Search

Background Microphthalmia, anophthalmia, and coloboma (MAC) spectrum disease encompasses a group of eye malformations which play a role in childhood visual impairment. Although the predominant cause of eye malformations is known to be heritable in nature, with 80% of cases displaying loss-of-function mutations in the ocular developmental genes OTX2 or SOX2, the genetic abnormalities underlying the remaining cases of MAC are incompletely understood. This study intended to identify the novel genes and pathways required for early eye development. Additionally, pathways involved in eye formation during embryogenesis are also incompletely understood. This study aims to identify the novel genes and pathways required for early eye development through systematic forward screening of the mammalian genome. Results Query of the International Mouse Phenotyping Consortium (IMPC) database (data release 17.0, August 01, 2022) identified 74 unique knockout lines (genes) with genetically associated eye defects in mouse embryos. The vast majority of eye abnormalities were small or absent eyes, findings most relevant to MAC spectrum disease in humans. A literature search showed that 27 of the 74 lines had previously published knockout mouse models, of which only 15 had ocular defects identified in the original publications. These 12 previously published gene knockouts with no reported ocular abnormalities and the 47 unpublished knockouts with ocular abnormalities identified by the IMPC represent 59 genes not previously associated with early eye development in mice. Of these 59, we identified 19 genes with a reported human eye phenotype. Overall, mining of the IMPC data yielded 40 previously unimplicated genes linked to mammalian eye development. Bioinformatic analysis showed that several of the IMPC genes colocalized to several protein anabolic and pluripotency pathways in early eye development. Of note, our analysis suggests that the serine-glycine pathway producing glycine, a mitochondrial one-carbon donator to folate one-carbon metabolism (FOCM), is essential for eye formation. Conclusions Using genome-wide phenotype screening of single-gene knockout mouse lines, STRING analysis, and bioinformatic methods, this study identified genes heretofore unassociated with MAC phenotypes providing models to research novel molecular and cellular mechanisms involved in eye development. These findings have the potential to hasten the diagnosis and treatment of this congenital blinding disease.

Gastro-intestinal stromal tumors and acute myeloid leukemia induced by activating stem cell factor receptor tyrosine kinase (KIT) mutations are highly malignant. Less clear is the role of KIT mutations in the context of breast cancer. Treatment success of KIT-induced cancers is still unsatisfactory because of primary or secondary resistance to therapy. Mouse models offer essential platforms for studies on molecular disease mechanisms in basic cancer research. In the course of the Munich N-ethyl-N-nitrosourea (ENU) mutagenesis program a mouse line with inherited polycythemia was established. It carries a base-pair exchange in the Kit gene leading to an amino acid exchange at position 824 in the activation loop of KIT. This KIT variant corresponds to the N822K mutation found in human cancers, which is associated with imatinib-resistance. C3H KitN824K/WT mice develop hyperplasia of interstitial cells of Cajal and retention of ingesta in the cecum. In contrast to previous Kit-mutant models, we observe a benign course of gastrointestinal pathology associated with prolonged survival. Female mutants develop mammary carcinomas at late onset and subsequent lung metastasis. The disease model complements existing oncology research platforms. It allows for addressing the role of KIT mutations in breast cancer and identifying genetic and environmental modifiers of disease progression.

Posttranslational modifications are essential for regulating the transcription factor p53, which binds DNA in a highly cooperative manner to control expression of a plethora of tumor-suppressive programs. Here we show at the biochemical, cellular, and organismal level that the cooperative nature of DNA binding is reduced by phosphorylation of highly conserved serine residues (human S183/S185, mouse S180) in the DNA-binding domain. To explore the role of this inhibitory phosphorylation in vivo, new phosphorylation-deficient p53-S180A knock-in mice were generated. Chromatin immunoprecipitation sequencing and RNA sequencing studies of S180A knock-in cells demonstrated enhanced DNA binding and increased target gene expression. In vivo, this translated into a tissue-specific vulnerability of the bone marrow that caused depletion of hematopoietic stem cells and impaired proper regeneration of hematopoiesis after DNA damage. Median lifespan was significantly reduced by 20% from 709 days in wild type to only 568 days in S180A littermates. Importantly, lifespan was reduced by a loss of general fitness and increased susceptibility to age-related diseases, not by increased cancer incidence as often seen in other p53-mutant mouse models. For example, S180A knock-in mice showed markedly reduced spontaneous tumorigenesis and increased resistance to Myc-driven lymphoma and Eml4–Alk-driven lung cancer. Preventing phosphorylation of S183/S185 in human cells boosted p53 activity and allowed tumor cells to be killed more efficiently. Together, our data identify p53 DNA-binding domain phosphorylation as a druggable mechanism that balances tumorigenesis and aging. Significance: These findings demonstrate that p53 tumor suppressor activity is reduced by DNA-binding domain phosphorylation to prevent aging and identify this phosphorylation as a potential target for cancer therapy. See related commentary by Horikawa, p. 5164

