Salt-inducible kinase 3, SIK3, is a new gene associated with hearing

Hearing function is known to be heritable, but few significant and reproducible associations of genetic variants have been identified to date in the adult population. In this study, genome-wide association results of hearing function from the G-EAR consortium and TwinsUK were used for meta-analysis. Hearing ability in eight population samples of Northern and Southern European ancestry (n = 4591) and the Silk Road (n = 348) was measured using pure-tone audiometry and summarized using principal component (PC) analysis. Genome-wide association analyses for PC1-3 were conducted separately in each sample assuming an additive model adjusted for age, sex and relatedness of subjects. Meta-analysis was performed using 2.3 million single-nucleotide polymorphisms (SNPs) tested against each of the three PCs of hearing ability in 4939 individuals. A single SNP lying in intron 6 of the salt-inducible kinase 3 (SIK3) gene was found to be associated with hearing PC2 (P = 3.7 710-8) and further supported by whole-genome sequence in a subset. To determine the relevance of this gene in the ear, expression of the Sik3 protein was studied in mouse cochlea of different ages. Sik3 was expressed in murine hair cells during early development and in cells of the spiral ganglion during early development and adulthood. Our results suggest a developmental role of Sik3 in hearing and may be required for the maintenance of adult auditory function

Mice deficient in H+-ATPase a4 subunit have severe hearing impairment associated with enlarged endolymphatic compartments within the inner ear

SUMMARY Mutations in the ATP6V0A4 gene lead to autosomal recessive distal renal tubular acidosis in patients, who often show sensorineural hearing impairment. A first Atp6v0a4 knockout mouse model that recapitulates the loss of H+-ATPase function seen in humans has been generated and recently reported (Norgett et al., 2012). Here, we present the first detailed analysis of the structure and function of the auditory system in Atp6v0a4−/− knockout mice. Measurements of the auditory brainstem response (ABR) showed significantly elevated thresholds in homozygous mutant mice, which indicate severe hearing impairment. Heterozygote thresholds were normal. Analysis of paint-filled inner ears and sections from E16.5 embryos revealed a marked expansion of cochlear and endolymphatic ducts in Atp6v0a4−/− mice. A regulatory link between Atp6v0a4, Foxi1 and Pds has been reported and we found that the endolymphatic sac of Atp6v0a4−/− mice expresses both Foxi1 and Pds, which suggests a downstream position of Atp6v0a4. These mutants also showed a lack of endocochlear potential, suggesting a functional defect of the stria vascularis on the lateral wall of the cochlear duct. However, the main K+ channels involved in the generation of endocochlear potential, Kcnj10 and Kcnq1, are strongly expressed in Atp6v0a4−/− mice. Our results lead to a better understanding of the role of this proton pump in hearing function

Multiple origins, migratory paths and molecular profiles of cells populating the avian interpeduncular nucleus

AbstractThe interpeduncular nucleus (IP) is a key limbic structure, highly conserved evolutionarily among vertebrates. The IP receives indirect input from limbic areas of the telencephalon, relayed by the habenula via the fasciculus retroflexus. The function of the habenulo-IP complex is poorly understood, although there is evidence that in rodents it modulates behaviors such as learning and memory, avoidance, reward and affective states. The IP has been an important subject of interest for neuroscientists, and there are multiple studies about the adult structure, chemoarchitecture and its connectivity, with complex results, due to the presence of multiple cell types across a variety of subnuclei. However, the ontogenetic origins of these populations have not been examined, and there is some controversy about its location in the midbrain-anterior hindbrain area. To address these issues, we first investigated the anteroposterior (AP) origin of the IP complex by fate-mapping its neuromeric origin in the chick, discovering that the IP develops strictly within isthmus and rhombomere 1. Next, we studied the dorsoventral (DV) positional identity of subpopulations of the IP complex. Our results indicate that there are at least four IP progenitor domains along the DV axis. These specific domains give rise to distinct subtypes of cell populations that target the IP with variable subnuclear specificity. Interestingly, these populations can be characterized by differential expression of the transcription factors Pax7, Nkx6.1, Otp, and Otx2. Each of these subpopulations follows a specific route of migration from its source, and all reach the IP roughly at the same stage. Remarkably, IP progenitor domains were found both in the alar and basal plates. Some IP populations showed rostrocaudal restriction in their origins (isthmus versus anterior or posterior r1 regions). A tentative developmental model of the structure of the avian IP is proposed. The IP emerges as a plurisegmental and developmentally heterogeneous formation that forms ventromedially within the isthmus and r1. These findings are relevant since they help to understand the highly complex chemoarchitecture, hodology and functions of this important brainstem structure

Results of the replication association study for the candidate SNPs of expressed genes.

