Mutation at the Evi1 locus in Junbo mice causes susceptibility to otitis media

Otitis media ( OM), inflammation of the middle ear, remains the most common cause of hearing impairment in children. It is also the most common cause of surgery in children in the developed world. There is evidence from studies of the human population and mouse models that there is a significant genetic component predisposing to OM, yet nothing is known about the underlying genetic pathways involved in humans. We identified an N-ethyl-N-nitrosourea-induced dominant mouse mutant Junbo with hearing loss due to chronic suppurative OM and otorrhea. This develops from acute OM that arises spontaneously in the postnatal period, with the age of onset and early severity dependent on the microbiological status of the mice and their air quality. We have identified the causal mutation, a missense change in the C-terminal zinc finger region of the transcription factor Evi1. This protein is expressed in middle ear basal epithelial cells, fibroblasts, and neutrophil leukocytes at postnatal day 13 and 21 when inflammatory changes are underway. The identification and characterization of the Junbo mutant elaborates a novel role for Evi1 in mammalian disease and implicates a new pathway in genetic predisposition to OM

Otitis Media in a New Mouse Model for CHARGE Syndrome with a Deletion in the Chd7 Gene

Otitis media is a middle ear disease common in children under three years old. Otitis media can occur in normal individuals with no other symptoms or syndromes, but it is often seen in individuals clinically diagnosed with genetic diseases such as CHARGE syndrome, a complex genetic disease caused by mutation in the Chd7 gene and characterized by multiple birth defects. Although otitis media is common in human CHARGE syndrome patients, it has not been reported in mouse models of CHARGE syndrome. In this study, we report a mouse model with a spontaneous deletion mutation in the Chd7 gene and with chronic otitis media of early onset age accompanied by hearing loss. These mice also exhibit morphological alteration in the Eustachian tubes, dysregulation of epithelial proliferation, and decreased density of middle ear cilia. Gene expression profiling revealed up-regulation of Muc5ac, Muc5b and Tgf-β1 transcripts, the products of which are involved in mucin production and TGF pathway regulation. This is the first mouse model of CHARGE syndrome reported to show otitis media with effusion and it will be valuable for studying the etiology of otitis media and other symptoms in CHARGE syndrome

A mutation in Nischarin causes otitis media via LIMK1 and NF-κB pathways

Otitis media (OM), inflammation of the middle ear (ME), is a common cause of conductive hearing impairment. Despite the importance of the disease, the aetiology of chronic and recurrent forms of middle ear inflammatory disease remains poorly understood. Studies of the human population suggest that there is a significant genetic component predisposing to the development of chronic OM, although the underlying genes are largely unknown. Using N-ethyl-N-nitrosourea mutagenesis we identified a recessive mouse mutant, edison, that spontaneously develops a conductive hearing loss due to chronic OM. The causal mutation was identified as a missense change, L972P, in the Nischarin (NISCH) gene. edison mice develop a serous or granulocytic effusion, increasingly macrophage and neutrophil rich with age, along with a thickened, inflamed mucoperiosteum. We also identified a second hypomorphic allele, V33A, with only modest increases in auditory thresholds and reduced incidence of OM. NISCH interacts with several proteins, including ITGA5 that is thought to have a role in modulating VEGF-induced angiogenesis and vascularization. We identified a significant genetic interaction between Nisch and Itga5; mice heterozygous for Itga5-null and homozygous for edison mutations display a significantly increased penetrance and severity of chronic OM. In order to understand the pathological mechanisms underlying the OM phenotype, we studied interacting partners to NISCH along with downstream signalling molecules in the middle ear epithelia of edison mouse. Our analysis implicates PAK1 and RAC1, and downstream signalling in LIMK1 and NF-κB pathways in the development of chronic OM

Learning to share knowledge for global agricultural progress

Web 2.0 tools combined with face-to-face methods offer new opportunities for better knowledge sharing across disciplines, languages and borders. This article comprises an overview and case studies – the personal accounts of six participants and one facilitator of a 2008 Workshop on Knowledge Sharing, sponsored by the Consultative Group on International Agricultural Research. It lays out the rationale for, and lessons learned from, those efforts, as well as from a second workshop hosted by the Food and Agriculture Organization of the United Nations. It explains why, in today's culture of self-directed learning, group experiences remain essential. The authors describe their learning trajectories and application of knowledge sharing tools and methods in their work.Online publication date: Wed, 17-Mar-2010<br/

The <i>Nisch</i> gene is mutated in <i>edison</i> mice.

