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

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 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.

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

Research 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.

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

PURPOSE: 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 characterization of the mouse mutant Aca47. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

A novel N-ethyl-N-nitrosourea-induced mutation in phospholipase Cγ2 causes inflammatory arthritis, metabolic defects, and male infertility in vitro in a murine model.

OBJECTIVE: It is difficult to identify a single causative factor for inflammatory arthritis because of the multifactorial nature of the disease. This study was undertaken to dissect the molecular complexity of systemic inflammatory disease, utilizing a combined approach of mutagenesis and systematic phenotype screening in a murine model. METHODS: In a large-scale N-ethyl-N-nitrosourea mutagenesis project, the Ali14 mutant mouse strain was established because of dominant inheritance of spontaneous swelling and inflammation of the hind paws. Genetic mapping and subsequent candidate gene sequencing were conducted to find the causative gene, and systematic phenotyping of Ali14/+ mice was performed in the German Mouse Clinic. RESULTS: A novel missense mutation in the phospholipase Cγ2 gene (Plcg2) was identified in Ali14/+ mice. Because of the hyperreactive external entry of calcium observed in cultured B cells and other in vitro experiments, the Ali14 mutation is thought to be a novel gain-of-function allele of Plcg2. Findings from systematic screening of Ali14/+ mice demonstrated various phenotypic changes: an abnormally high T cell:B cell ratio, up-regulation of Ig, alterations in body composition, and a reduction in cholesterol and triglyceride levels in peripheral blood. In addition, spermatozoa from Ali14/+ mice failed to fertilize eggs in vitro, despite the normal fertility of the Ali14/+ male mice in vivo. CONCLUSION: These results suggest that the Plcg2-mediated pathways play a crucial role in various metabolic and sperm functions, in addition to initiating and maintaining the immune system. These findings may indicate the importance of the Ali14/+ mouse strain as a model for systemic inflammatory diseases and inflammation-related metabolic changes in humans.

First mutation in the ßA2-crystallin encoding gene is associated with small lenses and age-related cataracts.

PURPOSE: 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. METHODS: 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. RESULTS: 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 ßA2-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. CONCLUSIONS: 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.

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

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 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.

Reduced corneal thickness and enlarged anterior chamber in a novel ColVIIIa2G257D mutant mouse.

PURPOSE: The purpose of this study was the morphologic and genetic characterization of the novel eye size mutant Aca23 in the mouse. METHODS: The eyes of the mutants were characterized in vivo by optical low-coherence interferometry, Scheimpflug imaging, and funduscopy. Visual acuity was examined using a virtual optomotor system. Morphology was studied by histology, in situ hybridization, and immunohistochemistry. Linkage analysis was performed using genomewide scans with single nucleotide polymorphisms and microsatellite markers. RESULTS: Aca23 is a new semidominant eye size mutant that was discovered in an ENU mutagenesis screen. The phenotype includes increased anterior chamber depths, extended axial lengths, and reduced thickness of corneal layers. Aca23 was mapped to chromosome 4. A G-->A point mutation was identified at cDNA position 770 of Col8a2 encoding collagen VIII alpha2. The transition results in a G257D amino acid exchange affecting a highly conserved glycine residue in the collagenous domain. Proliferation of corneal endothelium, eye fundus, and visual acuity are not affected. CONCLUSIONS: The mouse mutant Aca23 described here offers the first point mutation of the Col8a2 gene in the mouse. The results of this study suggest that a functional collagen VIII alpha2 is essential for the correct assembly of the Descemet's membrane and for corneal stability. Aca23 might be used as a novel model for keratoglobus.

N-ethyl-N-nitrosourea mutagenesis produced a small number of mice with altered plasma electrolyte levels.

BACKGROUND: Clinical chemical blood analysis including plasma electrolytes is routinely carried out for the diagnosis of various organ diseases. Phenotype-driven N-ethyl-N-nitrosourea (ENU) mouse mutagenesis projects used plasma electrolytes as parameters for the generation of novel animal models for human diseases. METHODS: Here, we retrospectively evaluated the use of the plasma electrolytes calcium, chloride, inorganic phosphorus, potassium and sodium in the Munich ENU mouse mutagenesis project where clinical chemical blood analysis was carried out on more than 20,000 G1 and G3 offspring of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to deviations in various plasma parameter levels. RESULTS: We identified a small number of animals consistently exhibiting altered plasma electrolyte values. Transmission of the phenotypic deviations to the subsequent generations led to the successful establishment of mutant lines for the parameters calcium and potassium. Published data from other phenotype-driven ENU projects also included only a small number of mutant lines which were generated according to altered plasma electrolyte levels. CONCLUSION: Thus, use of plasma electrolytes detected few mouse mutants in ENU projects compared to other clinical chemical blood parameters.

