Validation of TMX-inducible cCx26 null mice was described in our published paper [10
]. Here we describe the data validating the two new cCx26 null mouse models obtained by deleting the Gjb2
in a spatially-specific manner. Patterns of Cre recombinase expression in the cochlea of foxg1-Cre () and pax2-Cre () cCx26 null mice were examined by utilizing the R26R LacZ reporter mice [9
], which demonstrated strong Cre activities in the organ of Corti, spiral limbus and the spiral ganglia. In contrast, the Cre activation in the stria vascularis and lateral wall was weak (). Consistent with this pattern, Western blot quantification of Cx26 protein levels revealed that the largest reduction was in the organ of Corti and spiral limbus (, panel on the top). Comparing to the wild type (WT) controls, the Cx26 protein in the organ of Corti of foxg1-Cre and pax2-Cre cCx26 null mice was 11.2±3.3% and 9.1±4.2%, respectively (n=4). In contrast, changes of Cx26 protein in the spiral ligament and stria vascularis were both statistically insignificant (p>0.05) (, panels in the middle and bottom). This pattern of Cx26 reduction in the cochlea of cCx26 null mice was supported by immunolabeling data (). Since Cx26 and Cx30 are co-expressed in almost all cochlear GJs [1
], co-immunolabeling with an antibody against Cx30 was used as a control. Cx30 immunoreactivities were identified in the cell membrane of supporting cells in the organ of Corti (, top left panel) and spiral limbus (, top right panel), presumably forming homomeric GJs in the cochlea of cCx26 null mice. In contrast to the uninterrupted network of GJs suggested by Cx30 immunoreactivity (, top panels), the pattern of Cx26 showed absence of Cx26 in most of the cells (red fluorescence in , bottom two panels). The incomplete deletion of Gjb2
in some cells is demonstrated by arrows in showing examples of some cells in the spiral limbus where Cx26 was still detected.
Figure 1 Validation of cCx26 null mouse models by various methods. Cre activities in the cochlea driven by the foxg1 (A) and pax2 (B) were reported by the LacZ staining in R26R reporter mice. Abbreviations: SLi: spiral limbus; RM: Reissner’s membrane; (more ...)
The foxg1-Cre (n=60) and pax2-Cre (n=48) cCx26 null mice have normal body weight and are fertile with normal litter size. These mouse models for human Cx26 mutations were confirmed further by the non-syndromic deafness displayed in all the mutant mice (). ABRs measured across a frequency range of 4–32 kHz showed that hearing thresholds in these mutant mice were elevated by about 40–50 dB () comparing to their littermate-control mice that didn’t carry the Cre gene (n=17). In summary, four independent lines of evidence presented above support that we have obtained valid mouse models for studying deafness caused by conditional Cx26 null expression in the cochlea.
Using the cCx26 null mice, we first examined whether prenatal cell differentiation in the organ of Corti is completed. Immunolabeling with specific hair cell markers showed typical structure of one row of inner HCs (arrowhead in ), three rows of outer HCs (small arrows in ) in both WT () and mutant () mice. Patterns of supporting cell arrangements around the HCs were examined with antibodies against p75 (horizontal arrows in ) and Prox1 (green fluorescence in ). Results obtained from WT () and cCx26 null mice () were indistinguishable. These immunolabeling results suggested that hair cell and supporting cell differentiation in the organ of Corti of cCx26 null mice was not affected.
Figure 2 Cell differentiation and degeneration in the organ of Corti. (A–D) Cellular structures of the organ of Corti at P0 were compared between WT and cCx26 null mice. Hair cell and supporting cell specific markers were immunolabeled. (E–G) A (more ...)
Gross cochlear morphology in cCx26 null mice appeared to be normal, with no obvious collapse or expansion of the Reissner’s membrane. No apparent defects were observed in the development of the tectorial membrane, inner spiral tunnel and the stria vascularis (, and ). By observing cochlear samples obtained at a series of postnatal development stages, we found that the initial degeneration in the organ of Corti of cCx26 null mice was in the Claudius cells around P8 (arrows in ). In contrast, the HCs showed normal orientation and arrangement of their stereocilia at the same developmental stage (). compares morphology of the organ of Corti of WT and three cCx26 null mouse models obtained at a series of developmental stages. All three mouse models yielded consistent spatial and temporal morphological changes in the organ of Corti. Inner and outer HCs are present in both WT (, arrowheads in all the panels) and cCx26 null (, small arrows) mice at birth. General structures of the organ of Corti were indistinguishable between WT and mutant mice before P9. A major difference emerged at around P9 when the tunnel of Corti and the Nuel’s space in the middle cochlear turn opened in the WT (, panels in the first column) but not in any of the cCx26 null mice (, upward white arrows). The development of tectorial membrane () and the inner spiral tunnel () appeared to be normal in the mutant mice. Dramatic cell death occurred around P13 in the outer HCs and the surrounding supporting cells (big black arrowheads in ). Consistent with the results obtained from the whole-mount cochlear preparations (), Claudius cells in cochlear sections also show signs of degeneration before the outer HCs die (big arrowhead in ). The inner HCs, in contrast, were intact judged by the presence of stereocilia (pointed by small arrowheads in ) at the time when outer HCs were disintegrated (big arrowheads in ). The tunnel of Corti was still closed at P13. Presence of intact pillar cells was supported by the two nuclei (upward white arrows in ).
Figure 3 Comparison of the morphology of the organ of Corti at the middle turn in WT (panels in the first column) and three cCx26 null mouse models at P4 (top panels), P9 (middle panels) and P13 (bottom panels). Abbreviations: TC: tunnel of Corti; TM: tectorial (more ...)
Figure 4 Comparison of the morphology of the organ of Corti in WT (A&D) and cCx26 null mice at middle (B&E) and apical (C&F) turns. Cochlear samples obtained at P16 (A, B&C) and P30 (D, E&F) are given. Whole cochlear sections (more ...)
Only a layer of non-specific epithelial cells were left in the middle turn organ of Corti around P16 (). By P30, cells in a large segment of the middle turn in the organ of Corti were dead (). Cell degeneration progressed gradually from the middle turn to the basal turn. A dramatic contrast is that all types of cells in the apical turn organ of Corti survived for at least six months (longest period we have observed so far). Both the nuclei (white arrows in ) and stereocilia (horizontal arrowheads in ) of inner and outer HCs are present. The tunnel of Corti and the Nuel’s space (TC in ), however, were never opened in the mutant mice (upward arrowheads in ). The spiral ganglion neurons were also degenerated in the corresponding locations. While neurons in the apical turn were essentially intact (), most of them in the middle turn disappeared by P30 in the Rosenthal’s canal in the cCx26 null mice ().