Immunologically Defined Component of the Circumferential Ring Around the Cuticular Plate in Mammalian Hair Cells

Nishida, Yasuyoshi; Holley, Matthew C.

doi:10.1159/000259200

“…We have raised numerous monoclonal antibodies to mammalian hair cells with in vitro immunization by using antigen prepared from guinea pig organs of Corti (Holley and Richardson, 1994;Holley and Nishida, 1995;Nishida and Holley, 1996). Many of the antibodies raised by using this method react with antigens in the cuticular plate, stereocilia, and numerous additional components of the reticular lamina.…”

Section: Methodsmentioning

confidence: 99%

“…The immunization method yields many antibodies to inner ear antigens, but few of those that are effective for immunofluorescence can also be used for immunoblotting. Two exceptions are antibodies against the lateral cisternae of guinea pig outer hair cells (Holley and Richardson, 1994) and the circumferential actin ring (Nishida and Holley, 1996).…”

Section: Possible Antigens For Cp1 Sc1 and Sp1-sp3mentioning

confidence: 99%

Timed markers for the differentiation of the cuticular plate and stereocilia in hair cells from the mouse inner ear

Nishida¹,

Rivolta²,

Holley³

1998

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The differentiation of the cuticular plate and stereocilia in cochleovestibular hair cells from the mouse was traced with monoclonal antibodies raised by in vitro immunization. The cuticular plate is detected first from embryonic days 14-15 (E14-E15), before cell differentiation is apparent, either with scanning electron microscopy or with actin filament labeling. A flat disc of material forms beneath the apical membrane and subsequently expands, forming a fully shaped cuticular plate at postnatal stages 3-5 (P3-P5). A second antibody labels stereocilia from stage E16 to E18. In the cochlea, the label initially appears as a punctate disc on the cell apex and then follows the development of the stereocilia until the adult shape of the bundle forms at P4-P6. Additional antibodies label stereocilia from P4 to P6 and are apparently specific for the inner ear. They do not label the cuticular plate at any stage and do not cross react with tissues of muscle, kidney, eye, tongue, gut, skin, or brain. At stage P12-P14, coinciding with the functional maturity of the ear, they label the apical regions of Deiter's cells. The temporally overlapping sequence of antibody labeling sheds new light on the development of the hair cell apex and allows us to monitor the differentiation of hair cells from their last mitotic division to the initiation of organ function, a period of over 2 weeks.

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“…A blocking solution of 5% goat serum in PBS was used for 10 min before the antibody was added. The primary antibodies used for the characterization were as follows: anti-fimbrin antibody (rabbit polyclonal, dilution 1 : 25, a kind gift from Dr P. Matsudaira, Whitehead Institute for Biomedical Research, MIT, Cambridge, MA, USA); cuticular plate 1 (CP1) (dilution 1 : 100), stereocilia and pillar cells 1 (SP1) (dilution 1 : 100) and stereocilia 1 (SC1) (dilution 1 : 100), three mouse monoclonals raised against cochlear tissue (Nishida & Holley, 1996;Nishida et al, 1998); anti-myosin VIIa (rabbit polyclonal, dilution 1 : 100, a kind gift from Professor C. Petit, Institut Pasteur, Paris, France); anti-myosin VI (rabbit polyclonal, dilution 1 : 60, a gift of Dr T. Hasson, University of California at San Diego, USA); anti-espin antibody (rabbit polyclonal, dilution 1 : 7.6, a gift from Dr J. Bartles, Northwestern University, Chicago, USA) and anti-p27 kip1 (Abcam, Cambridge, UK). As a control for non-specific labelling, primary antibodies were replaced with either mouse or rabbit non-reactive immunoglobulins.…”

Section: Immunocytochemistrymentioning

confidence: 99%

“…To analyse the actin cytoskeleton more closely we used three monoclonal antibodies that recognize epitopes expressed by differentiating hair cells (Nishida & Holley, 1996;Nishida et al, 1998). During inner ear development in the mouse CP1 labels the cuticular plates of hair cells, SC1 labels stereocilia and SP1 labels both stereocilia and pillar cells.…”

