XB-ART-41886J Neurosci July 21, 2010; 30 (29): 9683-94.
The R109H variant of fascin-2, a developmentally regulated actin crosslinker in hair-cell stereocilia, underlies early-onset hearing loss of DBA/2J mice.
The quantitative trait locus ahl8 is a key contributor to the early-onset, age-related hearing loss of DBA/2J mice. A nonsynonymous nucleotide substitution in the mouse fascin-2 gene (Fscn2) is responsible for this phenotype, confirmed by wild-type BAC transgene rescue of hearing loss in DBA/2J mice. In chickens and mice, FSCN2 protein is abundant in hair-cell stereocilia, the actin-rich structures comprising the mechanically sensitive hair bundle, and is concentrated toward stereocilia tips of the bundle''s longest stereocilia. FSCN2 expression increases when these stereocilia differentially elongate, suggesting that FSCN2 controls filament growth, stiffens exposed stereocilia, or both. Because ahl8 accelerates hearing loss only in the presence of mutant cadherin 23, a component of hair-cell tip links, mechanotransduction and actin crosslinking must be functionally interrelated.
PubMed ID: 20660251
PMC ID: PMC2922854
Article link: J Neurosci
Genes referenced: abr actb actg1 actl6a ag1 fscn1 fscn2 notch1 pls1 tbx2
Antibodies: Fscn2 Ab1
Article Images: [+] show captions
|Figure 1. Rapid progression of hearing loss in DBA/2J mice. Average 16 kHz ABR thresholds are shown for five DBA-related strains tested from 4 to 28 weeks of age. The data points for DBA/2J and those for DBA/2NCrl, the most closely related strain, are connected by lines to contrast the difference in their hearing loss progression. Error bars represent the SDs of the means to illustrate the degree of variation observed at each time point. ABR thresholds for all auditory test stimuli, SEs, and numbers of mice tested for each strain and test age combination are given in supplemental Table 1 (available at www.jneurosci.org as supplemental material).|
|Figure 2. Rapid degeneration of cochlear hair cell bundles in DBA/2J mice. In the basal turn of the cochlea, bundles of outer hair cells (OHCs) and inner hair cells (IHCs) of DBA/2J mice (2J) appeared normal at 2 weeks of age ( a ), but at 1 month ( b ) and 3 months ( c ) of age, most OHC bundles already showed signs of degeneration, and some were missing (indicated by asterisks). By 6 months of age in the base ( d ), all OHCs were missing and the few remaining IHCs had degenerated bundles (indicated by arrow); in the middle turn ( e ), bundles were severely disheveled (arrowhead) and often had lost most rows of stereocilia (arrow). In contrast, all OHC and IHC bundles of the control DBA/2NCrl mice (2NCrl) at 3 months ( g ) and 6 months ( f , h ) of age appeared normal, and only a few OHC bundles were missing (asterisks in h ); the bundles of the remaining OHCs appeared normal. A fourth row of OHCs was seen at frequent intervals throughout the length of the cochlea in both DBA/2J and DBA/2NCrl mice (indicated by arrow in h ). Panel widths: a–d , g , h , 29 μm; e , f , 7.3 μm.|
|Figure 3. A SNP variant within the Fscn2 gene is unique to the DBA/2J strain and changes a highly conserved amino acid. a , Nucleotide variation among 20 inbred mouse strains for the rs26996001 SNP, which is located within exon 1 of the Fscn2 gene. DBA/2J is the only strain examined with an adenine (A) at this position; all other strains have guanine (G). The G to A nucleotide substitution causes an amino acid change from arginine (R) to histidine (H) at position 109 of the mouse FSCN2 protein (NP_766390). b , Evolutionary conservation of the FSCN2 arginine (R) residue that is mutated in DBA/2J mice. This residue (highlighted in blue) is conserved in fascin proteins of all mammalian species examined, as well as fascins of other vertebrate species. c , Genealogy of DBA-related inbred mouse strains and their association with the Fscn2 SNP. All are direct descendants of the original dba strain developed by C. C. Little in 1909. Shown are the approximate dates when these DBA-related inbred strains were separated from one another (Bailey, 1978; Zylstra et al., 2003). DBA/2J is the only strain with the G>A missense mutation in Fscn2; this mutation occurred sometime between 1951 and 1975, as deduced from archived DNA samples of DBA/2J strain mice. d , Molecular model of FSCN1 (Protein Databank Structure 1DFC) showing the β-trefoil domains (1–4), the regulatory phosphorylation target S39, and the predicted location of the R109 residue of FSCN2 (R110 in FSCN1) that is mutated in DBA/2J mice. e , Hearing loss in F2 intercross progeny of (DBA/2J × DBA/2NCrl) F1 hybrids strongly associates with homozygosity for the DBA/2J allele of Fscn2. Shown are the average 16 kHz ABR thresholds and SEs for each of the three Fscn2 genotypes of F2 mice: D2J/D2J (homozygous for the DBA/2J mutation, n = 9), D2J/D2NCrl (heterozygous for the mutation, n = 7), and D2NCrl/D2NCrl (homozygous for the wild-type DBA/2NCrl allele, n = 5), all tested at 6 weeks of age. The DBA/2J mutant allele appears recessive to the DBA/2NCrl wild-type allele.|