Mutations in the X chromosomal tRNA 2'‑O‑methyltransferase FTSJ1 cause intellectual disability (ID). Although the gene is ubiquitously expressed affected individuals present no consistent clinical features beyond ID. In order to study the pathological mechanism involved in the aetiology of FTSJ1 deficiency-related cognitive impairment, we generated and characterized an Ftsj1 deficient mouse line based on the gene trapped stem cell line RRD143. Apart from an impaired learning capacity these mice presented with several statistically significantly altered features related to behaviour, pain sensing, bone and energy metabolism, the immune and the hormone system as well as gene expression. These findings show that Ftsj1 deficiency in mammals is not phenotypically restricted to the brain but affects various organ systems. Re-examination of ID patients with FTSJ1 mutations from two previously reported families showed that several features observed in the mouse model were recapitulated in some of the patients. Though the clinical spectrum related to Ftsj1 deficiency in mouse and man is variable, we suggest that an increased pain threshold may be more common in patients with FTSJ1 deficiency. Our findings demonstrate novel roles for Ftsj1 in maintaining proper cellular and tissue functions in a mammalian organism.

ObjectivesHeterogeneity in outcomes measured across trials of glucose-lowering interventions for people with type 2 diabetes impacts on the ability to compare findings and may mean that the results have little importance to healthcare professionals and the patients that they care for. The SCORE-IT study (Selecting Core Outcomes for Randomised Effectiveness trials In Type 2 diabetes) has addressed this issue by establishing consensus on the most important outcomes for non-surgical interventions for hyperglycemia in type 2 diabetes.Research design and methodsA comprehensive list of outcomes was developed from registered clinical trials, online patient resources, qualitative literature and long-term studies in the field. This list was then scored in a two-round online Delphi survey completed by healthcare professionals, people with type 2 diabetes, researchers in the field and healthcare policymakers. The results of this online Delphi were discussed and ratified at a face-to-face consensus meeting.Results173 people completed both rounds of the online survey (116 people with type 2 diabetes, 37 healthcare professionals, 14 researchers and 6 policymakers), 20 of these attended the consensus meeting (13 people with type 2 diabetes and 7 healthcare professionals). Consensus was reached on 18 core outcomes across five domains, which include outcomes related to diabetes care, quality of life and long-term diabetes-related complications.ConclusionsImplementation of the core outcome set in future trials will ensure that outcomes of importance to all stakeholders are measured and reported, enhancing the relevance of trial findings and facilitating the comparison of results across trials.

Diabetes mellitus is a group of disorders characterized by prolonged high levels of circulating blood glucose. Type 1 diabetes is caused by decreased insulin production in the pancreas whereas type 2 diabetes may develop due to obesity and lack of exercise; it begins with insulin resistance whereby cells fail to respond properly to insulin and it may also progress to decreased insulin levels. The brain is an important target for insulin, and there is great interest in understanding how diabetes affects the brain. In addition to the direct effects of insulin on the brain, diabetes may also impact the brain through modulation of the inflammatory system. Here we investigate how perturbation of circulating insulin levels affects the expression of Hes3, a transcription factor expressed in neural stem and progenitor cells that is involved in tissue regeneration. Our data show that streptozotocin-induced β-cell damage, high fat diet, as well as metformin, a common type 2 diabetes medication, regulate Hes3 levels in the brain. This work suggests that Hes3 is a valuable biomarker helping to monitor the state of endogenous neural stem and progenitor cells in the context of diabetes mellitus. © 2018, The Author(s).

