Histological assessment of the middle ears of the <i>Enpp1</i> mutant and control mice.

<p>MEE: Middle Ear Effusion; EH: Epithelial Hyperplasia; IC: Inflammatory Cells; ETO: Eustahian Tube Opening; EM: Ectopic Mineralization; CGC: Cluster of Goblet Cells.</p

Progressive hearing loss in <i>Enpp1</i>^{<i>asj/asj</i>} mice.

<p>ABR threshold means are shown for <i>Enpp1</i>^{<i>asj/asj</i>} mice and littermate control mice, tested at the ages of 3 (n = 6 mutant ears/8 control ears), 6 (n = 10/10), 8 (n = 6/6), 12 (n = 22/16), 18 (n = 8/6), 26 (n = 10/8), and 30 (n = 12/14) weeks. Starting from 6 weeks of age, the mutant mice exhibit significantly higher mean ABR threshold values at all the stimulus frequencies tested (click, 8 kHz, 16 kHz, 32 kHz) compared to those of the littermate controls. Thresholds of mutant mice continue to increase with age and by 18 weeks most of the <i>Enpp1</i>^{<i>asj/asj</i>} mice are profoundly hearing impaired. The increase in 32 kHz ABR thresholds of the control mice is due to the B6 background, a strain known to exhibit age-related hearing loss starting at high frequencies. Error bars indicate standard errors of the mean.</p

Scanning electron micrographs of epithelium lining the middle ear cavity.

<p>Compared with littermate controls (A), the middle ear epithelium in 1-month-old <i>Enpp1</i>^<i>asj</i> mutant mice (B) shows an increased number of goblet cells (red asterisks). Scattered goblet cells (green asterisks) and cilia are seen in the mucociliary epithelia of control mice at 6-months of age (C); however, a layer of mucin obscures most cilia and goblet cells (arrows) in age-matched <i>Enpp1</i>^<i>asj</i> mutant mice (D). Scale bars = 25μm.</p

Otitis media in <i>Enpp1</i>^{<i>asj/asj</i>} mice.

<p>A, B: Representative images of pathological changes in the middle ears of <i>Enpp1</i>^<i>asj</i> mutant mice (B) compared with controls (A) at the age of 5 months. The middle ear cavity (MEC) of <i>Enpp1</i>^<i>asj</i> mutant mice is filled with effusions (black asterisk), fibroblastic and amorphous tissue masses (long arrows), and inflammatory cells (arrow head). Ectopic mineralization of the otic capsule is also evident in mutant mice (open arrow in B). Control mice show a clear middle ear cavity without fibroblastic proliferation (A). C, D: The thickness of the middle ear epithelium (double headed arrows) is greater in <i>Enpp1</i>^<i>asj</i> mutant mice (D) than controls (C). E, F: The stapedial artery wall (arrows) is thicker in <i>Enpp1</i>^<i>asj</i> mutant mice (F) than controls (E). G, H: Representative images of discharge in mutant mice (H) and littermate controls (G) at the age of 5 months. The external ear cavity of the mutant mice is filled with discharge (red asterisk), while control mice have a clear external ear canal and easily observable tympanic membrane (black asterisk). Scale bars: A, B = 200μm, C, D = 50μm, E, F = 80 μm.</p

ABR thresholds of <i>asj/asj</i> mutant mice

<p>ABR thresholds of <i>asj/asj</i> mutant mice</p

Calcification of middle ear structures (or tympanosclerosis) in <i>Enpp1</i>^{<i>asj/asj</i>} mice.

<p>In the control mice (A), the outer ear canal is clear without any discharge. The tympanic membrane appears to be transparent and malleus (blue asterisk) is clearly visible. In the age matched mutant mice (B), surrounding bone must be removed to expose the tympanic membrane, most of which is completely covered by discharge. White patches (inside black dashed lines) were observed on the tympanic membrane of the mutant mice (B). The ossicle bones of the age matched control and mutant mice appear to have similar morphology (C-D), but malleus and incus are fused in the mutant mice (D, red arrow). M: Malleus; I: Incus; S: Stapes. Alizarin red staining reveals extensive mineralization in the stapedial artery wall (green asterisk) in <i>Enpp1</i>^{<i>as/asjj</i>} mice (F), but not in the control mice (E).</p

Increased density of goblet cells in <i>Enpp1</i>^{<i>asj/asj</i>} mice.

<p>Mayer's mucincarmine method was used to visualize goblet cells (stained red) in the epithelia lining the Eustachian tube (A, B) and middle ear cavity (C, D) of mutant and control mice. A, B: Few goblet cells are seen in the epithelia lining the Eustachian tube of littermate control mice (A, empty arrow points to Eustachian tube, insert shows higher magnification of the Eustachian tube epithelia). By contrast, goblet cells are present at high density in the epithelium lining the Eustachian tube of <i>Enpp1</i>^<i>asj</i> mutant mice (B, empty arrow points to Eustachian tube, magnified inset shows goblet cells, marked by arrows, in the Eustachian tube epithelia). C, D: More goblet cells are present in the epithelia in the middle ear cavity (MEC) of the <i>asj</i> mutant mouse (arrows in D) than in the control (C). Scale bars: A, B = 200 μm, C, D = 50 μm, A and B inserts = 20 μm.</p

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