<p><b>(A)</b> SNP mapping of 10 <i>edsn</i> mutants identified an approximately 9 Mb interval on chromosome 14 delineated by marker rs30778552 and rs46823676. The grayed box indicates the chromosomal interval bearing the <i>edsn</i> mutation. <b>(B)</b> Sequence analysis of the <i>Nisch</i> locus in <i>Nisch</i>^<i>+/+</i> and <i>Nisch</i>^{<i>edsn/edsn</i>} DNA. A c.3079T>C transition is detected in <i>Nisch</i>^{<i>edsn/edsn</i>} mutants that is not present in <i>Nisch</i>^<i>+/+</i> DNA. <b>(C)</b> Conservation of the mutated leucine residue across species. <i>Mus musculus</i>, ENSMUSG00000021910; <i>Homo sapiens</i>, ENSG00000010322; <i>Pan troglodytes</i>, ENSPTRG00000015001; <i>Sus scrofa</i>, ENSSSCG00000011442; <i>Gallus gallus</i>, ENSGALG00000043825; <i>Anolis carolinensis</i>, ENSACAG00000006939. <b>(D)</b> Schematic of the full length NISCH peptide (1593 amino acids). The molecule consists of a phox-homology (PX) domain, six leucine-rich repeats, a coiled-coil (CC) domain and an alanine/proline-rich region. Both the PX and CC domains of Nischarin are essential for endosomal targeting and interaction with phosphatidylinositol 3-phosphate (PI3P) in PI3P-enriched endosomes. Amino acids 709–807 of Nischarin interact with the integrin α5 (ITGA5) cytoplasmic tail. Both LIMK1 and LKB1 interact with positions 661–869 of Nischarin. Residues 246–1047 of Nischarin interact with PAK1. Rab14 interacts with amino acids 1190–1593. Finally, Rac1 interacts with two regions of Nischarin, amino acids 246–1047 and 1190–1593. The positions of the <i>Nisch</i>^<i>edsn</i> and <i>Nisch</i>^<i>V33A</i> mutations are also indicated.</p

<i>Nisch</i>^{<i>edsn/edsn</i>} mice display embryonic and adult lung abnormalities.

