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

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

<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

<i>Elmod1</i> gene structure and expression.

<p><b>A.</b> The mouse <i>Elmod1</i> gene spans 63.84 kb and is comprised of 11 exons transcribed on the reverse strand of the NCBI genomic DNA reference sequence (Chr 9: 53,823,108 to 53,759,267 bp, Build 37). <b>B.</b> The 2605 nt reference mRNA sequence (NM_177769) encodes a 326 amino acid protein (NP_808437). Amino acids 132–305 (encoded by exons 6–11) comprise a conserved ELMO/CED-12 domain (pfam04727, IPR006816) characteristic of the ELMO protein family. Diagrams A and B were downloaded from the Ensembl website (<a href="http://www.ensembl.org/" target="_blank">http://www.ensembl.org/</a>). <b>C. </b><i>Elmod1</i> gene expression was examined by northern blot analysis. Commercially prepared blots from mouse embryos and adult tissues (MTN blots, Clontech, Palo Alto, CA) were hybridized with a mouse <i>Elmod1</i> cDNA probe corresponding to nucleotides 426–904 of the NM_177769 reference cDNA sequence. Blots contained purified Poly A+ RNA from 7 day (lane 1), 11 day (lane 2), 15 day (lane 3) and 17 day (lane 4) embryos and from heart (H), brain (B), spleen (S), lung (Lu), liver (Li), skeletal muscle (M), kidney (K), and testis (T) of adult mice. A single transcript of about 2,600 nucleotides was detected, most highly expressed in adult brain.</p

Deafness and cochlear pathology of <i>rda</i> and <i>rda^2J</i> mutant mice.

<p><b>A.</b> ABR thresholds (dB SPL) of <i>rda/rda</i> and <i>rda ^2J/rda^2J</i> mutant mice and non-mutant controls tested at 33–48 days of age. ABRs for 8, 16, and 32 kHz stimulus frequencies were not detected in any of the <i>rda/rda</i> (N = 9) and <i>rda^2J/rda^2J</i> (N = 8) mutant mice tested, even with the maximum stimulus presentation of 100 dB SPL. Heterozygous +/<i>rda</i> (N = 8) and +/<i>rda^2J</i> (N = 4) mice and homozygous +/+ B6 mice (N = 17) were combined as controls because their ABR thresholds did not significantly differ from one another. Error bars represent standard deviations of the threshold means. <b>B, C.</b> Cross sections through the basal turn of the cochlea from a +/<i>rda</i> heterozygous control mouse (B) and a littermate <i>rda/rda</i> mutant mouse (C) examined at 4 months of age. Note the complete degeneration of the organ of Corti (oc) and decreased density of spiral ganglion cells (sgc) in the <i>rda/rda</i> cochlea. Scale bars represent 100 microns. Cochlear cross sections of <i>rda^2J</i> mutant mice (not shown) exhibited this same pathology.</p

Molecular analysis of the E<i>lmod1^rda-2J</i> mutation.

<p><b>A.</b> Southern blots of genomic DNA from mice with +/+ (lanes marked 1), +/<i>rda^2J</i> (lanes marked 2) and <i>rda^2J/rda^2J</i> (lanes marked 3) genotypes, digested with <i>Eco</i>RI (Eco), <i>Pst</i>I (Pst), <i>Pvu</i>II (Pvu), and <i>Msp</i>I (Msp) restriction enzymes. The blot was hybridized with an <i>Elmod1</i> cDNA probe corresponding to exons 7–11. Although the same quantity of DNA was loaded in each lane, the intensity of <i>Elmod1</i>-hybridizing bands is greater in in <i>Eco</i>RI- and <i>Pvu</i>II-digested DNA samples from <i>rda^2J/rda^2J</i> mice than samples from +/+ mice and exhibit additional bands (indicated by arrows) in <i>Pst</i>I and <i>Msp</i>I digested DNA. <b>B.</b> A northern blot of total RNA extracted from brains of adult C57BL/6J control (lane 1), <i>rda^2J</i>/+ heterozygotes (lanes 2 and 3), <i>rda^2J/rda^2J</i> homozygotes (lanes 4 and 5), and <i>rda/rda</i> homozygote (lane 6; negative control) was hybridized with an <i>Elmod1</i> cDNA probe. Wildtype <i>Elmod1</i> transcript (∼2600 nt) was not detected in RNA from <i>rda^2J/rda^2J</i> mice; however, a 3200 nt mutant transcript (about 600 nucleotides larger than wildtype) was abundantly expressed. <b>C.</b> PCR products from <i>rda^2J</i>–derived cDNAs indicate an intragenic duplication. cDNAs from mice with +/+ (lanes marked 1), +/<i>rda^2J</i> (lanes marked 2) and <i>rda^2J/rda^2J</i> (lanes marked 3) genotypes were used as PCR templates in combination with primers specific to <i>Elmod1</i> exons. Expected wildtype PCR products are marked by asterisks, and unexpected large PCR products unique to <i>rda^2J</i> samples are indicated by arrows. 500 bp (lane marked L1) and 100 bp (lane marked L2) ladders were used to estimate PCR product sizes, and predicted sizes are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036074#pone.0036074.s004" target="_blank">Table S2B</a>. <b>D.</b> A western blot of protein extracts from adult brains of <i>rda/rda</i> (lane 1, negative control), <i>rda^2J/rda^2J</i> (lane 2), and +/+ B6 mice (lane 3), shows that the predicted 38 kDa wildtype ELMOD1 protein is absent from both <i>rda</i> and <i>rda^2J</i> mutant mice, but a larger 62 kDa mutant protein can be seen in <i>rda^2J</i> mutants (indicated by arrow in lane 2), corresponding to the predicted duplication of 202 amino acids. The polyclonal ELMOD1 antibody cross-reacted with other unknown proteins (non-specific bands, NS). <b>E.</b> The large PCR products obtained from <i>rda^2J</i>–derived cDNA (indicated by arrows in <b>C</b>) and multiple combinations of exon-specific primers were used to determine the DNA sequence and structure of the mutant <i>Elmod1</i> transcript, and this analysis indicated a duplication of exons 3–8, as shown in the diagram of the presumed mutant gene structure.</p

Hearing impairment in hypothyroid dwarf mice caused by mutations of the thyroid peroxidase gene.

