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. 2009 May;41(5):614-8.
doi: 10.1038/ng.369. Epub 2009 Apr 12.

An ENU-induced mutation of miR-96 associated with progressive hearing loss in mice

Morag A Lewis  1 , Elizabeth Quint, Anne M Glazier, Helmut Fuchs, Martin Hrabé De Angelis, Cordelia Langford, Stijn van Dongen, Cei Abreu-Goodger, Matias Piipari, Nick Redshaw, Tamas Dalmay, Miguel Angel Moreno-Pelayo, Anton J Enright, Karen P Steel
Affiliations

An ENU-induced mutation of miR-96 associated with progressive hearing loss in mice

Morag A Lewis et al. Nat Genet. 2009 May.

Abstract

Progressive hearing loss is common in the human population, but little is known about the molecular basis. We report a new N-ethyl-N-nitrosurea (ENU)-induced mouse mutant, diminuendo, with a single base change in the seed region of Mirn96. Heterozygotes show progressive loss of hearing and hair cell anomalies, whereas homozygotes have no cochlear responses. Most microRNAs are believed to downregulate target genes by binding to specific sites on their mRNAs, so mutation of the seed should lead to target gene upregulation. Microarray analysis revealed 96 transcripts with significantly altered expression in homozygotes; notably, Slc26a5, Ocm, Gfi1, Ptprq and Pitpnm1 were downregulated. Hypergeometric P-value analysis showed that hundreds of genes were upregulated in mutants. Different genes, with target sites complementary to the mutant seed, were downregulated. This is the first microRNA found associated with deafness, and diminuendo represents a model for understanding and potentially moderating progressive hair cell degeneration in hearing loss more generally.

