Headbobber: A Combined Morphogenetic and Cochleosaccular Mouse Model to Study 10qter Deletions in Human Deafness

<div><p>The recessive mouse mutant headbobber (<em>hb</em>) displays the characteristic behavioural traits associated with vestibular defects including headbobbing, circling and deafness. This mutation was caused by the insertion of a transgene into distal chromosome 7 affecting expression of native genes. We show that the inner ear of <em>hb/hb</em> mutants lacks semicircular canals and cristae, and the saccule and utricle are fused together in a single utriculosaccular sac. Moreover, we detect severe abnormalities of the cochlear sensory hair cells, the stria vascularis looks severely disorganised, Reissner's membrane is collapsed and no endocochlear potential is detected. Myo7a and Kcnj10 expression analysis show a lack of the melanocyte-like intermediate cells in <em>hb/hb</em> stria vascularis, which can explain the absence of endocochlear potential. We use Trp2 as a marker of melanoblasts migrating from the neural crest at E12.5 and show that they do not interdigitate into the developing strial epithelium, associated with abnormal persistence of the basal lamina in the <em>hb/hb</em> cochlea. We perform array CGH, deep sequencing as well as an extensive expression analysis of candidate genes in the headbobber region of <em>hb/hb</em> and littermate controls, and conclude that the headbobber phenotype is caused by: 1) effect of a 648 kb deletion on distal Chr7, resulting in the loss of three protein coding genes (<em>Gpr26</em>, <em>Cpmx2</em> and <em>Chst15</em>) with expression in the inner ear but unknown function; and 2) indirect, long range effect of the deletion on the expression of neighboring genes on Chr7, associated with downregulation of <em>Hmx3, Hmx2</em> and <em>Nkx1.2</em> homeobox transcription factors. Interestingly, deletions of the orthologous region in humans, affecting the same genes, have been reported in nineteen patients with common features including sensorineural hearing loss and vestibular problems. Therefore, we propose that headbobber is a useful model to gain insight into the mechanisms underlying deafness in human 10qter deletion syndrome.</p> </div

List of candidate genes derived from previously published Meta-Analysis [3] with inclusion criteria satisfied.

<p>Key: Sugg. p-value† = suggestive p-value: p∼10-5,10-6; H.Sugg. p-value‡ = highly suggestive p-value: p∼10-7 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085352#pone.0085352-Girotto1" target="_blank">[3]</a>.</p

Immunohistochemistry in the mouse cochlea at P5.

<p>Brown indicates positive staining. A, B, C) Ptprd is localized in hair cells of the organ of Corti (bracket in A, arrowheads in C), in the marginal cells of the stria vascularis (arrow in A, B), in the supporting cells of the Kölliker’s organ (marked by an asterisk in A) and in the spiral ganglions neurons (arrowhead in A); D, E) Cdh13 is expressed in cells of Claudius (red arrowhead in E), outer and inner hair cells (arrowheads in E), Deiters’ cells (bracket in E) and pillar cells (asterisk in E), cells of the Kolliker’s organ (arrows in E) in the organ of Corti. Staining was also noted in interdental cells (arrow in D), stria vascularis (asterisk in D), spiral prominence and external sulcus cells (bracket in D). F, G) Ank2 could be noted in the Hensen’s cells (bracket in G), Deiters’ cells (arrowheads in G)and pillar cells (asterisk in G) in the organ of Corti. Moreover, Ank2 is expressed in the Reissner’s membrane (arrowhead in F) and cells of the Kolliker’s organ (arrow in G). Scale bars: A–C: 20 µm. D, F: 20 µm; E,G: 10 µm. Rectangles label the regions shown in higher magnification.</p

Summary of the expression patterns of selected markers of inner ear development, performed on sagittal sections from <i>hb/hb</i> mutants and littermate controls at different stages of development.

<p><b>A–D:</b> RNA <i>In situ</i> hybridisation for <i>Bmp4</i> E12.5 in <i>hb/hb</i> and littermate controls. In control mice, <i>Bmp4</i> expression overlaps with the pattern detected for <i>Hmx3</i> and <i>Hmx2</i>, being expressed in the non-sensory cells adjacent to the organ of Corti and cristae (arrowheads in A and C). No <i>Bmp4</i> expression is detected in maculae at that stage. (mu in A) In <i>hb/hb</i>, <i>Bmp4</i> is expressed in two regions in the vestibular cyst (white arrowheads in B), which are definitely not adjacent to any sensory regions in the headbobber homozygotes at later stages. C,D<i>:</i> No difference in <i>Bmp4</i> RNA levels has been detected in <i>hb/hb</i> mutant cochleae compared to their littermate controls. <b>E–H: </b><i>Sox2</i> immunohistochemistry in <i>hb/hb</i> and littermate controls at E14.5. In control mice, <i>Sox2</i> is expressed in all the prosensory regions of the inner ear (arrows in E,G). In <i>hb/hb</i> vestibular system, <i>Sox2</i> shows normal expression in the fused maculae (fm in F), which is the only vestibular prosensory patch we detect in <i>hb/hb</i>. <i>Sox2</i> cochlear expression in <i>hb/hb</i> looks normal when compared with the littermate controls, suggesting a normal development of the organ of Corti at embryonic age E14.5. In addition, <i>Sox2</i> marks the nuclei of both vestibular and cochlear ganglia (vg in E,F and ag in G,H), and again no differences in <i>Sox2</i> expression have been detected in these cells at this stage of development. <b>I–J</b>: Expression of Calretinin at E14.5 of <i>hb/hb</i> and control littermates. At this stage Calretinin marks the developing hair cells. While the hair cells in the organ of Corti are not developing yet at this stage, a few hair cells start to develop in the maculae of normal mice (arrow in I). Calretinin expression analysis shows presence of a few normally developing hair cells in the fused maculae of <i>hb/hb</i> at this stage (arrow in J). <b>K–L:</b> p27^Kip1 expression on <i>hb/hb</i> and littermate controls at E14.5. P27^Kip1 at E14.5 is upregulated in cells of the sensory patch in the cochlea as they prepare to exit the cell cycle. Immunohistochemistry using P27^Kip1 antibody demonstrates that in <i>hb/hb</i> mutants prosensory cells this marker is expressed in the same way in the <i>hb/hb</i> organ of Corti, compared to littermate controls (arrows in K,L). Scale bars: 200 µm A: anterior; ag: acoustic ganglion; c: cochlea; cc: common crus; cy: vestibular cyst;D: distal; ed: endolymphatic duct; fm: fused maculae; ms: maculae sacculi; mu: maculae utriculi; oc:organ of Corti; pc: posterior semicircular canal; pcr: posterior cristae; vg: vestibular ganglion; s:saccule; sv: stria vascularis; u:utricle; s+u: utriculosaccular space; vd: vestibular diverticulum.</p