Mesenchymal TNF signaling is etiopathogenic for inflammatory diseases such as rheumatoid arthritis and spondyloarthritis (SpA). The role of Tnfr1 in arthritis has been documented; however, Tnfr2 functions are unknown. Here, we investigate the mesenchymal-specific role of Tnfr2 in the TnfΔARE mouse model of SpA in arthritis and heart valve stenosis comorbidity by cell-specific, Col6a1-cre-driven gene targeting. We find that TNF/Tnfr2 signaling in resident synovial fibroblasts (SFs) and valvular interstitial cells (VICs) is detrimental for both pathologies, pointing to common cellular mechanisms. In contrast, systemic Tnfr2 provides protective signaling, since its complete deletion leads to severe deterioration of both pathologies. SFs and VICs lacking Tnfr2 fail to acquire pathogenic activated phenotypes and display increased expression of antiinflammatory cytokines associated with decreased Akt signaling. Comparative RNA sequencing experiments showed that the majority of the deregulated pathways in TnfΔARE mesenchymal-origin SFs and VICs, including proliferation, inflammation, migration, and disease-specific genes, are regulated by Tnfr2; thus, in its absence, they are maintained in a quiescent nonpathogenic state. Our data indicate a pleiotropy of Tnfr2 functions, with mesenchymal Tnfr2 driving cell activation and arthritis/valve stenosis pathogenesis only in the presence of systemic Tnfr2, whereas nonmesenchymal Tnfr2 overcomes this function, providing protective signals and, thus, containing both pathologies.

Background— ADAMTS-7, a member of the disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family, was recently identified to be significantly associated genomewide with coronary artery disease. However, the mechanisms that link ADAMTS-7 and coronary artery disease risk remain elusive. We have previously demonstrated that ADAMTS-7 promotes vascular smooth muscle cell migration and postinjury neointima formation via degradation of a matrix protein cartilage oligomeric matrix protein. Because delayed endothelium repair renders neointima and atherosclerosis plaque formation after vessel injury, we examined whether ADAMTS-7 also inhibits re-endothelialization. Methods and Results— Wire injury of the carotid artery and Evans blue staining were performed in Adamts7 –/– and wild-type mice. Adamts-7 deficiency greatly promoted re-endothelialization at 3, 5, and 7 days after injury. Consequently, Adamts-7 deficiency substantially ameliorated neointima formation in mice at days 14 and 28 after injury in comparison with the wild type. In vitro studies further indicated that ADAMTS-7 inhibited both endothelial cell proliferation and migration. Surprisingly, cartilage oligomeric matrix protein deficiency did not affect endothelial cell proliferation/migration and re-endothelialization in mice. In a further examination of other potential vascular substrates of ADAMTS-7, a label-free liquid chromatography-tandem mass spectrometry secretome analysis revealed thrombospondin-1 as a potential ADAMTS-7 target. The subsequent studies showed that ADAMTS-7 was directly associated with thrombospondin-1 by its C terminus and degraded thrombospondin-1 in vivo and in vitro. The inhibitory effect of ADAMTS-7 on postinjury endothelium recovery was circumvented in Tsp1 –/– mice. Conclusions— Our study revealed a novel mechanism by which ADAMTS-7 affects neointima formation. Thus, ADAMTS-7 is a promising treatment target for postinjury vascular intima hyperplasia.

The human growth hormone (hGH) minigene used for transgene stabilization in mice has been recently identified to be locally expressed in the tissues where transgenes are active and associated with phenotypic alterations. Here we extend these findings by analyzing the effect of the hGH minigene in TgC6hp55 transgenic mice which express the human TNFR1 under the control of the mesenchymal cell-specific CollagenVI promoter. These mice displayed a fully penetrant phenotype characterized by growth enhancement accompanied by perturbations in metabolic, skeletal, histological and other physiological parameters. Notably, this phenotype was independent of TNF-TNFR1 signaling since the genetic ablation of either Tnf or Tradd did not rescue the phenotype. Further analyses showed that the hGH minigene was expressed in several tissues, also leading to increased hGH protein levels in the serum. Pharmacological blockade of GH signaling prevented the development of the phenotype. Our results indicate that the unplanned expression of the hGH minigene in CollagenVI expressing mesenchymal cells can lead through local and/or systemic mechanisms to enhanced somatic growth followed by a plethora of primary and/or secondary effects such as hyperphagia, hypermetabolism, disturbed glucose homeostasis, altered hematological parameters, increased bone formation and lipid accumulation in metabolically critical tissues. © 2017 The Author(s).