<p><b>(A-D)</b> At E16.5, E18.5 and P0, some <i>Nisch</i>^{<i>edsn/edsn</i>} embryos display lung abnormalities. H&E stained lung sections from (A) <i>Nisch</i>^<i>+/+</i> and (B) severely affected <i>Nisch</i>^{<i>edsn/edsn</i>} mice at P0, show thickened interstitial mesenchyme and smaller airspaces in <i>Nisch</i>^{<i>edsn/edsn</i>} lungs. (C) Number of airspaces was counted in three different 6.5 x 10⁵ μm² regions for all embryos and new born mice, with no significant difference between genotypes. (D) Airspace diameters were measured for 30 airspaces in three different regions for all embryos and new born mice. The mean airspace width in severely affected <i>Nisch</i>^{<i>edsn/edsn</i>} animals was significantly smaller. <i>Nisch</i>^<i>+/+</i> n = 6–9; <i>Nisch</i>^{<i>edsn/+</i>} n = 11–15; <i>Nisch</i>^{<i>edsn/edsn</i>} n = 6–10; mildly affected <i>Nisch</i>^{<i>edsn/edsn</i>} n = 4–7; severely affected <i>Nisch</i>^{<i>edsn/edsn</i>} n = 2–3. <b>(E-H)</b> Adult <i>Nisch</i>^{<i>edsn/edsn</i>} mice exhibit an emphysema-like phenotype. H&E stained lung sections from (E) <i>Nisch</i>^<i>+/+</i> and (F) <i>Nisch</i>^{<i>edsn/edsn</i>} animals at 20 wk, show enlargement of air spaces in <i>Nisch</i>^{<i>edsn/edsn</i>} lungs accompanied by disruption of normal alveolar architecture. (G) In <i>Nisch</i>^{<i>edsn/edsn</i>} lungs quantification of the number of airspaces indicated a significant decrease compared to wild-type and (H) the mean airspace width in <i>Nisch</i>^{<i>edsn/edsn</i>} lungs was significantly larger than in wild-type tissue. <i>Nisch</i>^<i>+/+</i> n = 10; <i>Nisch</i>^{<i>edsn/+</i>} n = 10; <i>Nisch</i>^{<i>edsn/edsn</i>} n = 18. <b>(I-M)</b> The severity of the emphysema-like lung phenotype observed in <i>Nisch</i>^{<i>edsn/edsn</i>} replicates the severity of the OM phenotype in the middle ear. H&E stained lung sections from <i>Nisch</i>^{<i>edsn/edsn</i>} animals at 20 wk show the increasing severity of the emphysema-like phenotype from mice with (I) no OM phenotype, to (J) unilateral OM and to (K) bilateral OM. (L) The mean number of airspaces in <i>Nisch</i>^{<i>edsn/edsn</i>} animals with no OM phenotype was significantly increased compared to <i>Nisch</i>^{<i>edsn/edsn</i>} animals with bilateral OM. (M) The mean airspace width in <i>Nisch</i>^{<i>edsn/edsn</i>} mice with no OM phenotype was significantly smaller than <i>Nisch</i>^{<i>edsn/edsn</i>} animals with bilateral OM. Bilateral OM n = 10; Unilateral OM n = 6; Clear n = 2. <b>(N, O)</b> Immunohistochemical staining of lung sections using an F4/80 antibody. Lung sections from (N) <i>Nisch</i>^<i>+/+</i> and (O) <i>Nisch</i>^{<i>edsn/edsn</i>} animals at 20 wk stained with F4/80 show collections of enlarged alveolar macrophages (brown) in <i>Nisch</i>^{<i>edsn/edsn</i>} lungs compared to wild-type littermates. Representative images from four mice per genotype. N, O scale bar = 200 μm; A, B, E, F, I, J, K scale bar = 100 μm. ns <i>P</i> > 0.05; * <i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> < 0.001. Error bars indicate standard error of mean. Embryonic lung data in panels C and D were analysed by one-way ANOVAs and Holm-Sidak’s multiple comparison procedures for post-hoc testing. Adult lung data (G, H, L, M) was not normally distributed and a Kruskall-Wallis test was performed followed by Dunn’s multiple comparison tests for post-hoc analysis.</p

Protein expression analysis of NISCH interacting partners and downstream pathways in middle ear by immunohistochemistry.

<p>Middle ear sections of <i>Nisch</i>^<i>+/+</i>, <i>Nisch</i>^{<i>edsn/edsn</i>} and <i>Itga5</i>^{<i>tm1Hyn/+</i>}; <i>Nisch</i>^{<i>edsn/edsn</i>} mice at 3 wk, stained with <b>(A)</b> NISCH, <b>(B)</b> ITGA5, <b>(C)</b> p-PAK1, <b>(D)</b> p-LIMK1/2, <b>(E)</b> RAC1, <b>(F)</b> NF-κB p65, <b>(G)</b> FAK and <b>(H)</b> p-SMAD2 antibodies. To quantify the results middle ear epithelial cells in wild-types and mutants were counted in four middle ears from each genotype. Scale bar = 100 μm. ns <i>P</i> > 0.05; * <i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> < 0.001. Error bars indicate standard error of mean. The data was analysed by one-way ANOVAs and Holm-Sidak’s multiple comparison procedures for post-hoc testing.</p

Deficiencies in <i>Nisch</i> and <i>Itga5</i> exacerbate the otitis media phenotype.

