ELMOD1, therefore, is not involved in mechanisms regulating ciliogenesis or planar cell polarity. ELMOD1, small GTPases, and actin dynamics of stereocilia In mammalian cells, ELMO1 forms a tertiary complex with RhoG and Dock180 to induce activation of Rac1 in a signaling cascade that mediates cell surface morphology [23]. Indirect evidence suggests that small GTPases like RhoG and Rac1 also may regulate actin cytoskeleton dynamics in hair cell stereocilia [11,12], but which GTPases are active at the hair cell surface and the molecules that control their timing and localization are unknown. We hypothesize that ELMOD1 functions to mediate the activation or inactivation of particular small regulatory GTPases in hair cells and that perturbation of this function is responsible for the altered hair bundle morphology seen in rda mutant mice, in which inner hair cell stereocilia are greatly elongated and fused. Dock4, a member of the Dock180 family, has been shown to form complexes with ELMO proteins to activate Rac1 and regulate such processes as cell migration [18] and neuronal dendrite development [46]. Dock4 has been localized to inner ear stereocilia [47], suggesting that a GTPase-DOCK4-ELMO signaling pathway may be involved in regulating the cytoskeletal dynamics of hair cells. ELMOD1, however, does not have the 200 amino acid C-terminus (plekstrin homology) characteristic of ELMO proteins 1–3 [22], and this C-terminus is the region shown to form a complex with DOCK180 [48]. ELMOD1, therefore, is not likely to interact with Dock4 and act as a GEF for Rac- mediated actin regulation in hair cell stereocilia. ERM proteins (ezrin, radixin, moesin) have been shown to interact with ELMO1 to promote RhoG-dependent apical cytoskeleton rearrangements in epithelial cells [49,50]. Radixin [51,52] and the ERM-interacting protein CLIC5 [53] are expressed in hair cell stereocilia and loss of these proteins causes deafness, suggesting that an ELMO-ERM interaction may mediate actin cytoskeleton organization in hair cell stereocilia. The ERM binding domain on ELMO1 was mapped to the N- terminal 280 amino acids [49]. The N-terminal region of ELMOD proteins, however, does not have this N-terminal armadillo-like domain; therefore, ELMOD1 is not expected to bind with ERM proteins. Recent evidence indicates that ELMO-family proteins may perform functions other than acting as GEFs to promote RAC- activation via their interaction with DOCK proteins. ELMOD1 and ELMOD2 have been shown to function as GAPs to inactivate members of the ADP-ribosylation factor (Arf) and ADP-ribosyla- tion factor-like (Arl) families of small regulatory GTPases [22]. Arf and Arl GTPases can regulate downstream activation of Rac1 and have been implicated in the regulation of membrane trafficking and the actin cytoskeleton [10,54,55]. Phosphoinositides are important regulators of actin cytoskeletal organization [56,57,58,59], and Arf-family GTPases and their effectors have been shown to be important mediators of this function [60,61]. For example, Arf6 localizes to the plasma membrane and facilitates actin cytoskeletal rearrangements by activating type 1 phosphatidylinositol 4-phosphate 5-kinase (PIP-5 kinase) to generate phosphatidylinositol-4,5-biphosphate (PIP2) [62]. Protein tyrosine phosphatase receptor Q (PTPRQ) is a phosphotidylinositol phosphatase that that can regulate the phosphoinositide content of membranes [63,64], and PTPRQ is required for normal cochlear hair bundle development [41]. Mice deficient in PTPRQ exhibit elongations of inner hair cell stereocilia [42]. The similarity of hair bundle phenotypes of Ptprq and Elmod1 mutant mice suggests the possibility that ELMOD1 Elmod1 Mutations Cause Inner Ear Dysfunction may be involved in PIP2 regulation of the actin cytoskeleton during hair bundle maturation, perhaps acting as a GAP for an Arf-family GTPase that mediates PIP2 production in hair cell membranes. Similar Arf-phosphoinositide interactions are known to influence cytoskeleton organization in other cell types [10,62]. Elmo-related proteins have both GEF and GAP promoting activities on a variety of small GTPases. In the amoeba Dictyostelium, an Elmo-related protein (ElmoA) inhibits actin polymerization either as a GAP or by interfering with localized GEF activity [21,65]. Cells lacking ElmoA display increased pseudopod formation and excessive F-actin polymerization within pseudopods. ElmoA is most closely related to the ELMOD1 and ELMOD2 members of the mammalian ELMO family [21], suggesting that ELMOD1 may also function as a negative regulator of actin polymerization in mammalian hair cell stereocilia. ELMOD1 is known to have GAP activity on Arf family GTPases [22] and may function as a GAP to deactivate an inner ear GTPase that specifically promotes the P7 to P13 rapid elongation of the tall stereocilia row of cochlear inner hair cells [44]. Loss of ELMOD1 thus would result in continued, excessive elongation, consistent with the striking increase in stereocilia length observed between P10 and P15 in inner hair cells of ELMOD1 deficient mice. The Elmod1 mutant mouse model presented here will provide a means to test these proposed molecular pathways and functions. Materials and Methods Mouse strains and husbandry Two independently arising recessive mutations named round- about (rda) and roundabout-2 Jackson (rda2J) were discovered and are being maintained at The Jackson Laboratory (Bar Harbor, Maine). The rda mutation occurred in a C57BL/6J (B6) colony of mice, and the rda2J mutation occurred in a B10.BR-H2kH2-T18a/ SgSnJ colony. The rda2J mutation was subsequently made congenic on a B6 strain. Mice with these two Elmod1 mutations are available from The Jackson Laboratory (http://jaxmice.jax. org/index.html); the strain carrying the rda mutation is designated C57BL/6J-Elmod1rda/JKjn (Stock #2745), and the strain carrying the rda2J mutation is designated B6.Cg-Elmod1rda-2J/JKjn (Stock #6018). Experimental mice were housed in the Research Animal Facility of The Jackson Laboratory, and all procedures involving their use were approved by the Institutional Animal Care and Use Committee. The Jackson Laboratory is accredited by the American Association for the Accreditation of Laboratory Animal Care. Auditory-evoked brainstem response (ABR) Hearing in mice was assessed by ABR threshold analysis, as previously described [66]. Mice were anesthetized with an intraperitoneal injection of tribromoethanol (0.2 ml of 12.5 mg/ ml stock per 10 g of body weight), and then placed on a heating pad in a sound-attenuating chamber. Needle electrodes were placed just under the skin, with the active electrode placed between the ears just above the vertex of the skull, the ground electrode between the eyes, and the reference electrode under- neath the left ear. High-frequency transducers were placed just inside the ear canal and computer-generated sound stimuli were presented at defined intervals. Thresholds were determined 8-, 16- , and 32-kHz pure-tone stimuli by increasing the sound pressure level (SPL) in 10-dB increments followed by 5-dB increases and decreases to determine the lowest level at which a distinct ABR wave pattern could be recognized. Stimulus presentation and data PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e36074