Elmod1 Mutations Cause Inner Ear Dysfunction Figure 5. Molecular analysis of the Elmod1rda-2J mutation. A. Southern blots of genomic DNA from mice with +/+ (lanes marked 1), +/rda2J (lanes marked 2) and rda2J/rda2J (lanes marked 3) genotypes, digested with EcoRI (Eco), PstI (Pst), PvuII (Pvu), and MspI (Msp) restriction enzymes. The blot was hybridized with an Elmod1 cDNA probe corresponding to exons 7–11. Although the same quantity of DNA was loaded in each lane, the intensity of Elmod1-hybridizing bands is greater in in EcoRI- and PvuII-digested DNA samples from rda2J/rda2J mice than samples from +/+ mice and exhibit additional bands (indicated by arrows) in PstI and MspI digested DNA. B. A northern blot of total RNA extracted from brains of adult C57BL/6J control (lane 1), rda2J/+ heterozygotes (lanes 2 and 3), rda2J/rda2J homozygotes (lanes 4 and 5), and rda/rda homozygote (lane 6; negative control) was hybridized with an Elmod1 cDNA probe. Wildtype Elmod1 transcript (,2600 nt) was not detected in RNA from rda2J/rda2J mice; however, a 3200 nt mutant transcript (about 600 nucleotides larger than wildtype) was abundantly expressed. C. PCR products from rda2J–derived cDNAs indicate an intragenic duplication. cDNAs from mice with +/+ (lanes marked 1), +/rda2J (lanes marked 2) and rda2J/rda2J (lanes marked 3) genotypes were used as PCR templates in combination with primers specific to Elmod1 exons. Expected wildtype PCR products are marked by asterisks, and unexpected large PCR products unique to rda2J samples are indicated by arrows. 500 bp (lane marked L1) and 100 bp (lane marked L2) ladders were used to estimate PCR product sizes, and predicted sizes are given in Table S2B. D. A western blot of protein extracts from adult brains of rda/rda (lane 1, negative control), rda2J/rda2J (lane 2), and +/+ B6 mice (lane 3), shows that the predicted 38 kDa wildtype ELMOD1 protein is absent from both rda and rda2J mutant mice, but a larger 62 kDa mutant protein can be seen in rda2J mutants (indicated by arrow in lane 2), corresponding to the predicted duplication of 202 amino acids. The polyclonal ELMOD1 antibody cross-reacted with other unknown proteins (non-specific bands, NS). E. The large PCR products obtained from rda2J–derived cDNA (indicated by arrows in C) and multiple combinations of exon-specific primers were used to determine the DNA sequence and structure of the mutant Elmod1 transcript, and this analysis indicated a duplication of exons 3–8, as shown in the diagram of the presumed mutant gene structure. doi:10.1371/journal.pone.0036074.g005 dysfunction could explain the hyperactive circling behavior of the Elmod1 mutant mice. Elmod1 is also expressed in Purkinje cells of the cerebellum (Fig. S2A). Cerebellar dysfunction in mice impairs balance and coordination and can result in ataxic behaviors that are similar to those caused by vestibular defects of the inner ear. It is possible that cerebellar function is compromised in Elmod1 mutant mice but that the behavioral evidence for it is masked by the overlapping phenotype of inner ear dysfunction; however, we found no overt cellular abnormalities or degeneration of Purkinje cells in mutant mice. Likewise, we found no evidence of retinal ganglion cell degeneration or eye dysfunction in Elmod1 mutant mice, although Elmod1 is expressed in retinal ganglion cells (Fig. S2C). More refined structural and functional analyses of these non-otic Elmod1-expressing cells might reveal subtle abnormalities in mutant mice. Elmod1 encodes a member of the ELMO family of effector molecules for small GTPases, many of which are known to regulate actin cytoskeleton organization [21]. The hair bundle phenotype of Elmod1 mutant mice suggests a similar role for ELMOD1 in regulating the actin dynamics that determine stereocilia length in mammalian inner hair cells. The concept that hair cell stereocilia exhibit a rapid treadmilling of their actin filament cores [26] and that the balance between actin polymerization and disassembly maintains the steady-state length of stereocilia [27,28] has recently been put in doubt. New evidence indicates that stereocilia are remarkably stable with rapid turnover occurring only at the tips and not by a treadmilling process [29]. Thus, stereocilia lengths are determined during hair bundle maturation and once formed do not require a continuous steady- state maintenance. The analyses of mouse deafness mutations have identified several proteins involved in the regulation of hair cell PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e36074