International Journal of Cancer Volume 136, Issue 10, 15 May 2015, Pages 2293-2303 MiR-34a deficiency accelerates medulloblastoma formation in vivo (Article) Thor, T.abc , Künkele, A.a, Pajtler, K.W.a, Wefers, A.K.d, Stephan, H.a, Mestdagh, P.e, Heukamp, L.f, Hartmann, W.g, Vandesompele, J.e, Sadowski, N.a, Becker, L.hi, Garrett, L.hj, Hölter, S.M.hj, Horsch, M.hk, Calzada-Wack, J.hl, Klein-Rodewald, T.hl, Racz, I.hm, Zimmer, A.hm, Beckers, J.hkn, Neff, F.hl, Klopstock, T.i, Antonellis, P.D.op, Zollo, M.op, Wurst, W.hjqrs, Fuchs, H.hk, Gailus-Durner, V.hk, Schüller, U.d, De Angelis, M.H.hkn, Eggert, A.t, Schramm, A.a, Schulte, J.H.abcuv a Department of Pediatric Oncology and Hematology, University Children's Hospital Essen, Hufelandstr. 55, Essen, Germany b German Cancer Consortium (DKTK), Heidelberg, Germany c German Cancer Research Center (DKFZ), Heidelberg, Germany View additional affiliations View references (61) Abstract Previous studies have evaluated the role of miRNAs in cancer initiation and progression. MiR-34a was found to be downregulated in several tumors, including medulloblastomas. Here we employed targeted transgenesis to analyze the function of miR-34a in vivo. We generated mice with a constitutive deletion of the miR-34a gene. These mice were devoid of mir-34a expression in all analyzed tissues, but were viable and fertile. A comprehensive standardized phenotypic analysis including more than 300 single parameters revealed no apparent phenotype. Analysis of miR-34a expression in human medulloblastomas and medulloblastoma cell lines revealed significantly lower levels than in normal human cerebellum. Re-expression of miR-34a in human medulloblastoma cells reduced cell viability and proliferation, induced apoptosis and downregulated the miR-34a target genes, MYCN and SIRT1. Activation of the Shh pathway by targeting SmoA1 transgene overexpression causes medulloblastoma in mice, which is dependent on the presence and upregulation of Mycn. Analysis of miR-34a in medulloblastomas derived from ND2:SmoA1(tg) mice revealed significant suppression of miR-34a compared to normal cerebellum. Tumor incidence was significantly increased and tumor formation was significantly accelerated in mice transgenic for SmoA1 and lacking miR-34a. Interestingly, Mycn and Sirt1 were strongly expressed in medulloblastomas derived from these mice. We here demonstrate that miR-34a is dispensable for normal development, but that its loss accelerates medulloblastomagenesis. Strategies aiming to re-express miR-34a in tumors could, therefore, represent an efficient therapeutic option.

Although various mouse inbred strains are widely used to investigate disease mechanisms and to establish new therapeutic strategies, sex-specific reference intervals for laboratory diagnostic analytes that are generated from large numbers of animals have been unavailable. In this retrospective study, we screened data from more than 12,000 mice phenotyped in the German Mouse Clinic from January 2006 through June 2014 and selected animals with the genetic background of C57BL/6J, C57BL/6N, or C3HeB/FeJ. In addition, we distinguished between the C57BL/6NTac substrain and C57BL/6N mice received from other vendors. The corresponding data sets of electrolytes (sodium, potassium, calcium, chloride, inorganic phosphate), lipids (cholesterol, triglyceride), and enzyme activities (ALT, AST, ALP, α-amylase) and urea, albumin, and total protein levels were analyzed. Significant effects of age and sex on these analytes were identified, and strain- or substrain- and sex-specific reference intervals for 90- to 135-d-old mice were calculated. In addition, we include an overview of the literature that reports clinical chemistry values for wild-type mice of different strains. Our results support researchers interpreting clinical chemistry values from various mouse mutants and corresponding wild-type controls based on the examined strains and substrains.

