Martin Hrabé de Angelis

Publications of Martin Hrabé de Angelis

• Cardiopulmonary dysfunction in the Osteogenesis Imperfecta mouse model Aga2 and human patients are caused by bone-independent mechanisms.

  Authors: Frank Thiele, Christian M Cohrs, Armando Flor, Thomas S Lisse, Gerhard K H Przemeck, Marion Horsch, Anja Schrewe, Valerie Gailus-Durner, Boris Ivandic, Hugo A Katus, Wolfgang Wurst, Catherine Reisenberg, Hollis Chaney, Helmut Fuchs, Wolfgang Hans, Johannes Beckers, Joan C Marini, Martin Hrabé de Angelis

  Human molecular genetics. 05/2012;

  Osteogenesis imperfecta (OI) is an inherited connective tissue disorder with skeletal dysplasia of varying severity, predominantly caused by mutations in the collagen I genes (COL1A1/COL1A2).Osteogenesis imperfecta (OI) is an inherited connective tissue disorder with skeletal dysplasia of varying severity, predominantly caused by mutations in the collagen I genes (COL1A1/COL1A2). Extraskeletal findings like cardiac and pulmonary complications are generally considered to be significant secondary features. Aga2, a murine model for human OI, was systemically analyzed in the German Mouse Clinic by means of in vivo and in vitro examinations of the cardiopulmonary system, to identify novel mechanisms accounting for perinatal lethality. Pulmonary and especially cardiac fibroblast of perinatal lethal Aga2/+ animals display a strong down-regulation of Col1a1 transcripts in vivo and in vitro, resulting in loss of extracellular matrix integrity. In addition, dysregulated gene expression of Nppa, different types of collagen and Agt in heart and lung tissue supports a bone-independent vicious cycle of heart dysfunction including hypertrophy, loss of myocardial matrix integrity, pulmonary hypertension, pneumonia and hypoxia leading to death in Aga2. These murine findings are corroborated by a pediatric OI cohort study, displaying significant progressive decline of pulmonary function and restrictive pulmonary disease independent of scoliosis. Most participants show mild cardiac valvular regurgitation, independent of pulmonary and skeletal findings. Data obtained from human OI patients and the mouse model Aga2 provide novel evidence for primary effects of type I collagen mutations on heart and lung. The findings will have potential benefits of anticipatory clinical exams and early intervention in OI patients.

• Negative Feedback Mechanisms Surpass the Effect of Intrinsic EGFR Activation during Skin Chemical Carcinogenesis.

  Authors: Maik Dahlhoff, Christian Rose, Martin Hrabé de Angelis, Eckhard Wolf, Marlon R Schneider

  The American journal of pathology. 02/2012;

  The negative feedback regulation of epidermal growth factor receptor (EGFR) and other tyrosine kinase receptors, including receptor dephosphorylation and endocytosis followed by degradation, isThe negative feedback regulation of epidermal growth factor receptor (EGFR) and other tyrosine kinase receptors, including receptor dephosphorylation and endocytosis followed by degradation, is becoming recognized as a major determinant of receptor function. To evaluate the significance of the negative regulation of EGFR during carcinogenesis in vivo, we subjected the mutant mouse line Dsk5, in which the intrinsic activation of the receptor due to a point mutation is normally counterbalanced by increased posttranslational receptor down-regulation, to skin chemical carcinogenesis. Dsk5 mice showed reduced tumor numbers and tumor burden compared with control littermates, and Dsk5-derived tumors showed a reduction in the activation and total levels of EGFR. Furthermore, the transcript levels of several molecules known to act as negative regulators of EGFR were significantly increased in Dsk5-derived tumors. Another intriguing observation was the appearance of tumors with sebaceous differentiation in the ears of Dsk5 mice after chemical carcinogenesis. Further studies are necessary to reveal whether these tumors represent a cell type-specific evasion from EGFR negative feedback machinery. In conclusion, this study reveals that several negative feedback regulators contribute to suppression of the intrinsic activation of mutant EGFR during skin carcinogenesis, stressing the potential exploitation of negative regulators as either therapeutic targets or diagnostic tools in cancer and other diseases.

• Pink1-deficiency in mice impairs gait, olfaction and serotonergic innervation of the olfactory bulb.

