In this study we positionally cloned the jc locus and show that truncating mutations in Jxc1 create a unique combination of phenotypes during cochlear development. Several lines of evidence indicate that the detected nucleotide changes in Jxc1 are causative mutations. First, a 10-bp deletion and a nonsense mutation in two independent alleles are predicted to truncate the protein; second, the jc and jc2J mutations perfectly correlated with hearing function and cochlear malformation and were absent in a number of inbred strains; third, no other nucleotide change was found in the coding and non-coding sequence of Jxc1 or other predicted exons in the jc interval.
mutation is associated with a shortened cochlea, supernumerary hair cells, mirror-image duplications of organ of Corti, and the presence of ectopic hair cells in Kölliker’s organ. In jc
, the stereocilia hair bundle of some OHCs is severely disoriented, but at most IHCs and OHCs along the cochlear duct the hair bundle was properly oriented and looked structurally well organized. A shortened cochlea with supernumerary apical hair cells is frequently observed in mutations causing PCP defects. It remains to be tested whether the PCP defect is a direct consequence of reduced Jxc1 function or is simply a concomitant appearance of the failure of the cochlea to grow. Our in situ
hybridization data show that Jxc1
transcription overlaps with the elongation of the cochlear duct (McKenzie et al., 2004
). Although the shortening of the cochlea by approximately 28% is similar to the result of mutations in planar polarity proteins such as Vangl2Lp
(Curtin et al., 2003
; Montcouquiol et al., 2003
), the planar polarity defects in these latter mutants were also expressed in portions of the sensory epithelium that had an otherwise normal cellular pattern. In jc
mutants, the planar polarity defects occurred only in those apical regions of the cochlea with the most severe patterning defects. This suggests that the PCP defects in jc
might be secondary to the elongation deficiency.
A shortened cochlea with abundant apical outer hair cells is also a phenotypic hallmark of the Foxg1tm1M
mutant cochlea (Ma et al., 2000
; Pauley et al., 2006
). However, both null mutants exhibit pathologies not observed in jc
such as absent sensory neurons (Neurog1tm1And
) and abnormal semicircular canals (Foxg1tm1M
). The function of Neurog1 (neurogenin1) during cochlear elongation and that of Foxg1 (forkhead box G1) during telencephalic neurogenesis was recently linked to cell-cycle control and proliferation (Martynoga et al., 2005
; Matei et al., 2005
expression coincides with the period during which the organ of Corti-forming cells become postmitotic (E12 – E14) (Ruben, 1967
). This phenotypic overlap suggests that Jxc1 function may intersect with pathways governing the transition to the post-mitotic state of hair cells and supporting cells.
The mirror-image duplication with the ectopic tunnel of Corti and IHC at the apical region is a unique characteristic of the jc
phenotype. Whether the appearance of these ectopic cells is the result of de novo
specification or due to massive cell movements at the constraint apex is not immediately apparent. The convergence of ectopic and naive rows seems to argue that cells are moved around the truncated apex. But this convergence is not seen in all samples suggesting ectopic differentiation. Such ectopic differentiation seems to occur unambiguously in the Kölliker’s organ by the formation of vestibular-like hair cells. This suggest that Jxc1
may play a role in cell fate perhaps by silencing target genes required for hair cell specification. Jxc1
expression in the entire cochlear epithelium, including inner and outer sulcus is consistent with this hypothesis. A role for Jxc1
in repressing transcription is supported by the predicted motif and domain structure, in particular by the presence of the FCS zinc finger domains. These zinc fingers are present in SCM and PHC proteins that are part of the polycomb repressive complex 1 (PRC1), which is best known for its role in transcriptional silencing of homeotic genes to maintain segment identity in Drosophila
(Pirrotta et al., 2003
; Lund and van Lohuizen, 2004
; Ringrose and Paro, 2004
). The FCS zinc finger domain of mouse PHC1 (also known as Rae28) was recently shown to bind DNA and RNA in non-sequence-specific manner (Zhang et al., 2004
) suggesting a similar DNA and/or RNA binding activity of Jxc1. We speculate that the sequence-specificity might be conferred by the unique Jxc1 motif I and/or motif II.
A role for Jxc1
in cell fate and patterning of the organ of Corti is also suggested by the function of its Drosophila
homologue sine oculis-binding protein (Sobp
) during eye development. Sobp
was identified in a yeast two-hybrid screen interacting with the Six domain of the Sine oculis (SO) protein (Kenyon et al., 2005
). In Drosophila
, ectopic expression of Sobp
leads to structural aberrations in the adult eye and it was suggested that Sobp forms a complex with the transcription factors Eya1 and SO (Kenyon et al., 2005
). In mammals Six1, a vertebrate homologue of Sine oculis, interacts with Eya1 to control the patterning of the otic vesicle (Zheng et al., 2003
; Ruf et al., 2004
). Given that the Six1
show overlapping expression pattern in the inner ear at E15.5, it is conceivable that Jxc1 acts as cofactor in the Six1/Eya1 pathway.
Hearing loss, cochlear malformation, and the retention of mutant isoform in the cytoplasm are less pronounced in the jc2J allele than in jc. These phenotypic differences seem to correlate with the location of the mutations in the gene, such that the 10-bp deletion in jc truncates motifs that are otherwise present and presumably functional in the jc2J mutant. Since the jc isoform still retains the amino-terminal nuclear localization signal, motif I, part of the proline-rich region, and the two FCS zinc finger domains, it is possible that jc might represent a hypo-functional allele. This is supported by the observation that Jxc1-expressing tissues such as the retina and olfactory epithelium appear morphologically normal in jc mutants (data not shown). Furthermore, jc homozygotes have a normal life span and reproduction, despite Jxc1 mRNA expression in all major adult tissues.
Auditory brain stem response measurements on young jc
mutants showed a considerable variation of thresholds ranging from 65 to 100 dB SPL with a mean threshold at the click stimulus of 83 ± 11 dB SPL (Calderon et al., 2006
). The hearing impairment is on average more severe at the higher than at the lower frequencies and does not change significantly with age. The absence of distortion-product-otoacoustic emissions (f2
frequency range: 7–50kHz) at 75 dB SPL is consistent with a loss of outer hair cell function along the entire cochlear duct. While loss of acoustic responsiveness at lower frequencies can be explained by the patterning defects, the hearing loss at higher frequencies seems to be at odds with the relatively normal morphology of the organ of Corti at the base. Possibly the jc
mutation has an adverse effect on the stiffness of the organ of Corti. The variability of hearing thresholds on the isogenic C57BL/6J background may be due to the hypomorphic nature of the jc
allele producing residual protein activity above threshold levels.
Jc mutants also exhibit erratic circling behavior and have no vestibular-evoked potentials (Jones et al., 2005
), which is likely the result of the deformed structure of the sensory epithelium of utricle and saccule. This structural deformation is qualitatively and quantitatively similar to the growth defect of the cochlea.
In summary, our study of the jc mutant identifies Jxc1 (synonym Sobp) as a nuclear protein with FCS-type zinc finger domains that is likely involved in transcriptional regulation and reveals a unique combination of developmental phenotypes suggesting that Jxc1 controls a critical step during cochlear growth, cell fate and cellular patterning of the mouse organ of Corti.