Thyroid hormone (TH) is essential for proper cochlear development and function, and TH deficiencies cause variable hearing impairment in humans and mice. Thyroid peroxidase (TPO) catalyzes key reactions in TH synthesis, and TPO mutations have been found to underlie many cases of congenital hypothyroidism in human patients. In contrast, only a single mutation of the mouse TPO gene has been reported previously (Tpo (R479C) ) but was not evaluated for auditory function. Here, we describe and characterize two new mouse mutations of Tpo with an emphasis on their associated auditory deficits. Mice homozygous for these recessive mutations have dysplastic thyroid glands and lack detectable levels of TH. Because of the small size of mutant mice, the mutations were named teeny (symbol Tpo (tee) ) and teeny-2 Jackson (Tpo (tee-2J) ). Tpo (tee) is a single base-pair missense mutation that was induced by ENU, and Tpo (tee-2J) is a 64 bp intragenic deletion that arose spontaneously. The Tpo (tee) mutation changes the codon for a highly conserved tyrosine to asparagine (p.Y614N), and the Tpo (tee-2J) mutation deletes a splice donor site, which results in exon skipping and aberrant transcripts. Mutant mice are profoundly hearing impaired with auditory brainstem response (ABR) thresholds about 60 dB above those of non-mutant controls. The maturation of cochlear structures is delayed in mutant mice and tectorial membranes are abnormally thick. To evaluate the effect of genetic background on auditory phenotype, we produced a C3.B6-Tpo (tee-2J) congenic strain and found that ABR thresholds of mutant mice on the C3H/HeJ strain background are 10-12 dB lower than those of mutant mice on the C57BL/6 J background. The Tpo mutant strains described here provide new heritable mouse models of congenital hypothyroidism that will be valuable for future studies of thyroid hormones\u27 role in auditory development and function. J Assoc Res Otolaryngol 2014 Feb; 15(1):45-55

Mutation of the Cyba gene encoding p22phox causes vestibular and immune defects in mice

In humans, hereditary inactivation of either p22phox or gp91phox leads to chronic granulomatous disease (CGD), a severe immune disorder characterized by the inability of phagocytes to produce bacteria-destroying ROS. Heterodimers of p22phox and gp91phox proteins constitute the superoxide-producing cytochrome core of the phagocyte NADPH oxidase. In this study, we identified the nmf333 mouse strain as what we believe to be the first animal model of p22phox deficiency. Characterization of nmf333 mice revealed that deletion of p22phox inactivated not only the phagocyte NADPH oxidase, but also a second cytochrome in the inner ear epithelium. As a consequence, mice of the nmf333 strain exhibit a compound phenotype consisting of both a CGD-like immune defect and a balance disorder caused by the aberrant development of gravity-sensing organs. Thus, in addition to identifying a model of p22phox-dependent immune deficiency, our study indicates that a clinically identifiable patient population with an otherwise cryptic loss of gravity-sensor function may exist. Thus, p22phox represents a shared and essential component of at least 2 superoxide-producing cytochromes with entirely different biological functions. The site of p22phox expression in the inner ear leads us to propose what we believe to be a novel mechanism for the control of vestibular organogenesis

A spontaneous mouse deletion in Mctp1 uncovers a long-range cis-regulatory region crucial for NR2F1 function during inner ear development.

Hundreds of thousands of cis-regulatory DNA sequences are predicted in vertebrate genomes, but unlike genes themselves, few have been characterized at the functional level or even unambiguously paired with a target gene. Here we serendipitously identified and started investigating the first reported long-range regulatory region for the Nr2f1 (Coup-TFI) transcription factor gene. NR2F1 is temporally and spatially regulated during development and required for patterning and regionalization in the nervous system, including sensory hair cell organization in the auditory epithelium of the cochlea. Analyzing the deaf wanderer (dwnd) spontaneous mouse mutation, we traced back the cause of its associated circling behavior to a 53 kb deletion removing five exons and adjacent intronic regions of the poorly characterized Mctp1 gene. Interestingly, loss of Mctp1 function cannot account for the hearing loss, inner ear dysmorphology and sensory hair cell disorganization observed in dwnd mutants. Instead, we found that the Mctp1dwnd deletion affects the Nr2f1 gene located 1.4 Mb away, downregulating transcription and protein expression in the embryonic cochlea. Remarkably, the Mctp1dwnd allele failed to complement a targeted inactivation allele of Nr2f1, and transheterozygotes or Mctp1dwnd homozygotes exhibit the same morphological defects observed in inner ears of Nr2f1 mutants without sharing their early life lethality. Defects include improper separation of the utricle and saccule in the vestibule not described previously, which can explain the circling behavior that first brought the spontaneous mutation to attention. By contrast, mice homozygous for a targeted inactivation of Mctp1 have normal hearing and inner ear structures. We conclude that the 53 kb Mctp1dwnd deletion encompasses a long-range cis-regulatory region essential for proper Nr2f1 expression in the embryonic inner ear, providing a first opportunity to investigate Nr2f1 function in postnatal inner ears. This work adds to the short list of long-range regulatory regions characterized as essential to drive expression of key developmental control genes