Elmod1 Mutations Cause Inner Ear Dysfunction mice (F). Scale bars: A–G, 10 microns; H–N, 5 microns. All hair cells shown in the figure are from the middle region of the cochlea, but similar bundle abnormalities were seen in other regions (not shown), consistent with the absence of an ABR at all test frequencies (Fig. 1A). doi:10.1371/journal.pone.0036074.g002 mutant controls. Hair bundle abnormalities were similar in rda2J mutants (Figure 2G). High-resolution genetic mapping To refine the map position of rda for positional cloning of the responsible gene, we produced a total of 1357 F2 mice from the intercross of (C57BL/6J-rda X CAST/EiJ) F1 hybrids. All F2 progeny produced from the linkage cross were genotyped at weaning for the D9Mit231 and D9Mit248 flanking markers, and 48 mice with informative recombinant chromosomes were further analyzed (Table S1). Of these recombinant mice, 30 had flanking markers with B6/CAST and B6/B6 genotypes and were evaluated for hearing impairment by ABR; 18 of these had significantly elevated ABR thresholds (.20 dB above normal) when tested at 4–6 weeks of age and were classified as rda/rda mutants; the other 12 mice had normal thresholds and were classified as +/rda. The 18 recombinant mice that had flanking markers with B6/CAST and CAST/CAST genotypes were progeny tested to distinguish between +/rda and +/+ genotypes. ABR testing was used to unambiguously classify homozygous mutant mice because circling behavior of rda/rda mice in the F2 linkage cross was not fully penetrant. An analysis of non- recombinant, obligate rda/rda mice from the intercross showed that all exhibited elevated ABR thresholds, but only about 70% showed circling behavior. Although all rda/rda F2 mice exhibited significant hearing impairment, the degree of impairment was variable, indicating the influence of strain-specific modifier gene or genes. DNA samples from recombinant mice were typed for additional markers within the original 6.12 Mb candidate interval, including microsatellite markers D9Mit334 and D9Mit100 and polymor- phisms within the Exph5, Atm, and Chrna5 genes. We developed four additional microsatellite markers by designing PCR primers that flank variable CA-repeats, which we identified from the reference genome sequence of C57BL/6J mice. Refined analysis of chromosome crossover positions in the recombinant mice, coupled with the deduced rda genotypes of these mice, enabled us to reduce the candidate gene interval to 1.14 Mb, between Exph5 at the 53.186 Mb position and a CA repeat marker at 54.326 Mb. Details of recombinant genotypes used to determine the 1.14 Mb candidate gene interval for the rda mutation are shown in Table S1. The rda candidate region contained 16 annotated genes: Kdelc2, 4930550C14Rik, Atm, Npat, Acat1, Cul5, Rab39, n-R5s83, Slc35f2, Sln, Elmod1, Tnfaip8l3, Cyp19a1, 1700104A03Rik, Gldn, and Dmxl2. The ELMO domain containing 1 gene (Elmod1) at the 53.8 Mb position of Chr 9 was considered the most plausible candidate gene for rda because it is primarily expressed in neural tissues (UniGene Mm.259791), and an ELMOD1 EST (AW022370.1) was identified in a human fetal cochlea library. Elmod1 gene structure and expression The 2605-nucleotide reference sequence for mouse Elmod1 mRNA (NM_177769) is transcribed from 11 exons and encodes a 326 amino acid protein (reference sequence NP_808437). We found that the predominant Elmod1 transcript in adult mouse brain corresponds with this reference sequence and its derivative exon structure (Figure 3A). Detailed results from our analyses of alternative transcripts and determination of the transcription start site of Elmod1 are presented in Figure S1. The protein encoded by Elmod1 (Fig. 3B) contains a conserved domain (ELMO/CED-12) that is characteristic of the ELMO family of proteins, whose members mediate the activation or inactivation of small G proteins that regulate actin cytoskeleton dynamics [19,21,23]. Dysregula- tion of actin-based hair cell stereocilia formation or maintenance caused by ELMOD1 deficiency might, therefore, account for the hair bundle abnormalities observed in rda and rda2J mutant mice (Figure 2). Northern blot analysis of wildtype mice (Figure 3C) showed that Elmod1 expression is barely detectable in 11-day embryos but progressively increases in 15 and 17 day-old embryos. Elmod1 is highly expressed in wildtype adult brain with low or undetectable expression in heart, spleen, lungs, liver, skeletal muscle, kidney, and testis. A single ,2600 nucleotide transcript was detected in RNA from embryos and adult brain. Though not quantitative, RT-PCR showed that Elmod1 is also expressed in tissues of the adult inner ear. Molecular analysis of the rda mutation of Elmod1 PCR amplification of genomic DNA from rda/rda mutant mice with primers flanking individual Elmod1 exons (Table S3A) revealed a deletion of exons 1–5. Southern blot analysis with different Elmod1 cDNA probes confirmed this partial deletion, showing missing restriction fragments in genomic DNA from rda/ rda mice that corresponded with the exon 1–5 region of the gene (Figure 4A). Northern blot analysis showed a complete loss of Elmod1 expression in rda/rda mutant mice (Figure 4B). The extent of the rda deletion was refined by PCR analysis with 27 additional primer pairs distributed throughout the genomic region surrounding the Elmod1gene. The presence or absence of PCR products delimited the extent of the deleted region. A 963 bp PCR product was amplified in rda/rda DNA using primers closely flanking the deleted region (Figure 4C, Table S3B), and sequence analysis of this PCR product revealed a deletion of 137,786 bp, corresponding to the 53,780,870 to 53,918,656 bp region of Chr 9. DNA sequence analysis also revealed a 757 bp insertion at the site of the deletion that corresponds to the inverted 53,699,332– 53,698,576 bp region of Chr 9, originating from within the single intron of the sarcolipin gene (Sln). The rda deletion includes exons 1–5 of the Elmod1 gene, which is transcribed on the reverse strand from 53,823,108 to 53,759,267 bp. The rda deletion also includes the tumor necrosis factor, alpha-induced protein 8-like 3 gene (Tnfaip8l3), which also is transcribed from the reverse strand (53,916,214 to 53,873,415 bp). Tnfaip8l3 is an uncharacterized gene with unknown function. A three-primer assay (Figure 4C, Table S3B) was developed to distinguish +/+, +/rda, and rda/rda genotypes. The molecular structure of the rda mutation is illustrated in Figure 4D. Molecular analysis of the rda2J mutation of Elmod1 Because rda2J was shown to be allelic with rda, we examined the Elmod1 gene in rda2J/rda2J mice. The exons and adjacent splice sites of Elmod1 were amplified from genomic DNA with PCR primers flanking individual exons (Table S3A), but no DNA sequence differences were found between the products of rda2J mutants and wildtype mice. Southern blot analysis with Elmod1 cDNA probes, however, indicated some type of genomic rearrangement in rda2J DNA compared with wildtype DNA (Figure 5A). Northern blot analysis of brain RNA with an Elmod1 cDNA probe showed that rda2J mutant transcripts (,3.2 kb) were PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e36074