  Authors: Lisa Glasl, Karina Kloos, Florian Giesert, Anne Roethig, Barbara Di Benedetto, Ralf Kühn, Jingzhong Zhang, Ulrich Hafen, Julia Zerle, Andreas Hofmann, Martin Hrabé de Angelis, Konstanze F Winklhofer, Sabine M Hölter, Daniela M Vogt Weisenhorn, Wolfgang Wurst

  Experimental neurology. 01/2012;

  Parkinson's Disease (PD) is the most common neurodegenerative movement disorder. Autosomal-recessive mutations in the mitochondrial protein kinase PINK1 (PTEN-induced kinase 1) account for 1-2% ofParkinson's Disease (PD) is the most common neurodegenerative movement disorder. Autosomal-recessive mutations in the mitochondrial protein kinase PINK1 (PTEN-induced kinase 1) account for 1-2% of the hereditary early-onset cases. To study the mechanisms underlying disease development, we generated Pink1-deficient mice. In analogy to other genetic loss-of-function mouse models, Pink1(-/-) mice did not show morphological alterations in the dopaminergic system. As a consequence, no gross motor dysfunctions were observed indicating that these mice do not develop the cardinal symptoms of PD. Nonetheless, symptoms which develop mainly before bradykinesia, rigidity and resting tremor were clearly evident in Pink1-deficient mice. These symptoms were gait alterations and olfactory dysfunctions. Remarkably in the glomerular layer of the olfactory bulb the density of serotonergic fibers was significantly reduced. Concerning mitochondrial morphology, neurons in Pink1(-/-) mice had less fragmented mitochondria. In contrast, upon acute knock-down of Pink1 increased mitochondrial fragmentation was observed in neuronal cultures. This fragmentation was, however, evened out within days. Taken together, we demonstrate that Pink1-deficient mice exhibit behavioral symptoms of early phases of PD and present systematic experimental evidence for compensation of Pink1-deficiency at the cellular level. Thus, Pink1-deficient mice represent a model for the early phases of PD in which compensation may still impede the onset of neurodegeneration. Consequently, these mice are a valuable tool for studying Pink1-related PD development, as well as for searching for reliable PD biomarkers.

• MAPK signaling determines anxiety in the juvenile mouse brain but depression-like behavior in adults.

  Authors: Benedikt Wefers, Christiane Hitz, Sabine M Hölter, Dietrich Trümbach, Jens Hansen, Peter Weber, Benno Pütz, Jan M Deussing, Martin Hrabé de Angelis, Till Roenneberg, Fang Zheng, Christian Alzheimer, Alcino Silva, Wolfgang Wurst, Ralf Kühn

  PloS one. 01/2012; 7(4):e35035.

  MAP kinase signaling has been implicated in brain development, long-term memory, and the response to antidepressants. Inducible Braf knockout mice, which exhibit protein depletion in principleMAP kinase signaling has been implicated in brain development, long-term memory, and the response to antidepressants. Inducible Braf knockout mice, which exhibit protein depletion in principle forebrain neurons, enabled us to unravel a new role of neuronal MAPK signaling for emotional behavior. Braf mice that were induced during adulthood showed normal anxiety but increased depression-like behavior, in accordance with pharmacological findings. In contrast, the inducible or constitutive inactivation of Braf in the juvenile brain leads to normal depression-like behavior but decreased anxiety in adults. In juvenile, constitutive mutants we found no alteration of GABAergic neurotransmission but reduced neuronal arborization in the dentate gyrus. Analysis of gene expression in the hippocampus revealed nine downregulated MAPK target genes that represent candidates to cause the mutant phenotype.Our results reveal the differential function of MAPK signaling in juvenile and adult life phases and emphasize the early postnatal period as critical for the determination of anxiety in adults. Moreover, these results validate inducible gene inactivation as a new valuable approach, allowing it to discriminate between gene function in the adult and the developing postnatal brain.

• Discovery of a novel enzyme mediating glucocorticoid catabolism in fish: 20beta-hydroxysteroid dehydrogenase type 2.

  Authors: Janina Tokarz, Rebekka Mindnich, William Norton, Gabriele Möller, Martin Hrabé de Angelis, Jerzy Adamski

  Molecular and cellular endocrinology. 10/2011; 349(2):202-13.

  Hydroxysteroid dehydrogenases (HSDs) are involved in metabolism and pre-receptor regulation of steroid hormones. While 17beta-HSDs and 11beta-HSDs are extensively studied in mammals, only fewHydroxysteroid dehydrogenases (HSDs) are involved in metabolism and pre-receptor regulation of steroid hormones. While 17beta-HSDs and 11beta-HSDs are extensively studied in mammals, only few orthologs are characterized in fish. We discovered a novel zebrafish HSD candidate closely related to 17beta-HSD types 3 and 12, which has orthologs in other species. The enzyme catalyzes the conversion of cortisone to 20beta-hydroxycortisone identified by LC-MS/MS. We named the new enzyme 20beta-HSD type 2. All 20beta-HSD type 2 orthologs localize in the endoplasmic reticulum. Zebrafish 20beta-HSD type 2 is expressed during embryonic development showing the same expression pattern as 11beta-HSD type 2 known to oxidize cortisol to cortisone. In adult tissues 20beta-HSD type 2 shows a ubiquitous expression pattern with some minor sex-specific differences. In contrast to other enzymes metabolizing C21-steroids and being mostly involved in reproduction we propose that novel type 2 20beta-HSDs in teleost fish are important enzymes in cortisol catabolism.

