Deafness is one of the most common types of congenital impairments, and at least half of the cases are caused by hereditary mutations. Mutations of the gene
Deafness is one of the most common types of congenital impairment, and at least half of all cases are caused by hereditary mutations. In some patients, hearing loss is progressive, with hearing loss developing gradually during childhood or youth after the acquisition of speech abilities. Mutation of the
Signal cascade analyses revealed that
Transgenic or knockout animal models are powerful tools for clarifying disease mechanisms. In many genetic disorders, including hereditary hearing loss, their mechanisms have been unveiled by using animal models, especially transgenic or knockout mouse models [
Fixed and decapitated cadaverous heads of newborn common marmoset (postnatal day 2) and 3–6-year-old common marmosets were kindly provided by Junichi Hata, Reona Kobayashi, Takahiro Kondo, Kimika Yoshino-Saito, and Seiji Shiozawa. Fixed skin samples were also kindly provided. The animal experiments were approved by the Ethics Committee of Keio University (number 11006) and were in accordance with the guidelines of the National Institutes of Health and the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
Temporal bones from newborn and young adult marmosets were dissected, fixed, decalcified with Decalcifying Solution B (Wako, Saitama, Japan) for 3-4 weeks, and embedded in Tissue-Tek OCT compound (Sakura Finetek, Tokyo, Japan) for cryosections. The 7
Temporal bones from 3-week-old mice (C57BL/6) and 3-month-old rats were dissected, fixed, decalcified with Decalcifying Solution B for 3–10 days, and embedded in Tissue-Tek OCT compound for cryosection. The 7
After a brief wash with phosphate-buffered saline (PBS) the sections were heated (80°C) in 10 mM citrate buffer (pH 6) for 1 h. After another brief wash, the sections were preblocked for 1 h at room temperature with 10% normal serum in PBS, incubated with primary antibodies at 4°C overnight, and then incubated with Alexa Fluor-conjugated secondary antibodies (Alexa488, Alexa555, and Alexa647) for 60 min at room temperature. The nuclei were counterstained with Hoechst 33342.
After 6 weeks of decalcifications of the cochlea of common marmoset, sensory epithelium containing organ of Corti was dissected. Sections were incubated with 10% normal serum in PBS for 1 h at room temperature and then with primary antibodies at 4°C overnight, followed by an incubation with Alexa Fluor-conjugated secondary antibodies (Alexa555 and Alexa647) and Phalloidin-conjugated Alexa488 (Thermo Fisher Scientific) for 60 min at room temperature. The nuclei were counterstained with Hoechst 33342.
The primary antibodies used in this study are as follows: anti-KIAA1199 (rabbit immunoglobulin G (IgG), Proteintech, Manchester, UK, 21129-1-AP, 1 : 50, rabbit IgG, Cosmo Bio, Tokyo, Japan, CNP-IP-208, 1 : 50), anti-SOX2 (goat IgG, Santa Cruz Biotechnology, Dallas, TX, US, sc17320, 1 : 100), anti-CTGF (goat IgG, Santa Cruz Biotechnology, sc14939, 1 : 100), anti-CALDESMON (Sigma-Aldrich, C0297, 1 : 100), anti-NKCC1 (goat IgG, Santa Cruz Biotechnology, sc21545, 1 : 300), anti-MYOSIN7a (mouse IgG, DSHB, Iowa City, IA, US, 138-1-s, 1 : 30), and anti-
Recently we reported a brief document about the basic morphology and feasible methods for immunohistochemical analysis of the cochlea of the common marmoset (
In the common marmoset, KIAA1199 protein expression is observed in the lateral wall spiral ligament, hair cells, supporting cells, spiral limbus, and spiral ganglion neurons (Figure
Expression of KIAA1199 in the cochlea of the common marmoset. (a and b) KIAA1199 expression is observed in the lateral wall of the cochlea, sensory epithelium, spiral limbus, and spiral ganglion neuron. No expression is observed in Reissner’s membrane. LW: lateral wall of cochlea, OC: organ of Corti, SL: spiral limbus, SGN: spiral ganglion neuron, and RM: Reissner’s membrane. The nuclei were counterstained with Hoechst (blue). Scale bar: 200
In the spiral ligament, KIAA1199 expression was observed in type I, II, III, and V fibrocytes (Figure
Expression of KIAA1199 in the lateral wall. (a) KIAA1199 expression is observed in type I, II, III, and V spiral fibrocytes. (b) KIAA1199 expression is observed in intermediate cells (arrow) and basal cells in the stria vascularis. No expression is observed in NKCC1 positive marginal cells. (c) KIAA1199 expression is markedly observed in outer sulcus cells (arrow head). No expression was observed in type IV spiral fibrocytes. StV: stria vascularis, I–V: type I–V spiral ligament fibrocytes, MC: marginal cells, IM: intermediate cells, and BC: basal cells. The nuclei were counterstained with Hoechst (blue). Scale bar: 100