Section: Retinoic Acid Alters the Actin Cytoskeletonmentioning

confidence: 99%

A model for mammalian cochlear hair cell differentiation in vitro: effects of retinoic acid on cytoskeletal proteins and potassium conductances

Helyer¹,

Cacciabue-Rivolta²,

Davies³

et al. 2007

Eur J of Neuroscience

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We have established a model for the in-vitro differentiation of mouse cochlear hair cells and have used it to explore the influence of retinoic acid on proliferation, cytoskeletal proteins and voltage-gated potassium conductances. The model is based on the conditionally immortal cell line University of Sheffield/ventral otocyst-epithelial cell line clone 36 (US/VOT-E36), derived from ventral otic epithelial cells of the mouse at embryonic day 10.5 and transfected with a reporter for myosin VIIa. Retinoic acid did not increase cell proliferation but led to up-regulation of myosin VIIa and formation of prominent actin rings that gave rise to numerous large, linear actin bundles. Cells expressing myosin VIIa had larger potassium conductances and did not express the cyclin-dependent kinase inhibitor p27(kip1). US/VOT-E36 endogenously expressed the voltage-gated potassium channel alpha-subunits Kv1.3 and Kv2.1, which we subsequently identified in embryonic and neonatal hair cells in both auditory and vestibular sensory epithelia in vivo. These subunits could underlie the embryonic and neonatal delayed-rectifiers recorded in nascent hair cells in vivo. Kv2.1 was particularly prominent on the basolateral membrane of cochlear inner hair cells. Kv1.3 was distributed throughout all hair cells but tended to be localized to the cuticular plates. US/VOT-E36 recapitulates a coherent pattern of cell differentiation under the influence of retinoic acid and will provide a convenient model for screening the effects of other extrinsic factors on the differentiation of cochlear epithelial cell types in vitro.

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“…Two exceptions are antibodies against the lateral cisternae of guinea pig outer hair cells (Holley and Richardson, 1994) and the circumferential actin ring (Nishida and Holley, 1996). Thus, we have not identified the antigens.…”

Section: Possible Antigens For Cp1 Sc1 and Sp1-sp3mentioning

confidence: 71%

Timed markers for the differentiation of the cuticular plate and stereocilia in hair cells from the mouse inner ear

Nishida

¹

,

Rivolta

²

,

Holley

³

1998

Self Cite

View full text Add to dashboard Cite

The differentiation of the cuticular plate and stereocilia in cochleovestibular hair cells from the mouse was traced with monoclonal antibodies raised by in vitro immunization. The cuticular plate is detected first from embryonic days 14-15 (E14-E15), before cell differentiation is apparent, either with scanning electron microscopy or with actin filament labeling. A flat disc of material forms beneath the apical membrane and subsequently expands, forming a fully shaped cuticular plate at postnatal stages 3-5 (P3-P5). A second antibody labels stereocilia from stage E16 to E18. In the cochlea, the label initially appears as a punctate disc on the cell apex and then follows the development of the stereocilia until the adult shape of the bundle forms at P4-P6. Additional antibodies label stereocilia from P4 to P6 and are apparently specific for the inner ear. They do not label the cuticular plate at any stage and do not cross react with tissues of muscle, kidney, eye, tongue, gut, skin, or brain. At stage P12-P14, coinciding with the functional maturity of the ear, they label the apical regions of Deiter's cells. The temporally overlapping sequence of antibody labeling sheds new light on the development of the hair cell apex and allows us to monitor the differentiation of hair cells from their last mitotic division to the initiation of organ function, a period of over 2 weeks.

show abstract

Immunologically Defined Component of the Circumferential Ring Around the Cuticular Plate in Mammalian Hair Cells

Cited by 5 publications

References 24 publications

Timed markers for the differentiation of the cuticular plate and stereocilia in hair cells from the mouse inner ear

Timed markers for the differentiation of the cuticular plate and stereocilia in hair cells from the mouse inner ear

A model for mammalian cochlear hair cell differentiation in vitro: effects of retinoic acid on cytoskeletal proteins and potassium conductances

Timed markers for the differentiation of the cuticular plate and stereocilia in hair cells from the mouse inner ear

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