Dominant negative connexin26 mutation R75W causing severe hearing loss influences normal programmed cell death in postnatal organ of Corti
© Inoshita et al.; licensee BioMed Central Ltd. 2014
Received: 11 March 2013
Accepted: 30 December 2013
Published: 3 January 2014
The greater epithelial ridge (GER) is a developmental structure in the maturation of the organ of Corti. Situated near the inner hair cells of neonatal mice, the GER undergoes a wave of apoptosis after postnatal day 8 (P8). We evaluated the GER from P8 to P12 in transgenic mice that carry the R75W + mutation, a dominant-negative mutation of human gap junction protein, beta 2, 26 kDa (GJB2) (also known as connexin 26 or CX26). Cx26 facilitate intercellular communication within the mammalian auditory organ.
In both non-transgenic (non-Tg) and R75W + mice, some GER cells exhibited apoptotic characteristics at P8. In the GER of non-Tg mice, both the total number of cells and the number of apoptotic cells decreased from P8 to P12. In contrast, apoptotic cells were still clearly evident in the GER of R75W + mice at P12. In R75W + mice, therefore, apoptosis in the GER persisted until a later stage of cochlear development. In addition, the GER of R75W + mice exhibited morphological signs of retention, which may have resulted from diminished levels of apoptosis and/or promotion of cell proliferation during embryogenesis and early postnatal stages of development.
Here we demonstrate that Cx26 dysfunction is associated with delayed apoptosis of GER cells and GER retention. This is the first demonstration that Cx26 may regulate cell proliferation and apoptosis during development of the cochlea.
KeywordsApoptosis Hereditary hearing loss Gjb2 Greater epithelial ridge Mouse Organ of corti
Hereditary deafness affects about 1 in 2,000 children, and mutations in the gap junction protein, beta 2, 26 kDa gene (GJB2), also known as connexin 26 (CX26), are the most common genetic causes of congenital bilateral non-syndromic sensorineural hearing loss. Gap junctions play important roles during the maturation and differentiation of developing tissues [1–5]. In the mammalian cochlea, we have demonstrated that a dominant-negative GJB2 mutation results in incomplete postnatal development of the cochlear organ of Corti by R75W + transgenic mice, which carry a dominant-negative mutation of human CX26[6, 7]. The organ of Corti in R75W + mice is reduced in height and has an increased midmodiolar-sectional area. In addition, several cochlear structures are absent in R75W + mice (including the tunnel of Corti, Nuel’s space, and spaces surrounding the outer hair cells), and the number of microtubules within the inner pillar cells is significantly reduced. Morphometric changes in R75W + mice likely result from collapse of the organ of Corti and enlargement of support cells causing impaired distortion product otoacoustic emission , although the underlying mechanisms remain unclear.
The greater epithelial ridge (GER) is a developmental structure that is important during maturation of the organ of Corti in mice. Present at birth, the GER typically disappears by postnatal day 10 (P10) [9, 10]. In the mouse, most apoptosis in the developing cochlear system occurs during embryogenesis . In the developing spiral ganglion cells of rats, however, 22% of programmed cell death was measured between P5 and P6 . We have shown that development of normal hearing function in C3H/HeJ mice requires a wave of programmed cell death in the GER from P7 to P12. We also observed mitosis in the GER after P7, indicating that GER cells are both degenerating and regenerating until their eventual elimination at P12 .
Here, we evaluated postnatal changes in the GER of Gjb2 mutant mice. We report that a dominant-negative Gjb2 mutation induced GER retention, indicating that Gjb2-dependant apoptosis is critical for formation of a functionally and morphologically normal auditory system.
Programmed cell death with Caspase-3 activation in GER
Histological analysis of the GER
Apical turns of the cochleae were observed with cross sections. Histological examinations of H-E-stained cochlear sections revealed no gross differences in GER morphology between R75W + mice and non-transgenic (non-Tg) controls at P8 (Figure 1A,B). In both R75W + and non-Tg mice, a number of cells within the GER exhibited apoptotic characteristics (e.g., chromatin condensation) at P8 (Figure 1A,B, arrows). At P12, GER cells were clearly present in non-Tg mice, but both the number of cells in the GER and the area of the GER were visibly reduced compared to P8 (Figure 1C), and no apoptotic cells were detected. In contrast, there was less reduction in the GER size and apoptotic cells were visible in R75W + mice at P12 (Figure 1D, arrow).
TEM studies also revealed differences between non-Tg and R75W + mice at P12 (Figure 1E,F). Nuclei with highly condensed chromatin were detected in retained GER cells only in R75W + mice (Figure 1F, arrow). TEM images also indicated that the GER of R75W + mice at P12 was larger, both in cell number and in total area, than that of non-Tg mice (Figure 1E,F region within dotted line).
Area of the GER
Number of cells in the GER
Apoptotic cells in the GER
In this study, we found elevated levels of programmed cell death in the GER of R75W + mice at P12 and the retention of GER at the same time.
The loss or impairment of gap junction-mediated intercellular communication has been associated with tumor progression in a number of contexts. Reduced expression of gap junction genes has been demonstrated in several human cancers, including gastric cancer (CX32) , prostatic adenocarcinoma (CX43) , brain glioma (CX43) , breast cancer (CX43) , and lung cancer (CX32 and CX43) . When these gene functions are restored, cell growth slows and more normal phenotypes arise [19, 20]. Thus, connexins are generally considered tumor suppressor genes . Transfection of CX26 into a tumor cell lines typically results in growth suppression [22–24].