The vertebrate Scube (Signal peptide, CUB and EGF-like domain-containing protein) family consists of three independent members Scube1-3, which encode secreted cell surface-associated membrane glycoproteins. Limited information about the general function of this gene family is available, and their roles during adulthood. Here, we present the first Scube3 mutant mouse line (Scube3N294K/N294K) that clearly shows phenotypic alterations by carrying a missense mutation in exon 8, and thus contributes to understand SCUBE3 functions. We performed a detailed phenotypic characterization in the German Mouse Clinic (GMC). Scube3N294K/N294K mutants showed morphological abnormalities of the skeleton, alterations of parameters relevant for bone metabolism, changes in renal function and hearing impairments. These findings correlate with characteristics of the rare metabolic bone disorder Paget disease of bone (PDB), associated with the chromosomal region of human SCUBE3. In addition, alterations in energy metabolism, behavior and neurological functions were detected in Scube3N294K/N294K mice. The Scube3N294K/N294K mutant mouse line may serve as a new model for further studying the effect of impaired SCUBE3 gene function.

The function of the majority of genes in the mouse and human genomes remains unknown. The mouse embryonic stem cell knockout resource provides a basis for the characterization of relationships between genes and phenotypes. The EUMODIC consortium developed and validated robust methodologies for the broad-based phenotyping of knockouts through a pipeline comprising 20 disease-oriented platforms. We developed new statistical methods for pipeline design and data analysis aimed at detecting reproducible phenotypes with high power. We acquired phenotype data from 449 mutant alleles, representing 320 unique genes, of which half had no previous functional annotation. We captured data from over 27,000 mice, finding that 83% of the mutant lines are phenodeviant, with 65% demonstrating pleiotropy. Surprisingly, we found significant differences in phenotype annotation according to zygosity. New phenotypes were uncovered for many genes with previously unknown function, providing a powerful basis for hypothesis generation and further investigation in diverse systems. Comment in : Genetic differential calculus. [Nat Genet. 2015] Comment in : Scaling up phenotyping studies. [Nat Biotechnol. 2015]

Uromodulin-associated kidney disease (UAKD) is a hereditary progressive renal disease which can lead to renal failure and requires renal replacement therapy. UAKD belongs to the endoplasmic reticulum storage diseases due to maturation defect of mutant uromodulin and its retention in the enlarged endoplasmic reticulum in the cells of the thick ascending limb of Henle's loop (TALH). Dysfunction of TALH represents the key pathogenic mechanism of UAKD causing the clinical symptoms of this disease. However, the molecular alterations underlying UAKD are not well understood. In this study, transcriptome profiling of whole kidneys of two mouse models of UAKD, UmodA227T and UmodC93F, was performed. Genes differentially abundant in UAKD affected kidneys of both Umod mutant lines at different disease stages were identified and verified by RT-qPCR. Additionally, differential protein abundances of SCD1 and ANGPTL7 were validated by immunohistochemistry and Western blot analysis. ANGPTL7 expression was down-regulated in TALH cells of Umod mutant mice which is the site of the mutant uromodulin maturation defect. SCD1 was expressed selectively in the S3 segment of proximal tubule cells, and SCD1 abundance was increased in UAKD affected kidneys. This finding demonstrates that a cross talk between two functionally distinct tubular segments of the kidney, the TALH segment and the S3 segment of proximal tubule, exists.