Dll1 haploinsufficiency in adult mice leads to a complex phenotype affecting metabolic and immunological processes.

BACKGROUND: The Notch signaling pathway is an evolutionary conserved signal transduction pathway involved in embryonic patterning and regulation of cell fates during development and self-renewal. Recent studies have demonstrated that this pathway is integral to a complex system of interactions, involving as well other signal transduction pathways, and implicated in distinct human diseases. Delta-like 1 (Dll1) is one of the known ligands of the Notch receptors. The role of the Notch ligands is less well understood. Loss-of-function of Dll1 leads to embryonic lethality, but reduction of Delta-like 1 protein levels has not been studied in adult stage. METHODOLOGY/PRINCIPAL FINDINGS: Here we present the haploinsufficient phenotype of Dll1 and a missense mutant Dll1 allele (Dll1(C413Y)). Haploinsufficiency leads to a complex phenotype with several biological processes altered. These alterations reveal the importance of Dll1 mainly in metabolism, energy balance and in immunology. The animals are smaller, lighter, with altered fat to lean ratio and have increased blood pressure and a slight bradycardia. The animals have reduced cholesterol and triglyceride levels in blood. At the immunological level a subtle phenotype is observed due to the effect and fine-tuning of the signaling network at the different levels of differentiation, proliferation and function of lymphocytes. Moreover, the importance of the proteolytic regulation of the Notch signaling network emphasized. CONCLUSIONS/SIGNIFICANCE: In conclusion, slight alterations in one player of Notch signaling alter the entire organism, emphasizing the fine-tuning character of this pathway in a high number of processes.

Short term regulation of aldosterone secretion after stimulation and suppression experiments in mice.

Aldosterone is synthesized acutely from the zona glomerulosa cells upon stimulation by the renin-angiotensin-aldosterone system. Several enzymes are involved in this steroidogenic process including the steroidogenic acute regulatory protein (StAR), P450 side chain cleavage enzyme (Cyp11a1), and aldosterone synthase (Cyp11b2) which has been demonstrated to be transcriptionally regulated by the nuclear transcription factors NGF1-B and Nurr1. We investigated the short time transcriptional regulation of these genes in wild-type mice at 10 min intervals for 1 h following application of 0.2 nmol angiotensin II (ANGII) or sodium chloride in comparison sham injections. Using real-time PCR a fast upregulation of adrenal Cyp11b2 expression (53+/-5% increase over baseline) could be observed 10 min after sham injection which was accompanied by a transient increase in aldosterone secretion while StAR and Cyp11a1 upregulation was delayed and more sustained. ANGII caused an increase of StAR and Cyp11a1 expression similar to that observed after sham injection while Cyp11b2 upregulation was more pronounced (10 min, 236+/-39%) and reflected ANGII induced stimulation of aldosterone output. Sodium challenge was followed by a sustained reduction of all three genes examined (Cyp11b2, 20 min, -63+/-6%) which was accompanied by a significant suppression of aldosterone secretion detectable after 60 min. While increases in NGF1-B mRNA levels were similar between the treatment groups, Nurr1 expression levels were induced only upon ANGII administration. These data suggest that acute regulation of aldosterone synthesis is accompanied by fast transcriptional modulation of steroidogenic enzymes and transcription factors that are likely to be involved in aldosterone secretion.

Genome-wide search for genes that modulate inflammatory arthritis caused by Ali18 mutation in mice.

Many of inflammatory diseases, including inflammatory arthritis, are multifactorial bases. The Ali18 semidominant mutation induced by N-ethyl-N-nitrosourea in the C3HeB/FeJ (C3H) genome causes spontaneous inflammation of peripheral limbs and elevated immunoglobulin E (IgE) levels in mice. Although the Ali18 locus was mapped to a single locus on chromosome 4, the arthritic phenotype of Ali18/+ mice was completely suppressed in F1 hybrid genetic backgrounds. To determine the chromosomal locations of the modifier loci affecting the severity of arthritis, an autosomal genome scan of 22 affected Ali18/+ F2 mice was conducted using C57BL/6J as a partner strain. Interestingly, regions on chromosomes 1 and 3 in C3H showed significant genetic interactions. Moreover, 174 N2 (backcross to Ali18/Ali18) and 267 F2 animals were used for measurement of arthritis scores and plasma IgE levels, and also for genotyping with 153 genome-wide single nucleotide polymorphism (SNP) markers. In N2 populations, two significant trait loci for arthritis scores on chromosomes 1 and 15 were detected. Although no significant scores were detected in F2 mice besides chromosome 4, a suggestive score was detected on chromosome 3. In addition, a two-dimensional genome scan using F2 identified five suggestive scores of chromosomal combinations, chromosomes 1 x 10, 2 x 6, 3 x 4, 4 x 9, and 6 x 15. No significant trait loci affecting IgE levels were detected in both N2 and F2 populations. Identification of the Ali18 modifier genes by further detailed analyses such as congenic strains and expression profiling may dissect molecular complexity in inflammatory diseases.

