Pedigree production

We established a large-scale mouse screen for recessive mutations causing age-related disease incorporating an ENU mutagenesis4 and phenotype-driven approach. We screened G 3 mice in large pedigrees of up to 100 mice4 (Supplementary Fig. 1a). Briefly, male C57BL/6J mice were mutagenized with ENU and mated to C3H.Pde6b+ mice11 to generate G 1 founder males. G 1 males were subsequently bred to C3H.Pde6b+ mice to generate G 2 offspring. Next, large G 3 pedigrees of at around 100 individuals were produced by two rounds of mating of the founder G 1 male to ⩾8 G 2 female offspring. Large pedigrees ensured sufficient homozygous G 3 individuals were available to map a phenotype directly from the G 3 cohort. Each G 3 pedigree was generated as two cohorts, 2–3 months apart. Across an entire G 3 pedigree comprising some 100 mice, we might expect to identify on average 12 affected individuals homozygous for an individual recessive mutation inherited from the G 1 founder male. The mixed genetic background assisted the mapping of mutations underlying the affected individuals identified in the G 3 pedigrees.

Wild-type C3H strains exhibit retinal degeneration, due to a recessive mutation in the Pde6b gene (Pde6brd1), and hence we employed the line, C3H.Pde6b+ (ref. 11), congenic for the BALB/c region encompassing Pde6b and which does not exhibit retinal degeneration enabling screening for visual abnormalities. C57BL/6J also carries a recessive mutation in the Cdh23 gene resulting in age-related hearing loss (Cdh23ahl) (ref. 12). Therefore, in our protocol G 2 female mice were genotyped for the Cdh23ahl allele and only offspring from G 2 females wild-type for Cdh23 were used for auditory assessment.

Phenotyping pipeline and mutant detection

We developed and implemented a high-throughput phenotyping pipeline to examine the G 3 mice generated in the ageing screen (Table 1 and summarized in Supplementary Fig. 1b). This incorporated many of the established standard operating procedures that have been utilized in the International Mouse Phenotyping Consortium (IMPC, www.impc.org) phenotyping pipeline13. The IMPC has implemented IMPReSS (www.mousephenotype.org/impress), a database of standardized phenotyping pipelines that encompasses a large number of disease systems and ensures data reproducibility and integrity. The design of the pipeline considered carefully the timing and order of the phenotyping tests to ensure that we minimized the impact of individual tests on other phenotype outcomes. For each G 3 pedigree the production of dual cohorts provided an opportunity to explore in more detail phenotypes detected in the first cohort and to add complementary phenotype tests. The scale of the screen precluded a complete pathological examination of tissues from all G 3 mice produced, but this was carried out on selected mice to assist in the confirmation or determination of a phenotype. We have analysed 157 pedigrees to date, out of which 134 are complete and 23 are currently active (Table 2). The average pedigree size was 102 mice and a total of 13,601 G 3 animals have been generated and phenotyped from the 157 pedigrees.

Table 1 The Harwell Ageing screen phenotyping pipeline. Full size table

Table 2 Summary of output from the ENU screen including number of pedigrees analysed along with mutant discovery and mutations cloned. Full size table

Phenotype detection and MouseBook

We devised a high-throughput automated phenotype-detection strategy, alongside a resource to visualize the phenotyping data. We defined a mutant line as one where multiple affected G 3 mice (⩾3) in a pedigree display very similar phenotypes. All raw data from the phenotyping platforms was initially captured into local laboratory information management systems according to the data standards defined in the IMPReSS database. Following quality control procedures, a step-wise strategy was devised to detect outliers from temporal data in ENU pedigrees. Briefly, we used a reference range approach that established percentile values for the entire data set that provides high and low critical values, approximately equating to ±2 s.d. Parameter values outside of these critical values for three or more animals in a pedigree led to a phenodeviancy call. Subsequently, phenodeviant mice were annotated with the appropriate phenotype (MP term) as specified by IMPReSS. All raw data and automatically annotated phenotype data are available via the publically accessible data portal MouseBook (http://www.mousebook.org/).

