarified. Thus, this study for the first time provides an insight into the pathogenesis of congenital hearing loss caused by impairment of Ednrb(-/-) by deletion of exon 3 in mice.
Our results suggest that 60-70 dB SPL of hearing levels could be maintained even if there are no melanocytes in the SV in inner ears of Ednrb(-/-);DBH-Ednrb-mice. Since a previous study has shown that a transgene driven by the Dct promoter is expressed in melanocytes (31), further study is needed to determine the concurrent rescue effect of Ednrb transgene driven by the Dct promoter and the DBH promoter on congenital deafness in Ednrb(-/-)-mice.
In summary, this study demonstrates a novel role of Ednrb expression in SGNs distinct from that in melanocytes in the SV contributing partially to postnatal hearing development via survival of SGNs. A therapeutic strategy for congenital hearing loss in WS-IV patients has not been established. Enhancement of EDNRB expression in SGNs could be a novel potential therapeutic strategy for congenital hearing loss in WS-IV patients.