High-throughput measurement of fibroblast rhythms reveals genetic heritability of circadian phenotypes in diversity outbred mice and their founder strains.

Circadian variability is driven by genetics and Diversity Outbred (DO) mice is a powerful tool for examining the genetics of complex traits because their high genetic and phenotypic diversity compared to conventional mouse crosses. The DO population combines the genetic diversity of eight founder strains including five common inbred and three wild-derived strains. In DO mice and their founders, we established a high-throughput system to measure cellular rhythms using in vitro preparations of skin fibroblasts. Among the founders, we observed strong heritability for rhythm period, robustness, phase and amplitude. We also found significant sex and strain differences for these rhythms. Extreme differences in period for molecular and behavioral rhythms were found between the inbred A/J strain and the wild-derived CAST/EiJ strain, where A/J had the longest period and CAST/EiJ had the shortest. In addition, we measured cellular rhythms in 329 DO mice, which displayed far greater phenotypic variability than the founders-80% of founders compared to only 25% of DO mice had periods of ~ 24 h. Collectively, our findings demonstrate that genetic diversity contributes to phenotypic variability in circadian rhythms, and high-throughput characterization of fibroblast rhythms in DO mice is a tractable system for examining the genetics of circadian traits

Behavioral phenotypes revealed during reversal learning are linked with novel genetic loci in diversity outbred mice.

Impulsive behavior and impulsivity are heritable phenotypes that are strongly associated with risk for substance use disorders. Identifying the neurogenetic mechanisms that influence impulsivity may also reveal novel biological insights into addiction vulnerability. Our past studies using the BXD and Collaborative Cross (CC) recombinant inbred mouse panels have revealed that behavioral indicators of impulsivity measured in a reversal-learning task are heritable and are genetically correlated with aspects of intravenous cocaine self-administration. Genome-wide linkage studies in the BXD panel revealed a quantitative trait locus (QTL) on chromosome 10, but we expect to identify additional QTL by testing in a population with more genetic diversity. To this end, we turned to Diversity Outbred (DO) mice; 392 DO mice (156 males, 236 females) were phenotyped using the same reversal learning test utilized previously. Our primary indicator of impulsive responding, a measure that isolates the relative difficulty mice have with reaching performance criteria under reversal conditions, revealed a genome-wide significant QTL on chromosome 7 (max LOD score = 8.73, genome-wide corrected

Mutations of the Mouse ELMO Domain Containing 1 Gene (Elmod1) Link Small GTPase Signaling to Actin Cytoskeleton Dynamics in Hair Cell Stereocilia

Stereocilia, the modified microvilli projecting from the apical surfaces of the sensory hair cells of the inner ear, are essential to the mechanoelectrical transduction process underlying hearing and balance. The actin-filled stereocilia on each hair cell are tethered together by fibrous links to form a highly patterned hair bundle. Although many structural components of hair bundles have been identified, little is known about the signaling mechanisms that regulate their development, morphology, and maintenance. Here, we describe two naturally occurring, allelic mutations that result in hearing and balance deficits in mice, named roundabout (rda) and roundabout-2J (rda2J). Positional cloning identified both as mutations of the mouse ELMO domain containing 1 gene (Elmod1), a poorly characterized gene with no previously reported mutant phenotypes. The rda mutation is a 138 kb deletion that includes exons 1–5 of Elmod1, and rda2J is an intragenic duplication of exons 3–8 of Elmod1. The deafness associated with these mutations is caused by cochlear hair cell dysfunction, as indicated by conspicuous elongations and fusions of inner hair cell stereocilia and progressive degeneration of outer hair cell stereocilia. Mammalian ELMO-family proteins are known to be involved in complexes that activate small GTPases to regulate the actin cytoskeleton during phagocytosis and cell migration. ELMOD1 and ELMOD2 recently were shown to function as GTPase-activating proteins (GAPs) for the Arf family of small G proteins. Our finding connecting ELMOD1 deficiencies with stereocilia dysmorphologies thus establishes a link between the Ras superfamily of small regulatory GTPases and the actin cytoskeleton dynamics of hair cell stereocilia

Hyperspin Mouse Mutation as a Model for Human SHFM1D Syndrome

The identification of mouse mutations that result in deafness is often the initial characterization of a genes involvement in hearing and inner ear development, providing a mouse model to study the molecular nature of the hearing process. A newly described mouse mutation named hyperspin (hspn) induces profound hearing loss and vestibular dysfunction in mice. Inner ear paint-fill analysis from embryonic hspn mice and wildtype controls reveal severe malformations in ears of hspn mice. Loss of the endolymphatic duct as well as anterior and posterior semicircular canals are observed as well as the variable loss of the lateral semicircular canal. This observation is highly similar to inner ear defects described in DIx5 -/- mice. The hspn mutation is localized to chromosome 6, approximately 840 kb away from Dlx5, and is a 123.4 kb deletion within the Slc25a13 gene. This gene is not known to play a role in inner ear development or hearing loss, mice that lack Slc25al3 transcripts are not described as having hearing loss. The protein encoded by Slc25al3, is a calcium-activated mitochondrial transporter called Citrin, and its absence contributes to varying forms of neonatal and adult onset citrullinemia. Another disorder, Split hand/split foot malformation 1 with sensorineural hearing loss (SHFM1D), is a complex disorder with degrees of severity involving deafness and ectrodactyly. Within the hspn deletion region also resides a putative DIx5 non-coding enhancer element eDlx#23, which may underlie Dlx5 dysregulation and thus the hspn phenotype observed. In this study we present the close association between the hspn phenotype and those observed in Dlx5 -/- mice, including phenotypic characterization and gene expression, to demonstrate the plausibility that the loss of Citrin is not a factor in the hearing loss phenotype, but rather it is an alternate genetic element, likely eDlx#23, that is responsible for the detected deafness, and that loss of such element may underlie the sensorineural deafness in SHFM1D; thus, creating a new mouse model to study deafness and elucidate the regulation of otic specific Dlx5