• Systematic selection of housekeeping genes for gene expression normalization in chicken embryo fibroblasts infected with Newcastle disease virus.

  Authors: Renfu Yin, Xinxin Liu, Chang Liu, Zhuang Ding, Xiaodong Zhang, Furong Tian, Weihong Liu, Jinghai Yu, Lin Li, Martin Hrabé de Angelis, Tobias Stoeger

  Biochemical and biophysical research communications. 09/2011; 413(4):537-40.

  Gene expression analysis is frequently used to analyze the response to viral infection, and 18S RNA, SHDA and GAPDH represent popular house keeping genes (HKGs) often used to normalize geneGene expression analysis is frequently used to analyze the response to viral infection, and 18S RNA, SHDA and GAPDH represent popular house keeping genes (HKGs) often used to normalize gene expression. Here we describe the first systematic selection and evaluation of suitable HKGs for gene expression analysis in chicken embryo fibroblasts (CEF) infected with NDV adapted to the guidelines from Gorzelniak and Ferguson. Our results indicate that ACTB, HPRT1 and HMBS were valuable and stable HKGs, while 18S RNA, GAPDH and SHDA are considerably regulated during the course of infection and thus precluded for normalization. Normalizing the infection dependent gene IFN-a and the infection independent gene B2M to inappropriate HKGs consequently misleads to significant errors in estimating their regulations. Our study emphasizes that even the most popular HKGs like 18S RNA and GAPDH can lead to divergent and inaccurate data interpretation of significant magnitude if not carefully analyzed for stability before.

• Human metabolic individuality in biomedical and pharmaceutical research.

  Authors: Karsten Suhre, So-Youn Shin, Ann-Kristin Petersen, Robert P Mohney, David Meredith, Brigitte Wägele, Elisabeth Altmaier, Panos Deloukas, Jeanette Erdmann, Elin Grundberg [......] Werner Römisch-Margl, Nilesh J Samani, Kerrin S Small, H-Erich Wichmann, Guangju Zhai, Thomas Illig, Tim D Spector, Jerzy Adamski, Nicole Soranzo, Christian Gieger

  Nature. 09/2011; 477(7362):54-60.

  Genome-wide association studies (GWAS) have identified many risk loci for complex diseases, but effect sizes are typically small and information on the underlying biological processes is oftenGenome-wide association studies (GWAS) have identified many risk loci for complex diseases, but effect sizes are typically small and information on the underlying biological processes is often lacking. Associations with metabolic traits as functional intermediates can overcome these problems and potentially inform individualized therapy. Here we report a comprehensive analysis of genotype-dependent metabolic phenotypes using a GWAS with non-targeted metabolomics. We identified 37 genetic loci associated with blood metabolite concentrations, of which 25 show effect sizes that are unusually high for GWAS and account for 10-60% differences in metabolite levels per allele copy. Our associations provide new functional insights for many disease-related associations that have been reported in previous studies, including those for cardiovascular and kidney disorders, type 2 diabetes, cancer, gout, venous thromboembolism and Crohn's disease. The study advances our knowledge of the genetic basis of metabolic individuality in humans and generates many new hypotheses for biomedical and pharmaceutical research.

• Monocarboxylate transporter 8 deficiency: altered thyroid morphology and persistent high triiodothyronine/thyroxine ratio after thyroidectomy.

  Authors: Eva K Wirth, Sien-Yi Sheu, Jazmin Chiu-Ugalde, Remy Sapin, Marc O Klein, Ilona Mossbrugger, Leticia Quintanilla-Martinez, Martin Hrabĕ de Angelis, Heiko Krude, Thomas Riebel, Karin Rothe, Josef Köhrle, Kurt W Schmid, Ulrich Schweizer, Annette Grüters

  European journal of endocrinology / European Federation of Endocrine Societies. 08/2011; 165(4):555-61.