Generation of N-ethyl-N-nitrosourea-induced mouse mutants with deviations in plasma enzyme activities as novel organ-specific disease models.

Measurement of plasma enzyme activities is part of routine medical examination protocols and provides valuable parameters for the diagnosis of various organ diseases. In the phenotype-driven Munich N-ethyl-N-nitrosourea (ENU) mouse mutagenesis project, clinical chemical blood analysis was carried out on more than 20,000 G1 and G3 offspring of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to deviations in the plasma enzyme activities of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, alpha-amylase and creatine kinase. We identified a large number of animals that consistently exhibited altered plasma enzyme activities. Transmission of the phenotypic deviations to the subsequent generations led to the successful establishment of mutant lines for each parameter. Breeding experiments in selected lines detected the linkage of the causative mutations to defined chromosomal regions. Subsequently, identification of the mutated genes was successfully carried out in chosen lines, resulting in a novel alkaline phosphatase liver/bone/kidney (Alpl) alteration in one line and the strong indication for a dystrophin (Dmd) alteration in another line. The mouse mutants with abnormal plasma enzyme activities recovered in the Munich ENU project are novel tools for the systematic dissection of the pathogenesis of organ diseases.

A phenotype-driven ENU mutagenesis screen for the identification of dominant mutations involved in alcohol consumption.

The aim of this study was the application of a phenotype-driven N-ethyl-N-nitrosourea (ENU) mutagenesis screen in mice for the identification of dominant mutations involved in the regulation and modulation of alcohol-drinking behavior. The chemical mutagen ENU was utilized in the generation of 131 male ENU-mutant C57BL/6J mice (G0). These ENU-treated mice were paired with wild-type C57BL/6J mice to generate G1 and subsequent generations. In total, 3327 mice were generated. Starting with G1, mice were screened for voluntary oral self-administration of 10% (v/v) alcohol vs. water in a two-bottle paradigm. From these mice, after a total period of 5 weeks of drinking, 43 mutants fulfilled the criteria of an "alcohol phenotype," that is, high or low ethanol intake. They were then selected for breeding and tested in a "confirmation cross" (G2-G4) for inheritance. Although we did not establish stable high or low drinking lines, several results were obtained in the context of alcohol consumption. First, female mice drank more alcohol than their male counterparts. Second, the former demonstrated greater infertility. Third, all animals displayed relatively stable alcohol intake, although significantly different in two different laboratories. Finally, seasonal and monthly variability was observed, with the highest alcohol consumption occurring in spring and the lowest in autumn. In conclusion, it seems difficult to identify dominant mutations involved in the modulation or regulation of voluntary alcohol consumption via a phenotype-driven ENU mutagenesis screen. In accordance with the findings from knockout studies, we suggest that mainly recessive mutations contribute to an alcohol-drinking or alcohol-avoiding phenotype.

A genetic screen for modifiers of the delta1-dependent notch signaling function in the mouse.

The Notch signaling pathway is an evolutionarily conserved transduction pathway involved in embryonic patterning and regulation of cell fates during development. Recent studies have demonstrated that this pathway is integral to a complex system of interactions, which are also involved in distinct human diseases. Delta1 is one of the known ligands of the Notch receptors. Mice homozygous for a loss-of-function allele of the Delta1 gene Dll1(lacZ/lacZ) die during embryonic development. Here, we present the results of two phenotype-driven modifier screens. Heterozygous Dll1(lacZ) knockout animals were crossed with ENU-mutagenized mice and screened for dysmorphological, clinical chemical, and immunological variants that are dependent on the Delta1 loss-of-function allele. First, we show that mutagenized heterozygous Dll1(lacZ) offspring have reduced body weight and altered specific clinical chemical parameters, including changes in metabolites and electrolytes relevant for kidney function. In our mutagenesis screen we have successfully generated 35 new mutant lines. Of major interest are 7 mutant lines that exhibit a Dll1(lacZ/+)-dependent phenotype. These mutant mouse lines provide excellent in vivo tools for studying the role of Notch signaling in kidney and liver function, cholesterol and iron metabolism, cell-fate decisions, and during maturation of T cells in the immune system.