Type I IFN signaling amplifies the secretion of LPS-induced proinflammatory cytokines such as TNF-α or IL-6 and might thus contribute to the high mortality associated with Gram-negative septic shock in humans. The underlying molecular mechanism, however, is ill defined. In this study, we report the generation of mice deficient in IFN-induced protein with tetratricopeptide repeats 2 (Ifit2) and demonstrate that Ifit2 is a critical signaling intermediate for LPS-induced septic shock. Ifit2 expression was significantly upregulated in response to LPS challenge in an IFN-α receptor- and IFN regulatory factor (Irf)9-dependent manner. Also, LPS induced secretion of IL-6 and TNF-α by bone marrow-derived macrophages (BMDMs) was significantly enhanced in the presence of Ifit2. In accordance, Ifit2-deficient mice exhibited significantly reduced serum levels of IL-6 and TNF-α and reduced mortality in an endotoxin shock model. Investigation of the underlying signal transduction events revealed that Ifit2 upregulates Irf3 phosphorylation. In the absence of Irf3, reduced Ifn-β mRNA expression and Ifit2 protein expression after LPS stimulation was found. Also, Tnf-α and Il-6 secretion but not Tnf-α and Il-6 mRNA expression levels were reduced. Thus, IFN-stimulated Ifit2 via enhanced Irf3 phosphorylation upregulates the secretion of proinflammatory cytokines. It thereby amplifies LPS-induced cytokine production and critically influences the outcome of endotoxin shock.

Background: Nuclear myosin I (NM1) is a nuclear isoform of the well-known "cytoplasmic" Myosin 1c protein (Myo1c). Located on the 11th chromosome in mice, NM1 results from an alternative start of transcription of the Myo1c gene adding an extra 16 amino acids at the N-terminus. Previous studies revealed its roles in RNA Polymerase I and RNA Polymerase II transcription, chromatin remodeling, and chromosomal movements. Its nuclear localization signal is localized in the middle of the molecule and therefore directs both Myosin 1c isoforms to the nucleus. Methodology/Principal Findings: In order to trace specific functions of the NM1 isoform, we generated mice lacking the NM1 start codon without affecting the cytoplasmic Myo1c protein. Mutant mice were analyzed in a comprehensive phenotypic screen in cooperation with the German Mouse Clinic. Strikingly, no obvious phenotype related to previously described functions has been observed. However, we found minor changes in bone mineral density and the number and size of red blood cells in knock-out mice, which are most probably not related to previously described functions of NM1 in the nucleus. In Myo1c/NM1 depleted U2OS cells, the level of Pol I transcription was restored by overexpression of shRNA-resistant mouse Myo1c. Moreover, we found Myo1c interacting with Pol II. The ratio between Myo1c and NM1 proteins were similar in the nucleus and deletion of NM1 did not cause any compensatory overexpression of Myo1c protein. Conclusion/Significance: We observed that Myo1c can replace NM1 in its nuclear functions. Amount of both proteins is nearly equal and NM1 knock-out does not cause any compensatory overexpression of Myo1c. We therefore suggest that both isoforms can substitute each other in nuclear processes.

The mammalian Interferon induced transmembrane protein 1 (Ifitm1) gene was originally identified as a member of a gene family highly inducible by type I and type II interferons. Based on expression analyses, it was suggested to be required for normal primordial germ cell migration. The knockdown of Ifitm1 in mouse embryos provided evidence for a role in somitogenesis. We generated the first targeted knockin allele of the Ifitm1 gene to systematically reassess all inferred functions. Sperm motility and the fertility of male and female mutant mice are as in wild type littermates. Embryonic somites and the adult vertebral column appear normal in homozygous Ifitm1 knockout mice, demonstrating that Ifitm1 is not essential for normal segmentation of the paraxial mesoderm. Proportions of leucocyte subsets, including granulocytes, monocytes, B-cells, T-cells, NK-cells, and NKT-cells, are unchanged in mutant mice. Based on a normal immune response to Listeria monocytogenes infection, there is no evidence for a dysfunction in downstream IFNγ signaling in Ifitm1 mutant mice. Expression from the Ifitm1 locus from E8.5 to E14.5 is highly dynamic. In contrast, in adult mice, Ifitm1 expression is highly restricted and strong in the bronchial epithelium. Intriguingly, IFITM1 is highly overexpressed in tumor epithelia cells of human squamous cell carcinomas and in adenocarcinomas of NSCLC patients. These analyses underline the general importance of targeted in vivo studies for the functional annotation of the mammalian genome. The first comprehensive description of the Ifitm1 expression pattern provides a rational basis for the further examination of Ifitm1 gene functions. Based on our data, the fact that IFITM1 can function as a negative regulator of cell proliferation, and because the gene maps to chromosome band 11p15.5, previously associated with NSCLC, it is likely that IFITM1 in man has a key role in tumor formation.

