Deafness and cochlear pathology of <i>rda</i> and <i>rda^2J</i> mutant mice.

<p><b>A.</b> ABR thresholds (dB SPL) of <i>rda/rda</i> and <i>rda ^2J/rda^2J</i> 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 <i>rda/rda</i> (N = 9) and <i>rda^2J/rda^2J</i> (N = 8) mutant mice tested, even with the maximum stimulus presentation of 100 dB SPL. Heterozygous +/<i>rda</i> (N = 8) and +/<i>rda^2J</i> (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>B, C.</b> Cross sections through the basal turn of the cochlea from a +/<i>rda</i> heterozygous control mouse (B) and a littermate <i>rda/rda</i> 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 <i>rda/rda</i> cochlea. Scale bars represent 100 microns. Cochlear cross sections of <i>rda^2J</i> mutant mice (not shown) exhibited this same pathology.</p

<i>Elmod1</i> gene structure and expression.

<p><b>A.</b> The mouse <i>Elmod1</i> 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>B.</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 (<a href="http://www.ensembl.org/" target="_blank">http://www.ensembl.org/</a>). <b>C. </b><i>Elmod1</i> 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 <i>Elmod1</i> 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.</p

Molecular analysis of the E<i>lmod1^rda-2J</i> mutation.

<p><b>A.</b> Southern blots of genomic DNA from mice with +/+ (lanes marked 1), +/<i>rda^2J</i> (lanes marked 2) and <i>rda^2J/rda^2J</i> (lanes marked 3) genotypes, digested with <i>Eco</i>RI (Eco), <i>Pst</i>I (Pst), <i>Pvu</i>II (Pvu), and <i>Msp</i>I (Msp) restriction enzymes. The blot was hybridized with an <i>Elmod1</i> cDNA probe corresponding to exons 7–11. Although the same quantity of DNA was loaded in each lane, the intensity of <i>Elmod1</i>-hybridizing bands is greater in in <i>Eco</i>RI- and <i>Pvu</i>II-digested DNA samples from <i>rda^2J/rda^2J</i> mice than samples from +/+ mice and exhibit additional bands (indicated by arrows) in <i>Pst</i>I and <i>Msp</i>I digested DNA. <b>B.</b> A northern blot of total RNA extracted from brains of adult C57BL/6J control (lane 1), <i>rda^2J</i>/+ heterozygotes (lanes 2 and 3), <i>rda^2J/rda^2J</i> homozygotes (lanes 4 and 5), and <i>rda/rda</i> homozygote (lane 6; negative control) was hybridized with an <i>Elmod1</i> cDNA probe. Wildtype <i>Elmod1</i> transcript (∼2600 nt) was not detected in RNA from <i>rda^2J/rda^2J</i> mice; however, a 3200 nt mutant transcript (about 600 nucleotides larger than wildtype) was abundantly expressed. <b>C.</b> PCR products from <i>rda^2J</i>–derived cDNAs indicate an intragenic duplication. cDNAs from mice with +/+ (lanes marked 1), +/<i>rda^2J</i> (lanes marked 2) and <i>rda^2J/rda^2J</i> (lanes marked 3) genotypes were used as PCR templates in combination with primers specific to <i>Elmod1</i> exons. Expected wildtype PCR products are marked by asterisks, and unexpected large PCR products unique to <i>rda^2J</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036074#pone.0036074.s004" target="_blank">Table S2B</a>. <b>D.</b> A western blot of protein extracts from adult brains of <i>rda/rda</i> (lane 1, negative control), <i>rda^2J/rda^2J</i> (lane 2), and +/+ B6 mice (lane 3), shows that the predicted 38 kDa wildtype ELMOD1 protein is absent from both <i>rda</i> and <i>rda^2J</i> mutant mice, but a larger 62 kDa mutant protein can be seen in <i>rda^2J</i> 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). <b>E.</b> The large PCR products obtained from <i>rda^2J</i>–derived cDNA (indicated by arrows in <b>C</b>) and multiple combinations of exon-specific primers were used to determine the DNA sequence and structure of the mutant <i>Elmod1</i> transcript, and this analysis indicated a duplication of exons 3–8, as shown in the diagram of the presumed mutant gene structure.</p

<i>In situ</i> expression of <i>Elmod1</i> mRNA in the inner ear.

<p>Expression was detected in inner hair cells (IHC) and outer hair cells (OHCs) of the cochlea (<b>A</b>) and vestibular hair cells (VHCs) of the crista ampullaris (<b>B</b>) in inner ears from wildtype (+/+) mice at postnatal day 7 (P7). Mutant <i>rda/rda</i> mice served as negative controls for probe specificity, as seen by the lack of detectable <i>Elmod1</i> expression in cochlear hair cells of P7 <i>rda/rda</i> mice (<b>C</b>). <i>Elmod1</i> expression was not detected above background staining in inner ears of P2 wildtype mice (<b>D</b>) or in P15 mice (not shown). The general location of hair cells (HCs) is indicated in the P2 cochlea (D), because inner and outer hair cells are not clearly distinguishable in cryosections at this age. For reference, the Reissner's membrane (RM) and stria vascularis (SV) of the cochleae are indicated with arrows. All panels are at the same magnification; the scale bar represents 50 micrometers.</p

Cochlear hair cell abnormalities in <i>rda</i> and r<i>da^2J</i> mutant mice.

<p>Examination of the apical surfaces of cochlear hair cells by scanning electron microscopy showed normal bundle morphology in both inner hair cells (IHC) and outer hair cells (OHC) of <i>rda/rda</i> mutant mice at P0 (<b>A</b>). At P7 the stereocilia adjacent to the kinocilium (marked by arrow in <b>H</b>) had degenerated in some of the OHC bundles (marked by asterisks in <b>B, D</b>) of <i>rda/rda</i> mice, compared with the fully intact bundles of wildtype (+/+) controls (<b>C</b>). IHC bundles of <i>rda/rda</i> mice retained a normal appearance at P10 (<b>D, K</b>), but by P15 (<b>F, L</b>) all IHCs of mutant mice exhibited stereocilia elongations and fusions as compared with the normal IHC bundle morphology of age-matched controls (<b>E</b>). IHCs at P35 illustrate the striking degree of stereocilia elongations and fusions seen in <i>rda/rda</i> mutants (<b>N</b>) compared with age-matched controls (<b>M</b>). In contrast to IHCs, the OHC stereocilia of <i>rda/rda</i> mice did not elongate or fuse, but they did degenerate over time. By P15, nearly all OHCs had lost stereocilia at the bundle peak and a few (marked by asterisks) had lost the entire bundle (<b>F, I</b>), as compared with the intact OHC bundles of control mice (<b>J</b>). The IHC and OHC bundle abnormalities of <i>rda^2J/rda^2J</i> mutant mice (<b>G</b>) were very similar to those of <i>rda/rda</i> mice (<b>F</b>). 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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036074#pone-0036074-g001" target="_blank">Fig. 1A</a>).</p