Supporting cell cytoskeleton during development of the organ of Corti in rat JOHNEN Nicolas, THELEN Nicolas, MALGRANGE Brigitte, THIRY Marc Introduction In mammals, the perception of sound is mediated by an epithelial sensory patch located in the cochlear region of the inner ear named the organ of Corti (OC) [Kelley and Bianchi, 2002 ; Kelley, 2006]. The latter is componed of mechanosensory hair cells and nonsensory supporting cell types. The hair cells are modified epithelial cells that utilise a group of derived microvilli, referred to as stereocilia, to perceive pressure waves induced through sound. Based on their morphology and physiology, two types of hair cells can be distinguished: inner and outer hair cells. Likely, at least four types of supporting cells can be identified in the OC: inner pillar cell, outer pillar cell, inner phalangeal cell and Deiters’ cells. These cells are arranged in a regular mosaic pattern running along the length of the snail-like cochlea from base to apex. One of the most striking aspects of this mosaic is that specific cell types are arranged in discreet rows. The edge of the OC located closest to the modiolus is composed of a single row of alternating inner hair cells and inner phalangeal cells. The edge of the Corti's organ located closest to the stria vascularis is composed of three rows of outer hair cells and Deiters’cells that are also arranged into a regular alternating mosaic. Finally, the single row of inner hair cells and the three rows of outer hair cells are separated by the tunnel of Corti that is a space delimited by a single row of inner pillar cells and a single row of outer pillar cells. The formation of Corti’s tunnel appears after birth in rat [Roth and Bruns, 1992]. Although the structure of the auditory organ in mature mammals, the organ of Corti, is clearly established, its development is far to be elucidated. Using antibodies against different proteins cytoskeleton (tubulin (tubulin beta IV), cytokeratins pan (CK 1, 4, 5, 6, 8, 10, 13, 18, and 19) and vimentin (V9)),we investigated by confocal microscopy the setting up of supporting cells' cytoskeleton during the differentiation of the OC in rat from the embryonic day 18 (E18) to postnatal day 15 (P15). Results Tubulin beta IV Cytokeratins pan Vimentin V9 Conclusions We showed that inner pillar cells are labelled with an anti-tubulin beta IV antibody from P0. Using an antibody to cytokeratins, a labelling appeared in Deiters' cells from E22. We also revealed that during the development of the OC, supporting cells were labelled with anti-vimentin antibody from P0. Surprinsingly, supporting cells, which are epithelial cells, are labelled with vimentin at P15. Further investigations will be necessary to understand the development of the supporting cell cytoskeleton. Figures A-G: Spatiotemporal distribution of tubulin beta IV during the mammalian auditory organ development from E22 to P15 in the basal part of the cochlea. At E22, small tubulin beta IV could only be detected in the cytoplasm of inner hair cell by confocal microscopy. At P0, moreover the inner hair cell, the inner pillar cell and the inner phallangeal cell are labelled. At P4-P5, the tubulin beta IV is important within the inner pillar cells and it dissapear of the inner hair cell. At P8 to P14-P15, there is the tubulin beta IV in the pillar cells and at the periphery of the Deiters' cells. I: Inner hair cell; O: Outer hair cell; Ip: Inner Pillar cell; Op: Outer Pillar cell; D: Deiters' cell. Bar: 10µm. Red: Myosin VI; Green: Tubulin beta IV; Blue: Dapi. Figures A-F: Spatiotemporal distribution of Cytokeratin pan during the mammalian auditory organ development from E22 to P15 in the basal part of the cochlea. At E22, small cytokeratins could only be detected in the base of the inner inner phalangeal cell, outer pillar cell and Deiters' cells by confocal microscopy. At P0 to P4-P5, it desappear of the inner phalangeal cell, the inner and outer pillar cell and the Deiter's cells are labelled. At P8 to P10, there is the cytokeratin in the pillar cells, the Deiters' cells and near the phalangeal and bording cells. At P14-P15, the basal parts of the pillar cell and of the Deiters' cells, and the phalangeal cells are labelled. I: Inner hair cell; O: Outer hair cell; Ip: Inner Pillar cell; Op: Outer Pillar cell; D: Deiters' cell. Bar: 10µm. Red: Myosin VI; Green: Tubulin beta IV; Blue: Dapi. Figures A-F: Spatiotemporal distribution of vimentin V9 during the mammalian auditory organ development from E22 to P15 in the basal part of the cochlea. At E22, no label of vimentin is observed. At P0, there are vimentin in the cytoplasm of outer pillar cells and in the basal part of inner pillar cell and Deiters' cells it desappear of the inner phalangeal cell, the inner and outer pillar cell and the Deiter's cells are labelled. At P10, the vimentin is present in the pillar cells, the Deiters' cells and near the phalangeal and bording cells. At P11-12 to P14-P15, the pillar cells and Deiter's cell are labelled. I: Inner hair cell; O: Outer hair cell; Ip: Inner Pillar cell; Op: Outer Pillar cell; D: Deiters' cell. Bar: 10µm. Red: Myosin VI; Green: Tubulin beta IV; Blue: Dapi. GIGA Neurosciences, CNCM, University of Liège, Belgium