<p><b>(A)</b> Click-evoked ABR thresholds across a time course show <i>Itga5</i>^{<i>tm1Hyn/+</i>}; <i>Nisch</i>^{<i>edsn/edsn</i>} mice exhibit significantly elevated auditory thresholds compared to <i>Itga5</i>^<i>+/+</i>; <i>Nisch</i>^{<i>edsn/edsn</i>} mice. Additionally, a mild late-onset hearing deficit is observed in <i>Itga5</i>^{<i>tm1Hyn/+</i>}; <i>Nisch</i>^{<i>edsn/+</i>} mice, with onset at 12 wk. Expected ABR threshold range for normal hearing was between 15–30 dB SPL (dashed red lines). <i>Itga5</i>^<i>+/+</i>; <i>Nisch</i>^<i>+/+</i> n = 14; <i>Itga5</i>^<i>+/+</i>; <i>Nisch</i>^{<i>edsn/+</i>} n = 14; <i>Itga5</i>^{<i>tm1Hyn/+</i>}; <i>Nisch</i>^<i>+/+</i> n = 15; <i>Itga5</i>^{<i>tm1Hyn/+</i>}; <i>Nisch</i>^{<i>edsn/+</i>} n = 13; <i>Itga5</i>^<i>+/+</i>; <i>Nisch</i>^{<i>edsn/edsn</i>} n = 8; <i>Itga5</i>^{<i>tm1Hyn/+</i>}; <i>Nisch</i>^{<i>edsn/edsn</i>} n = 12. <b>(B-D)</b> Visual inspection of the tympanic membrane was used as a semi-quantitative measure for the prevalence of OM. (B) <i>Itga5</i>^<i>+/+</i>; <i>Nisch</i>^<i>+/+</i> mice show a very low prevalence of unilateral OM at 4 wk and 6 wk only. (C) <i>Itga5</i>^<i>+/+</i>; <i>Nisch</i>^{<i>edsn/edsn</i>} mice show a progressive increase in prevalence of OM, whereas (D) <i>Itga5</i>^{<i>tm1Hyn/+</i>}; <i>Nisch</i>^{<i>edsn/edsn</i>} mice display a consistently high prevalence of bilateral OM throughout the time course. <b>(E-J)</b> H&E stained transverse sections of the MEC and mucoperiosteum, in 20 wk (E, F) <i>Itga5</i>^<i>+/+</i> <i>Nisch</i>^<i>+/+</i>, (G, H) <i>Itga5</i>^<i>+/+</i>; <i>Nisch</i>^{<i>edsn/edsn</i>} and (I, J) <i>Itga5</i>^{<i>tm1Hyn/+</i>}; <i>Nisch</i>^{<i>edsn/edsn</i>} mice. Both <i>Itga5</i>^<i>+/+</i>; <i>Nisch</i>^{<i>edsn/edsn</i>} and <i>Itga5</i>^{<i>tm1Hyn/+</i>}; <i>Nisch</i>^{<i>edsn/edsn</i>} mice demonstrate chronic inflammation with an exudate. Inflammation of mucosa was more severe in sections from <i>Itga5</i>^{<i>tm1Hyn/+</i>}; <i>Nisch</i>^{<i>edsn/edsn</i>} ears, with increased polypoid exophytic growths and a thick cellular effusion. <b>(K)</b> Blinded assessment of mean mucosal thickness demonstrates significant increases in <i>Itga5</i>^{<i>tm1Hyn/+</i>}; <i>Nisch</i>^{<i>edsn/+</i>}, <i>Itga5</i>^<i>+/+</i>; <i>Nisch</i>^{<i>edsn/edsn</i>} and <i>Itga5</i>^{<i>tm1Hyn/+</i>}; <i>Nisch</i>^{<i>edsn/edsn</i>} mice compared to wild-type. Both <i>Itga5</i>^<i>+/+</i>; <i>Nisch</i>^{<i>edsn/edsn</i>} and <i>Itga5</i>^{<i>tm1Hyn/+</i>}; <i>Nisch</i>^{<i>edsn/edsn</i>} mice exhibit significant increases in mucosal thickness compared to <i>Itga5</i>^{<i>tm1Hyn/+</i>}; <i>Nisch</i>^{<i>edsn/+</i>} mice. <i>Itga5</i>^<i>+/+</i>; <i>Nisch</i>^<i>+/+</i> n = 12; <i>Itga5</i>^<i>+/+</i>; <i>Nisch</i>^{<i>edsn/+</i>} n = 8; <i>Itga5</i>^{<i>tm1Hyn/+</i>}; <i>Nisch</i>^<i>+/+</i> n = 10; <i>Itga5</i>^{<i>tm1Hyn/+</i>}; <i>Nisch</i>^{<i>edsn/+</i>} n = 18; <i>Itga5</i>^<i>+/+</i>; <i>Nisch</i>^{<i>edsn/edsn</i>} n = 10; <i>Itga5</i>^{<i>tm1Hyn/+</i>}; <i>Nisch</i>^{<i>edsn/edsn</i>} n = 10. <b>(L)</b> To account for the disparities in OM prevalence, the mean mucosal thickness was additionally assessed for OM ears only. <i>Itga5</i>^{<i>tm1Hyn/+</i>}; <i>Nisch</i>^{<i>edsn/edsn</i>} OM ears exhibit significant increases in mucosal thickness compared to <i>Itga5</i>^<i>+/+</i>; <i>Nisch</i>^{<i>edsn/edsn</i>} OM ears. OM only: <i>Itga5</i>^{<i>tm1Hyn/+</i>}; <i>Nisch</i>^{<i>edsn/+</i>} n = 3; <i>Itga5</i>^<i>+/+</i>; <i>Nisch</i>^{<i>edsn/edsn</i>} n = 8; <i>Itga5</i>^{<i>tm1Hyn/+</i>}; <i>Nisch</i>^{<i>edsn/edsn</i>} n = 9. C, cochlea; ET, eustachian tube; E, exudate; MEC, middle ear cavity; MP, mucoperiosteum (arrowheads); TB, temporal bone; TM, tympanic membrane. E, G, I scale bar = 2 mm; F, H, J scale bar = 200 μm. ns <i>P</i> > 0.05; * <i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> < 0.001. Error bars indicate standard error of mean. A Kruskall-Wallis test was performed followed by Dunn’s multiple comparison tests for post-hoc analysis.</p

