A, From top to bottom is shown the genetic region on chromosome 10 (MMU10) containing the 0.9 cM jc interval defined by markers D10Mit108 and D10Mit55. The physical map shows the location of D10Ntra--- markers used for genetic mapping, the approximate location of recombination events (filled oval) and the 170kb jc minimal interval. Physical coordinates obtained from the USCS Genome Browser (mouse assembly Jul. 2007) are shown on the left and right and given in kb. Location and names of BAC clones used to generate transgenic lines are shown and approximately location of predicted exons and open-reading frames. Location and size of three genes (Scml4, sex comb on midleg like 4) indicating the direction of transcription is given. Intron/exon structure of Jxc1 (BC059851), coding exons (filled boxes), untranslated regions (UTR), length of exons in bp, number of exons (1–7), and location of jc and jc^2J mutations are shown. B, Sequencing chromatograms of C57BL/6J-+/+ and jc/jc mutant genomic DNA across the jc mutation in exon 6 is shown. The 10-bp deletion (c1346-1355del) is indicated. Re-sequencing genomic DNA of C57BL/6J-+/jc heterozygotes (n=15) and C57BL/6J-jc/jc homozygotes (n=20) demonstrated a perfect correlation between the genotype and hearing function. The deletion also segregated with the hearing status in the critical recombinants and was absent in nine common inbred strains. Transgenic mice harboring BAC clones RP23-204F11 and RP23-128J2, which were introgressed on the C57BL/6J-jc/jc mutant background failed to rescue the jc phenotype, consistent with the location of the jc mutation. C, Sequence traces of a C57BL/6J-+/+ an affected jc^2J mutant mouse is shown. The location of the G to T substitution is indicated by an arrow. The missense mutation segregated with the cochlea malformation (n=5) and was not detected in nine inbred strains including the B6.129S6-Il6^tm1Kopf strain, on which the mutation occurred.

A, Shown is a diagram depicting schematically the design of the fusion proteins and the location of the Jxc1 epitope. B, Shown are COS cells after transfection with GFP-tagged wild-type Jxc1 protein and mutant constructs expressing the jc and jc^2J truncated GFP fusion protein. Fixed COS cells were also stained with antibody against Jxc1 (Pb544). Nuclei appear in blue, the GFP domain is shown in green, and the antibody staining is shown in the red channel.

A–H, shown are confocal images of organ of Corti whole mount preparations. Apical region at age P0 stained for S100A1 (green) and phalloidin (red, A) shows a disorganized pattern of OHC rows (oh, bracket) followed by a stretch if IHCs located ectopically (eih, bracket) at the lateral side of the organ of Corti (L, lateral). Location of IHCs at the medial side is depicted (ih, bracket; M, medial). Higher magnification of the medial location of four IHCs (ih, bracket and arrows) is shown in B and the ectopically located IHCs at the lateral side of the organ of Corti (eih, bracket, arrows) are depicted in C. Shown is an immunostaining of the mid-apical (D) and apical region (E–H) of jc/jc at P3 for p75^NTR (red; phalloidin, green). Location of pillar cells at medial side is indicated (pc, bracket, D–F). A row of ectopic pillar cells at the lateral side of the organ of Corti is stained (epc, bracket, E). F–G, Higher zoom images of pc and epc as shown in E. I–K, shown are plastic sections followed by Toluidine Blue O staining. Normal position of IHCs (ih), inner (ip) and outer (op) pillar cells, tunnel of Corti (tc) and Hensen cells (hc) in a jc heterozygote is shown (G). In jc mutants an immature tunnel of Corti is shown at the medial side (J) and ectopic tunnel of Corti (etc) formed by ectopic outer (eop) and inner (eip) pillar cells followed by an IHC at the lateral side is shown (K). Note the stereociliary hair bundle of the ectopic IHC (arrow). Scale bars: A – H, 10μm; I – K, 50μm.

Shown are mRNA in situ hybridizations using Jxc1 antisense (A–F, H–K) and sense (i) probes. se, sensory epithelium; sg, spiral ganglion; oc, organ of Corti; sv, stria vascularis; ger, greater epithelial ridge; ler, lesser epithelial ridge; mu, macula utricle; ac, cristae ampullaris; onl, puter nuclear layer; inl, inner nuclear layer; tg, trigemninal ganglion; hf, hair fillicle; oe, olfactory epithelium. Staining with the sense Jxc1 probe produced no significant signal (I). We used the lunatic fringe gene (Lfng) as marker to identify the sensory regions in the cochlea (G).