  Thyroid hormone transport across the plasma membrane depends on transmembrane transport proteins, including monocarboxylate transporter 8 (MCT8). Mutations in MCT8 (or SLC16A2) lead to a severe formThyroid hormone transport across the plasma membrane depends on transmembrane transport proteins, including monocarboxylate transporter 8 (MCT8). Mutations in MCT8 (or SLC16A2) lead to a severe form of X-linked psychomotor retardation, which is characterised by elevated plasma triiodothyronine (T(3)) and low/normal thyroxine (T(4)). MCT8 contributes to hormone release from the thyroid gland. To characterise the potential impact of MCT8-deficiency on thyroid morphology in a patient and in Mct8-deficient mice. Thyroid morphology in a patient carrying the A224V mutation was followed by ultrasound imaging for over 10 years. After thyroidectomy, a histopathological analysis was carried out. The findings were compared with histological analyses of mouse thyroids from the Mct8(-/y) model. We show that an inactivating mutation in MCT8 leads to a unique, progressive thyroid follicular pathology in a patient. After thyroidectomy, histological analysis revealed gross morphological changes, including several hyperplastic nodules, microfollicular areas with stromal fibrosis and a small focus of microfollicular structures with nuclear features reminiscent of papillary thyroid carcinoma (PTC). These findings are supported by an Mct8-null mouse model in which we found massive papillary hyperplasia in 6- to 12-month-old mice and nuclear features consistent with PTC in almost 2-year-old animals. After complete thyroidectomy and substitution with levothyroxine (l-T(4)), the preoperative, inadequately low T(4) and free T(4) remained, while increasing the l-T(4) dosage led to T(3) serum concentrations above the normal range. Our results implicate peripheral deiodination in the peculiar hormonal constellation of MCT8-deficient patients. Other MCT8-deficient patients should be closely monitored for potential thyroid abnormalities.

• Identification of a potential biomarker for FABP4 inhibition: the power of lipidomics in preclinical drug testing.

  Authors: Karsten Suhre, Werner Römisch-Margl, Martin Hrabé de Angelis, Jerzy Adamski, Gerd Luippold, Robert Augustin

  Journal of biomolecular screening. 06/2011; 16(5):467-75.

  The fatty acid binding protein 4 (FABP4) belongs to the family of lipid chaperones that control intracellular fluxes and compartmentalization of their respective ligands (e.g., fatty acids). FABP4,The fatty acid binding protein 4 (FABP4) belongs to the family of lipid chaperones that control intracellular fluxes and compartmentalization of their respective ligands (e.g., fatty acids). FABP4, which is almost exclusively expressed in adipocytes and macrophages, contributes to the development of insulin resistance and atherosclerosis in mice. Lack of FABP4 protects against the development of insulin resistance associated with genetic or diet-induced obesity in mice. Furthermore, total or macrophage-specific FABP4 deficiency is protective against atherosclerosis in apolipoprotein E-deficient mice. The FABP4 small-molecule inhibitor BMS309403 has demonstrated efficacy in mouse models for type 2 diabetes mellitus and atherosclerosis, resembling phenotypes of mice with FABP4 deficiency. However, despite the therapeutically attractive long-term effects of FABP4 inhibition, an acute biomarker for drug action is lacking. The authors applied mass spectrometry lipidomics analysis to in vitro and in vivo (plasma and adipose tissue) samples upon inhibitor treatment. They report the identification of a potential biomarker for acute in vivo FABP4 inhibition that is applicable for further investigations and can be implemented in simple and fast-flow injection mass spectrometry assays. In addition, this approach can be considered a proof-of-principle study that can be applied to other lipid-pathway targeting mechanisms.

• Generation of N-ethyl-N-nitrosourea-induced mouse mutants with deviations in hematological parameters.

  Authors: Bernhard Aigner, Birgit Rathkolb, Martina Klempt, Sibylle Wagner, Dian Michel, Matthias Klaften, Jürgen Laufs, Boris Schneider, Reinhard Sedlmeier, Martin Hrabé de Angelis, Eckhard Wolf

  Mammalian genome : official journal of the International Mammalian Genome Society. 05/2011; 22(9-10):495-505.

  Research on hematological disorders relies on suitable animal models. We retrospectively evaluated the use of the hematological parameters hematocrit (HCT), hemoglobin (HGB), mean corpuscularResearch on hematological disorders relies on suitable animal models. We retrospectively evaluated the use of the hematological parameters hematocrit (HCT), hemoglobin (HGB), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), red blood cell count (RBC), white blood cell count (WBC), and platelet count (PLT) in the phenotype-driven Munich N-ethyl-N-nitrosourea (ENU) mouse mutagenesis project as parameters for the generation of novel animal models for human diseases. The analysis was carried out on more than 16,000 G1 and G3 offspring of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to deviations in the levels of the chosen parameters. Identification of animals exhibiting altered values and transmission of the phenotypic deviations to the subsequent generations led to the successful establishment of mutant lines for the parameters MCV, RBC, and PLT. Analysis of the causative mutation was started in selected lines, thereby revealing a novel mutation in the transferrin receptor gene (Tfrc) in one line. Thus, novel phenotype-driven mouse models were established to analyze the genetic components of hematological disorders.

• Missing-in-metastasis MIM/MTSS1 promotes actin assembly at intercellular junctions and is required for integrity of kidney epithelia.

  Authors: Juha Saarikangas, Pieta K Mattila, Markku Varjosalo, Miia Bovellan, Janne Hakanen, Julia Calzada-Wack, Monica Tost, Luise Jennen, Birgit Rathkolb, Wolfgang Hans [......] Eckhard Wolf, Martin Hrabé de Angelis, Mikko J Frilander, Harri Savilahti, Hannu Sariola, Kirsi Sainio, Sanna Lehtonen, Jussi Taipale, Marjo Salminen, Pekka Lappalainen

  Journal of cell science. 03/2011; 124(Pt 8):1245-55.