To assess the false positive rate (FPR) of the reference range method, we calculated the FPR of wild-type C57BL/6NTac animal data from the IMPC project as a test data set. IMPC uses similar phenotype procedures to the ageing project. We then modelled the ageing pedigrees by sampling random IMPC baseline animals to produce a simulated pedigree and employed the reference range method on each procedure. Wild-type animals should not be called as outliers therefore any positive calls from the method are classed as false positive calls. Using simulated pedigrees, we detected between 0.58 and 1.22% phenodeviance, suggesting a low average FPR of 0.71% across the procedures examined (Supplementary Table 1).

Categorical data, such as X-ray abnormalities, were scored as potential mutants when observed in multiple affected G 3 mice (⩾3) in a pedigree. We also, on occasion, considered mice as phenodeviant when outliers were <2 s.d. from the mean but where deviations were sustained over multiple time points or, alternatively, additional phenotyping data confirmed a phenotype was present. Each putative mutant was confirmed by one or all of the following; appropriate statistical analyses of primary phenotyping data, comparison of data from other relevant phenotypic tests, inheritance testing and mapping data.

Mutant lines

In total, 105 distinct mutant lines were identified in 72 out of 157 pedigrees completed or currently undergoing analysis (Supplementary Table 2). The mean number of affected G 3 mice for each line was 6.1, with a range from 2 to 17 (Fig. 1a). Figure 1b demonstrates the distribution of the number of mutations detected per pedigree. Some pedigrees had multiple segregating mutations, which may reflect the size of the pedigrees and the inherent chance of detecting multiple affected mice for distinct genetic lesions.

Figure 1: Summary statistics for mutation discovery in the Harwell ageing Screen. (a) The distribution of the numbers of affected mice observed for each mutation within the G 3 pedigrees. For lines with low numbers of affected mice, putative mutations were confirmed by inheritance testing and/or cloning of the underlying mutation. (b) The distribution of the number of mutations identified per pedigree from a total of 105 mutant lines identified from a total of 157 pedigrees. (c) The distribution of mutation discovery over time. The time point of the initial identification of an individual phenotype is represented here for a total of 105 mutant lines identified in the screen to date. Where mutants were outliers in more than one phenotypic test the time point of the first test to reveal an abnormality is shown. Full size image

Abnormal phenotypes were identified throughout the lifespan of the pedigrees (Fig. 1c). Visible anomalies such as coat colour variations, dysmorphologies and extreme behavioural abnormalities were often detected during initial husbandry practices early in the pipeline before scheduled screens took place. Between 3 and 6 months the first round of tests uncovered a large number of additional phenotypes. Most importantly, of the 105 mutant lines identified, 27 were only detected after 6 months (Fig. 1c).

Mapping and mutation detection

Individual mutations were mapped utilizing the Illumina Medium Density SNP mapping panel, which is informative at over 900 single-nucleotide polymorphisms (SNPs) for the C3H.Pde6b+ and C57BL/6J strains. The genotypes of affected animals were compared with those of unaffected individuals from the same pedigree, generally resulting in a map location of around ∼20 Mb. Following mapping, we carried out whole-genome sequencing (WGS) of the G 1 founder male, or, on occasion, an affected G 3 . A local database was developed to manage the WGS data from G 1 mice. All WGS data was analysed through a standard next-generation sequencing (NGS) pipeline. The MouseBook portal holds the complete range of ENU mutations identified in G 1 mice (noncoding and coding mutations) and within noncoding regulatory regions (such as lincRNAs, miRNAs and promoter regions) annotated from WGS analysis pipeline. This archive includes all ENU mutations identified during NGS, including all incidental mutations that have not been associated with a phenotype. This data set enabled rapid identification of all mutations segregating within the sequenced pedigrees and this information, combined with the map location of the phenotype, allowed us to determine the causative ENU-induced genetic lesion with high confidence.

For the 105 lines identified we have currently mapped 81. WGS of 28 G 1 and 8 G 3 mice identified the gene lesion underlying the observed phenotype in 44 mutant lines (Table 2 and Supplementary Table 2). For individual pedigrees that contained multiple segregating phenotypes (Fig. 1b) we were able to identify several causative mutations from a single-G 1 sequence. Some obvious candidate genes are undergoing individual gene sequencing and some phenotypes were not of sufficient impact to warrant WGS. For three of the mapped mutations analysed (all early phenotypes) we failed to uncover a coding lesion, and these may represent functional changes in noncoding DNA. One coding lesion was identified as an intracisternal A-particle insertion that appears to have occurred spontaneously on the C3H.Pde6b+ background pedigree MPC-59, disrupting the Mcr1 gene and resulting in a yellow coat colour (Supplementary Table 2). Out of the 27 ageing phenotypes, 23 have been mapped and for 12 late-onset mutant lines we have identified and confirmed the underlying mutation (Table 3).