Cochlear expression of genes in the headbobber deletion on Chr7.

<p><b>A–B:</b> Immunohistochemistry on ear sections at postnatal day five shows Gpr26 expression in spiral ligament fibrocytes (black arrowheads in A). We did not detect presence of Gpr26 in <i>hb/hb</i> cochleae at this stage (B). Scale bar: 10 µm. <b>C–D:</b> Immunohistochemistry on ear sections at postnatal day five shows Cpmx2 expression specifically in intermediate cells of stria vascularis (arrowheads in C). No protein was detected in <i>hb/hb</i> (B). Scale bars: 10 µm. <b>E–H:</b> Immunohistochemistry for Chst15 in <i>hb/hb</i> and control littermates at P5. At this stage, Chst15 shows expression in hair cells (arrowheads in E), marginal cells (black arrowheads in G) and their interdigitation to intermediate cells of stria vascularis (red arrowheads in G). As shown in F and H, we cannot detect any significant expression of Chst15 in <i>hb/hb</i> compared to littermate controls in the organ of Corti (F) and stria vascularis (H), although we can still detect a few protein dots (black arrowheads in F,H), which can either be an artifact or due to non-specific staining. Scale bar: 10 µm. <b>I–J:</b> Whole mount immunofluorescence of organ of Corti at postnatal day five shows expression of Chst15 (green) in the basal region of stereocilia of both inner and outer hair cells (arrows in I). We do not detect any Chst15 expression in the <i>hb/hb</i> disorganized hair bundles (arrowheads in J). Phalloidin (red) stains actin filaments of stereocilia. Scale bars: 10 µm. ohc: outer hair cells; ihc: inner hair cells.</p

Analysis flow chart.

<p>The diagram illustrates the 4 steps defining our strategy. Relevant details for each step are given: GWAS meta-analysis description, expression studies in the mouse cochlea, replication association study in Silk Road cohort and genotype-phenotype relationship.</p

Results of the gene-based association test for candidate expressed genes.

<p><b>Key:</b> Total N. SNPs = total number of intragenic SNPs for each gene; N. Selected PCs = number of principal components explaining the largest amount of variance and used for the analysis (see Materials and Methods); Variance Explained = Total variance accounted for by the selected principal components.</p

Further details of shortlisted candidate genes.

<p>Expression in the ear reported in Mouse Genome Informatics (MGI, <a href="http://www.informatics.jax.org/" target="_blank">http://www.informatics.jax.org/</a>); the Morton cDNA human foetal cochlea cDNA library (Morton, <a href="http://brighamandwomens.org/Research/labs/BWH_Hearing/Cochlear_ESTs.aspx" target="_blank">http://brighamandwomens.org/Research/labs/BWH_Hearing/Cochlear_ESTs.aspx</a>); the SHIELD database (SHIELD: Shared Harvard Inner-Ear Laboratory Database, <a href="https://shield.hms.harvard.edu/" target="_blank">https://shield.hms.harvard.edu/</a>); plus localisation within a reported human deafness locus and phenotypic reports from MGI of any mouse mutations of the gene.</p><p>Key: SHIELD* = SHIELD - enriched in hair cells compared to supporting cells; SHIELD** = SHIELD - auditory/vestibular ganglia; DFN = within a human deafness locus?; N/A = data not available.</p

Expression pattern of Nkx1.2 in <i>hb/hb</i> and control littermates at postnatal day 5.

<p>Nkx1.2 shows expression in hair cells of organ of Corti (arrowhead in A), marginal cells and marginal cells processes in stria vascularis (white and red arrowheads in B, respectively) and hair cells in maculae (C) at P5. Moreover, Nkx1.2 is detected in the perinuclear area of melanoblasts in developing stria vascularis (arrowheads in D). Nkx1.2 staining is reduced in <i>hb/hb</i> compared to littermate controls (arrowheads in E–H). Scale bars: A,B,C,D,E,G,H: 10 µm; F: 20 µm.</p

Results of the replication association study for the candidate SNPs of expressed genes.

<p><b>Key</b>: Eff.All = effect allele; *from published work <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085352#pone.0085352-Girotto1" target="_blank">[3]</a>; Repl. = replication; Maf = minor allele frequency; Str = strand; imp = imputed; gen = genotype; Info Score = imputation quality score form IMPUTE2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085352#pone.0085352-Delaneau1" target="_blank">[6]</a>, Conc = concordance for the region encompassing the SNP (chunk dimension = 5 Mb); HWE = Hardy-Weinberg Equilibrium; add = additive; dom = dominant.</p