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

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

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

Current recording in OHCs from adult Tc1 mice.

<p><b>A</b> and B, Typical current responses from apical-coil adult control (P14) and Tc1 (P13) OHCs. Currents were elicited by depolarizing voltage steps (10 mV nominal increments) from −124 mV (holding potential of −84 mV). <b>C</b>, Steady-state I–V curves for control (P13–P14, <i>n</i> = 9) and Tc1 (P13–14, <i>n</i> = 3) OHCs. <b>D</b>, Average size of the total K⁺ current at the steady-state (<i>I</i>_K+<i>I</i>_K,n) and the isolated I_K,n.</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

ABR Thresholds in control and Tc1 mice.

<p>ABR thresholds for individual control (black circles) and Tc1 (red circles) mice are shown on the left and right panels respectively. The middle panel indicates the mean (± standard deviation, SD) thresholds for control (black) & Tc1 (red) mice, respectively. Tone-evoked ABRs were recorded over the left side of the head only whereas click-evoked ABRs were recorded bilaterally.</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

Expression analysis of <i>Hmx3</i> and <i>Hmx2</i> homeobox transcription factors in <i>hb/hb</i> and control littermates.

<p><b>A–B: </b><i>Hmx3</i> expression in <i>hb/hb</i> mutants tested by whole mount RNA <i>in situ</i> hybridisation at E10.5, showing decreased expression of <i>Hmx3</i> in the dorsal part of the otocyst of <i>hb/hb</i> mutants compared to littermate controls (arrows) <b>C,D:</b> RNA <i>in situ</i> hybridisation for <i>Hmx3</i> at E12.5 in vestibular system sagittal sections. In control littermates, <i>Hmx3</i> RNA is detected in the canal fusion plate (black arrowhead in C), semicircular canals (red arrowheads in C), and in the utricle and saccule (not shown) but its expression is always observed in non-sensory epithelial cells as previously reported (C). In <i>hb/hb</i> mutants we still detect <i>Hmx3</i> mRNA in the vestibular non-sensory regions compared to the littermate controls (arrowhead in D). <b>E,F: </b><i>Hmx2</i> expression in vestibular system detected by <i>in situ</i> hybridisation on sagittal sections from <i>hb/hb</i> and littermate controls at E12.5. In control mice, as previously reported, <i>Hmx2</i> shows a similar expression pattern to <i>Hmx3</i> in the non-sensory cells and in the canal plate, in the utricle (arrow in <i>E</i>) and in the canals (not shown). In <i>hb/hb</i> we detect <i>Hmx2</i> expression only in a few cells in the non-sensory regions of the structurally abnormal vestibular system, compared to the littermate controls (arrowheads in <i>F</i>). Scale bars: A,B, 0.5 mm; C–F, 100 µm. <b>G:</b> Quantitative real-time PCR of cDNA generated from RNA from E12.5 littermate embryo half heads. Only <i>Hmx3, Hmx2</i> and <i>Nkx1.2</i> mRNA levels are significantly downregulated in <i>hb/hb</i> compared to littermate controls. Error bars, s.d. Quantity normalised to <i>Hprt1</i> levels. N = 3. *:p<0.05; **: p<0.01. <b>H–I:</b> Expression of Hmx3 in <i>hb/hb</i> cochlear sections and control littermates at postnatal day five. After birth, Hmx3 is expressed in intermediate cells in stria vascularis (arrowhead in H, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056274#pone.0056274.s002" target="_blank">Figure S2</a>). As it is clear in I, no Hmx3 staining is detected in <i>hb/hb</i> cochlea at P5, consistent with the loss of intermediate cells (arrowhead in I). Scale bars: 20 µm. a: anterior, asc: anterior semicircular canal, cr:cristae, cy: vestibular cyst, D: distal; ed: endolymphatic duct fm: fused maculae lsc: lateral semicircular canal mu: maculae utriculi ms: maculae sacculi; oc: organ of corti, ov: otic vescicle; rm: Reissner's membrane, s: saccule scp: superior canal plate; sd: semicircular duct; s+u: utriculosaccular space, sv: stria vascularis, vd: vestibular diverticulum.</p

Cleared Inner Ears from Control and Tc1 mice.

<p>Gross morphology of the inner ears of a control and Tc1 mouse are shown in <b>A</b> and <b>B</b>, respectively. The cochlear duct (C) is visible as a spiral structure towards the top of each image. The oval (O) and round (R) windows can be seen in the middle of each panel. The semicircular canals of each inner ear are found in the wider region at the bottom of each image. No abnormalities can be detected in Tc1 inner ears.</p