Fimbrin expression in the developing rat cochlea

doi:10.1016/0378-5955(95)00088-L

Hearing Research

Volume 87, Issues 1–2, July 1995, Pages 165-169

https://doi.org/10.1016/0378-5955(95)00088-L Get rights and content

Abstract

The expression of fimbrin in the developing rat cochlea was analyzed using an immunohistochemical technique with fimbrin antibody. The cochlea displayed temporal and lateral-longitudinal gradients for fimbrin expression during development. Fimbrin immunoreactivity first appeared in the inner hair cell stereocilia of the basal turn on the first gestational day studied (day 18). At birth, both inner (IHC) and outer hair cell (OHC) stereocilia of the basal turn showed positive labeling with fimbrin antibody. The progression of appearance was always from IHCs to OHCs and fimbrin immunostaining appeared in the apical hair cells by postnatal day 6. Immunostaining was restricted to stereocilia and the cuticular plate, and no immunoreactivity was observed in neighboring structures of the epithelium. Double labeling using both fimbrin antibody and phalloidin binding revealed similar chronological expression from the earliest stage studied. Increasing fimbrin immunoreactivity was observed in hair cells until late postnatal and adult stages. This study suggests that fimbrin is expressed with F-actin during development and fimbrin together with actin may constitute the two basic molecules that participate in stereocilia formation. We speculate that fimbrin may help maintain the parallel growth of actin filaments within the stereocilia. These data additionally support previous findings that hair cell maturation occurs from the base to the apex and from IHCs to OHCs.

