Deiters' cells in thin sections. (A) P25 control mouse. Plasma membranes of the adjacent cells are closely parallel with no space between. Inset shows higher magnification of a region of the apposition (boxed) that reveals the typical septilaminar appearance of a gap junction plaque in section. Arrows indicate limits of gap-junction-like structure. (B) Cx30 null mice at P17. There are numerous separations of the adjacent plasma membranes. Where the membranes meet over short distances (arrows), there is no space between them as in gap junction plaques. Inset shows region of close apposition at higher magnification in tissue from P30 animal; the appearance is similar to that of a gap junction. (C) Cx26 conditional knockout mouse at P60. Adjacent membranes are closely parallel with no space between them. Arrows indicate limits of gap-junction-like structure. Inset shows detail of gap-junction-like structure in the boxed region of the apposition. Scale bars: 0.5 µm (A), 50 nm (inset A); 1 µm (B), 0.25 µm (C).

Gap junction plaques exposed by freeze-fracture. (A,B) Plasma membranes of wild-type Deiters' cells. (A) Full face view: the gap junction plaques are outlined. The plaques are very large and appear as closely packed particles on the protoplasmic face or pits on the exoplasmic face where the fracture plane has jumped from the membrane of one cell to that of the other with which it is coupled. (B) Partially side-on view of the membrane. The gap junction plaque extends the full length of the membrane profile. Exoplasmic and protoplasmic faces are indicated. (C–E) Deiters' cells in Cx30 null mouse. (C,D) Similar views of the membrane fracture faces as shown in A and B, respectively. There are several small plaques (indicated by arrows in C), each consisting of a small number of connexons. (E) Arrows point to rows of closely packed particles of a size equivalent to connexons, indicating small linear junction plaques. (F) Hensen's cell in Cx30 null mouse. A large gap junction plaque is indicated by the asterisk. The arrows indicate square arrays of particles and pits that are a characteristic of the membrane fracture faces of Hensen's cells. e, exoplasmic; p, protoplasmic. Scale bars: 100 nm (A–E), 200 nm (F).

Gap junctional dye transfer is restricted in cochlear slice preparations from Cx30 null mice. (A) DIC image of organ of Corti at P15. Arrows indicate OHC. (B) Distribution of neurobiotin (Nbn) after its injection into a single P15 Deiters' cell is indicated by the asterisk. Nbn transfers to all other Deiters' cells and in to the outer pillar cell, as well as to Hensen's cells and the cells to the outer side. Dye does not pass to OHC, the positions of which are indicated by the arrows, demonstrating that hair cells are not coupled to supporting cells. (C,D) Cochlear slice from Cx30 null mouse at P15. (C). Nbn injected into a single Deiters' cell (indicated by the asterisk) transfers to only a few other Deiters' cells. It does not transfer to Hensen's cells. (D) Nbn injected into a single Hensen's cell (indicated by the asterisk) transfers to many other Hensen's cells and to the other cells on the lateral side, but it does not transfer medially to Deiters' cells. Dc1-Dc3, Deiters' cells; Hc, Hensen's cell; bm, basilar membrane. Scale bars: 10 µm.

Aberrant repair by supporting cells following hair cell loss in Cx30 null mice. (A) Normal, undamaged organ of Corti in control mouse. There are three rows of OHC, each one separated from its neighbour by the intervening heads of supporting cells. The cells to the outer side have broken away to reveal the body of the organ of Corti. Dieters' cells consist of a cell body region, and a thin phalangeal process that rises up to the luminal surface where the head expands to fill the space between OHC. Within the body of the organ of Corti are large extracellular spaces around the body of the OHC and the phalangeal processes of the supporting cells. (B,C) Stereotypical pattern of repair in organ of Corti following hair cell loss in control mice. (B) Repaired organ of Corti following OHC loss induced by combined administration of kanamycin and bumetanide (48 hours post-treatment). Stereotypical regular mosaic-like pattern of cells at the luminal surface is created by expansion of the heads of supporting cells into the sites from which OHC have been lost. The position where an OHC in each row once was is outlined in green. The participation of the supporting cells that surround each OHC in the lesion closure is outlined in red. Arrow indicates initiation of retraction of the heads of inner pillar cells exposing the apical surface of the outer pillar cell. (C) Retraction of heads of inner pillar cells in Ptprq mutant mouse. (D–G) Repair in Cx30 null mice. The site of a lost OHC is filled by the head of only a single supporting cell. The border of the head of the supporting cell effecting lesion closure is outlined in some instances. Examples of the head of the inner pillar cell replacing 1st row OHC are shown in D and G. There is no consistency in which of the supporting cells that contacted an OHC in a particular row expands to fill the space. (H,I) Phalloidin labeling of F-actin outlining the cell junctions. (H) Continuity of the cell border of an inner pillar cell that has expanded into the space of a lost first row OHC. (I) Single 1st row Deiters' cell that expanded to replace an OHC in the second row. Dc1-Dc3, Deiters' cells in rows 1, 2 and 3; ip, inner pillar cell; op, outer pillar cell. Scale bars: 10 µm (A–C), 2 µm (D–F), 10 µm (G–I).

Lack of supporting cell expansion following hair cell loss in Cx30 null mice. (A,B) Control mouse. The body of the organ of Corti after hair cell loss (48 hours following kanamycin-bumetanide treatment). (A) Viewed from the lateral side, cells on the outer side have broken away revealing bodies of Deiters' cells. (B) Radial view of a break across the organ of Corti. The phalangeal processes of Deiters' cells have widened and the large extracellular spaces normally present in the undamaged tissue have been occluded. (C) Conditional Cx26 knockout mouse. Surface view of the flat, squamous-like epithelium generated by migration of cells from the outer-side of the organ of Corti across the region normally occupied by the columnar supporting cells. Some heads of surviving inner pillar cells, and some surviving inner hair cells are evident. (D–E) Cx30 null mice. Views of the body of the organ of Corti, similar to those shown in A and B, after hair cell loss, at 2 months old (D) and 6 months old (E). There is no expansion of the phalangeal processes of Deiters' cells and the large extracellular spaces remain. (F) Cx30 null mice. Views of the body of the organ of Corti, similar to those shown in E, at 6 months of age. The layer of cells to the outer side of organ of Corti has moved upwards to approach the Reissner's membrane, creating enlarged extracellular spaces within the organ of Corti, in which the thin phalangeal processes of supporting cells persist. IHC, inner hair cell; ip, inner pillar cell; op, outer pillar cell; Rm, Reissner's membrane; tC, tunnel of Corti. Scale bars: 10 µm.