Proposed mechanism.

<p>Model of the mechanism of action in wild-type mice (<i>Itga5</i>^<i>+/+</i><i>; Nisch+</i>^<i>/+</i><b>),</b> during the onset OM in <i>edison</i> mice (<i>Itga5</i>^<i>+/+</i><i>; Nisch</i>^{<i>edsn/edsn</i>}<b>)</b> and in double mutants (<i>Itga5</i>^{<i>tm1Hyn/+</i>}<i>; Nisch</i>^{<i>edsn/edsn</i>}). Nisch, Nischarin; Nisch^edsn, Nischarin with the <i>edison</i> mutation; ITGA5, integrin α5; ITGB1, integrin β1; FAK, Focal adhesion kinase; PAK1, p21-activated kinase 1; LIMK1, LIM domain kinase 1; Rac1, Ras-related C3 botulinum toxin substrate 1; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; SRC, Proto-oncogene tyrosine-protein kinase; VEGF, Vascular endothelial growth factor. Red capped lines represent inhibition, yellow capped lines represent reduced inhibition and grey capped lines represent low inhibition. The blue arrow indicates the role of ITGA5 in enhancing binding of NISCH to PAK1. Green arrows indicate direct activation of downstream members of the pathway and dashed green arrows indicate indirect activation. Grey arrows indicate slightly raised levels of proteins, while yellow indicates raised levels and red indicates highly raised levels.</p