Table 3 Examples of phenotypes and mutations identified as part of the Harwell Ageing Screen. Full size table

Genetic and phenotypic analysis of mutants

We carried out a high-level ontological analysis of the distribution of phenotypes across disease areas investigated by our phenotyping pipeline. The total number of phenotypes observed across different phenotypic areas is shown in Fig. 2. Where a single line exhibited more than one phenotype resulting from the pleiotropic effects of the underlying mutation we have listed all the phenotypes detected. In the majority of phenotypic areas we have identified late-onset phenotypes, but for some categories we have uncovered no late-onset models. For example, dysmorphology mutants are overt abnormalities normally associated with developmental defects, and hence are unlikely to develop late in life.

Figure 2: The distribution of early and late phenotypes across a variety of disease and biological categories. A histogram of the distribution of early and late phenotypes across different phenotypic categories. Late phenotypes are defined as those phenotypes where affected mice were identified at 7 months or later. Note that we catalogue the number of individual ‘phenotypes’ in each disease or biological category, and that individual mutant lines may demonstrate multiple phenotypes. Full size image

Table 3 summarizes the genes that have been identified for late-onset phenotypes. In each case, we have either uncovered a gene for which there was no prior functional information, or alternatively, we have assigned novel functionality to a gene with known functions. For these mutants there is only a single medium or high-confidence coding mutation in the minimal mapping region and for laminin alpha 5 (Lama5) and tryptophanyl tRNA synthetase 2, mitochondrial (Wars2) (pedigrees MPC-205 and MPC-151, respectively) we have carried out complementation studies using a knockout (KO) allele to confirm these mutations as the causative allele (Table 3). In most cases, we also have identified functional alterations associated with the mutation that could account for the observed phenotype but, as these studies are ongoing, in several cases the causal link between mutation and phenotype are yet to be proven conclusively. Several examples in diverse biological areas serve to illustrate this powerful discovery platform for models of age-related disease.

We identified a missense mutation (E884G) in the Lama5 gene that results in a progressive nephrotic syndrome. G 3 mice were identified at 6 months of age with elevated plasma urea and creatinine levels, and with reduced plasma albumin (homozygotes, n=6, versus wild-type, n=33, Urea: 32.9±19.9 versus 5.9±1.1 mmol l−1, P<0.05, creatinine: 50.4±37.1 versus 11.45±3.6 μmol l−1, P<0.05, Albumin 16.6±1.6 versus 32.2±1.8 g l−1, P<0.05, Tukey’s multiple comparison test). Mice reached end-stage renal failure between 7 and 10 months of age. Lama5 has previously been shown to be critical in organ development14 and is associated with polycystic kidney disease15, but this is the first report of its involvement in a chronic renal phenotype. Recent sequencing data has identified Lama5 mutations in focal segmental glomerular sclerosis patients16. Thus, this novel mutant provides supporting evidence for a role for LAMA5 in nephrotic syndromes and will enable the investigation of the pathogenic processes involved.

Our studies also revealed a mutation in the aggrecan (Acan) gene in pedigree MPC-227, causing an A1946V substitution in the C-type-lectin domain, which results in late-onset joint deterioration and obesity, both novel phenotypic associations with this locus (Supplementary Fig. 2). By 12 months of age mutant mice have an altered body composition with a significantly higher percentage fat mass (Supplementary Fig. 2a). In contrast to the late-onset obesity, throughout life homozygous AcanA1946V mice have significantly lower absolute lean mass (Supplementary Fig. 2b). So, while metabolically obese, mutant mice are not heavier overall than littermates (Supplementary Fig. 2c). At 18 months of age mutant mice exhibit on X-ray bony deposits, particularly in the knee joints (Supplementary Fig. 2d,e). Existing mouse mutants17,18 have not recapitulated the early-onset osteoarthritis observed in patients with mutations in this gene19,20. In addition to the quantitative differences in fat mass, histopathology revealed qualitative differences in fat tissues. Adipocytes from white adipose tissue in AcanA1946V mice are enlarged and there is evidence of inflammatory cell inflammation (Supplementary Fig. 2f,g). Conversely, the brown adipose tissue shows reduced accumulation of fat in the mutant mice (Supplementary Fig. 2h,i). Aggrecan expression has been identified in several cell types within adipose tissue, including preadipocytes and pericytes, and can influence adipogenesis21.