  MIM/MTSS1 is a tissue-specific regulator of plasma membrane dynamics, whose altered expression levels have been linked to cancer metastasis. MIM deforms phosphoinositide-rich membranes through itsMIM/MTSS1 is a tissue-specific regulator of plasma membrane dynamics, whose altered expression levels have been linked to cancer metastasis. MIM deforms phosphoinositide-rich membranes through its I-BAR domain and interacts with actin monomers through its WH2 domain. Recent work proposed that MIM also potentiates Sonic hedgehog (Shh)-induced gene expression. Here, we generated MIM mutant mice and found that full-length MIM protein is dispensable for embryonic development. However, MIM-deficient mice displayed a severe urinary concentration defect caused by compromised integrity of kidney epithelia intercellular junctions, which led to bone abnormalities and end-stage renal failure. In cultured kidney epithelial (MDCK) cells, MIM displayed dynamic localization to adherens junctions, where it promoted Arp2/3-mediated actin filament assembly. This activity was dependent on the ability of MIM to interact with both membranes and actin monomers. Furthermore, results from the mouse model and cell culture experiments suggest that full-length MIM is not crucial for Shh signaling, at least during embryogenesis. Collectively, these data demonstrate that MIM modulates interplay between the actin cytoskeleton and plasma membrane to promote the maintenance of intercellular contacts in kidney epithelia.

• Huge splicing frequency in human Y chromosomal UTY gene.

  Authors: Ingeborg Laaser, Fabian J Theis, Martin Hrabé de Angelis, Hans-Jochem Kolb, Jerzy Adamski

  Omics : a journal of integrative biology. 02/2011; 15(3):141-54.

  Over 90% of human genes produce more than one mRNA by alternative splicing (AS). Human UTY (ubiquitously transcribed tetratricopeptide repeat protein on the chromosome Y) has six mRNA-transcripts.Over 90% of human genes produce more than one mRNA by alternative splicing (AS). Human UTY (ubiquitously transcribed tetratricopeptide repeat protein on the chromosome Y) has six mRNA-transcripts. UTY is subject to interdisciplinary approaches such as Y chromosomal genetics or development of leukemia immunotherapy based on UTY-specific peptides. Investigating UTY expression in a normal and leukemic setting we discovered an exceptional splicing phenomenon fostering huge transcript diversity. Transcript sequencing identified 90 novel AS-events being almost randomly combined in 284 new transcripts. We uncovered a novel system of transcript architecture and genomic organization in UTY. On a basis of a new UTY-splicing multigraph including a mathematical model we calculated the theoretical yield to exceed 1.3 billion distinct transcripts. To our knowledge, this is the greatest estimated transcript diversity by AS. On protein level we demonstrated interaction of AS-derived proteins with new interactors by yeast-two-hybrid assay. For translational research we predicted new UTY-peptide candidates for leukemia therapy development. Our study provides new insights into the complexity of human alternative splicing and its potential contribution to the transcript diversity of the transcriptome.

• Impaired resolution of inflammatory response in the lungs of JF1/Msf mice following carbon nanoparticle instillation.

  Authors: Koustav Ganguly, Swapna Upadhyay, Martin Irmler, Shinji Takenaka, Katrin Pukelsheim, Johannes Beckers, Martin Hrabé De Angelis, Eckard Hamelmann, Tobias Stoeger, Holger Schulz

  Respiratory research. 01/2011; 12:94.

