Research in the Section on Neurogenetics focuses on the identification and characterization of genes that are critical for structure and function of sensory hair cells in the cochlea. Spontaneous mutations that cause hearing loss are ideal tools to identify genes important for the functioning of the inner ear and to elucidate their role in these sensory systems. Towards this end research efforts concentrated on three lines of experimental investigation: (1) the ahl5 quantitative trait locus in the Black Swiss strain, (2) a high-frequency hearing loss phenotype in the NIH Swiss line and (3) hearing loss in the Tail short (Ts) mutant mouse. (1) Heterogeneous strains represent a genetic spectrum different from the common inbred strains and show a greater degree of allelic heterogeneity. They are models to isolate genetic interactions and to identify new deafness alleles. We found that mice of the Black Swiss strain develop early-onset slowly progressing hearing loss. The segregation pattern in intercrosses and backcrosses was consistent with a polygenic inheritance. Genome-wide linkage analyses on backcross and intercross populations localized two quantitative trait loce (QTLs) underlying hearing loss in Black Swiss mice. A major QTL localized to chromosome 10 (named ahl5) and a second small-effect QTL localized to chromosome 18 (ahl6). At four and eight weeks of age Black Swiss mice show ABR threshold shifts of 30 and 40 dB SPL at the 16 kHz stimulus, respectively. The gross morphology of the major structures in the scala media appeared normal. In particular the stria vascularis, spiral ligament and organ of Corti showed no pathology at this level. In addition, the spiral ganglion was densely packed with normal-appearing neurons. To ascertain the morphology of the organ of Corti at higher resolution, whole mount surface preparations were stained with phalloidin. At postnatal days (P) 3 through P7, we consistently observed a marked stereociliary hair bundle defect, which was most obvious on outer hair cells. Most pronounced at mid-apical regions, the hair bundle was bent, twisted, displayed a mild polarity defect and was compressed at its lateral edges and rounded at its apical pole. The phenotype is reminiscent to the hair bundle defects observed in the BUB/BnJ and Vlgr1-/- mutant suggesting that a pathway that regulates the structure of the hair bundle is affected in Black Swiss hair cells. Consistent with the stereocilia defects, we observed absent DPOAE's in eight-week old Black Swiss mice. To fine-map ahl5, (Black Swiss x CAST/Ei) F1 hybrids were backcrossed to Black Swiss for ten consecutive generations. Recombination events in two congenic lines delimit the ahl5 critical interval to a 2-Mb region. (2) The NIH Swiss outbred strain was originally derived from a population of Swiss albino mice in Lausanne, Switzerland. Previous ABR measurements in a population of thirty NIH Swiss mice showed a wide range of thresholds from normal to completely absent responses. This wide range of thresholds offered the opportunity to select for specific phenotypes such as hearing loss affecting only the higher frequencies. The cochlea is tonotopically organized from the base to the apex, where higher frequencies are recognized at the base and lower frequencies at the apex. The exact molecular mechanisms that underlies this frequency-recognition gradient is not known although some data suggest that location-specific alternative splice-forms of the Ca2+-activated K+ channel Kcnma1 might play a role. As most types of hearing impairment ultimately affect all frequencies, a mouse model that exhibits a frequency-specific hearing loss could provide insights into how frequency-selectivity in the cochlea is attained. Starting from a phenotypically mixed population, we selected mice that showed a high-frequency hearing loss at eight weeks of age and developed an inbred line, referred to as HF. At each brother-sister intercross generation a cohort of 10 - 20 mice were ABR-tested. At four, eight, and 26 weeks of age these mice show a hearing loss only at the 32-kHz stimulus, but not at the click or 8-kHz frequency. At the 16-kHz frequency the thresholds appear slightly increased. The standard deviation ranges from 6 to 10 dB SPL. Hence, after nine generations of inbreeding the phenotype is very robust. In parallel, we developed a line that shows hearing loss across all frequencies (AF). To obtain a second independent measure of the hearing phenotype in these lines, we investigated the structure of the stereociliary hair bundle using phalloidin staining. We found that in animals of the HF line the stereociliary hair bundle had a normal appearance, but it was deformed in the AF line. The AF-phenotype showed the characteristics of the Black Swiss hair bundle. (3) Tail Short is a semi-dominant mutation, which was discovered by Walter Morgan in 1950. The mutation arose spontaneously on the BALB/c background and maps to distal chromosome 11. Homozygous mutants die before or at the time of gestation between 3.5 and 5.5 days post-coitum. The most obvious phenotype in heterozygotes is the short, kinked, and curled tail. This is the result of skeletal abnormalities that occur along the vertebral column, which include fusion of two or three successive vertebrae, dyssymphyses, and additional vertebrae and ribs. These skeletal malformations can be traced back to an absent or dysmorphic notochord, a thinner neural tube and a marked anemia, which is first observed in 8-day old embryos. Embryonic lethality in heterozygotes is due to neural tube defects including exencephaly and spina bifida. The known effect of the neural tube on inner ear development and the previously recognized link between neural tube defects and planar cell polarity defects of cochlea hair cells prompted us to study the hearing phenotype in the Ts mutant. By genetic means, we show that the Ts phenotypes arise from an 18kb deletion/insertion of the Rpl38 gene, encoding a ribosomal protein of the large subunit. We show that Ts mutants exhibit significantly elevated auditory-brain stem response (ABR) thresholds and reduced distortion-product otoacoustic emissions (DPOAEs), in the presence of normal endocochlear potentials and typical inner ear histology suggestive of a conductive hearing impairment. We locate the cause of the hearing impairment to the middle ear, demonstrating over-ossification at the round window ridge, ectopic deposition of cholesterol crystals in the middle ear cavity, enlarged Eustachian tube, and chronic otitis media with effusion all beginning at around three weeks after birth. Using specific antisera, we demonstrate that Rpl38 is an 8 kDa protein that is predominantly expressed in mature erythrocytes. Finally, using an Rpl38 cDNA transgene we rescue the Ts phenotypes. Together, these data present a previously uncharacterized combination of interrelated middle ear pathologies and suggest Rpl38-deficiency as a model to dissect the causative relationships between neo-ossification, cholesterol crystal deposition, and Eustachian tube in the etiology of otitis media.
