Identification of a rat model for usher syndrome type 1B by N-ethyl-N-nitrosourea mutagenesis-driven forward genetics

Abstract

The rat is the most extensively studied model organism and is broadly used in biomedical research. Current rat disease models are selected from existing strains and their number is thereby limited by the degree of naturally occurring variation or spontaneous mutations. We have used ENU mutagenesis to increase genetic variation in laboratory rats and identified a recessive mutant, named tornado, showing aberrant circling behavior, hyperactivity, and stereotypic head shaking. More detailed analysis revealed profound deafness due to disorganization and degeneration of the organ of Corti that already manifests at the onset of hearing. We set up a single nucleotide polymorphism (SNP)-based mapping strategy to identify the affected gene, revealing strong linkage to the central region of chromosome 1. Candidate gene resequencing identified a point mutation that introduces a premature stopcodon in Myo7a. Mutations in human MYO7A result in Usher syndrome type 1B, a severe autosomal inherited recessive disease that involves deafness and vestibular dysfunction. Here, we present the first characterized rat model for this disease. In addition, we demonstrate proof of principle for the generation and cloning of human disease models in rat using ENU mutagenesis, providing good perspectives for systematic phenotypic screens in the rat.

Figure 1.—

Overview of the crosses that resulted in the identification of the tornado mutant. Four ENU mutagenized males were used to generate F₁ animals by crossing to untreated females. Sixteen breeding pairs were selected from this progeny to produce the F₂ generation. Brother-sister matings were set up for these animals to breed induced mutations to homozygosity to reveal potential recessive phenotypes. The figure represents all matings set up for a specific F₁ pair (the others did not result in any visually apparent aberrant phenotype). One of the three F₂ matings resulted in progeny with an aberrant phenotype that was named tornado. Genetic inheritance of the phenotype was confirmed in a second mating. Squares and circles represent males and females, respectively. Solid symbols indicate animals with the tornado phenotype.

Figure 2.—

SNP-based mapping of the tornado mutation. (a) Distribution on the rat physical map of the 84 verified SNP markers used for mapping the tornado mutation. SNPs were selected from equally sized genomic segments (represented in alternating open and shaded bars). The mapping cross showed that the mutation is inherited in a normal autosomal recessive manner, which made it unnecessary to include markers on the X chromosome. (b) Representation of linkage of the mutant phenotype to the central region of chromosome 1. The polymorphic SNP markers are plotted on the x-axis ordered by chromosome location (the numbers on the bottom represent the chromosomes). The graph shows the percentage of BN alleles, calculated from 67 genotyped tornado animals obtained from the mapping cross. The tornado mutation was introduced in the Wistar background.

Figure 3.—

The tornado phenotype is caused by a premature stopcodon in Myo7a. (a) Schematic organization of Myo7a in the rat and the position and context of the identified tornado mutation (Myo7a^tnd-1Hubr). The exons encoding the myosin head are indicated at the top of the graph. (b) Sequence traces of the mutated position in exon 5 of a homozygous wild-type (top), heterozygous (middle), and mutant (bottom) animal.

Figure 4.—

Degeneration of the organ of Corti in tornado rats. SEM micrographs of the surface of the organ of Corti of tornado animals (Myo7a^{tnd-1Hubr/tnd-1Hubr}: a, b, d, and f and nonmutant littermates (Myo7a^tnd-1Hubr/+or Myo7a^+/+: c, e, and g) at 6 days (a), 10 days (b and c), 20 days (d and e), and 13 weeks (f and g) of age. At 6 days of age, the outer hair cell stereocilia are already disorganized and fused (a) and degenerate progressively (b, d, and f) even on the inner hair cells (f). Eventually, an irregular epithelium remains in the organ of Corti region (f). Bars, 10 μm.

Figure 5.—

Profound deafness in tornado rats. Typical auditory brain-stem responses from mutant tornado animals (Myo7a^{tnd-1Hubr/tnd-1Hubr}) and wild-type heterozygous littermates (Myo7a^tnd-1Hubr/+). (a) Four weeks of age, wild type; (b) 4 weeks of age, mutant; (c) 10 days of age, wild type; (d) 10 days of age, mutant. Horizontal axis: time window of 15 msec (1.5 msec/division); vertical axis: amplitude in microvolts. Stimulus onset at 1.5 msec. (Left, a and c) Good brain-stem responses obtained at different stimulation levels according to standard audiometrical descending top-down procedures until the level that no reproducible responses are recognized (i.e., auditory threshold); (right, b and d) all responses reveal no reproducible brain-stem responses at a maximum stimulation level of 83 dB (SPL), including soundfield correction.

Figure 6.—

Melanosome migration defect in tornado rats. Histological staining (hematoxylin/eosin) of the eyes of pigmented nonmutant (a) and tornado (b) animals, revealing the absence of melanosomes in the apical processes of the retinal pigment epithelium. Bars, 10 μm.