Elmod1 Mutations Cause Inner Ear Dysfunction Figure 6. In situ expression of Elmod1 mRNA in the inner ear. Expression was detected in inner hair cells (IHC) and outer hair cells (OHCs) of the cochlea (A) and vestibular hair cells (VHCs) of the crista ampullaris (B) in inner ears from wildtype (+/+) mice at postnatal day 7 (P7). Mutant rda/rda mice served as negative controls for probe specificity, as seen by the lack of detectable Elmod1 expression in cochlear hair cells of P7 rda/rda mice (C). Elmod1 expression was not detected above background staining in inner ears of P2 wildtype mice (D) or in P15 mice (not shown). The general location of hair cells (HCs) is indicated in the P2 cochlea (D), because inner and outer hair cells are not clearly distinguishable in cryosections at this age. For reference, the Reissner’s membrane (RM) and stria vascularis (SV) of the cochleae are indicated with arrows. All panels are at the same magnification; the scale bar represents 50 micrometers. doi:10.1371/journal.pone.0036074.g006 stereocilia length, including espin [30,31], myosin XVa [32], whirlin [33,34,35], and EPS8 [36,37], whose absence results in shortened stereocilia, and myosin VIIa [38], myosin VI [39,40] and PTPRQ [41,42] whose absence results in elongated stereocilia. Mice lacking gelsolin also show elongated stereocilia, but only in outer hair cells at the cochlear apex [43]. The striking stereocilia elongations and fusions we observed in Elmod1 mutant mice (Fig. 2) are more extensive than those reported in other mutant mice [36–41], and in contrast to these other mutants, are limited to cochlear inner hair cells. Measurements of stereocilia length during development in mice reveal that the tallest stereocilia row of cochlear inner hair cells elongates by 60% from P7 to P13 [44]. This period of rapid stereocilia elongation, which occurs during normal hair bundle maturation, coincides with the onset time of the abnormal over- elongations and fusions of inner hair cell stereocilia observed in Elmod1 mutant mice between P10 and P15. It also coincides with our in situ hybridization results, where Elmod1 expression in hair cells could be detected at P7 but not at P2 or P15. ELMOD1 thus may be involved in a regulatory process that establishes the mature staircase architecture of the inner hair cell bundle. The actin- capping protein twinfilin 2 is thought to prevent elongation of the shorter stereocilia rows during this final phase of hair bundle maturation [44], and ELMOD1 may be involved in regulating the final length of the tallest row of stereocilia. An uncoupling of actin polymerization and plasma membrane turnover rates may underlie the stereocilia fusions observed in inner hair cell bundles of Elmod1 mutant mice, whereby the plasma membrane is unable to fully form along the entire lengths of individual, rapidly expanding stereocilia. Outer hair cells mature earlier and do not undergo the final rapid burst of stereocilia elongation characteristic of inner hair cells [45], and this difference in maturation pattern may relate to the different inner and outer hair cell phenotypes observed in Elmod1 mutant mice (Figure 2). The hair bundles of outer hair cells, though not elongated, show other abnormalities in Elmod1 mutant mice. Bundles appear normal at P0, but by P15 stereocilia in the region adjacent to the site of the kinocilium have degenerated in all outer hair cells of mutant mice. Stereocilia near the kinocilium are the first to develop, and they grow into the longest stereocilia [45]; however, it is not clear how these characteristics relate to the localized loss of stereocilia at the bundle vertex in outer but not inner hair cells. Kinocilia of both outer and inner cochlear hair cells in Elmod1 mutant mice appear normal and disappear as expected by P15, and hair bundle polarity is maintained throughout development. PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e36074