Slc4a10 is a novel late-onset hearing loss gene

We have uncovered several novel genes associated with progressive and/or late-onset hearing loss, including solute carrier family 4, sodium bicarbonate transporter, member 10 (Slc4a10), Wars2, protein tyrosine phosphatase, receptor type, Q (Ptprq) and zinc finger, FYVE domain containing 26 (Zfyve26). Here we elaborate the phenotype and characterization of the Slc4a10 late-onset hearing loss mutant, which exemplifies the novel insights into gene function and molecular mechanisms associated with late-onset disease. Auditory phenotyping of pedigree MPC-96 at 3, 6, 9 and 12 months of age found all mice displayed a normal response to a clickbox stimulus. However, when assessed using auditory-evoked brainstem response (ABR) testing six mice were found to have mildly elevated hearing thresholds at 9 months of age thus indicating an impaired hearing function (Supplementary Fig. 3a). Subsequent screening of this pedigree at 12 months of age found these six mice had a further increase in their hearing thresholds suggesting a progressive phenotype (Supplementary Fig. 3b). After further breeding no hearing impaired mice were observed in the backcrossed G 4 litters. However in the inter-crossed G 5 litters mice with reduced hearing were identified, indicating a recessive inheritance.

A genome scan of G 3 mice showed linkage to a ∼63 Mb region on chromosome 2 containing 936 genes. Subsequent mapping narrowed the critical interval to ∼12.5 Mb (Supplementary Fig. 4a). Analysis of the WGS data identified only a single high-confidence non-synonymous coding change within the mapped interval, consisting of a T-to-C transition at nucleotide 1940 of the Slc4a10 gene (Ensembl transcript ID ENSMUST00000112480) causing a leucine-to-proline substitution at residue 647 (Supplementary Fig. 4b) A list of noncoding mutations in the minimal mapping region identified through WGS is shown in Supplementary Table 3. The presence of this lesion was confirmed using Sanger sequencing (Supplementary Fig. 4b). Only mice showing late-onset hearing impairment were homozygous for the L647P ENU-induced mutation. The L647 residue is within the transmembrane helix-containing domain of the protein (Supplementary Fig. 4c) and is conserved across species (Supplementary Fig. 4d). This mutation was predicted to be deleterious: SIFT, affect protein function (0.00); PolyPhen-2, probably damaging (0.986); and Mutation Taster, disease causing (0.999) (refs 22, 23, 24). This mouse line was named trombone (trmb).

To further investigate the auditory phenotype in the trombone model, additional mice were bred and assessed by clickbox and ABR at 2, 6, 9 and 12 months of age. All genotypes (Slc4a10+/+, Slc4a10+/trmb, and Slc4a10trmb/trmb) displayed a normal Preyer reflex in response to a clickbox stimulus at all ages tested. In addition, Slc4a10+/+ and Slc4a10+/trmb mice have ABR thresholds within the normal range at all ages tested. However, while Slc4a10trmb/trmb mice have similar ABR thresholds to their Slc4a10+/+ and Slc4a10+/trmb controls at 2 and 6 months of age, by 9 months they have mildly elevated ABR thresholds at all tested frequencies (Fig. 3). At 12 months of age the auditory thresholds of Slc4a10trmb/trmb mice are further elevated, displaying an increase of ⩾10 dB sound pressure level (SPL) at 8, 16 and 32 kHz, indicating a progressive late-onset auditory impairment (Fig. 3). In addition, no overt vestibular dysfunction (for example, circling, head bob, abnormal swim), craniofacial dysmorphology or weight phenotype were observed.

