Elmod1 Mutations Cause Inner Ear Dysfunction Figure 3. Elmod1 gene structure and expression. A. The mouse Elmod1 gene spans 63.84 kb and is comprised of 11 exons transcribed on the reverse strand of the NCBI genomic DNA reference sequence (Chr 9: 53,823,108 to 53,759,267 bp, Build 37). B. The 2605 nt reference mRNA sequence (NM_177769) encodes a 326 amino acid protein (NP_808437). Amino acids 132–305 (encoded by exons 6–11) comprise a conserved ELMO/CED-12 domain (pfam04727, IPR006816) characteristic of the ELMO protein family. Diagrams A and B were downloaded from the Ensembl website (http:// www.ensembl.org/). C. Elmod1 gene expression was examined by northern blot analysis. Commercially prepared blots from mouse embryos and adult tissues (MTN blots, Clontech, Palo Alto, CA) were hybridized with a mouse Elmod1 cDNA probe corresponding to nucleotides 426–904 of the NM_177769 reference cDNA sequence. Blots contained purified Poly A+ RNA from 7 day (lane 1), 11 day (lane 2), 15 day (lane 3) and 17 day (lane 4) embryos and from heart (H), brain (B), spleen (S), lung (Lu), liver (Li), skeletal muscle (M), kidney (K), and testis (T) of adult mice. A single transcript of about 2,600 nucleotides was detected, most highly expressed in adult brain. doi:10.1371/journal.pone.0036074.g003 abundantly expressed but were much larger than wildtype transcripts (,2.6 kb), and that no wildtype transcripts were produced in rda2J/rda2J mutant mice (Figure 5B). PCR analysis of brain-derived cDNAs using primers corre- sponding to different exons of the Elmod1 gene (Table S2) revealed the presence of extra, large amplification products in cDNAs of +/ rda2J and rda2J/rda2J but not +/+ mice (Figure 5C). Not all of the combinations of exon-specific primers produced additional PCR products in mutant cDNAs, indicating that Elmod1 is only partially duplicated. Sequence analysis of the large PCR products obtained from rda2J/rda2J cDNA with multiple combinations of Elmod1 exon-specific primers indicated that the rda2J mutant transcript contains a 606 nt intragenic duplication of exons 3–8, which corresponds to a predicted 11.5 to 25.5 kb duplication in genomic DNA. The altered transcript and intragenic duplication of the Elmod1rda-2J mutation is illustrated in Figure 5E. The 606 nt duplication in the rda2J transcript is in-frame and predicted to encode a 202 amino acid duplication, which is supported by western blot results (Figure 5D). The estimated mass of the wildtype protein is about 36 kDa, consistent with its predicted size of 326 amino acids, and the estimated mass of the rda2J mutant protein is about 62 kDa, consistent with its predicted size of 528 amino acids. Western blot results also demonstrate the absence of wildtype ELMOD1 protein in both rda/rda and rda2J/ rda2Jmutant mice (Figure 5D). Because the Tnfaip813 gene, as well as Elmod1, was deleted by the rda mutation, we examined DNA from rda2J mutant mice for possible alterations of Tnfaip813, although the spontaneous occurrence and coisogenic nature of the rda2J mutation would argue against any additional gene mutations. As expected, we found no evidence for a Tnfaip813 mutation in rda2J/rda2J mutant mice; there were no exon, splice site, or cDNA sequence differences between wildtype controls and mutant mice. We conclude, therefore, that the common phenotype of rda and rda2J mutant mice is solely the result of Elmod1 inactivating mutations. Cellular localization of Elmod1 expression We localized Elmod1 mRNA expression by in situ hybridization with an antisense RNA probe. Using tissues of rda/rda mutant mice as negative controls, we confirmed Elmod1-specific expression in cells of the inner ear, brain, and eyes of wildtype mice at various time points. In the inner ears of postnatal day 7 (P7) mice, Elmod1- specific expression was restricted to cochlear and vestibular hair cells (Figure 6A, B). Inner ear expression was not detected above background staining levels in younger mice examined at E14.5 (not shown) and P2 (Figure 6D) nor in older mice examined at P15 (not shown). In the brain, Elmod1-specific expression was detected in Purkinje cells of the cerebellum and in granule cell and pyramidal neurons of the hippocampus examined at P7 and P15 (not shown) and at P45 (Figure S2A, B). Expression also was PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e36074