Search

The development of the verterbrate inner ear depends on the precise expression of fibroblast growth factors. In a mutagenisis screen for zebrafish with abnormalities of inner-ear development and behavior, we isolate a mutant line, ru622, whose phenotypic characteristics resembled those of null mutants for the gene encoding fibroblast growth factor 8 (Fgf8): an inconsistent startle response. circular swimming, fused otoliths, and abnormal semi-circular canals. Positional cloning disclosed thE the mutant gene encodes that the transcriptional corepressor ATrophin2. Both the Fgf8 itself , were found to be overexpressed in ru622 mutants. We therefore hyphothesized that an excess of Sef eliminates Fgf8 signals and produced a Fgf8 null phenotype in ru622 mutants. In support of this idea we could rescue larvae whose atrophin2 plays a role in the feedback regulation of Fgf8 signaling. When mutation of the atrophin2 gene results in the overexpression of both Fgf8 and Sef, the excessive Sef inhibits Fgf8 signaling. The resultant imbalance of Fgf8 and Sef signals then underlies the abnormal aural development observed in ru622. auditory system | hearing | vestibular system

Amplification of auditory stimuli by hair cells augments the sensitivity of the vertebrate inner ear. Cell-body contractions of outer hair cells are thought to mediate amplification in the mammalian cochlea. In vertebrates that lack these cells, and perhaps in mammals as well, active movements of hair bundles may underlie amplification. We have evaluated a mathematical model in which amplification stems from the activity of mechanoelectrical-transduction channels. The intracellular binding of [Ca.sup.2+] to channels is posited to promote their closure, which increases the tension in gating springs and exerts a negative force on the hair bundle. By enhancing bundle motion, this force partially compensates for viscous damping by cochlear fluids. Linear stability analysis of a six-state kinetic model reveals Hopf bifurcations for parameter values in the physiological range. These bifurcations signal conditions under which the system's behavior changes from a damped oscillatory response to spontaneous limit-cycle oscillation. By varying the number of stereocilia in a bundle and the rate constant for [Ca.sup.2+] binding, we calculate bifurcation frequencies spanning the observed range of auditory sensitivity for a representative receptor organ, the chicken's cochlea. Simulations using prebifurcation parameter values demonstrate frequency-selective amplification with a striking compressive nonlinearity. Because transduction channels occur universally in hair cells, this active-channel model describes a mechanism of auditory amplification potentially applicable across species and hair-cell types.

The L-type voltage-gated [Ca.sup.2+] channels that control tonic release of neurotransmitter from hair cells exhibit unusual electrophysiological properties: a low activation threshold, rapid activation and deactivation, and a lack of [Ca.sup.2+]-dependent inactivation. We have inquired whether these characteristics result from cell-specific splicing of the mRNA for the L-type [[Alpha].sub.1D] subunit that predominates in hair cells of the chicken's cochlea. The [[Alpha].sub.1D] subunit in hair cells contains three uncommon exons: one encoding a 26-aa insert in the cytoplasmic loop between repeats I and II, an alternative exon for transmembrane segment IIIS2, and a heretofore undescribed exon specifying a 10-aa insert in the cytoplasmic loop between segments IVS2 and IVS3. We propose that the alternative splicing of the [[Alpha].sub.1D] mRNA contributes to the unusual behavior of the hair cell's voltage-gated [Ca.sup.2+] channels.

The voltage-gated [Ca.sup.2+] channels that effect tonic release of neurotransmitter from hair cells have unusual pharmacological properties: unlike most presynaptic [Ca.sup.2+] channels, they are sensitive to dihydropyridines and therefore are L-type. To characterize these [Ca.sup.2+] channels, we investigated the expression of L-type [[Alpha].sub.1] subunits in hair cells of the chicken's cochlea. In PCRs with five different pairs of degenerate primers, we always obtained [[Alpha].sub.1D] products, but only once an [[Alpha].sub.1C] product and never an [[Alpha].sub.1S] product. A full-length [[Alpha].sub.1D] mRNA sequence was assembled from overlapping PCR products: the predicted amino acid sequence of the [[Alpha].sub.1D] subunit was about 90% identical to those of the mammalian [[Alpha].sub.1D] subunits. In situ hybridization confirmed that the [[Alpha].sub.1D] mRNA is present in hair cells. By using a quantitative PCR assay, we determined that the [[Alpha].sub.1D] mRNA is 100-500 times more abundant than the [[Alpha].sub.1C] mRNA. We conclude that most, if not all, voltage-gated [Ca.sup.2+] channels in hair cells contain an [[Alpha].sub.1D] subunit. Furthermore, we propose that the [[Alpha].sub.1D] subunit plays a hitherto undocumented role at tonic synapses.

Bacteriophage display of antibodies provides a method for the generation of immunological reagents against rare and uncharacterized antigens. To ascertain the usefulness of this approach for the characterization of inner-ear proteins, we produced a bacteriophage-displayed antibody-fragment library directed against proteins from the bullfrog's sacculus. This library was probed for bacteriophage that bound to proteins present in a lysate of hair cells, the sensory receptors of the inner ear. The predominant bacteriophage clone after selection expressed an antibody fragment that recognized a single protein in the inner ear. This antigen occurred in both the nonsensory and sensory epithelia of the sacculus. The specificity of the antibody fragment indicates that our bacteriophage-displayed library provides a useful source of immunological tools that should facilitate the identification and biochemical characterization of novel proteins in the inner ear.