Figure 3: ABR phenotyping of trombone mice from 2 to 12 months of age. Minimum auditory detection thresholds (decibel SPL (dB SPL)) were determined using ABR and scored independently by two operators. (a) At 2 months of age, Slc4a10trmb/trmb (homozygous) mice (n=44) show only a very mild elevation in ABR thresholds in comparison with Slc4a10+/trmb (heterozygous) (n=54) and Slc4a10+/+ (wild-type) (n=18) littermates. (b) At 6 months of age, the very mild elevation of ABR thresholds is still observed in Slc4a10trmb/trmb mice (n=10) compared with Slc4a10+/trmb (n=21) and Slc4a10+/+ (n=13) littermates. (c) By 9 months of age, Slc4a10trmb/trmb mice (n=26) show significantly elevated ABR thresholds (∼20 dB shift) across all frequencies tested compared with Slc4a10+/trmb (n=25) and Slc4a10+/+ (n=6) littermates. (d) At 12 months of age, Slc4a10trmb/trmb mice (n=17) continue to have elevated thresholds compared with the normal hearing Slc4a10+/trmb (n=13) and Slc4a10+/+ (n=7) control mice. In addition, the average hearing thresholds of Slc4a10trmb/trmb mice increase between 9 and 12 months of age. The Slc4a10+/trmb and Slc4a10+/+ control mice do not show any auditory decline during the first 12 months of life, nor do they show any significant differences between their auditory thresholds at any frequency or age tested. Black circle/line, Slc4a10+/+; grey square/line, Slc4a10+/trmb; red triangle/line, Slc4a10trmb/trmb. Data shown are mean ±s.e.m. and significance determined using a one-way ANOVA with Tukey’s multiple comparisons test comparing Slc4a10trmb/trmb threshold data with the corresponding Slc4a10+/+ control data: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Full size image

To assess the ultrastructure of the cochlear sensory epithelium scanning electron microscopy was undertaken (Fig. 4). Slc4a10+/+ and Slc4a10+/trmb mice display the expected complement of inner hair cells and outer hair cells (OHCs) up to 12 months of age, the latest age tested. At 2 and 6 months of age Slc4a10trmb/trmb mice also display the expected number of inner hair cells and OHCs, with no differences in shape or organization of their stereocilia bundles (Fig. 4a). However, by 9 months of age OHC bundle loss is evident in Slc4a10trmb/trmb mutant mice, and this loss progresses such that by 12 months of age <50% of OHC bundles remain (Fig. 4a). To quantify the loss, counts were made in the apical, mid and basal turns of the cochlea (Fig. 4b). This shows that in Slc4a10trmb/trmb mice OHC bundle loss is occurring along the length of the cochlear spiral. There is no evidence of OHC bundle loss in Slc4a10+/+ and Slc4a10+/trmb mice up to 12 months of age.

Figure 4: Ultrastructural analyses of the cochlear sensory epithelium reveals progressive loss of hair cell bundles in trombone mutant mice. (a) Scanning Electron Micrographs of the mid-basal coil of the cochlear sensory epithelium from Slc4a10+/+, Slc4a10+/trmb, Slc4a10trmb/trmb mice at 2, 6, 9 and 12 months of age. At 2 and 6 months of age, the number and appearance of the inner and outer hair cell stereocilia bundles are is as expected and similar across all three genotypes. At 9 months, there is some loss of outer hair cell bundles in the Slc4a10trmb/trmb mutant mice, which is not observed in the Slc4a10+/+ and Slc4a10+/trmb control mice. By 12 months of age, there is a substantial loss of outer hair cell bundles in the Slc4a10trmb/trmb mutant mice, which is not observed in the Slc4a10+/+ and Slc4a10+/trmb control mice. Magnification × 2,500. Scale bars, 10 μm (b) Outer hair cell bundle counts in the apical, mid and basal turns of the cochlear spiral in trombone mice. At 2 months of age Slc4a10+/+ (apex n=5, mid n=7, base n=5), Slc4a10+/trmb (apex n=3, mid n=6, base n=3), Slc4a10trmb/trmb (apex n=5, mid n=4, base n=3) mice all have similar numbers of OHC bundles. At 6 months of age Slc4a10+/+ (apex n=3, mid n=3, base n=3), Slc4a10+/trmb (apex n=4, mid n=3, base n=4), Slc4a10trmb/trmb (apex n=3, mid n=3, base n=3) mice all still have similar numbers of OHC bundles. However, by 9 months of age Slc4a10trmb/trmb (apex n=3, mid n=3, base n=3) mice have a reduced number of OHC bundles in all cochlear regions compared with Slc4a10+/+ (apex n=3, mid n=3, base n=3), Slc4a10+/trmb (apex n=2, mid n=3, base n=3) mice. At 12 months of age Slc4a10trmb/trmb (apex n=5, mid n=3, base n=6) mice show a further loss of OHC bundles in all cochlear regions compared with Slc4a10+/+ (apex n=2, mid n=3, base n=2), Slc4a10+/trmb (apex n=4, mid n=6, base n=3) mice. Although Slc4a10trmb/trmb mice show a progressive loss of OHC bundles throughout the cochlear spiral, no significant OHC bundle loss is observed in the Slc4a10+/+ or Slc4a10+/trmb mice up to 12 months of age. Slc4a10+/+ (black bars), Slc4a10+/trmb (grey bars), Slc4a10trmb/trmb (red bars). Data shown are mean ±s.e.m. and significance determined using a two-way ANOVA with Tukey’s multiple comparisons test: **P<0.01, ***P<0.001, ****P<0.0001. Full size image