A hypomorphic mutation of the gamma-1 adaptin gene (Ap1g1) causes inner ear, retina, thyroid, and testes abnormalities in mice.

Adaptor protein (AP) complexes function in the intracellular sorting and vesicular transport of membrane proteins. The clathrin-associated AP-1 complex functions at the trans-Golgi network and endosomes, and some forms of this complex are thought to mediate the sorting of proteins in plasma membranes of polarized epithelial cells. A null mutation of the mouse Ap1g1 gene, which encodes the gamma-1 subunit of the AP-1 complex, causes embryonic lethality when homozygous, indicating its critical importance in early development but precluding studies of its possible roles during later stages. Here, we describe our analyses of a new spontaneous mutation of Ap1g1 named figure eight (symbol fgt) and show that it is an in-frame deletion of 6 bp, which results in the elimination of two amino acids of the encoded protein. In contrast to Ap1g1 (-/-) null mice, mice homozygous for the recessive fgt mutation are viable with adult survival similar to controls. Although Ap1g1 is ubiquitously expressed, the phenotype of Ap1g1 (fgt) mutant mice is primarily restricted to abnormalities in sensory epithelial cells of the inner ear, pigmented epithelial cells of the retina, follicular epithelial cells of the thyroid gland, and the germinal epithelium of the testis, suggesting that impaired AP-1 sorting and targeting of membrane proteins in these polarized cells may underlie the observed pathologies. Ap1g1 (fgt) mutant mice provide a new animal model to study the in vivo roles of gamma-1 adaptin and the AP-1 complex throughout development and to investigate factors that underlie its associated phenotypic abnormalities. Mamm Genome 2016 Jun; 27(5-6):200-1

A QTL on Chr 5 modifies hearing loss associated with the fascin-2 variant of DBA/2J mice.

Inbred mouse strains serve as important models for human presbycusis or age-related hearing loss. We previously mapped a locus (ahl8) contributing to the progressive hearing loss of DBA/2J (D2) mice and later showed that a missense variant of the Fscn2 gene, unique to the D2 inbred strain, was responsible for the ahl8 effect. Although ahl8 can explain much of the hearing loss difference between C57BL/6J (B6) and D2 strain mice, other loci also contribute. Here, we present results of our linkage analyses to map quantitative trait loci (QTLs) that modify the severity of hearing loss associated with the D2 strain Fscn2 (ahl8) allele. We searched for modifier loci by analyzing 31 BXD recombinant inbred (RI) lines fixed for the predisposing D2-derived Fscn2 (ahl8/ahl8) genotype and found a statistically significant linkage association of threshold means with a QTL on Chr 5, which we designated M5ahl8. The highest association (LOD 4.6) was with markers at the 84-90 Mb position of Chr 5, which could explain about 46 % of the among-RI strain variation in auditory brainstem response (ABR) threshold means. The semidominant nature of the modifying effect of M5ahl8 on the Fscn2 (ahl8/ahl8) phenotype was demonstrated by analysis of a backcross involving D2 and B6.D2-Chr11D/LusJ strain mice. The Chr 5 map position of M5ahl8 and the D2 origin of its susceptibility allele correspond to Tmc1m4, a previously reported QTL that modifies outer hair cell degeneration in Tmc1 (Bth) mutant mice, suggesting that M5ahl8 and Tmc1m4 may represent the same gene affecting maintenance of stereocilia structure and function during aging. Mamm Genome 2015 Aug; 26(7-8):338-47

Association of a citrate synthase missense mutation with age-related hearing loss in A/J mice.

We previously mapped a locus (ahl4) on distal Chromosome 10 that contributes to the age-related hearing loss of A/J strain mice. Here, we report on a refined genetic map position for ahl4 and its association with a mutation in the citrate synthase gene (Cs). We mapped ahl4 to the distal-most 7 megabases (Mb) of chromosome 10 by analysis of a new linkage backcross and then further narrowed the interval to 5.5 Mb by analysis of 8 C57BL/6J congenic lines with different A/J-derived segments of chromosome 10. A nucleotide variant in exon 3 of Cs is the only known DNA difference within the ahl4 candidate gene interval that is unique to the A/J strain and that causes a nonsynonymous codon change. Multiple lines of evidence implicate this missense mutation (H55N) as the underlying cause of ahl4-related hearing loss, likely through its effects on mitochondrial adenosine trisphosphate (ATP) and free radical production in cochlear hair cells. The A/J mouse thus provides a new model system for in vivo studies of mitochondrial function and hearing loss

Effects of Cdh23 single nucleotide substitutions on age-related hearing loss in C57BL/6 and 129S1/Sv mice and comparisons with congenic strains.

