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

Expression of mast cell tryptase in <i>hb/hb</i> and control littermates stria vascularis at postnatal day five.

<p><b>A,B:</b> At this stage, our immunohistochemistry detects serine protease expression in proximity of blood vessels and intermediate cells (black arrowheads in A) and nuclei of marginal cells (red arrowhead in A), in line with the mast cell tryptase being involved in DNA synthesis stimulation in some cell types <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056274#pone.0056274-Molino1" target="_blank">[66]</a>. In <i>hb/hb</i> stria we detect reduced protein levels of mast cell tryptase compared to littermate controls, mostly close to its small capillaries (black arrowheads) and marginal cells (red arrowhead). Scale bar: 10 µm. <b>C:</b> Extracts from <i>hb/hb</i> and control littermate inner ears at P5 were subjected to 10% SDS-PAGE. Western blots were probed with anti-mast cell tryptase antibody. GAPDH is used as loading control.</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

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

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

Figure 7

<p><b>A–E:</b> Trp2 expression performed on inner ear sagittal sections in <i>hb/hb, Hmx3^KO/Hmx3^KO</i> and control littermates at E12.5 and E16.5. <b>A–C</b>: Trp2 marks neural crest-derived melanoblasts at E12.5 (arrowheads in A). As shown by the arrowheads in B and C, we detect melanoblasts migrating from the neural crest to developing stria is detected in <i>hb/hb</i>, <i>Hmx3^KO/Hmx3^KO</i>, and littermate controls. D–E: While in control mice at E16.5 some intermediate cells precursors are already in the process of interdigitation in the developing stria vascularis (arrowheads in D), none are in the same process in the <i>hb/hb</i> mutants with only a couple of them lying outside the epithelium (arrowheads E). Moreover, Hematoxylin and Eosin counterstaining shows that the epithelium of developing stria looks immature in <i>hb/hb</i> compared to littermate controls (arrows in D,E). Scale bars: 10 µm. <b>F</b>: Cartoon of the cellular structure of stria vascularis. Adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056274#pone.0056274-Steel7" target="_blank">[77]</a>. <b>G–I</b>: Immunohistochemistry showing Laminin expression in cochlea and general cochlear morphology of <i>hb/hb, Hmx3^KO/Hmx3^KO</i> and control littermates at P5. Laminin is expressed in all cochlear basal lamina <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056274#pone.0056274-Rodgers1" target="_blank">[63]</a>, including stria vascularis, Reissner's membrane, root cell processes and spiral prominence (arrows in G). <b>J–L</b>: Laminin expression in stria vascularis of <i>hb/hb, Hmx3^KO/Hmx3^KO</i> and control littermates at P5. At this stage, we detect basal lamina in blood vessel endothelia (arrowhead in J) and in very small pockets below marginal cells (black arrowhead in J) in control mice. In <i>hb/hb</i> we detect a much stronger laminin expression (denser basal lamina) around the immature stria vascularis (arrow in K). Moreover, we observe fewer and smaller blood vessels in <i>hb/hb</i> compared to littermate controls (examples of blood vessels are labeled with transparent arrowheads in J,K,L). We did not detect any difference in laminin expression in <i>Hmx3^KO/Hmx3^KO</i> compared to littermate controls (arrowhead in L). <b>M–O</b>: Kcnj10 expression in the stria vascularis of <i>hb/hb, Hmx3^KO</i> mutants and control littermates at P5. Kcnj10 is an inward potassium channel of intermediate cells. We detect only some intermediate cells in <i>hb/hb</i> mutants (arrowheads in N) compared to their littermate controls at this stage (M). These intermediate cells are just outside the undifferentiated strial epithelium (the black arrowhead in N points to the immature marginal cell layer in <i>hb/hb</i>, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056274#pone.0056274.s001" target="_blank">Figure S1</a>). No difference in Kcnj10 expression is detected in <i>Hmx3^KO/Hmx3^KO</i> at this stage compared to control littermates (arrowhead in O), consistent with their EP values being close to normal. Boxes delimit regions in higher magnification. Scale bars: A–E: 20 µm; G,I: 10 µm; J–O; 20 µm. bc: basal cells, bv: blood vessels, ic: intermediate cells, imc: immature marginal cells, isv: immature stria vascularis, oc: organ of Corti, rc: root cell processes, Rm: Reissner's membrane, sp: spiral prominence, sv: stria vascularis.</p

Transcription factors binding with high affinity in the region of the headbobber deletion on Chr7, identified with the use of the TRANSFAC database.

<p>Transcription factors binding with high affinity in the region of the headbobber deletion on Chr7, identified with the use of the TRANSFAC database.</p

The headbobber stria vascularis remains immature with lack of intermediate cells and impaired basal lamina degradation.

<p><b>A–D:</b> Trp2 expression in the vestibular system at E16.5 is analysed by immunohistochemistry in the <i>hb/hb</i> and control littermates on sagittal sections. At this stage, melanoblasts have already reached all the different vestibular structures (arrowheads in B, see E). We can still detect amelanoblasts in <i>hb/hb</i> vestibular system (arrowheads in D). Scale bars: A,C: 100 µm; B,D: 20 µm. Boxes delimit regions in higher magnification. <b>E:</b> Schematic drawings of the flattened vestibular membranes of normally pigmented mice showing distribution of melanocytes in the vestibule viewed on medial (E1) and lateral (E2) position, adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056274#pone.0056274-Cable1" target="_blank">[7]</a>. <b>F:</b> Quantitative real-time PCR on cDNA generated from RNA from E12.5 littermate embryo half heads. <i>Netrin-1</i> mRNA levels are significantly lower in <i>hb/hb</i> than in littermate controls. Error bars, s.d. Quantity normalised to <i>Hprt1</i> levels. N = 3. **: p<0.01. <b>G–J:</b> Laminin staining in the vestibular system of <i>hb/hb</i> and littermate controls at P5. In controls, we find melanocytes between the common crus and the other vestibular structures (black arrowheads in G) as well as on the saccular wall (red arrowhead in G) and adjacently to cristae (black arrowhead in H). We show that basal lamina is breaking close to melanocytes (red arrowhead in H). We cannot detect any distinct structure nor melanocytes in the vestibular system of <i>hb/hb</i> mutants at this stage (I,J), but we detect the presence of an immature and tight basal lamina all around the vestibular cyst wall, where melanocytes are supposed to be positioned. (black arrowheads in J compared to H). Scale bars: 10 µm. A: anterior, cc: common crus, cy: vestibular cyst, D: dorsal, es: endolymphatic sac, lsc: lateral semicircular canal, pcr: posterior cristae, psc: posterior semicircular canal,,s: saccule, s+u: utriculosaccular space, u: utricle.</p