Jxc1/Sobp, Encoding a Nuclear Zinc Finger Protein, Is Critical for Cochlear Growth, Cell Fate, and Patterning of the Organ of Corti

Domain and motif structure of Jxc1. 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. The scale bar is in amino acids. The Jxc1 GenBank accession number is DQ157775. B , Shown is a ClustalW alignment of the predicted zinc finger regions. Conserved cysteine (C) and FCS residues are highlighted in blue and red, respectively. A 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 (GenBank accession number Q969R5); PHC1–PHC3, mouse polyhomeotic-like 1–3 (GenBank accession numbers Q64208, Q9QWH1, and Q8CHP6, respectively), SCM, mouse sex-comb on midleg (GenBank accession number Q9VHA0); and ZFP198, human Zfp198 (GenBank accession number CAH7013).

Jxc1 is targeted to the nucleus. A , Diagram depicting schematically the design of the fusion proteins and the location of the Jxc1 epitope. B , 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.

Cochlea dysplasia in jc mutants. A , 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 (arrowhead). B , C , Cochlear ducts from P12 (+/jc, jc/jc) and P14 (jc^2J /jc^2J ) 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 stained by toluidine blue O are shown. The organ of Corti is normally developed in 3-week-old +/jc mice ( D ). The locations of IHCs (ih), OHCs (oh), Deiter's cells (dc), inner (ip) and outer (op) pillar cells, tectorial membrane (tm), and tunnel of Corti (tc) are depicted. A section from the midapical region of a jc shows four rows of OHCs ( E ), an ectopic IHC (eih), and an ectopic outer pillar cell (eop) at the lateral edge. 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. 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 midbase 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 midapical 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 midapical 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 , 500 μm; D–F , 50 μm; G–K , 10 μm.

Ectopic tunnel of Corti and inner hair cells. A–H , 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 (L) side of the organ of Corti. Location of IHCs at the medial (M) side is depicted (ih, bracket). B , Higher magnification of the medial location of four IHCs (ih, bracket, and arrows). C , The ectopically located IHCs at the lateral side of the organ of Corti (eih, bracket, and arrows). D–H , Immunostaining of the midapical ( D ) and apical region ( E–H ) of jc/jc at P3 for p75^NTR (red; phalloidin, green). D–F , Location of pillar cells at the medial side is indicated (pc, arrowhead). E , A row of ectopic pillar cells at the lateral side of the organ of Corti is stained (epc, arrowhead). F , G , Higher-magnification images of pc and epc as shown in E . I–K , Plastic sections stained by toluidine blue O. I , Normal position of IHCs (ih), inner (ip) and outer (op) pillar cells, tunnel of Corti (tc), and Hensen's cells (hc) in a jc heterozygote. J , K , 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 is shown at the lateral side ( K ). Note the stereociliary hair bundle of the ectopic IHC (arrow). Scale bars: A–H , 10 μm; I–K , 50 μm.

Vestibular-like hair cells in Kölliker's organ. A , A low-magnification view of organ of Corti (bracket) and a group of ectopic hair cells in Kölliker's organ (arrow, inset) stained with phalloidin. B , 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). C , A higher magnification of a hair cell depicts the stereociliary hair bundle reminiscent of vestibular hair cells (arrowhead). D , E , Phalloidin stainings of utricle +/jc ( D ) and jc/jc ( E ) at P0. F , G , Toluidine-stained plastic sections of +/jc and jc/jc saccule ( F ) and utricle ( G ) are shown. Brackets indicate the thickening of the neuroepithelium. H , The graph shows the mean ± SEM thickness of the macule of saccule and utricle of jc heterozygous and homozygous mutants. The asterisk 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 , 5 μm; D , E , 50 μm; F , G , 300 μm.

Jxc1 expression in sensory and neuronal epithelia. A–F , H–K , 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, outer nuclear layer; inl, inner nuclear layer; tg, trigeminal ganglion; hf, hair follicle; oe, olfactory epithelium; nfl, nerve fiber layer. I , Staining with the sense Jxc1 probe produced no significant signal. G , We used Lfng as marker to identify the sensory regions in the cochlea.