Progression of inner ear pathology in Ames waltzer mice and the role of protocadherin 15 in hair cell development

Abstract

The Ames waltzer (av) mouse mutant exhibits auditory and vestibular abnormalities resulting from mutation of protocadherin 15 (Pcdh15). Ames waltzer has been identified as an animal model for inner ear pathology associated with Usher syndrome type 1F. Studies correlating anatomical phenotype with severity of genetic defect in various av alleles are providing better understanding of the role played by Pcdh15 in inner ear development and of sensorineural abnormalities associated with alterations in Pcdh15 protein structure as a result of gene mutation. In this work we present new findings on inner ear pathology in four alleles of av mice with differing mutations of Pcdh15 as well as varying alterations in inner ear morphology. Two alleles with in-frame deletion mutations (Pcdh15 (av-J) and Pcdh15 (av-2J)) and two presumptive functional null alleles (Pcdh15 (av-3J) and Pcdh15 (av-Tg)) were studied. Light and electron microscopic observations demonstrated that the severity of cochlear and vestibular pathology in these animals correlates positively with the extent of mutation in Pcdh15 from embryonic day 18 (E18) up to 12 months. Electron microscopic analysis of immature ears indicated early abnormalities in the arrangement of stereocilia and the inner and outer hair cell cuticular plates, stereocilia rootlets, and the actin meshwork within the cuticular plate. In severe cases, displacement of the kinocilium and alterations in the shape of the cuticular plate was also observed. Mice harboring in-frame deletion mutations showed less disorganization of stereocilia and cuticular plates in the organ of Corti than the presumptive functional null alleles at P0-P10. A slower progression of pathology was also seen via light microscopy in older animals with in-frame deletions, compared to the presumptive functional null mutations. In summary, our results demonstrate that mutation in Pcdh15 affects the initial formation of stereocilia bundles with associated changes in the actin meshwork within the cuticular plate; these effects are more pronounced in the presumed null mutation compared to mutations that only affect the extracellular domain. The positive correlation of severity of effects with extent of mutation can be seen well into adulthood.

Fig. 1

Mutation in the four alleles of Pcdh15 used in this study.

Fig. 2

Scanning electron micrographs of the surface of the organ of Corti in immature mice aged P0 (A–C), P5 (D–F), and P10 (G–I). The normal arrangement of stereocilia and kinocillia can be seen on the surface of the inner hair cells (IHCs, bottom row) and three rows of outer hair cells (OHCs, upper three rows) in tissues from heterozygous control mice (A, D, and G). The outline of the hair cells at the surface of the organ of Corti changes from a circular shape at P0 (A) to a triangular shape by P5 for OHCs (D) and an oval shape by P10 for IHCs (G). A more severe phenotype can be seen in the organs of Corti from mice with presumed functional null mutations (C, F, and I) than in in-frame deletions (B, F, and H). The OHC stereocilia bundles are more disorganized, and kinocilia placement is more affected (F arrow) compared to mice with in-frame deletions (E arrow). The stereocilia bundles of both the OHCs and IHCs are disorganized by P0 (compare C to B), but the extent of displacement is greater for OHCs and it becomes more apparent as the surface of the cells matures (F and I). The outline of the surface of OHCs is abnormal in all three rows in Pcdh15^av-3J mice by P5 (F), whereas the IHC outline is relatively unaffected up to P10 (I). Some alterations in the arrangement of the stereocilia and the position of the kinocillia can be seen at P0 in Pcdh15^av-J mice. By P5, the abnormal arrangement of stereocilia becomes more apparent (E) and an abnormal shape of the cuticular plate is seen Pcdh15^av-J by P10 (H). The arrangement of stereocilia on IHCs and the cell outline are less affected than that of OHCs. Bar = 5 μm.

Fig. 3

Scanning electron micrographs of the surface of the organ of Corti in immature mice aged E18. Microvilli and stereocilia cover the surface of the hair cells in heterozygous controls (A). Bare areas of the cell surface can be seen in many of the hair cells in Pcdh15^av-3J mice (B, D) with accompanying disorganization of stereocilia and microvilli. (C and D) Higher magnification view of A and B, respectively. At this age, kinocillia (arrows) can be located either in the center or at the periphery of the cell in either the control or the mutant mice. Bar = 5 μm.