A single nucleotide variant (SNV) of the cadherin 23 gene (Cdh23(c.753A)), common to many inbred mouse strains, accelerates age-related hearing loss (AHL) and can worsen auditory phenotypes of other mutations. We used homologous recombination in C57BL/6 NJ (B6N) and 129S1/SvImJ (129S1) embryonic stem cells to engineer mouse strains with reciprocal single base pair substitutions (B6-Cdh23(c.753A\u3eG) and 129S1-Cdh23(c.753G\u3eA)). We compared ABR thresholds and cochlear pathologies of these SNV mice with those of congenic (B6.129S1-Cdh23(Ahl+) and 129S1.B6-Cdh23(ahl)) and parental (B6N and 129S1) strain mice. Results verified the protective effect of the Cdh23(c.753G) allele, which prevented high frequency hearing loss in B6 mice to at least 18 months of age, and the AHL-inducing effect of the Cdh23(c.753A) allele, which worsened hearing loss in 129S1 mice. ABR thresholds differed between 129S-Cdh23(c.753A) SNV and 129S1.B6-Cdh23(ahl) congenic mice, and a linkage backcross involving these strains localized a Chr 10 QTL contributing to the difference. These results illustrate the large effects that strain background and congenic regions have on the hearing loss associated with Cdh23(c.753)alleles. Importantly, the B6-Cdh23(c.753G)strain can be used to eliminate the confounding influence of the Cdh23(c.753A)variant in hearing studies of B6 mice and mutant mice on the B6 background. Sci Rep 2017 Mar 13; 7:44450

<i>In situ</i> expression of <i>Elmod1</i> mRNA in the inner ear.

<p>Expression was detected in inner hair cells (IHC) and outer hair cells (OHCs) of the cochlea (<b>A</b>) and vestibular hair cells (VHCs) of the crista ampullaris (<b>B</b>) in inner ears from wildtype (+/+) mice at postnatal day 7 (P7). Mutant <i>rda/rda</i> mice served as negative controls for probe specificity, as seen by the lack of detectable <i>Elmod1</i> expression in cochlear hair cells of P7 <i>rda/rda</i> mice (<b>C</b>). <i>Elmod1</i> expression was not detected above background staining in inner ears of P2 wildtype mice (<b>D</b>) or in P15 mice (not shown). The general location of hair cells (HCs) is indicated in the P2 cochlea (D), because inner and outer hair cells are not clearly distinguishable in cryosections at this age. For reference, the Reissner's membrane (RM) and stria vascularis (SV) of the cochleae are indicated with arrows. All panels are at the same magnification; the scale bar represents 50 micrometers.</p

Cochlear hair cell abnormalities in <i>rda</i> and r<i>da^2J</i> mutant mice.

<p>Examination of the apical surfaces of cochlear hair cells by scanning electron microscopy showed normal bundle morphology in both inner hair cells (IHC) and outer hair cells (OHC) of <i>rda/rda</i> mutant mice at P0 (<b>A</b>). At P7 the stereocilia adjacent to the kinocilium (marked by arrow in <b>H</b>) had degenerated in some of the OHC bundles (marked by asterisks in <b>B, D</b>) of <i>rda/rda</i> mice, compared with the fully intact bundles of wildtype (+/+) controls (<b>C</b>). IHC bundles of <i>rda/rda</i> mice retained a normal appearance at P10 (<b>D, K</b>), but by P15 (<b>F, L</b>) all IHCs of mutant mice exhibited stereocilia elongations and fusions as compared with the normal IHC bundle morphology of age-matched controls (<b>E</b>). IHCs at P35 illustrate the striking degree of stereocilia elongations and fusions seen in <i>rda/rda</i> mutants (<b>N</b>) compared with age-matched controls (<b>M</b>). In contrast to IHCs, the OHC stereocilia of <i>rda/rda</i> mice did not elongate or fuse, but they did degenerate over time. By P15, nearly all OHCs had lost stereocilia at the bundle peak and a few (marked by asterisks) had lost the entire bundle (<b>F, I</b>), as compared with the intact OHC bundles of control mice (<b>J</b>). The IHC and OHC bundle abnormalities of <i>rda^2J/rda^2J</i> mutant mice (<b>G</b>) were very similar to those of <i>rda/rda</i> mice (<b>F</b>). Scale bars: A–G, 10 microns; H–N, 5 microns. All hair cells shown in the figure are from the middle region of the cochlea, but similar bundle abnormalities were seen in other regions (not shown), consistent with the absence of an ABR at all test frequencies (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036074#pone-0036074-g001" target="_blank">Fig. 1A</a>).</p