Immunolabelling following the progressive re-modelling of the repaired epithelium.

<p><b>A.</b> Immunolabelling for acetylated tubulin (green) reveals microtubules in cells across the Deiters' cell region approaching the inner pillar cells. At the arrow, a nucleus in the position of the tunnel of Corti is associated with microtubules extending back into the Deiters' cell region. The heads of some inner pillar cells (ip) are intensely labelled for acetylated tubulin whereas in others the labelling is significantly reduced or absent (larger arrows) suggesting depolymerisation of the microtubule bundles in pillar cell heads. <b>B.</b> Labelling for KCC4 (green) is apparent in cells in the location of, and entering, the tunnel of Corti (arrows) Inner hair cells are labelled for calretinin (red). <b>C.</b> Cell labelled for GLAST (arrow) in the location of the tunnel of Corti. <b>D.</b> Labelling for Cx26. Large plaques of labelling around the borders of cells across the entire outer side of the organ of Corti up to the level of the inner pillar cells. The pattern of labelling is consistent with that of Hensen's and Claudius' cells. Some inner hair cells labelled for calretenin survive and these cells retain innervation (arrows), also immunolabelled by antibodies to calretinin. Scale bars: 5 µm.</p

Morphological characteristics of the squamous-like epithelium.

<p><b>A. C57BL/6 mouse at 6 weeks post-treatment.</b> Actin labelling in whole mount preparation of the apical coil. The arrow indicates the apical tip of the organ of Corti. A patch of the flat epithelium, indicated by the larger arrows, interrupts the strip of repaired epithelium. Scale bar: 100 µm. <b>B. C57BL/6 mouse at 4 weeks post-treatment.</b> Mid-basal region of the cochlea viewed towards the lateral wall (from inside outwards). Pillar cells (ip) are still erect either side of a patch of flattened epithelium. The sheet of flattened epithelial cells is continuous with the cells of the outer sulcus (os). Scale bar: 10 µm. <b>C.</b> Edge of squamous patch where it cuts across the repaired epithelium. The enlarged surfaces of cells that normally reside on the outerside of Deiters' cells curve round the end of the strip containing Deiters' cells (Dc) and pillar cells (ip). IHC still present (arrow). Scale bar: 10 µm. <b>D.</b> The apicalmost tip of the organ of Corti. The surfaces of cells either side of the strip of cells containing Deiters' and pillar cells, curve across the termination point of that strip in a similar pattern to that at the edges of the patches of squamous-like epithelium. The surface features of those cells are also similar to those that create the flat epithelium. Scale bar: 10 µm. <b>E.</b> Thin section through a region of flattened epithelium. The cells across the basilar membrane are all similar in morphology and show relatively few organelles or other cytoplasmic specialisations. Their features are reminiscent of those of Claudius' cells. Scale bar: 2 µm. <b>F</b> and <b>G.</b> Details of cell forming the flat epithelium. The cell cytoplasm is relatively unstructured and there are few organelles. Large gap junction plaques occupy much of the plasma membrane along the contact between adjacent cells. The extent of two gap junctions are defined by the pairs of arrows in panel F, the lower contact region viewed at higher power showing the characteristic thin section morphology of a gap junction. Annular gap junctions are also evident in the cell cytoplasm (arrow in panel F), suggesting continuing turnover of gap junction plaques. Scale bars: 1 µm in F; 0.1 µm in G.</p

Primary antibodies used in this study.

<p>Primary antibodies used in this study.</p

Macrophages in thin sections.

<p><b>A. CBA/Ca mouse 4 weeks post-treatment.</b> All OHC lost, debris cleared and Deiters' cell expanded. A cell with morphological characteristics of a macrophage in the tunnel of Corti closely abuts the inner pillar cell. The region of close contact indicated by the arrow is shown at higher power in panel B. Scale bar: 2 µm. <b>B.</b> Detail of the contact region between the macrophage and the inner pillar cell. The plasma membranes of the cells are closely apposed and parallel to each other. Endocytotic opening on the inner pillar side (upper arrow) and protrusion from inner pillar cell enclosed by macrophage (lower arrow) indicate activity reminiscent of an immune synapse between the cells. Scale bar: 0.5 µm. <b>C.</b> Region of the habenula perforata – the opening through the bony lip by which neural dendrites enter the organ of Corti - below the inner phalangeal cells (iph) and IHC. A cell which is pleomorphic in shape with an irregularly shaped nucleus, characteristics of macrophages, is present within the habenula (arrow). This may indicate that macrophages can gain access to the organ of Corti along the nerve tract. Scale bar: 2 µm.</p

The organ of Corti of the mouse.

