Genetic modification of hearing in tubby mice: evidence for the existence of a major gene (moth1) which protects tubby mice from hearing loss

Abstract

Quantitative trait locus (QTL) analysis of genetic crosses has proven to be a useful tool for identifying loci associated with specific phenotypes and for dissecting genetic components of complex traits. Inclusion of a mutation that interacts epistatically with QTLs in genetic crosses is a unique and potentially powerful method of revealing the function of novel genes and pathways. Although we know that a mutation within the novel tub gene leads to obesity and cochlear and retinal degeneration, the biological function of the gene and the mechanism by which it induces its phenotypes are not known. In the current study, a QTL analysis for auditory brainstem response (ABR) thresholds, which indicates hearing ability, was performed in tubby mice from F(2)intercrosses between C57BL/6J- tub / tub and AKR/J-+/+ F(1)hybrids (AKR intercross) and between C57BL/6J- tub / tub and CAST/Ei.B6- tub / tub F(1)hybrids (CAST intercross). A major QTL, designated asmodifieroftubbyhearing1 ( moth1 ), was identified on chromosome 2 with a LOD score of 33.4 ( P < 10(-33)) in the AKR intercross (181 mice) and of 6.0 ( P < 10(-6)) in the CAST intercross (46 mice). This QTL is responsible for 57 and 43% of ABR threshold variance, respectively, in each strain combination. In addition, a C57BL/6J congenic line carrying a 129/Ola segment encompassing the described QTL region when made homozygous for tubby also exhibits normal hearing ability. We hypothesize that C57BL/6J carries a recessive mutation of the moth1 gene which interacts with the tub mutation to cause hearing loss in tub / tub mice. A moth1 allele from either AKR/J, CAST/Ei or 129/Ola is sufficient to protect C57BL/6J- tub / tub mice from hearing loss.

Figure 1

Frequency distribution of click ABR threshold values from F₂ mice homozygous for tubby. (a) F₂ mice from the AKR/J × C57BL/6J-tub/tub intercross. (b) F₂ mice from the CAST.B6-tub/tub × C57BL/6J-tub/tub intercross. The horizontal bar indicates the range of click ABR thresholds observed in mice from the parental strains C57BL/6J, AKR/J and CAST/Ei tested at the same age.

Figure 2

LOD score distributions for associations of click ABR threshhold with loci on chromosome 2 in the AKR intercross (a) and in the CAST intercross (b). The dotted line represents highly significant linkage as assessed by permutation testing (P < 0.001).

Figure 3

The ABR thresholds in C57BL/6J-tub/tub.moth1^129/Ola/moth1^B6 and CAST/Ei.B6-tub/tub mice. The mean ± SD of the sound pressure threshold is represented by a bar and shown for each genotype and stimulus as indicated. Five to six mice at 6–8 weeks of age were tested for each column. C57BL/6J-tub/tub and C57BL/6J-tub/+ strains were used as an abnormal and normal control, respectively. C57BL/6J-tub/tub.moth1^129/Ola/moth1^B6 and CAST/Ei.B6-tub/tub mice were generated as described in Materials and Methods.

Figure 4

Map position of the moth1 gene and the syntenic human genetic regions. Bar A represents the confidence intervel including the moth1 gene. Bar B represents the 129/Ola segment which the C57BL/6J.129/Ola congenic line carries.

Figure 5

Distribution of tubby immunoreactivity (A, C, E and F) and light microscopic morphology (B and D) of the cochlea of C57BL/6J (wt) and C57BL/6J-tub/tub (tub) mice at 10 weeks of age. The TUB protein is strongly expressed (green) in the outer and inner hair cells (OHC/IHC), the adjacent external phalangeal cells of Deiters (DC), cochlear nerve fibers (CN) and in the spiral ganglion cells (SC). Note that there are no obvious differences in distribution or expression of the tubby protein between normal and mutant mice (A and C). At this 8–10 weeks time point, no histopathological changes in the organ of Corti could be detected (B and D). Higher magnifications of representative sections of the Corti organ of C57BL/6J mice reveal the subcellular distribution of TUB. A cytoplasmic localization pattern can be observed in the outer and inner hair cells, depicted by arrowheads (E and F), whereas there is a distinct nucleolar immunoreactivity detectable in the external phalangeal cells (marked by arrows in F). TM, tectorial membrane, Scale bars: (A–D) 100 µm; (E) 5 µm; (F) 20 µm.