Cdc42-dependent structural development of auditory supporting cells is required for wound healing at adulthood

Abstract

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.

How to Bury the Dead: Elimination of Apoptotic Hair Cells from the Hearing Organ of the Mouse

“…By analyzing the Cdc42 loxP/loxP ; Fgfr3-iCre-ER T2 mutant mice, we have previously shown that inactivation of the Rho GTPase Cdc42 impairs structural maturation of the actin-rich apices of auditory supporting cells (Anttonen et al 2012). This is consistent with the fact that Cdc42 is a major regulator of actin dynamics (Ridley 2006).…”

“…In addition, from the same crossings, mice carrying one wild-type Cdc42 allele were used as control animals. The generation and genotyping of Cdc42 loxP/loxP ; Fgfr3-iCre-ER T2 and Cdc42 loxP/wt ; Fgfr3-iCre-ER T2 mice has been previously described (Wu et al 2006;Young et al 2010;Anttonen et al 2012). This mouse line was maintained in a mixed background.…”

“…Tamoxifen (Sigma, T5648) was prepared and intraperitoneally injected (50 μg/g body weight) once daily between P2 and P4 as previously described (Anttonen et al 2012). That study also described the characteristics of Fgfr3-iCre-ER T2 -mediated recombination.…”

“…OHC loss was induced at P22 by a single subcutaneous injection of 1 mg/g kanamycin (Sigma) in phosphatebuffered saline (PBS), pH 7.4, followed 45 min later by intraperitoneal injection of 0.4 mg/g furosemide (Fresenius Kabi) as previously described (Oesterle et al 2008;Taylor et al 2008;Anttonen et al 2012). Mice were killed 36 and 48 h after the ototoxic challenge.…”

“…Synergism between these compounds triggers rapid, selective, and reproducible OHC loss that proceeds in a base-to-apex gradient along the cochlear duct (Oesterle et al 2008;Taylor et al 2008;Anttonen et al 2012); 36 h after injection, the wave of OHC loss had proceeded to the middle coil of the cochlea. We term this region where approximately half of OHCs are lost as the acute lesion site, and the immediate lower part of this coil showing total OHC loss as the late lesion site.…”