<p>Labelling for all figures: OHC = outer hair cell, IHC = inner hair cell; Dc = Deiters' cell, ip = inner pillar cell, op = outer pillar cell, Hc = Hensen's cell, Cc = Claudius cell, Bc = Boettcher's cell, os = outer sulcus, iph = inner phalangeal cell; ib = inner border cell, is = inner sulcus; tunC = tunnel of Corti, otun = outer tunnel; bm = basilar membrane. <b>A</b>. SEM of the apical surface of the organ of Corti (the “reticular lamina”). Each hair cell is separated from its neighbours by intervening supporting cells. The luminal surfaces of the Hensen's cells (HC) are distinguished by numerous microvilli. Scale bar: 10 µm. <b>B</b>. Confocal projection series image at the level of the reticular lamina of phalloidin-FITC labelled whole mount preparation. Phalloidin labels filamentous actin of the hair cell stereocilia, and the cuticular plate beneath the hair bundle in the apical cytoplasm of the hair cell, as well as the filamentous actin associated with the intercellular junctions. The arrow indicates the prominent, thick band of actin associated with the junctions between adjacent supporting cells in the OHC region. Scale bar: 5 µm. <b>C</b>. Montage of low power TEM images of thin section across the organ of Corti of the basal turn to show locations of all the cell types referred to in the text. The central strip of the sensory region containing the hair cells interspersed with specialised columnar supporting cells, is flanked either side with less specialised cells; the cuboidal inner sulcus cells to the inner side, and various cells types to the outer side. Scale bar: 10 µm. <b>D</b>. Thin section of sensory region of the organ of Corti showing some of the structural specialisations of the different supporting cell types. Arrow points to microtubule bundle in Deiters' cell. Bundles of microtubules are especially prominent in the pillar cells. Scale bar: 5 µm. <b>E</b>. The reticular lamina in thin section showing features at intercellular junctions. The head of the inner pillar and outer pillar cell are filled with microtubules, whereas the junction between an outer hair cell (OHC) and a Deiters' cell is characterised by microfilament assemblies, appearing as electron dense structures running the depth of the junction and widely into the supporting cell. Scale bar: 1 µm.</p

Immunolabelling of Macrophages.

<p><b>A–C Undamaged organ of Corti.. </b><b>A A'.</b> Immunolabelling for CD45 (red) and for prestin (green) to mark OHC. Focus at the level of the body of the organ of Corti in A and the basilar membrane in A'. In A, macrophages in the nerve tract but none within the organ of Corti itself. Macrophages on the underside of the basilar membrane in A'. Scale bar: 50 µm. <b>B,C.</b> F4-80 in frozen sections. B. Macrophages (indicated by arrows) within the ligament of the lateral wall and in the nerve tract (arrows). C. Macrophages (arrows) on the underside of the basilar membrane. Scale bars: 20 µm. <b>D–F. Following hair cell damage.. </b><b>D D'. C57BL/6 at 24 hours post treatment.</b> D. Focus at the level of the body the organ of Corti shows debris of degenerating OHC labelled for prestin (green). There are no cells labelled with macrophage marker CD45 at this level indicating they are absent from the body of the organ of Corti at the time when hair cell degeneration is occurring. D'. Focus at the level below the organ of Corti reveals CD45 positive cells (red), remain on the underside of the basilar membrane. Scale bar: 20 µm. <b>E. CBA/Ca mouse; 48 hours post treatment.</b> Frozen section labelled for cells expressing F4-80 (green). Macrophages are present on the underside of the basilar membrane and in the nerve tract (arrows) but are absent from the body of the organ of Corti at a time when OHC loss is on-going. Scale bar: 10 µm. <b>F. CBA/Ca mouse 7 days post-treatment.</b> Frozen section labelled for cells expressing F4-80. All OHC have been lost and all debris cleared by this time. Large cell of irregular shape expressing the macrophage marker F4-80 (green) is within the tunnel of Corti. Scale bar: 10 um.</p

Initial progression of re-modelling of the repaired epithelium.