Fig. 4

Transmission electron micrographs of sections through the hair cells in immature hair cells in heterozygous, control, and functional null mutant mice. (A, B) Cross sections through the surface of the inner hair cells of mice aged P2 showing some derangement in the cell from the functional null mutant. (C–F) Longitudinal sections through the cuticular plate and stereocilia bundle of P5 heterozygous and functional null mutant mice. (E, F) Higher magnification views from two other representative cells. No clear ultrastructural differences were found using this method of observation. Lateral links can be seen in both samples (E, F arrows).

Fig. 5

Transmission electron micrographs of cross sections through the surface of the organ of Corti in immature mice aged P0 (A, B, and C), P2 (D, E, and F) and P10 (G–L) near the level of insertion of the stereocilia into the cuticular plate. Normal arrangement of stereocilia and kinocilia can be seen in outer hair cells at P0 (A), P2 (D), and P10 (G) from heterozygous control mice. A dense staining mesh within the actin of the cuticular plate can be seen surrounding the stereocilia and kinocilia in the samples from heterozygous and in-frame deletion mice at P10 (J, K and L arrows). The actin mesh within the cuticular plate is not as well developed by P2 (D and E arrows). (B, E, and H) Tissue from homozygous mice with in-frame deletions in Pcdh15. A slight derangement can be seen in the rows of stereocilia near the surface of the cuticular plate at P0 (B) and P2 (E). The P10 specimen appears fairly normal, which was often seen in this group (H). (C, F and I) Tissue from homozygous mice with presumed functional null mutations in Pcdh15. The stereocilia near the surface are displaced to a greater degree than those seen in the tissue from the in-frame deletion. The rows of stereocilia on the surface of OHCs are frequently significantly disorganized in this group; often the kinocilium is also out of position (F and I arrows). At P10 (I), the actin mesh is present but out of place, still oriented to the abnormal positions of the stereocilia and kinocilium. I and L Bar = 500 nm.

Fig. 6

Light micrographs of cross sections of P15 mouse inner ears. Normal morphology can be seen in scala media cross sections from midbasal turns of cochleae from heterozygous control mice (A) and from mice homozygous for an in-frame deletion of Pcdh15 (D). Swelling of the supporting cells and shortening of outer hair cells with loss of the spaces of Nuel are seen in cross sections from mice homozygous for a presumed functional null mutation (G, arrow). Normal morphology can be seen in cross sections of the utricular (top) and saccular (bottom) maculi in control mice (B), mice homozygous for in-frame deletions (E), and mice homozygous for a presumed functional null mutations in Pcdh15 (H) at this age. A normal compliment of slightly immature spiral ganglion cells can be seen in cross sections of the modiolus of control mice (C), mice homozygous for in-frame deletions (F), and mice homozygous for presumed functional null mutations in Pcdh15 (I). Bar = 0.1 mm for A, D, G, and 0.2 mm for rest. Arrows point to upper basal spiral ganglion; * = artifact.

Fig. 7

Light micrographs of cross sections of inner ears from 6.5-month-old mice. Normal morphology can be seen in scala media cross sections from the midbasal region of the cochlea (A, arrow = organ of Corti), sections from the utricular (B, top) and saccular (B, bottom) maculi, and from the spiral ganglion (C) of ears from heterozygous control mice. Damage can be seen in the lateral wall, nerve fibers, and organs of Corti (D and G arrows) in cross sections from mice homozygous for in-frame deletions (D) or homozygous for presumptive functional null mutations in Pcdh15 (G), with a simple cuboidal epithelium often replacing the organ of Corti in the functional null group. Normal morphology can be seen in the utricular (E, top) and saccular (E, bottom) maculi from mice carrying the in-frame deletions. In the mice carrying the presumed functional null mutation (H), hair cell loss can be seen in areas of the saccular macula with partial or complete loss of saccular otoconia (arrow), whereas the morphology of the utricular macula is normal. Spiral ganglion cross sections from control ears (C) look normal from the midbasal turn (arrow) to apex, with only slight cell loss in the far basal end of the cochlea. Both mice homozygous for in-frame deletions in (F) and mice homozygous for presumptive null mutations in Pcdh15 (I) have lost the majority of their spiral ganglion cells at this age. Bar = 0.1 mm for A, D, G, and 0.2 mm for rest; * = artifact.