|Figure 5. Identification and quantitation of FSCN2 in chicken vestibular stereocilia. a , Structure of FSCN2 protein. Colors indicate identity of residues in three-way comparison of chicken, mouse, and Xenopus FSCN2. Peptides used for antibody generation are indicated by black bars and antibody numbers; the position of the mutation in DBA/2J is indicated with an asterisk. The positions of the four β-trefoil domains are indicated below. b , Representative MS2 spectrum of a FSCN2 peptide identified by tandem mass spectrometry from a chicken hair bundle preparation. The intensity of peptide fragments, typically fragmented at peptide bonds during collision-induced dissociation, is plotted against the mass-to-charge ratio (m/z). Note the nearly complete y- and b-ion ladders, representing fragmentation sequentially at successive peptide bonds from the N and C termini. c , Distribution of FSCN2 peptides identified by X! Tandem algorithm from hair bundles. The width of each bar indicates the length of the identified peptide, the position of the bar along the x-axis corresponds to its position in the FSCN2 sequence, the height of the bar indicates the number of identical peptides identified, and the color of the bar corresponds to log(e), the statistical significance for peptide identification, averaged over all identical peptides. Note that the peptide analyzed in b was one of those represented by the black bar at ∼380 residues. d , Crosslinker/actin abundance ratios in E20 chick and P5 rat vestibular hair bundles. e , Immunoblot detection of FSCN2 in purified hair bundles with antibody 36. Bu, 100 ng of total hair-bundle protein; Ag, amount of agarose equivalent to that in bundles lane; Ut, 2 μg of peeled utricular epithelium; BP, 2 μg of peeled basilar papilla epithelium; Re, 1.5 μg of retina; and Br, 2 μg of total brain protein. f , g , Profile scans of bundle and utricular epithelium lanes from e .|
|Figure 6. FSCN2 in chick and Xenopus hair bundles. a , Low-power view of actin and FSCN2 in E20 chick utricle. Areas magnified in b and c are indicated. b , High-power view of single bundle. Note high levels of FSCN2 in rows of tallest stereocilia, adjacent to kinocilium notch. c , Reslice profile image of bundles showing localization of FSCN2 near stereocilia tips; tallest stereocilia are crushed against the coverslip. Note FSCN2 absence from a short, presumably immature bundle (asterisk). d , Reslice profile image showing FSCN2 localization near tips of adult Xenopus saccule bundles. e , Immunogold electron microscopy localization of FSCN2 in E20 chick utricle. All gold particles in micrograph are circled. Inset, magnification of region indicated with dashed lines. f , Differential localization of crosslinkers in E20 chick utricle. Note concentration of FSCN2 toward tips and PLS1 in middle of bundles. Neither crosslinker is abundant at bundle base. g . Quantitation of normalized actin signal, FSCN2/actin ratio, and PLS1/actin ratio in 14 cells from three utricle preparations. Diagram behind data depicts typical bundle morphology; gray box indicates region chosen for profile analysis. h . Scanning electron micrograph showing typical chick utricle hair bundles; note the length of the tallest stereocilia. KC, Kinocilium; SC, stereocilia. Panel heights: a , 52 μm; b , 8 μm; c , 12 μm; d , 12 μm; f , 32 μm. Scale bars: e , 500 nm; e , inset, 100 nm; h , 1 μm.|
|Figure 7. FSCN2 in mouse cochlea and vestibular system. a–c , Developmental appearance of FSCN2 around P10 in bundles of inner hair cells and by P30 in bundles of outer hair cells. d , FSCN2 in P10 cochlea; some labeling is seen in bundles of outer hair cells. The image is a maximum projection of 10 images, taken at 0.3 μm z-intervals. e , Magnified view of two inner hair cell (IHC) bundles from d . FSCN2 is primarily in the tallest stereocilia, near tips (asterisk). f , Similar distribution of FSCN2 in P30 DBA/2J mice. g , FSCN2 in mouse utricle hair bundles; note localization toward tips. The image is a z-projected (maximum) reslice image from a stack acquired axially through the utricular epithelium. h , ACTG1 developmental appearance around P10 in mouse cochlea. i , Quantitative RT-PCR showing elevation of FSCN2 transcripts around P10. No increase in Actg1 or Actb transcript levels was seen. Panel heights: a–c , f , h , 70 μm; d , 60 μm; e , g , 19 μm.|