It is still unclear why disruption of Gjb2 results in hearing loss, particularly when other cochlear connexins (e.g., Cx30) can compensate for Cx26 dysfunction. One possibility is that Cx26 may be more involved in regulating cell proliferation and apoptosis in the organ of Corti than other connexins. Cx26 disruption, therefore, may delay apoptosis in the GER, collapse the tunnel of Corti, and ultimately disrupt the cyto-architecture of the organ of Corti. Additional experiments are required to further elucidate the mechanisms by which the inner ear develops.
The present findings strongly suggest that the dominant-negative R75W + mutation of Cx26 delays programmed cell death around the organ of Corti. This results in collapse of the organ of Corti. These findings indicate that CX26 may play a critical role in regulating cell proliferation and apoptosis during cochlear development.
All mice were obtained from a breeding colony of R75W + mice which were heterozygous  and maintained at the Institute for Animal Reproduction (Ibaraki, Japan). R75W + mice were maintained in a mixed C57BL/6 background and intercrossed to generate R75W + animals. Littermates of wild type mice were used as controls.
The animals were genotyped using DNA obtained from tail clips and amplified using a Tissue PCR Kit (Sigma, Saint Louis, MO, USA) as described . All experimental protocols were approved by the Institutional Animal Care and Use Committee at Juntendo University, and were conducted in accordance with the US National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. All reasonable efforts were taken to minimize the number of animals used, as well as their suffering. Five mice were examined in each age group.
Mice were anesthetized, killed and inner-ear tissues were removed. The cochleae were further dissected and fixed in 4% paraformaldehyde”. Immunofluorescence staining with antibody against cleaved Caspase-3 (C-Casp3, rabbit IgG, Promega) was performed on whole-mount preparations of the finely dissected organ of Corti. We incubated the tissues in the antibody solutions for 1 h after blocking. As secondary antibodies, we used Cy3–conjugated anti rabbit IgG (Sigma Aldrich). F-actin was visualized with Alexa 633–conjugated Phalloidin (Life technologies). Fluorescence confocal images were obtained with a LSM510-META confocal microscope (Carl Zeiss). Some of the green fluorescence in Cx26R75W + mice indicated the pseudocolor obtained from the signal of Alexa 633–conjugated secondary antibodies (Invitrogen), because these mice have ubiquitous EGFP expression from their transgene. z-stacks of images were collected at 0.5 μm intervals, and the single image stacks were constructed with LSM Image Browser (Zeiss); three-dimensional images and videos were constructed with IMARIS software (Bitplane). We analyzed at least six samples from three animals, and representative images are shown. The compared images were photographed and processed using identical parameters. Three-dimensional images were constructed with z-stacked confocal images by IMARIS (Bitplane).
Animals were deeply anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). They were then perfused intracardially with 0.1 M phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 1.47 mM KH2PO4, 8.1 mM Na2HPO4, pH 7.2) followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB, 0.2 M Na2HPO4, 0.2 M NaH2PO4, pH 7.4). The mice were decapitated and their cochleae dissected under a microscope. Dissected cochleae were fixed in 4% PFA at room temperature overnight, decalcified in 0.12 M ethylenediaminetetraacetic acid in PBS (pH 7.0) for 1 week, dehydrated in a graded ethanol series, and finally embedded in paraffin. Serial sections (6 μm) were stained with hematoxylin and eosin (H-E).
Transmission electron microscopy (TEM)
Animals were deeply anesthetized and perfused intracardially with PBS followed by 4% PFA and 2% glutaraldehyde (GA) in PB. Cochleae were opened, flushed with buffered 4% PFA 2% GA, and fixed an additional 2 h at room temperature. After washing, specimens were post-fixed in 2% OsO4 in PB for 1.5 h, then dehydrated in a graded ethanol series and embedded in Epon. Serial sections (1 μm) were stained using uranyl acetate and lead citrate, and examined by electron microscopy (H-7100, Hitachi, Tokyo, Japan).
Quantification and statistical analysis
We measured the area, the number of cells, and the number of apoptotic cells in the GER of both mutant and wild-type littermates. Measurements were performed at P8, P10, and P12. All counts were performed using mid-modiolar sections of H-E-stained cochleae.
To measure GER area, digital light micrograph images of the organ of Corti were captured using NIS Elements-D software (Nicon, Tokyo, Japan). Cell counts were obtained by analyzing a 50-μm segment of the GER extending from the inner hair cell toward the modiolus using a 100× objective. The middle turns of the cochlea were analyzed. Results are expressed as mean ± SEM. Statistical significance between two groups was analyzed using the Mann–Whitney U test. Analyses between three groups were performed using the Kruskal-Wallis test followed by Dunn’s Multiple Comparison test. A p value ≤0.05 was considered significant. Three animals from P8 and P10, and four animals from P12, were analyzed. Five sections were analyzed for each mouse.
Greater epithelial ridge
Hematoxylin and eosin
Transmission electron microscopy
This work was supported in part by research grants from the Ministry of Education, Science and Culture (to K.K.); the Ministry of Health, Labor and Welfare of Japan (to K.K.); and the MEXT-support program for the Strategic Research Foundation at Private Universities, 2011–2012 (to K.I.). We thank M.Yoshida in the Laboratory of Ultrastructure Research for help with TEM, and J. Onoda for experimental assistance.
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