  Declined lung function is a risk factor for particulate matter associated respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD). Carbon nanoparticles (CNP) are a prominentDeclined lung function is a risk factor for particulate matter associated respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD). Carbon nanoparticles (CNP) are a prominent component of outdoor air pollution that causes pulmonary toxicity mainly through inflammation. Recently we demonstrated that mice (C3H/HeJ) with higher than normal pulmonary function resolved the elicited pulmonary inflammation following CNP exposure through activation of defense and homeostasis maintenance pathways. To test whether CNP-induced inflammation is affected by declined lung function, we exposed JF1/Msf (JF1) mice with lower than normal pulmonary function to CNP and studied the pulmonary inflammation and its resolution. 5 μg, 20 μg and 50 μg CNP (Printex 90) were intratracheally instilled in JF1 mice to determine the dose response and the time course of inflammation over 7 days (20 μg dosage). Inflammation was assessed using histology, bronchoalveolar lavage (BAL) analysis and by a panel of 62 protein markers. 24 h after instillation, 20 μg and 50 μg CNP caused a 25 fold and 19 fold increased polymorphonuclear leucocytes (PMN) respectively while the 5 μg represented the 'no observable adverse effect level' as reflected by PMN influx (9.7 × 10E3 vs 8.9 × 10E3), and BAL/lung concentrations of pro-inflammatory cytokines. Time course assessment of the inflammatory response revealed that compared to day1 the elevated BAL PMN counts (246.4 × 10E3) were significantly decreased at day 3 (72.9 × 10E3) and day 7 (48.5 × 10E3) but did not reach baseline levels indicating slow PMN resolution kinetics. Strikingly on day 7 the number of macrophages doubled (455.0 × 10E3 vs 204.7 × 10E3) and lymphocytes were 7-fold induced (80.6 × 10E3 vs 11.2 × 10E3) compared to day1. At day 7 elevated levels of IL1B, TNF, IL4, MDC/CCL22, FVII, and vWF were detected in JF1 lungs which can be associated to macrophage and lymphocyte activation. This explorative study indicates that JF1 mice with impaired pulmonary function also exhibits delayed resolution of particle mediated lung inflammation as evident from elevated PMN and accumulation of macrophages and lymphocytes on day 7. It is plausible that elevated levels of IL1B, IL4, TNF, CCL22/MDC, FVII and vWF counteract defense and homeostatic pathways thereby driving this phenomenon.

• First mutation in the βA2-crystallin encoding gene is associated with small lenses and age-related cataracts.

  Authors: Oliver Puk, Nafees Ahmad, Sibylle Wagner, Martin Hrabé de Angelis, Jochen Graw

  Investigative ophthalmology & visual science. 01/2011; 52(5):2571-6.

  A new mouse mutant with small lenses was identified within a mutagenesis screen. The aim of the study was to determine its molecular and morphologic characterization. The offspring of paternallyA new mouse mutant with small lenses was identified within a mutagenesis screen. The aim of the study was to determine its molecular and morphologic characterization. The offspring of paternally N-ethyl-N-nitrosourea (ENU)-treated C57BL/6J mice were analyzed for eye-size parameters by noninvasive in vivo laser interference biometry. A new mutant characterized by a clear, but significantly smaller lens without any changes for cornea thickness, anterior chamber depth, or aqueous humor size, was identified. The smaller size of the lens was more pronounced in the homozygous mutants, which were fully fertile and viable. The mutation was mapped to chromosome 1 between the markers D1Mit251 and D1Mit253. Using a positional candidate approach, the βA2-crystallin encoding gene Cryba2 was sequenced; a T→C exchange at cDNA position 139 led to a p.S47P amino-acid alteration. The eyes of newborn homozygous mutants showed no gross changes. At the age of three weeks, some clefts appeared at the cornea, but the lens and retina appeared without major changes. At the age of 25 weeks, the lenses of the heterozygous mutants develop a subcapsular cortical cataract, but the lenses of homozygous mutants were completely opaque. These findings demonstrate the first mutation in the Cryba2 gene. In contrast to the closely linked Cryg gene cluster, no congenital cataract mutation could be attributed to the Cryba2 gene. Therefore, the human CRYBA2 gene should be considered as a strong candidate gene for age-related cataracts, and the slightly smaller size of the lens might be recognized as an early biomarker for age-related cataracts.

• Microphakia and congenital cataract formation in a novel Lim2(C51R) mutant mouse.

  Authors: Oliver Puk, Nafees Ahmad, Sibylle Wagner, Martin Hrabé de Angelis, Jochen Graw

  Molecular vision. 01/2011; 17:1164-71.

  Within a mutagenesis screen, we identified the new mouse mutant Aca47 with small lenses and reduced axial eye lengths. The aim of the actual study was the molecular and morphological characterizationWithin a mutagenesis screen, we identified the new mouse mutant Aca47 with small lenses and reduced axial eye lengths. The aim of the actual study was the molecular and morphological characterization of the mouse mutant Aca47. We analyzed the offspring of paternally N-ethyl-N-nitrosourea (ENU) treated C57BL/6J mice for eye-size parameters by non-invasive in vivo laser interference biometry. Linkage analysis of the eye size mutant Aca47 was performed using single nucleotide polymorphisms and microsatellite markers. The Aca47 mutation was identified by sequence analysis of positional candidate genes. A general polymorphism at the mutated site was excluded by restriction analysis. Eyes of the Aca47 mouse mutant were characterized by histology. Visual properties were examined in the virtual drum. We identified a new mutant characterized by a significantly smaller lens and reduced axial eye length without any changes for cornea thickness, anterior chamber depth or aqueous humor size. The smaller size of lens was more pronounced in the homozygous mutants, which further developed congenital cataracts in the lens nucleus. The mutation was mapped to chromosome 7 between the markers D7Mit247 and D7Mit81. Using a positional candidate approach, the lens intrinsic integral membrane protein MP19 encoding gene Lim2 was sequenced; a T → C exchange at cDNA position 151 leads to a cysteine-to-arginine substitution at position 51 of the Lim2 protein. Eye histology of adult heterozygous mutants did not show alterations on the cellular level. However, homozygous lenses revealed irregularly arranged lens fiber layers in the cortex. Virtual vision tests indicated that visual properties are not affected by reduced eye size of heterozygous individuals. These findings demonstrate a novel missense mutation in the Lim2 gene that affects lens development in a semidominant manner. Since homozygous mutants develop congenital lens opacities, this line can be used as a model for inherited cataract formation in humans.