In the organ of Corti, KIAA1199 expression was broadly observed in supporting cells between the inner and outer sulcus cells as well as the inner and outer hair cells (Figure
Expression of KIAA1199 in the organ of Corti. (a) KIAA1199 expression is observed in the organ of Corti. (b) KIAA1199 expression is observed in MYOSIN7a-positive hair cells and supporting cells between the outer and inner sulcus cells, including SOX2-positive supporting cells. (c) Whole-mount immunofluorescence also showed broad expressions of KIAA1199 in the organ of Corti. ISC: inner sulcus cells, OSC: outer sulcus cells, IHC: inner hair cells, and OHC: outer hair cells. The nuclei were counterstained with Hoechst (blue). Scale bar: (a) and (b): 100
Expression of KIAA1199 in the spiral ganglion neurons. (a and b) KIAA1199 expression is observed in the spiral ganglion neurons. KIAA1199 expression is observed in
We also examined the expression of KIAA in the newborn of common marmoset. Immunoreactivities for KIAA1199 were observed in the lateral wall spiral ligament, hair cells, supporting cells, spiral limbus, and spiral ganglion neurons (Figure
Expression of KIAA1199 in the newborn common marmoset. KIAA1199 expression is observed in the cochlea of newborn common marmoset (postnatal day 2). Immunoreactivities of KIAA1199 in the cochlear epithelial cells including hair cells, supporting cells, and outer sulcus cells were observed. Expressions were also detected in the lateral wall fibrocytes and stria vascularis. The nuclei were counterstained with Hoechst (blue). Scale bar: 100
To validate the anti-KIAA1199 antibody (Proteintech) used in this study, we performed immunostaining in the skin of the common marmoset where both immunostaining and in situ hybridization were previously performed [
Validations of anti-KIAA1199 antibodies used in this study. (a) KIAA1199 expression is detected in the skin of the common marmoset. (b) Another anti-KIAA1199 antibody (Cosmo Bio: CB) revealed the same expression patterns of KIAA1199 in the common marmoset cochlea. (c) Immunohistochemistry with the cochleae of mouse. The immune reactivities in the lateral wall fibrocytes and spiral limbus were detected as reported previously. (d) Immunohistochemistry with the cochleae of rat. The immune reactivities were restricted in the supporting cells as reported previously. The nuclei were counterstained with Hoechst (blue). Scale bar: 100
The expression of KIAA1199 in the common marmoset was more widespread than that in the mouse or rat. Expression of KIAA1199 has been reported in the fibrocytes of the spiral ligament and the spiral limbus with a transient expression in Deiter’s cells at P0 in the mouse [
Differential expression of KIAA1199 in marmosets and rodents.
Expression patterns | Ref. | ||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Hair cells | Supporting cells | ||||||||||||||
Inner hair cell | Outer hair cell | Inner sulcus cells | Inner pillar cells | Outer pillar cells | Deiter’s cells | Hensen cells | Claudius cells | Outer sulcus cells | Spiral ligament | Spiral limbus | Stria vascularis | Reissner’s membrane | SGN | ||
Mouse | − | − | − | − | − | + (~P7) | − | − | − | + | + | − | − | − | Abe et al. 2003 [ |
Rat | − | − | − | + | + | + | − | − | − | NA | NA | NA | NA | NA | Usami et al. 2008 [ |
Marmoset | + | + | + | + | + | + | + | + | + | + | + | + | − | + |
To understand the pathophysiology of a progressive hereditary hearing loss in patients, it is necessary to understand the roles of the deafness gene in the maintenance of hearing. Recently, in cancer research, a relationship between the Wnt/
In a future study, functional analysis of KIAA1199 in the inner ear is necessary for understanding
KIAA1199 showed a primate-specific expression pattern in the cochlea. Future functional as well as mutation screening studies using primates will be crucial to understanding the mechanisms of
Hideyuki Okano is a founding scientist and a paid member in Scientific Advisory Board of SanBio Co., Ltd.
The authors thank Ayano Mitsui for their technical support and Junichi Hata, Reona Kobayashi, Takahiro Kondo, Kimika Yoshino-Saito, and Seiji Shiozawa for materials. Research was supported by JSPS Research Fellowships for Young Scientists (DC) to Makoto Hosoya, Research on Sensory and Communicative Disorders, MEXT, Grants-in-Aid for Scientific Research (C) and (B) (24592560, 15H04991), and Takeda Science Foundation to Masato Fujioka.