To assess the expression of Slc4a10 within the cochlea, mid-modiolar histological sections were prepared and immunostained using an anti-Slc4a10 antibody. For Slc4a10+/+ and Slc4a10+/trmb cochlear sections, immunohistochemical staining was observed in the spiral ligament (SL) fibrocytes throughout all cochlear turns at 2, 6, 9 and 12 months of age (Fig. 5a). The strongest staining was detected in the type II fibrocytes beneath the spiral prominence and the type V fibrocytes in the suprastrial region. There was no staining of additional cochlear structures, for example, organ of Corti (OoC), Reissner’s membrane (RM), or spiral ganglion neurons (SGN) (Fig. 5a). For Slc4a10trmb/trmb cochlear sections no staining of the SL was detected at any of the time points tested, implying a loss-of-function mutation (Fig. 5a). In addition, assessment of the cochlear sections identified a similar number of SGNs present across all genotypes at each time point investigated (2, 6, 9 and 12 months of age) (Fig. 5b).

Figure 5: Cochlear expression of Slc4a10 morphology of the lateral wall and endocochlear potential in trombone mice. (a) Immunohistochemical DAB staining of cochlear sections identifies Slc4a10 expression in the SL fibrocytes of Slc4a10+/+ and Slc4a10+/trmb mice. No DAB staining of Slc4a10trmb/trmb sections is observed at any of the time points tested. Scale bar, 600 μm. Number of cochleae imaged (one per mouse) for each genotype at 2-, 6-, 9- and 12 months of age were: Slc4a10+/+ n=5, 5, 6, 6; Slc4a10+/trmb n=7, 4, 5, 6; and Slc4a10trmb/trmb n=4, 3, 5, 6, respectively. (b) SGN counts in Slc4a10+/+, Slc4a10+/trmb and Slc4a10trmb/trmb mice shows there are no differences across genotype, or with age. Counts averaged from individual mice (N=3) for each genotype at each age. (c) Analysis of the cross-sectional surface area of the SL shows there are no differences across genotypes, or with age. (d) Analysis of the SV show there is a significant reduction in the cross-sectional surface area in Slc4a10trmb/trmb mice compared with Slc4a10+/+ and Slc4a10+/trmb mice, and this is consistent across the ages tested. The SL and SV surface areas are averaged from individual mice for each genotype at 2-, 6-, 9- and 12 months of age: Slc4a10+/+ n=5, 3, 5, 5; Slc4a10+/trmb n=7, 5, 8, 5; and Slc4a10trmb/trmb n=5, 6, 7, 6, respectively. (e,f) Comparison of SL and SV nuclei counts shows there are no differences across genotype, or with age. The nuclei counts are averaged from individual mice for each genotype at 2-, 6-, 9- and 12 months of age: Slc4a10+/+ n=5, 3, 3, 3; Slc4a10+/trmb n=5, 4, 4, 3; and Slc4a10trmb/trmb n=6, 5, 4, 3, respectively. (g) The endocochlear potential is chronically reduced in Slc4a10trmb/trmb mice compared with controls. Measurements averaged for each genotype at 2-, 9- and 12 months of age: Slc4a10+/+ n=6, 6, 8; Slc4a10+/trmb n=10, 12, 11; and Slc4a10trmb/trmb n=8, 6, 9, respectively. Slc4a10+/+ (black circles/bars), Slc4a10+/trmb (grey circles/bars), Slc4a10trmb/trmb (red circles/bars). Data correspond to mean±s.e.m., and statistical significance determined using two-way ANOVA with Tukey’s multiple comparisons test. *P<0.05; **P<0.01; ***P<0.001, ****P<0.0001. ANOVA, analysis of variance. Full size image