<p><b>Key</b>: Eff.All = effect allele; *from published work <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085352#pone.0085352-Girotto1" target="_blank">[3]</a>; Repl. = replication; Maf = minor allele frequency; Str = strand; imp = imputed; gen = genotype; Info Score = imputation quality score form IMPUTE2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085352#pone.0085352-Delaneau1" target="_blank">[6]</a>, Conc = concordance for the region encompassing the SNP (chunk dimension = 5 Mb); HWE = Hardy-Weinberg Equilibrium; add = additive; dom = dominant.</p

Analysis flow chart.

<p>The diagram illustrates the 4 steps defining our strategy. Relevant details for each step are given: GWAS meta-analysis description, expression studies in the mouse cochlea, replication association study in Silk Road cohort and genotype-phenotype relationship.</p

Results of the gene-based association test for candidate expressed genes.

<p><b>Key:</b> Total N. SNPs = total number of intragenic SNPs for each gene; N. Selected PCs = number of principal components explaining the largest amount of variance and used for the analysis (see Materials and Methods); Variance Explained = Total variance accounted for by the selected principal components.</p

Immunohistochemistry in the mouse cochlea at P5.

<p>Brown indicates positive staining. A, B, C) Ptprd is localized in hair cells of the organ of Corti (bracket in A, arrowheads in C), in the marginal cells of the stria vascularis (arrow in A, B), in the supporting cells of the Kölliker’s organ (marked by an asterisk in A) and in the spiral ganglions neurons (arrowhead in A); D, E) Cdh13 is expressed in cells of Claudius (red arrowhead in E), outer and inner hair cells (arrowheads in E), Deiters’ cells (bracket in E) and pillar cells (asterisk in E), cells of the Kolliker’s organ (arrows in E) in the organ of Corti. Staining was also noted in interdental cells (arrow in D), stria vascularis (asterisk in D), spiral prominence and external sulcus cells (bracket in D). F, G) Ank2 could be noted in the Hensen’s cells (bracket in G), Deiters’ cells (arrowheads in G)and pillar cells (asterisk in G) in the organ of Corti. Moreover, Ank2 is expressed in the Reissner’s membrane (arrowhead in F) and cells of the Kolliker’s organ (arrow in G). Scale bars: A–C: 20 µm. D, F: 20 µm; E,G: 10 µm. Rectangles label the regions shown in higher magnification.</p

Further details of shortlisted candidate genes.

<p>Expression in the ear reported in Mouse Genome Informatics (MGI, <a href="http://www.informatics.jax.org/" target="_blank">http://www.informatics.jax.org/</a>); the Morton cDNA human foetal cochlea cDNA library (Morton, <a href="http://brighamandwomens.org/Research/labs/BWH_Hearing/Cochlear_ESTs.aspx" target="_blank">http://brighamandwomens.org/Research/labs/BWH_Hearing/Cochlear_ESTs.aspx</a>); the SHIELD database (SHIELD: Shared Harvard Inner-Ear Laboratory Database, <a href="https://shield.hms.harvard.edu/" target="_blank">https://shield.hms.harvard.edu/</a>); plus localisation within a reported human deafness locus and phenotypic reports from MGI of any mouse mutations of the gene.</p><p>Key: SHIELD* = SHIELD - enriched in hair cells compared to supporting cells; SHIELD** = SHIELD - auditory/vestibular ganglia; DFN = within a human deafness locus?; N/A = data not available.</p

List of candidate genes derived from previously published Meta-Analysis [3] with inclusion criteria satisfied.

<p>Key: Sugg. p-value† = suggestive p-value: p∼10-5,10-6; H.Sugg. p-value‡ = highly suggestive p-value: p∼10-7 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085352#pone.0085352-Girotto1" target="_blank">[3]</a>.</p

Immunohistochemistry in the mouse cochlea at P5 (P4 for Slc16a6 gene).

<p>Brown indicates positive staining. A, B) Expression of Dclk1, showing intense staining in the marginal cells including projections towards the basal cells of the stria vascularis (arrows in A,B); C, D) Expression of Arsg is localized at the top of sensory hair cells in the organ of Corti (bracket in C, arrowheads in D). E, F), Apical hair cells at P4, showing staining in outer hair cells of Slc16a6 of the organ of Corti (bracket in E, arrowheads in F). Note that these samples are from the C3HeB/FeJ strain, which is pigmented. Gabrg3 shows a striking specific expression in the outer and inner hair cells (arrowheads in H). In particular, the inner hair cells have the strongest staining. H, I) Expression of Csmd1 is localized at the top of sensory hair cells in the organ of Corti (arrowheads in H; I) confocal expression shows a strong staining localized in the stereocilia of inner hair cells and weak staining is also present in the stereocilia of outer hair cells. Csmd1 is labelled in green while rhodamine/phalloidin labels actin filaments of stereocilia in red. Merged image (yellow) indicates colocalization of Csmd1 with actin in stereocilia hair bundles. Scale bars: A–H: 20 µm; I: 5 µm.</p