Elmod1 Mutations Cause Inner Ear Dysfunction Figure 1. Deafness and cochlear pathology of rda and rda2J mutant mice. A. ABR thresholds (dB SPL) of rda/rda and rda 2J/rda2J mutant mice and non-mutant controls tested at 33–48 days of age. ABRs for 8, 16, and 32 kHz stimulus frequencies were not detected in any of the rda/rda (N = 9) and rda2J/rda2J (N = 8) mutant mice tested, even with the maximum stimulus presentation of 100 dB SPL. Heterozygous +/rda (N = 8) and +/rda2J (N = 4) mice and homozygous +/+ B6 mice (N = 17) were combined as controls because their ABR thresholds did not significantly differ from one another. Error bars represent standard deviations of the threshold means. B, C. Cross sections through the basal turn of the cochlea from a +/rda heterozygous control mouse (B) and a littermate rda/rda mutant mouse (C) examined at 4 months of age. Note the complete degeneration of the organ of Corti (oc) and decreased density of spiral ganglion cells (sgc) in the rda/rda cochlea. Scale bars represent 100 microns. Cochlear cross sections of rda2J mutant mice (not shown) exhibited this same pathology. doi:10.1371/journal.pone.0036074.g001 The mammalian engulfment and cell motility protein ELMO1 can form a complex with Dock proteins to activate Rac1 and promote cytoskeletal reorganization [17,18]. In C. elegans, CED-12 (an ortholog of ELMO1) interacts with CED-5 (an ortholog of Dock180) to activate CED-10 (an ortholog of Rac1) and promote cytoskeletal rearrangements [19,20], indicating that this signaling pathway is evolutionarily conserved. In mammals, six proteins have been identified that contain the conserved ELMO domain (Prosite PDOC51335): ELMO1, ELMO2, and ELMO3 (orthologs of C. elegans CED-12) and three more distantly related proteins – ELMODl, ELMOD2, and ELMOD3 [21]. While ELMO1, ELMO2, and ELMO3 are known to act as guanine nucleotide exchange factors (GEFs) to activate Rac1 [19], ELMOD1 and ELMOD2 were shown to function as GTPase-activating proteins (GAPs) for the Arf family of small G proteins [22]. GEFs and GAPs serve as molecular switches for small G proteins. When in their inactive GDP-bound form, small regulatory GTPases can be activated by GEFs, which replace the bound GDP with free GTP, and when in their active GTP-bound form, they can be inactivated by GAPs, which increase the rate of hydrolysis of GTP to GDP. Here, we describe two independent spontaneous inactivating mutations of the mouse Elmod1 gene that cause deafness and balance defects. The inner ear dysfunction caused by these mutations is associated with dysmorphic hair cell stereocilia and implicates ELMOD1 in a signaling pathway that regulates the maturation and stability of these actin-based structures. In particular, the stereocilia of inner hair cells become greatly elongated and fused in ELMOD1 deficient mice. The Elmod1 mutations described here thus provide the first evidence for the involvement of small GTPases in regulation of hair bundle maturation. Results Phenotypes of the allelic rda and rda2J mutations Both roundabout (rda) and roundabout-2 Jackson (rda2J) mutations, when homozygous, cause circling behavior and loss of acoustic startle response in mice, phenotypes consistent with auditory and vestibular hair cell degeneration. Both mutations mapped to the same region of Chromosome (Chr) 9. We analyzed 123 mutant (rda/rda) F2 progeny from an intercross of (C57BL/6J- rda X CAST/EiJ) F1 hybrids and mapped the rda mutation between markers D9Mit231 and D9Mit248, an interval of 6.12 mega bases (Mb), corresponding to the 52.09–58.21 Mb region of Chr 9 (NCBI Build 37). We analyzed 79 mutant (rda2J/rda2J) mice from an intercross of (B6.Cg-rda2J X CAST/EiJ) F1 hybrids and mapped the rda2J mutation between markers D9Mit231 and D9Mit141, a 5.82 Mb interval (52.09–57.91 Mb region of Chr 9). Because of their nearly identical phenotypes and genetic map positions, the rda and rda2J mutations were tested for allelism. Separate matings of two +/rda females with two rda2J/rda2J males produced four litters consisting of 12 mutant and 11 non-mutant progeny. The presence of mutant progeny in the expected 1:1 Mendelian ratio indicated non-complementation and confirmed that the two mutations are allelic. Hearing in rda and rda2J mutant mice and controls was further assessed by auditory brainstem response (ABR) measurements (Figure 1A). The lack of detectable ABRs for pure tone 8 kHz, 16 kHz, and 32 kHz stimuli, even at the maximum sound pressure level presented (100 dB), demonstrated that rda/rda and rda2J/rda2J mutant mice on the C57BL/6J (B6) strain background are profoundly deaf by 5 weeks of age - the youngest age tested. ABR thresholds of +/rda and +/rda2J heterozygotes were the same as +/+ controls of the B6 background strain. Cross sections through the basal turns of cochleae from mutant mice at 4 months of age showed a complete degeneration of the organ of Corti and partial loss of spiral ganglion cells (Figure 1B). Because hair bundle dysmorphology often precedes hair cell death, we examined stereocilia of young mutant and control mice by scanning electron microscopy (SEM). Stereocilia bundle morphology of inner hair cells (IHC) and outer hair cells (OHC) appeared normal in newborn (P0) rda/rda mutant mice (Figure 2A), but abnormalities became apparent at older ages. At P7 and P10, a few OHCs of rda/rda mice exhibited abnormal hair bundles, wherein the stereocilia directly adjacent to the kinocilium had degenerated (Figure 2B, D, H), and by P15 all OHCs exhibited noticeable degrees of stereocilia degeneration with a few completely lacking bundles (Figure 2F, I). IHC bundle morphol- ogy appeared normal at P7 and P10 (Figure 2B, D, K), but by P15 a striking elongation and fusion of stereocilia was apparent in all IHCs (Figure 2F, L, N). SEM length estimates of the tallest stereocilia from medial turn inner hair cells of P15 and P35 mutants were 6–10 microns compared with 3–4 microns for non- PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e36074