Calcium signalling plays a critical role in the pathogenesis of heart failure. Here we describe a cardiac protein named Myoscape/FAM40B/STRIP2, which directly interacts with the L-type calcium channel. Knockdown of Myoscape in cardiomyocytes decreases calcium transients associated with smaller Ca(2+) amplitudes and a lower diastolic Ca(2+) content. Likewise, L-type calcium channel currents are significantly diminished on Myoscape ablation, and downregulation of Myoscape significantly reduces contractility of cardiomyocytes. Conversely, overexpression of Myoscape increases global Ca(2+) transients and enhances L-type Ca(2+) channel currents, and is sufficient to restore decreased currents in failing cardiomyocytes. In vivo, both Myoscape-depleted morphant zebrafish and Myoscape knockout (KO) mice display impairment of cardiac function progressing to advanced heart failure. Mechanistically, Myoscape-deficient mice show reduced L-type Ca(2+)currents, cell capacity and calcium current densities as a result of diminished LTCC surface expression. Finally, Myoscape expression is reduced in hearts from patients suffering of terminal heart failure, implying a role in human disease.

PURPOSE: Within a mutagenesis screen, we identified the new mouse mutant Aey80 with small eyes; homozygous mutants were not obtained. The aim of the study was its molecular characterization. METHODS: We analyzed the offspring of paternally N-ethyl-N-nitrosourea (ENU)-treated C3HeB/FeJ mice for dysmorphology parameters, which can be observed with the naked eye. The Aey80 mutant (abnormality of the eye) was further characterized with laser interference biometry, Scheimpflug imaging, and optical coherence tomography. Linkage analysis of the Aey80 mutant was performed using a panel of single nucleotide polymorphisms different among C3HeB/FeJ and C57BL/6J mice. The Aey80 mutation was identified with sequence analysis of the positional candidate gene. RESULTS: We identified a new mutant characterized by an obvious small-eye phenotype; homozygotes are not viable after birth. Embryos at embryonic day 15.5 demonstrate a clear gene-dosage effect: Heterozygotes have small eyes, whereas homozygous mutants do not have eyes. In adult mice, the lenses and the entire eyes of the heterozygous mutants were significantly smaller than those of the wild-types (pA exchange four bases downstream of the end of the alternative exon. The alternative exon in the mutants is predicted to encode 30 novel amino acids and three stop codons. This alternative exon kept the paired domain intact but led to a loss of the homeodomain and the C-terminal proline-serine-threonine (PST) domain. The mutation cosegregated in the mutant line, since all five additional small-eyed mice from this line showed the same mutation. A general polymorphism at the mutated site was excluded with sequence analysis of seven other wild-type mouse strains different from C3HeB/FeJ. CONCLUSIONS: These findings demonstrate a novel allele of the paired box gene 6 (Pax6) that affects lens development in a semidominant manner leading to a classical small-eye phenotype. However, the site of the mutation more than 1 kb downstream of exon 7 and resulting in an alternative exon is quite unusual. It indicates the importance of sequence analysis of cDNA for mutation detection; mutations like this are unlikely to be identified by analyzing genomic sequences only. Moreover, this particular mutation demonstrates how a novel exon can be created by only a single base-pair exchange. &nbsp