To assess the consequence of the trombone mutation on the structure of the cochlear lateral wall morphometric analyses were performed. Utilizing hematoxylin and eosin stained mid-modiolar cochlear sections the cross-sectional surface area of the SL, and the closely apposed stria vascularis (SV), were measured in the mid-basal cochlear turn. This identified that the cross-sectional surface area of the SL was consistent across all genotypes, at all ages (Fig. 5c). Assessment of the SV identified that the cross-sectional surface area was similar between Slc4a10+/+ and Slc4a10+/trmb mice at each time point. However, the cross-sectional surface area of the SV was reduced by >20% in Slc4a10trmb/trmb mice (Fig. 5d). Interestingly, SL and SV nuclei counts demonstrate there is not a statistically significant difference in the total number of cells within either of these structures across genotype (Slc4a10+/+, Slc4a10+/trmb and Slc4a10trmb/trmb) or age (2, 6, 9 and 12 months) (Fig. 5e,f).

The SV is critical for generating the extracellular fluid (endolymph) found in the scala media, which bathes the apical surface of the auditory sensory cells. By pumping K+ into the scala media the SV generates a high K+ concentration and large electrical potential, known as the endocochlear potential (EP), in this extracellular space compared with that of the perilymph-containing scala tympani. The EP is essential for the process of auditory transduction, establishing an electrochemical gradient that drives cations from the endolymph into the sensory cells via mechanically gated channels to cause depolarization of, and subsequent neurotransmitter release from, the sensory cells in a process known as mechanoelectrical transduction. To assess if SV function is compromised, the EP was measured in trombone mice at 2, 9 and 12 months of age. At 2 months of age the averaged EP values for Slc4a10+/+ and Slc4a10+/trmb mice were similar at 76 mV (range 62–89 mV) and 77 mV (range 53–89 mV), respectively. However, the averaged EP value for age-matched Slc4a10trmb/trmb mice was significantly lower at 40 mV (range 29–55 mV) (Fig. 5g). At 9 months of age the averaged EP values for Slc4a10+/+ and Slc4a10+/trmb mice were 73 mV (range 57–82 mV) and 59 mV (range 40–69 mV), respectively. Again, the averaged EP value for age-matched Slc4a10trmb/trmb mice was significantly lower at 32 mV (range 27–41 mV) (Fig. 5g). At 12 months of age, the averaged EP values for Slc4a10+/+ and Slc4a10+/trmb mice were 65 mV (range 42–82 mV) and 55 mV (range 44–81 mV), respectively; whereas the averaged EP value for age-matched Slc4a10trmb/trmb mice was significantly lower at 28 mV (range 15–46 mV) (Fig. 5g). The observed age-related decline in EP is only significant for the Slc4a10+/trmb mice (P<0.0001).

Recent studies of an Slc4a10 targeted KO mouse mutant (Slc4a10−/−) have identified a role for Slc4a10 in maintaining intracellular chloride and bicarbonate concentration in retinal neurons, demonstrating that loss of Slc4a10 in the retina leads to impaired visual function in the Slc4a10−/− KO mouse25. To ascertain if the Slc4a10trmb/trmb mice also display a retinal phenotype electroretinography (ERG) was undertaken. To enable this, the trombone allele was rederived on, and backcrossed to, C57BL/6J, a strain suitable for ERG studies. The ERG analysis showed that trombone mice display a very similar, albeit milder, retinal phenotype to the Slc4a10−/− KO mice (Supplementary Fig. 5a). Overall, compared with wild-type littermate mice, Slc4a10trmb/trmb mice showed: grossly similar dark-adapted irradiance-response curves for a- and b-waves (Supplementary Fig. 5b,c) significant differences in phase/timing of flicker frequency responses (delayed in mutant) especially in 7–15 Hz responses (Supplementary Fig. 5d); and, smaller and slower light-adapted responses showing significant differences in amplitude and implicit time at higher flash intensities (Supplementary Fig. 5e–i). The similar phenotype supports our hypothesis that the trombone allele causes loss-of-function. In summary, the trombone mutant (Slc4a10L647P) underlines the utility of characterising mutations with late-onset phenotypes, uncovering novel genes and insights into the underlying molecular mechanisms. Age-related hearing loss in trombone mice is preceded by changes to the structure of the SV and by defects in endocochlear potential.