A, A schematic diagram shows location of exons, mutations, motifs and domains of Jxc1. NLS, nuclear localization signal; ZF, zinc finger; PR, proline-rich region; jc and jc^2J mutations are indicated in red; scale bar in amino acids; Jxc1 GenBank accession number: DQ157775. B, Shown is a Clustal W alignment of the predicted zinc finger regions. Conserved cysteine (C) and phenylalanine-cysteine-serine (FCS) residues are highlighted in blue and red, respectively. Hyphen indicates a gap in the sequence. An asterisk indicates a conserved residue. ZF1, zinc-finger domain 1 of JXC1, L3MBTL2, human l(3)mbt-like 2 (Acc. no. Q969R5), PHC1-3, mouse polyhomeotic-like 1–3 (Acc. no. Q64208, Q9QWH1, Q8CHP6, respectively), SCM, mouse sex-comb on midleg (Acc. no. Q9VHA0), ZFP198, human Zfp198 (Acc. no. CAH7013).

A, Shown are inner ears injected with latex paint of +/jc and jc/jc at embryonic day 15.5. Note the bulbous structure at the apex of the shortened cochlea (arrow). B–C, Cochlear ducts from P12 (+/jc, jc/jc) and P14 (jc^2J/jc^2J) old mice are shown. Note that the cochlear duct in the jc^2J mutant is less shortened than in jc. The cochlear coil is indicated by the dotted lines. D–F, Plastic sections of the organ of Corti followed by Tolouidine Blue O staining are shown. The organ of Corti is normally developed in three-weeks old +/jc mice (D). The location of IHCs (ih), OHC (oh), Deiter’s cells (dc), inner (ip) and outer pillar (op) cell, tectorial membrane (tm), and tunnel of Corti (tc) are depicted. A section from the mid-apical region of a jc shows four rows of OHCs (E) and an ectopic IHC (eih) and ectopic outer pillar cell (eop) at the lateral edge is shown. At the apical region supernumerary rows of OHCs (oh, bracket) and Deiter’s cells (dc, bracket) are seen (F). G–M, Confocal images of organ of Corti surface preparations are shown. Staining with S100A1 (green) and phalloidin shows a normally developed organ of Corti in jc heterozygotes (G) with one row of IHCs (ih, bracket) and three rows of OHCs (oh, bracket). At mid-base in jc homozygotes short stretches of only two rows of OHCs appeared (H). Staining with an anti-myosin VI antibody (a marker of differentiated hair cells; red) identifies four rows of OHCs at the mid-apical region and up to six rows of OHCs at the more apical region (J). Phalloidin staining (red) of jc^2J organ of Corti shows four rows of OHCs at the mid-apical region (compare +/jc^2J with jc^2J/jc^2J; K, L) and a disorganized pattern of OHCs at the apex (M). Scale bars: A – C, 500μm; D – F, 50μm; G – K, 10μm.

Shown is a low magnification view of organ of Corti (A, bracket) and a group of ectopic hair cells in Kölliker’s organ (arrow, insert) stained with phalloidin. B, Shown is a high magnification view of a patch of ectopic hair cells in the Kölliker’s organ. Note the vestibular-like stereociliary hair bundle (arrowheads), the accumulation of F-actin at the apical surface, and the rosette-like formation of cells (star). A higher zoom of a hair cell depicts the stereociliary hair bundle reminiscent of vestibular hair cells (C, arrowhead). Shown are phalloidin stainings of utricle +/jc (D) and jc/jc (E) at P0. Tolouidin-stained plastic sections of +/jc and jc/jc saccule (F) and utricle (G) are shown. Brackets indicate the thickening of the neuroepithelium. H, Graph shows the mean ± SEM thickness of the macule of saccule and utricle of jc heterozygous and homozygous mutants. Star denotes a significant difference (p<0.05; t test). Ten representative sections from three +/jc and two jc/jc ears were measured. Scale bars: A, B, 10 μm; C, 5μm; D, E, 50μm. F, G, 300μm.

A, Jxc1 mRNA levels in adult and whole embryo tissue. Histogram shows the expression level of Jxc1 in each indicated tissue relative to its level in the cochlea. Bars represent the mean and standard deviation of six real-time PCR amplifications. We found Jxc1 mRNA in all major tissues tested albeit at different levels. Relative to mRNA levels in the cochlea, Jxc1 expression was strongest in the brain (1.03x), followed by testis (0.84x) and eye (0.76x); expression was weakest in prostate (0.44x) and liver (0.44x). Alternatively spliced transcripts include exon 5 and were detected in the cochlea and the majority of analyzed tissues (data not shown). B, Real-time PCR analyses of Jxc1 expression in indicated tissues. E, day after gestation; M, molecular weight marker. One of six representative experiments is shown. C, Quantitative comparison of mRNA levels in cochlea and brain of C57BL/6J (B6) and jc heterozygous and homozygous mutants. Bars are mean ± SEM of at least four tests run in parallel.