<p><b>A. C57BL/6 mouse, 2 weeks post-treatment.</b> Extension of tectal cell head into the repaired epithelium (arrow). The regular cellular pattern of the repaired reticular lamina is disrupted (asterisk). Scale bar: 5 µm. <b>B. C57BL/6 mouse 2 weeks post-treatment.</b> Phalloidin labelling indicates actin assembly at leading edge of the cell advancing into the Deiters' cell region (arrow). IHC are still present (arrowhead). Scale bar; 5 µm. <b>C. C57BL/6 mouse, 4 weeks post-treatment.</b> Head of tectal cell expanded (arrow) as far the first row Deiters' cell head. IHC are still present (arrowhead). Scale bar: 5 µm. <b>D. CBA/Ca mouse, 6 months post-treatment.</b> Hensen's cells, identifiable by the dense microvilli, cover Deiters'cells (Dc) and contact with the inner pillar cells (ip). IHC are still present (arrowhead). Scale bar: 10 µm. <b>E. CBA/Ca mouse, 6 months post-treatment.</b> The apical heads of the Deiters' cells (Dc) appear to be extruding from the apical surface (arrow). Scale bar: 5 µm. <b>F. C57BL/6 mouse; 13days post-treatment.</b> Hensen's cell (Hc) extends towards the pillar cell region. The third row Deiters' cell (arrow) appears to be fragmented; its basal aspect show features of cellular degeneration (shown at higher power in panel G). The bodies of the Deiters' cells within the corpus of the epithelium appear intact with prominent microtubule bundles. Scale bar: 5 µm. <b>G.</b> The apical fragment of Deiters' cell indicated by the arrow in F. The cytoplasmic continuity of the most apical junctional region with more basal denser region enclosed within convoluted plasma membrane is apparent. Series of thin sections through this structure confirmed this as a fragment of one cell. Scale bar: 1 µm. <b>H.</b> Section through the reticular lamina in a region where Hensen's cell is approaching the head of an outer pillar cell (op). The head of the outer pillar cell is recognised by the microtubule bundles. Only a single microfilament assembly (arrow) characteristic of the junction of a Deiters' cell is evident, suggesting withdrawal, or loss, of Deiters' cell heads from the apical surface of the epithelium (compare with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030577#pone-0030577-g001" target="_blank">Figure 1E</a>). Scale bar: 2 µm.</p

Repaired organ of Corti following hair cell loss.

<p><b>A. CBA/Ca mouse; 7 days post-treatment.</b> All OHC lost but all IHC remain. The heads of Deiters' cells and of the outer pillar cells have expanded into the regions where OHC used to be. A regular pattern of cells is created at the epithelial surface. Scale bar: 10 µm. <b>B. C57BL/6; 14 days post-treatment.</b> Phalloidin-FITC labelled whole mount. Wide bands of actin persist at junctions between adjacent supporting cells. Inner hair cells have been lost and the heads of inner border and inner phalangeal cells have expanded to close the lesions. Scale bar: 10 µm. <b>C. CBA/Ca; 24 h post-treatment.</b> OHC lost and Deiters' cell heads have expanded to close the lesions at the luminal surface. There is some widening of the phalangeal processes but extracellular spaces are still open. Arrows indicate dense, microfilament assemblies at the junctions between the adjacent cells that are present in each of the three Deiters' cells. Larger arrowhead denotes microtubule bundle in Deiters' cell. Scale bar: 5 µm. <b>D. CBA/Ca; 14 days post-treatment.</b> Deiters' cells have generally expanded. The outer tunnel is closed but some extracellular spaces in the Deiters' cell region and the tunnel of Corti are still open. The overall architecture is preserved in the repaired epithelium. Scale bar: 10 µm. <b>E. CBA/Ca; 48 h post-treatment.</b> Expansion of the 3^rd row Deiters' cell closes the outer tunnel. Microtubule bundles (large arrowhead) and microfilament assemblies at the intercellular junctions are maintained in Deiters' cells. Scale bar: 5 µm. <b>F. CBA/Ca; 3 months post-treatment.</b> IHC as well as OHC are lost, but the tunnel of Corti is open and the cellular arrangement of the repaired organ of Corti is preserved. Scale bar 10 µm. <b>G. C57BL/6; 4 weeks post-treatment.</b> Both IHC and OHC are missing. The regular pattern of the cells at the surface of the repaired epithelium is maintained. Scale bar: 10 µm.</p