Einleitung: Die German Mouse Clinic (GMC) erforscht mithilfe genetisch modifizierter Tiermodelle humane Erkrankungen, um neue Diagnose und Therapieansätze zu etablieren. Den Abschluss eines über 550 Parameter erfassenden Primärscreens bildet die makroskopische und histopathologische Untersuchung. Dabei ist es das Ziel, beobachtete funktionelle Abweichungen mit den morphologischen Alterationen zu korrelieren und Erkenntnisse über die mögliche Rolle eines spezifischen Gens für humane Erkrankungen zu erlangen. Material und Methoden: Jeweils 50 Wildtyp-Tiere verschiedener Stämme (C57BL/6J, C3He/FeJ, Balb/cAnPt und S129/J) im Alter von 14–22 Wochen wurden mit CO2 euthanasiert, einer vollständigen Sektion mit makroskopischer Beurteilung und Erfassung von Körper- und Organgewichten sowie der Tibialänge unterzogen. Ein Standardset von 28 Organen wurde in 4% Formalin fixiert, für die lichtmikroskopische Untersuchung in Paraffin eingebettet und 4 μm dicke, Hämatoxylin-Eosin-gefärbte Schnittpräparate angefertigt. Das Gehirn wird standardmäßig in drei Transversalebenen geschnitten. Befunde: Eine Agenesie des Corpus callosum trat bei 60% der S129/J-Mäuse auf, daneben wurden in allen Stämmen sporadische Fälle von zerebralen Epidermoidzysten, Hydrocephalus internus sowie lipomatösen Hamartomen beobachtet. Schlussfolgerung: Der pathologischen Untersuchung kommt im Rahmen des Screenings von Mausmodellen an der GMC eine besondere Bedeutung in der Einordnung klinischer Befunde zu. Dabei ist eine Interpretation nur im Vergleich mit Wildtyp-Tieren und mit Kenntnis stammtypischer Organveränderungen möglich.

Thyroid hormone transport into cells requires plasma membrane transport proteins. Mutations in one of these, monocarboxylate transporter 8 (MCT8), have been identified as underlying cause for the Allan-Herndon-Dudley syndrome, an X-linked mental retardation in which the patients also present with abnormally high 3 ',3,5-triiodothyronine (T-3) plasma levels. Mice deficient in Mct8 replicate the thyroid hormone abnormalities observed in the human condition. However, no neurological deficits have been described in mice lacking Mct8. Therefore, we subjected Mct8-deficient mice to a comprehensive immunohistochemical, neurological, and behavioral screen. Several behavioral abnormalities were found in the mutants. Interestingly, some of these behavioral changes are compatible with hypothyroidism, whereas others rather indicate hyperthyroidism. We thus hypothesized that neurons exclusively dependent on Mct8 are in a hypothyroid state, whereas neurons expressing other T-3 transporters become hyperthyroid, if they are exposed directly to the high plasma T-3. The majority of T-3 uptake in primary cortical neurons is mediated by Mct8, but pharmacological inhibition suggested functional expression of additional T-3 transporter classes. mRNAs encoding six T-3 transporters, including L-type amino acid transporters (LATs), were coexpressed with Mct8 in isolated neurons. We then demonstrated Lat2 expression in cultured neurons and throughout murine brain development. In contrast, LAT2 is expressed in microglia in the developing human brain during gestation, but not in neurons. We suggest that lack of functional complementation by alternative thyroid hormone transporters in developing human neurons precipitates the devastating neurodevelopmental phenotype in MCT8-deficient patients, whereas Mct8-deficient mouse neurons are functionally complemented by other transporters, for possibly Lat2.

It is unclear what role vision plays in guiding mouse behaviour, since the mouse eye is of comparably low optical quality, and mice are considered to rely primarily on other senses. All C3H substrains are homozygous for the Pde6b(rd1) mutation and get blind by weaning age. To study the impact of the Pde6b(rd1) mutation on mouse behaviour and physiology, sighted C3H (C3H.Pde6b+) and normal C3H/HeH mice were phenotyped for different aspects. We confirmed retinal degeneration 1 in C3H/HeH mice, and the presence of a morphologically normal retina as well as visual ability in C3H.Pde6b+ mice. However, C3H.Pde6b+ mice showed an abnormal retinal function in the electroretinogram response, indicating that their vision was not normal as expected. C3H.Pde6b+ mice showed reduced latencies for several behaviours without any further alterations in these behaviours in comparison to C3H/HeH mice, suggesting that visual ability, although impaired, enables earlier usage of the behavioural repertoire in a novel environment, but does not lead to increased activity levels. These results emphasize the importance of comprehensive behavioural and physiological phenotyping.