• The pathologic effect of a novel neomorphic Fgf9(Y162C) allele is restricted to decreased vision and retarded lens growth.

  Authors: Oliver Puk, Gabriele Möller, Arie Geerlof, Kathrin Krowiorz, Nafees Ahmad, Sibylle Wagner, Jerzy Adamski, Martin Hrabé de Angelis, Jochen Graw

  PloS one. 01/2011; 6(8):e23678.

  Fibroblast growth factor (Fgf) signalling plays a crucial role in many developmental processes. Among the Fgf pathway ligands, Fgf9 (UniProt: P54130) has been demonstrated to participate inFibroblast growth factor (Fgf) signalling plays a crucial role in many developmental processes. Among the Fgf pathway ligands, Fgf9 (UniProt: P54130) has been demonstrated to participate in maturation of various organs and tissues including skeleton, testes, lung, heart, and eye. Here we establish a novel Fgf9 allele, discovered in a dominant N-ethyl-N-nitrosourea (ENU) screen for eye-size abnormalities using the optical low coherence interferometry technique. The underlying mouse mutant line Aca12 was originally identified because of its significantly reduced lens thickness. Linkage studies located Aca12 to chromosome 14 within a 3.6 Mb spanning interval containing the positional candidate genes Fgf9 (MGI: 104723), Gja3 (MGI: 95714), and Ift88 (MGI: 98715). While no sequence differences were found in Gja3 and Ift88, we identified an A→G missense mutation at cDNA position 770 of the Fgf9 gene leading to an Y162C amino acid exchange. In contrast to previously described Fgf9 mutants, Fgf9(Y162C) carriers were fully viable and did not reveal reduced body-size, male-to-female sexual reversal or skeletal malformations. The histological analysis of the retina as well as its basic functional characterization by electroretinography (ERG) did not show any abnormality. However, the analysis of head-tracking response of the Fgf9(Y162C) mutants in a virtual drum indicated a gene-dosage dependent vision loss of almost 50%. The smaller lenses in Fgf9(Y162C) suggested a role of Fgf9 during lens development. Histological investigations showed that lens growth retardation starts during embryogenesis and continues after birth. Young Fgf9(Y162C) lenses remained transparent but developed age-related cataracts. Taken together, Fgf9(Y162C) is a novel neomorphic allele that initiates microphakia and reduced vision without effects on organs and tissues outside the eye. Our data point to a role of Fgf9 signalling in primary and secondary lens fiber cell growth. The results underline the importance of allelic series to fully understand multiple functions of a gene.

• Utilization of a mutagenesis screen to generate mouse models of hyperaldosteronism.

  Authors: Ariadni Spyroglou, Sibylle Wagner, Celso Gomez-Sanchez, Birgit Rathkolb, Eckhard Wolf, Jenny Manolopoulou, Martin Reincke, Martin Bidlingmaier, Martin Hrabé de Angelis, Felix Beuschlein

  Endocrinology. 11/2010; 152(1):326-31.

  Primary aldosteronism is considered to be responsible for almost 10% of all cases of arterial hypertension. The genetic background of this common disease, however, has been elucidated only for thePrimary aldosteronism is considered to be responsible for almost 10% of all cases of arterial hypertension. The genetic background of this common disease, however, has been elucidated only for the rare familial types, whereas in the large majority of sporadic cases, underlying mechanisms still remain unclear. In an attempt to define novel genetic loci involved in the pathophysiology of primary aldosteronism, a mutagenesis screen after treatment of mice with the alkylating agent N-ethyl-N-nitrosourea was established for the parameter aldosterone. As the detection method we used a time-resolved fluorescence immunoassay that allows the measurement of aldosterone in very small murine sample volumes. Based on this assay, we first determined the normal aldosterone values for wild-type C3HeB/FeJ mice under baseline conditions [92 ± 6 pg/ml for females (n = 69) and 173 ± 16 pg/ml for males (n = 55)]. Subsequently, aldosterone measurement was carried out in more than 2800 F(1) offspring of chemically mutagenized C3HeB/FeJ mice, and values were compared with aldosterone levels from untreated animals. Persistent hyperaldosteronism (defined as levels +3 sd above the mean of untreated animals) upon repeated measurements was present in seven female and two male F(1) offspring. Further breeding of these founders gave rise to F(2) pedigrees from which eight lines with different patterns of inheritance of hyperaldosteronism could be established. These animals will serve for detailed phenotypic and genetic characterization in the future. Taken together, our data demonstrate the feasibility of a phenotype-driven mutagenesis screen to detect and establish mutant mouse lines with a phenotype of chronic hyperaldosteronism.