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Hair cells and supporting cells of the mammalian cochlea terminally differentiate during development. Recent in vitro evidence suggests the presence of hair cell progenitors in the postnatal cochlea. Phenotypic properties of these cells and factors that promote their ability to generate spheres in aggregate cultures have not been reported. We define an in vitro system that allows stem/progenitor cells harvested from the early postnatal cochlea to develop into spheres. These spheres contain Abcg2, Jagged1 and Notch1 positive progenitor cells that can divide and generate new hair cell-like cells, i.e. immunopositive for specific hair cell markers, including Myosin VI, Myosin VIIa, Math1 and ability to uptake FM1-43.
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The number of identified proteins required for hair bundle development and function is rapidly expanding. Proteins like harmonin (Verpy et al., 2000; Siemens et al., 2002), whirlin (Mburu et al., 2003; Belyantseva et al., 2005), espins (Zheng et al., 2000; Li et al., 2004; Sekerkova et al., 2004, 2006; Rzadzinska et al., 2005), cadherins (Di Palma et al., 2001; Siemens et al., 2002, 2004; Sollner et al., 2004; Michel et al., 2005), and fimbrin (Tilney et al., 1989; Zine et al., 1995) have important roles as scaffolding and cross‐linking elements, but their function in terms of hair cell mechanotransduction remain to be elucidated. To this point, three mechanical bundle movements have been identified and related to MET currents.
Hair cells are capable of detecting mechanical vibrations of molecular dimensions at frequencies in the 10s to 100s of kHz. This remarkable feat is accomplished by the interplay of mechanically gated ion channels located near the top of a complex and dynamic sensory hair bundle. The hair bundle is composed of a series of actin-filled stereocilia that has both active and passive mechanical components as well as a highly active turnover process, whereby the components of the hair bundle are rapidly and continually recycled. Hair bundle mechanical properties have significant impact on the gating of the mechanically activated channels, and delineating between attributes intrinsic to the ion channel and those imposed by the channel's microenvironment is often difficult. This chapter describes what is known and accepted regarding hair-cell mechanotransduction and what remains to be explored, particularly, in relation to the interplay between hair bundle properties and mechanotransducer channel response. The interplay between hair bundle dynamics and mechanotransduction are discussed.
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Two other major cytoskeletal proteins of the inner ear epithelium are fimbrin and tubulin. Fimbrin is present exclusively in the hair bundle [6,8,40,41,60], where it cross-links actin filaments [8,22]. The specific isoform of β-tubulin, class III β-tubulin, is also localised to hair cells in the post-embryonic avian sensory epithelium [33,46].
In birds, spontaneous recovery of the hair cells of the inner ear can occur after damage induced by aminoglycoside antibiotics. The factors that influence this recovery and the process of hair cell regeneration itself have until recently been investigated largely by morphological and histological methods. The aim of this work was to use a molecular biological approach to the analysis of hair cell regeneration by measuring the changes that occur in expression of mRNA for hair cell-specific cytoskeletal proteins fimbrin and class III β-tubulin, along with that for β-actin, in the utricle of chicks after hair cell damage both in vitro and in vivo. Utricles were removed from 1-day-old chicks and incubated with the aminoglycoside antibiotics gentamicin or neomycin (both 1 mM), or chicks were injected intraperitoneally with 100 mg/kg gentamicin or neomycin for 4 consecutive days. At the end of the treatment periods, total RNA was extracted from single utricles, reverse transcribed to cDNA and the cDNA amplified by PCR with primers for β-actin, fimbrin and class III β-tubulin. Co-amplification allowed quantitative comparison of mRNA between fimbrin, or class III β-tubulin and β-actin from the same utricle. Both aminoglycosides, either after 48 h in vitro or immediately after treatment in vivo, caused a significant decrease in the expression of fimbrin mRNA and class III β-tubulin mRNA, relative to β-actin mRNA, which itself increased. Light and electron microscopy confirmed that this corresponded to loss of, and damage to, hair cells. The relative expression of fimbrin and class III β-tubulin mRNAs was partly restored almost to control levels 4 days after cessation of treatment in vivo and fully normalised by 4 weeks, by which time hair cells appeared normal. However, their relative expression remained depressed 4 days after removal of antibiotic in vitro. The iron chelator desferrioxamine (100 μM) in vitro prevented the aminoglycoside-induced reduction in relative expression of mRNA for both fimbrin and class III β-tubulin. Neither insulin (5 μM) nor a combination of dibutyryl cyclic AMP (5 mM) and the phosphodiesterase inhibitor IBMX (0.5 mM) prevented the decrease in relative expression of the mRNAs for the hair cell-specific proteins, but both treatments allowed their partial recovery in vitro during the 4-day-period after removal of aminoglycoside. It is concluded that the cells of the sensory epithelium of the chick utricle subjected to aminoglycoside-induced damage undergo a process in which mRNA expression is switched away from the production of functional proteins and towards proteins necessary for structural re-organisation. The restoration of mRNA expression to a normal pattern is promoted by the growth factor insulin and by cyclic AMP.
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Damaged hair cells in the avian basilar papilla are replaced by regenerative proliferation of supporting cells and transdifferentiation of supporting cells into hair cells. In the mammalian vestibular system, transdifferentiation and, possibly, the repair of damaged hair cells appear to play significant roles. Several growth factors have been found to be associated with the regeneration/repair process: insulin, insulin-like growth factor 1 (IGF-1), and fibroblast growth factors are important for avian inner ear regeneration/repair, whereas epidermal growth factor, transforming growth factor α, insulin, IGF-1, and IGF-2 are important for regeneration/repair in the mammalian labyrinth. Increasing evidence suggests that regeneration/repair of mammalian auditory hair cells is possible during the early neonatal period and may exist to a very limited degree at later times.
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There is substantial evidence that the motility of mammalian outer hair cells is generated close to or within the plasma membrane. Several analogies between the outer hair cell cortical lattice and the membrane-related cytoskeleton of erythrocytes have been noted. In erythrocytes a member of the anion exchanger protein family, AE1, also known as Band 3, is involved in membrane-cytoskeleton linkage via Protein 4.1. In the following paper, the presence of these two proteins in gerbilline outer hair cells is confirmed by western blot. Furthermore, co-localization of these two proteins was detected in the lateral wall of outer hair cells by immunofluorescence and postembedding electron immunohistochemistry. Band 3 is restricted to this region, whereas Protein 4.1 has a somewhat more dispersed distribution.
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Fimbrin expression in the developing rat cochlea

Abstract

Hearing Res.

J. Immunol. Meth.

Cell

Hearing Res.

Hearing Res.

Hearing Res.

Hearing Res.

Localization of actin and microfilament-associated proteins in the microvilli and terminal web of the intestinal brush border by immunofluorescence microscopy

J. Cell Biol.

Fimbrin, a new microfilament-associated protein present in microvilli and other cell surface structures

J. Cell Biol.

Fimbrin is cytoskeletal protein that crosslinks F-actin in vitro

Fimbrin is homologue of cytoplasmic phosphoprotein plastin and domains homologous with calmodulin and actin gelation proteins

J. Cell Biol.

Actin in the inner ear: the remarkable structure of the stereocilium

Nature

Actin, myosin and associated proteins in the vertebrate auditory and vestibular organs: immunocytochemical and biochemical studies

Three different actin filament assemblies occur in every hair cell: each contains a specific actin crosslinking protein

J. Cell Biol.

Differential localization of villin and fimbrin during development of the mouse visceral endoderm and intestinal epithelium

Development

Transducing mechanisms in the lateral line canal organ receptors