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Figures

Figure 1
Figure 1. Scanning electron micrographs of diminuendo inner ear
a, the heterozygote (Dmdo/+), and b, the homozygote (Dmdo/Dmdo) at postnatal day 5, showing irregular hair bundles and ectopic stereocilia. Scale bars = 2 μm. c, d, Stereocilia in homozygotes at P4 showing tip links (c; arrowheads) and lateral links (d; arrowheads). Scale bars = 500nm. e-h, Stereocilia bundles of inner hair cells (e, f) and outer hair cells (g, h) in heterozygotes (f, h) and wildtype littermates (e, g) at postnatal day 28. Scale bars = 3μm (e, g, h) and 2μm (f). Heterozygous outer hair cells (h) show irregular stereocilia bundles, a smaller apical surface, and are more widely spaced than in wildtypes. Inner hair cells also appear to be more widely separated, and show smaller bundles with fewer stereocilia organised in 4-5 rows (f). All Dmdo/+ stereocilia have rounded tips.
Figure 2
Figure 2. miR-96, miR-182 and miR-183 in littermates at postnatal day 5
a, Alignment of DNA sequences from wildtype mouse, diminuendo homozygote, rat, human, platypus, frog, zebrafish and pufferfish. The mature miRNA sequence for each species is shaded purple, and the seed region critical for target binding is bracketed. The mutation, indicated by the red letter, falls within the seed region. The mature sequence is absolutely conserved between the species shown, and also between cow, dog, horse, macaque, opossum, chimpanzee, orang-utan, ground squirrel, tree shrew, mouse lemur, bushbaby, cat, armadillo, tenrec, medaka, rabbit, stickleback and tetraodon (sequences obtained from Ensembl v50; http://www.ensembl.org). b, e, expression of miR-96 in wildtype (b) and homozygote (e). c, f, expression of miR-182 in wildtype (c) and homozygote (f). d, g, expression of miR-183 in wildtype (d) and homozygote (g). No specific staining was observed using the control probe (data not shown). Probes designed against the mature miRNA sequence have been shown incapable of detecting the precursor transcript, so these show the location of only the mature miRNA. Hair cells are marked by arrowheads. Scale bars = 10μm.
Figure 3
Figure 3. Ocm, Slc26a5, Pitpnm1, Ptpr1 and Gfi1 expression in diminuendo
a, Quantitative real-time PCR on cDNA generated from normalised RNA from the organs of Corti of 4 day old littermates. Ocm, Slc26a5, Ptprq and Gfi1 are downregulated in heterozygotes and homozygotes. Error bars represent standard deviation. Quantities normalised to Hprt1 levels; Ngfr is expressed in support cells adjacent to hair cells and was used to assess the quantity of sensory material. Ngfr: Wildtype, n=12, mean=1.01±0.12 (s.d.); heterozygote, n=12, mean=0.89±0.35 (s.d.); homozygote, n=12, mean=0.99±0.17 (s.d.) Ocm: Wildtype, n=9, mean=1.01±0.15 (s.d.); heterozygote, n=9, mean=0.07±0.04 (s.d.); homozygote, n=9, mean=0.003±0.001 (s.d.) Slc26a5: Wildtype, n=9, mean=1.01±0.14 (s.d.); heterozygote, n=9, mean=0.22±0.11 (s.d.); homozygote, n=9, mean=0.02±0.01 (s.d.) Gfi1: Wildtype, n=9, mean=1.01±0.12 (s.d.); heterozygote, n=9, mean=0.88±0.12 (s.d.); homozygote, n=9, mean=0.66±0.14 (s.d.) Ptprq: Wildtype, n=9, mean=1.01±0.15 (s.d.); heterozygote, n=9, mean=0.62±0.18 (s.d.); homozygote, n=9, mean=0.56±0.20 (s.d.) Pitpnm1: Wildtype n=8, mean=1.00±0.09 (s.d.); heterozygote, n=9, mean=0.80±0.29 (s.d.); homozygote, n=9, mean=0.78±0.27 (s.d.). Three animals were used for each genotype and DNA from each was run in triplicate. T-tests: Ngfr heterozygote p=0.25 (Welch's t-test), homozygote p=0.75 (Student's t-test); Ocm heterozygote p=1.51×10−8 (Welch's t-test), homozygote p=3.46×10−8 (Welch's t-test); Slc26a5 heterozygote p=7.73×10−10 (Student's t-test), homozygote p=3.37×10−8 (Welch's t-test); Gfi1 heterozygote p=0.038 (Student's t-test), homozygote p=3.39×10−5 (Student's t-test); Ptprq heterozygote p=1.37×10−4 (Student's t-test), homozygote p=6.46×10−5 (Student's t-test); Pitpnm1 heterozygote p=0.084 (Welch's t-test), homozygote p=0.35 (Student's t-test); α=0.05. b-k, location of oncomodulin (b, c), prestin (d, h), Pitpnm1 (e, i), Ptprq (f, j) and Gfi1 (g, k) in 5-day old wildtype (b, d-g) and homozygote (c, h-k) littermates. Scale bars = 10μm.
Figure 4
Figure 4. Microarray analysis showing enrichment and depletion of heptamers in 3′UTRs
a, Microarray analysis showing enrichment and depletion of heptamers in 3′UTRs using Sylamer. The x-axis represents the sorted gene list from most up-regulated (left) to most down-regulated (right). The y-axis shows the hypergeometric significance for enrichment or depletion of heptamers in 3′UTRs at leading parts of the gene list. Positive values indicate enrichment (−log10(P-value)) and negative values depletion (log10(P-value)). Heptamers that are depleted in the initial part are accordingly enriched in the complementary part with the same P-value, as a consequence of the hypergeometric distribution. For each miRNA (including the diminuendo mutant miR-96) the two heptamers matching the 5′ seed region, starting at positions 1 and 2, were considered. Each heptamer was tested at regularly placed rank cutoffs in the gene list. The P-value indicates the significance of the enrichment or depletion of the heptamer in the set of 3′UTRs in the initial part of the gene list when compared to the 3′UTRs in the complementary set. The horizontal dotted lines represent an E-value threshold (P-value corrected for multiple testing) of 0.01. Vertical dotted lines indicate Fold Change cutoffs of >1.5, >1.2, and >1.1, and the parts of the gene lists defined by these cutoffs. b, The same analysis as in (a), where each 3′UTR has been replaced by the concatenation of its orthologous 3′UTRs in Human and Rat. The seed match for the wild type miR-96 shows similar enrichment as compared with the analysis in (a). In contrast, the enrichment of the miR-96 diminuendo mutant binding sites in the down-regulated genes is barely above background (dotted line).
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