Hair cell death is a major cause of hearing impairment. Preservation of surface barrier upon hair cell loss is critical to prevent leakage of potassium-rich endolymph into the organ of Corti and to prevent expansion of cellular damage. Understanding of wound healing in this cytoarchitecturally complex organ requires ultrastructural 3D visualization. Powered by the serial block-face scanning electron microscopy, we penetrate into the cell biological mechanisms in the acute response of outer hair cells and glial-like Deiters' cells to ototoxic trauma in vivo. We show that Deiters' cells function as phagocytes. Upon trauma, their phalangeal processes swell and the resulting close cellular contacts allow engulfment of apoptotic cell debris. Apical domains of dying hair cells are eliminated from the inner ear sensory epithelia, an event thought to depend on supporting cells' actomyosin contractile activity. We show that in the case of apoptotic outer hair cells of the organ of Corti, elimination of their apices is preceded by strong cell body shrinkage, emphasizing the role of the dying cell itself in the cleavage. Our data reveal that the resealing of epithelial surface by junctional extensions of Deiters' cells is dynamically reinforced by newly polymerized F-actin belts. By analyzing Cdc42-inactivated Deiters' cells with defects in actin dynamics and surface closure, we show that compromised barrier integrity shifts hair cell death from apoptosis to necrosis and leads to expanded hair cell and nerve fiber damage. Our results have implications concerning therapeutic protective and regenerative interventions, because both interventions should maintain barrier integrity.
Cdc42 regulates the initial establishment of cytoskeletal and junctional structures, but only little is known about its role at later stages of cellular differentiation. We studied Cdc42′s role in vivo in auditory supporting cells, epithelial cells with high structural complexity. Cdc42 inactivation was induced early postnatally using the Cdc42loxP/loxP;Fgfr3-iCre-ERT2 mice. Cdc42 depletion impaired elongation of adherens junctions and F-actin belts, leading to constriction of the sensory epithelial surface. Fragmented F-actin belts, junctions containing ectopic lumens and misexpression of a basolateral membrane protein in the apical domain were observed. These defects and changes in aPKCλ/ι expression suggested that apical polarization is impaired. Following a lesion at adulthood, supporting cells with Cdc42 loss-induced maturational defects collapsed and failed to remodel F-actin belts, a process that is critical to scar formation. Thus, Cdc42 is required for structural differentiation of auditory supporting cells and this proper maturation is necessary for wound healing in adults.
Hair cells of the organ of Corti (OC) of the cochlea exhibit distinct planar polarity, both at the tissue and cellular level. Planar polarity at tissue level is manifested as uniform orientation of the hair cell stereociliary bundles. Hair cell intrinsic polarity is defined as structural hair bundle asymmetry; positioning of the kinocilium/basal body complex at the vertex of the V-shaped bundle. Consistent with strong apical polarity, the hair cell apex displays prominent actin and microtubule cytoskeletons. The Rho GTPase Cdc42 regulates cytoskeletal dynamics and polarization of various cell types, and, thus, serves as a candidate regulator of hair cell polarity. We have here induced Cdc42 inactivation in the late-embryonic OC. We show the role of Cdc42 in the establishment of planar polarity of hair cells and in cellular patterning. Abnormal planar polarity was displayed as disturbances in hair bundle orientation and morphology and in kinocilium/basal body positioning. These defects were accompanied by a disorganized cell-surface microtubule network. Atypical protein kinase C (aPKC), a putative Cdc42 effector, colocalized with Cdc42 at the hair cell apex, and aPKC expression was altered upon Cdc42 depletion. Our data suggest that Cdc42 together with aPKC is part of the machinery establishing hair cell planar polarity and that Cdc42 acts on polarity through the cell-surface microtubule network. The data also suggest that defects in apical polarization are influenced by disturbed cellular patterning in the OC. In addition, our data demonstrates that Cdc42 is required for stereociliogenesis in the immature cochlea.
p53 is a key component of a signaling network that protects cells against various stresses. As excess p53 is detrimental to cells, its levels are tightly controlled by several mechanisms. The E3 ubiquitin ligase Mdm2 is a major negative regulator of p53. The significance of balanced p53 levels in normal tissues, at different stages of lifetime, is poorly understood. We have studied in vivo how the disruption of Mdm2/p53 interaction affects the early-embryonic otic progenitor cells and their descendants, the auditory supporting cells and hair cells. We found that p53 accumulation, as a consequence of Mdm2 abrogation, is lethal to both proliferative progenitors and non-proliferating, differentiating cells. The sensitivity of postmitotic supporting cells to excess p53 decreases along maturation, suggesting that maturation-related mechanisms limit p53′s transcriptional activity towards pro-apoptotic factors. We have also investigated in vitro whether p53 restricts supporting cell’s regenerative capacity. Unlike in several other regenerative cellular models, p53 inactivation did not alter supporting cell’s proliferative quiescence nor transdifferentiation capacity. Altogether, the postmitotic status of developing hair cells and supporting cells does not confer protection against the detrimental effects of p53 upregulation. These findings might be linked to auditory disturbances observed in developmental syndromes with inappropriate p53 upregulation.
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