• Selection and evaluation of stable housekeeping genes for gene expression normalization in carbon nanoparticle-induced acute pulmonary inflammation in mice.

  Authors: Renfu Yin, Furong Tian, Birgit Frankenberger, Martin Hrabé de Angelis, Tobias Stoeger

  Biochemical and biophysical research communications. 09/2010; 399(4):531-6.

  Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is a highly specific and sensitive technique for the quantification of gene expression on the mRNA levels. But use ofQuantitative reverse transcription-polymerase chain reaction (qRT-PCR) is a highly specific and sensitive technique for the quantification of gene expression on the mRNA levels. But use of unconfirmed housekeeping genes (HKGs) could lead to misinterpretation of the expression of genes of interest (GOI). In this study, the stability and suitability of 11 frequently used housekeeping genes, namely 18S rRNA, ACTB, B2M, CYPA, GADPH, GUSB, HMBS, HPRT1, RPL13A, SDHA and TBP in 36 lung tissues isolated from either wild-type (WT) mice or p50 knock out (p50-/-) mice or p105 knock-out (p105-/-) mice which were treated with either carbon nanoparticle (CNP) or H(2)O or non-treated, have been validated by geNorm, NormFinder and BestKeeper programs. The expression levels of ACTB, GUSB and RPL13A were the most constant in lung tissues across three genotypes and three kinds of treatments. A set of three most stable genes is found sufficient to be used as housekeeping genes for lung tissues in studies of similar design.

• Combination of in silico and in situ hybridisation approaches to identify potential Dll1 associated miRNAs during mouse embryogenesis.

  Authors: Bastian Hoesel, Zambarlal Bhujabal, Gerhard K H Przemeck, Annerose Kurz-Drexler, Daniela M Vogt Weisenhorn, Martin Hrabé de Angelis, Johannes Beckers

  Gene expression patterns : GEP. 09/2010; 10(6):265-73.

  MicroRNAs (miRNAs) have regulatory functions during vertebrate embryogenesis. They are short approximately 21bp long endogenously expressed single-stranded RNAs, which preferentially bind toMicroRNAs (miRNAs) have regulatory functions during vertebrate embryogenesis. They are short approximately 21bp long endogenously expressed single-stranded RNAs, which preferentially bind to complementary sequences in the 3' untranslated regions (UTR) of mRNAs and typically down-regulate the respective target mRNAs by translational repression or enhanced mRNA degradation. The Notch ligand Delta-like 1 (Dll1) is expressed in a highly dynamic pattern and has pleiotropic functions during embryogenesis and in adult tissues. Here, we report an interspecies in silico analysis to identify 16 miRNAs, which potentially bind to the mouse, human and chicken Dll1 3'UTRs. To analyze whether these miRNAs could regulate Dll1 gene expression during somitogenesis and neurogenesis, we performed a systematic whole mount in situ hybridisation screen, followed by radioactive in situ hybridisation on sections, using LNA modified DNA probes in mouse embryos. We find that 7 miRNAs (miR-34a, miR-103, miR-107, miR-130a, miR-130b, miR-449a and miR-449c) are expressed in developing somites, limbs, restricted regions of the brain and neural tube between 9.5 dpc and 12.5 dpc. This suggests that these miRNAs could possibly target the Dll1 3'UTR in these regions. The other miRNAs are not expressed or below the detection limit and thus are unlikely to regulate Dll1 at the analyzed embryonic stages.

• Gene targeting by homologous recombination in mouse zygotes mediated by zinc-finger nucleases.

  Authors: Melanie Meyer, Martin Hrabé de Angelis, Wolfgang Wurst, Ralf Kühn

  Proceedings of the National Academy of Sciences of the United States of America. 08/2010; 107(34):15022-6.

  Gene targeting by homologous recombination in embryonic stem cells is extensively used to generate specific mouse mutants. However, most mammalian species lack tools for targeted gene manipulation.Gene targeting by homologous recombination in embryonic stem cells is extensively used to generate specific mouse mutants. However, most mammalian species lack tools for targeted gene manipulation. Since double-strand breaks strongly increase the rate of homologous recombination at genomic loci, we explored whether gene targeting can be directly performed in zygotes by the use of zinc-finger nucleases. Here we report that gene targeting is achieved in 1.7-4.5% of murine one-cell embryos upon the coinjection of targeting vectors with zinc-finger nucleases, without preselection. These findings enable the manipulation of the mammalian germ line in a single step in zygotes, independent of ES cells.