Deiters' cell migration.

<p><b>A.</b> Tectal cell (tc, dense cytoplasm) followed by Hensen's cell (Hc, microvilli at the surface) extends across the Deiters' cell to contact the head of the outer pillar (recognised by the microtubule bundles parallel to luminal surface, mt). The Deiters' cell bodies (Dc) fill the entire epithelium up to the outer pillar cell phalangeal processes, but retain their cytoplasmic characteristics, including organised microtubule bundles at the cell base and within the cell body (arrows), but they appear to be entirely enclosed within the epithelium with no exposure to the apical surface. Scale bar: 5 µm. <b>B</b> and <b>C.</b> Cells filling the tunnel of Corti in repaired epithelium with intact pillar cells. In B, the angle of section has revealed the phalangeal process of one outer pillar cell (op1) and the cell body of its neighbour (op2). A cell is filling the tunnel of Corti and spreads to the outer side through the gap that normally exists between adjacent outer pillar cells. Its cytoplasmic characteristics are consistent with those of a Deiters' cell. In C, the epithelium is cut at a larger angle relative to the perpendicular through the epithelium to reveal several outer and inner pillar cells. The cell (asterisk in nucleus) occupying the space between the inner and outer pillar shows the charactersitics of a normal Deiters' cell (Dc). Its nucleus appears normal with no evident apoptotic or necrotic features. In both B and C, IHC are lost and expansion of the inner border and inner phalangeal cells has closed the lesion (arrowhead). Scale bars: 5 µm.</p

A–C. Further progression of re-modelling on the lateral side of the pillar cell region.

<p><b>A.</b> The outer (lateral) side of the organ of Corti during re-modelling to show relative positions of different cell types. Boettcher's cells (Bc) are in position at the lateralmost side and appear to form the outer border. Deiters' cells (Dc) are flattened and covered by Claudius' and Hensen's cells (Cc, Hc) that have expanded medially (towards the pillar cells), but they retain their specialised microtubule bundles (arrows). Scale bar: 5 µm. <b>B.</b> The body of the outer pillar (op) is flattened and covered by Deiters' cells, but prominent, organised microtubule bundles are retained (arrows). The head of the outer pillar appears to have separated from the cell body as Deiters' cell spreads through to the tunnel of Corti. Scale bar: 5 µm. <b>C.</b> Cell with numerous microvilli at the apical surface and the cytoplasmic characteristics of Hensen's cell (Hc) in contact with inner phalangeal cell (identified by the dense cytoplasm) at the approximate site where the IHC used to be. A cell with cytoplasmic characteristics of Claudius' cell (Cc) expands across the apical surface of the epithelium. One inner phalangeal cell encloses cell debris (arrow). Scale bar: 5 µm. <b>D–F. Inner pillar cell during remodelling. </b><b>D.</b> Heads of inner pillar cells (ip) retract to expose the apical surfaces of the outer pillar cells beneath. Scale bar: 5 µm. <b>E.</b> The retracting tip of the head of the inner pillar cell is associated with a sub-membrane density that resembles the thin section appearance of a microfilament assembly. Scale bar: 2 µm. <b>F.</b> The inner pillar cell becomes flattened and covered by the expanding inner phalangeal and inner border cells but it retains prominent organised microtubule bundles (arrows). Scale bar: 2 µm.</p