adds to the growing series of Pcdh15 alleles (), some of which carry mutations similar to those reported in humans (Ahmed et al., 2001
). Characterizing these alleles has helped us understand which cell types in the mouse inner ear require Pcdh15 and whether Pcdh15 is required for normal hair cell development. Findings from morphological studies and physiological testing have raised some important questions (as mentioned previously in the introduction) regarding the role for Pcdh15 in hair cell function and maintenance. Identification of new alleles of Pcdh15
may help to answer some of these questions.
The av6J allele is maintained in the B6 inbred genetic background. It is well known that the B6 mouse develops premature presbycusis due to the recessive Ahl
(age-related hearing loss) allele (Erway et al., 1993
; Keithley et al., 2004
). B6 mice develop normal hearing function and show no inner ear pathology until they are several months old; mice homozygous for the av6J mutation fail to develop hearing function and show hair cell pathology several weeks after birth. Further, a previous report showed that mutation in Pcdh15
causes a similar ear phenotype in the FVB/N inbred background (Alagramam et al., 1999
). Therefore, the early hearing loss and associated hair cell pathology in the homozygotes are due to the recessive, loss-of-function Pcdh15
mutation and not due to the B6 background.
This study demonstrates that in the av6J mutant postnatal degeneration of the organ of Corti occurs over a time course similar to that observed in other av alleles, starting with alterations in outer hair cell stereocilia a few days after birth and progressing to severe neuroepithelial degeneration affecting all cochlear turns by P40. Alterations in configuration of outer hair cell stereocilia were observed at the earliest time point included in this study (2 days postnatal). At least up to P10, abnormalities of the organ of Corti appear to be limited largely to the stereocilia and cuticular plates of hair cells. Light microscopic examination of cochlear cross sections from animals aged P2 through P10 showed normal development of the sensory epithelium without apparent defects of the cell bodies of hair cells or supporting cells. This observation is consistent with the pattern of degeneration seen in other av
alleles in which alterations of OHC stereocilia are the first observable abnormalities occurring in the organ of Corti. Although stereocilia defects are a prominent feature during the initial stages of neuroepithelial degeneration in the av6J allele, alterations in the arrangement of stereocilia do not appear to be as severe as those previously observed in av
alleles with presumptive null mutations (Alagramam et al., 1999
; Hampton et al., 2003
; Raphael et al., 2001
). Loss of outer hair cell stereocilia, most often seen in the central portion of individual hair bundles and first observed at P10, is likely to be indicative of early-stage degenerative changes in the sensory cells. By P20 those changes advanced to degeneration and loss of both inner and outer hair cells, which occurred in a scattered, patchy fashion throughout the organ of Corti. In addition to sensory cell loss, scattered, focal lesions were seen at P20 in which there was also degeneration of supporting cells, leading to collapse of the entire sensory epithelium in the affected areas. At P40 there was more widespread loss of sensory and supporting cells, together with a reduction in the spiral ganglion cell population in the most severely affected areas. Although there were severe degenerative changes at P40, the overall configuration of the cochlear duct remained normal. Reissner’s membrane was in normal position and no abnormalities were apparent in the stria vascularis. This finding is consistent with all other av
alleles examined to date and is characteristic of cochlear degeneration of the neuroepithelial type.
The av6J allele, like the avJ and av2J alleles described earlier (Pawlowski et al., 2006
), is an inframe deletion mutation affecting the cadherin domains of the Pcdh15 molecule. All three inframe deletion mutants show hair cell phenotypes that are mild compared to presumptive null mutants av3J or avTg at a young age (P0–P10). Slight differences in phenotype can be detected between the three inframe deletions; avJ mutants tend to have more abnormal cells which demonstrate rotated or clumped stereocilia bundles and only a few abnormal cells that have missing stereocilia at the center of the bundle, whereas the latter anomaly is more commonly seen in av6J’s. The av2J phenotype varies from a phenotype similar to the avJ and av6J mutants to one similar to that of the functional nulls, where most of the hair cells have abnormal stereocilia bundles (Alagramam et al., 1999
; Hampton et al., 2003
; Raphael et al., 2001
). Therefore, the mutations affecting various cadherin domains have similar but slightly different effects on the development and integrity of the stereocilia bundle.
The av6J allele harbors an intragenic deletion that disrupts the expression of exon 22. The genotype–phenotype correlation was 100% since all mice with auditory and vestibular deficiency failed to express exon 22. Splicing of exon 21 into 23 is predicted to result in the deletion of most of the amino acid coding for the nineth cadherin domain, but the rest of the Pcdh15 protein is predicted to be inframe. The hair cell defect observed in av6J during the first two weeks after birth is less severe compared to hair cells from presumptive null alleles at the same time point (ex. Pcdh15av-3J or Pcdh15av-nmf19). The hair cell phenotype observed in av6J is similar to, but slightly less severe than, those observed in the Pcdh15av-J allele, which is consistent with the nature of the mutations present in the Pcdh15av-J and Pcdh15av-6J alleles. Mice homozygous for the 6J mutation fail to develop normal hearing or balance function, similar to that observed for Pcdh15alleles reported previously. These results suggest that the cadherin domains of Pcdh15 are not dispensable and that even small changes in the Pcdh15 protein could lead to hair cell dysfunction.
Six mouse mutants carrying various mutations in Pcdh15
have been reported thus far, all demonstrating loss of auditory function early in life. The absence of hair cell function, as measured by lack of transduction currents and dye-uptake studies is thought to be a consequence of defects in stereocilia bundle morphogenesis in Pcdh15-deficient mice (Alagramam et al., 2005
; Senften et al., 2006
). However, since hair bundle morphogenesis is dramatically affected in most Pcdh15
mutants, none of the studies discussed above excludes the possibility of a direct role for Pcdh15 in mechanotransduction. Pcdh15 mutants demonstating hearing and balance dysfunction but with stereocilia relatively intact at a young age may provide useful models to test for a direct role for Pcdh15 in mechanotransduction.