A genetic screen in zebrafish identifies the mutants vps18, nf2 and foie gras as models of liver disease

Hepatomegaly is a sign of many liver disorders. To identify zebrafish mutants to serve as models for hepatic pathologies, we screened for hepatomegaly at day 5 of embryogenesis in 297 zebrafish lines bearing mutations in genes that are essential for embryonic development. Seven mutants were identified, and three have phenotypes resembling different liver diseases. Mutation of the class C vacuolar protein sorting gene vps18 results in hepatomegaly associated with large, vesicle-filled hepatocytes, which we attribute to the failure of endosomal-lysosomal trafficking. Additionally, these mutants develop defects in the bile canaliculi and have marked biliary paucity, suggesting that vps18 also functions to traffic vesicles to the hepatocyte apical membrane and may play a role in the development of the intrahepatic biliary tree. Similar findings have been reported for individuals with arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome, which is due to mutation of another class C vps gene. A second mutant, resulting from disruption of the tumor suppressor gene nf2, develops extrahepatic choledochal cysts in the common bile duct, suggesting that this gene regulates division of biliary cells during development and that nf2 may play a role in the hyperplastic tendencies observed in biliary cells in individuals with choledochal cysts. The third mutant is in the novel gene foie gras, which develops large, lipid-filled hepatocytes, resembling those in individuals with fatty liver disease. These mutants illustrate the utility of zebrafish as a model for studying liver development and disease, and provide valuable tools for investigating the molecular pathogenesis of congenital biliary disorders and fatty liver disease.     

Novel phenotypes identified by plasma biochemical screening in the mouse

We used ENU mutagenesis in the mouse for the rapid generation of novel mutant phenotypes for both gene function studies and use as new animal models of human disease (Nolan et al. 2000b). One focus of the program was the development of a blood biochemistry screen. At 8-12 weeks of age, approximately 300 ml of blood was collected from F1 offspring of ENU mutagenized male mice. This yielded approximately 125 ml of plasma, used to perform a profile of 17 standard biochemical tests on an Olympus analyzer. Cohorts of F1 mice were also aged and then retested to detect late onset phenotypes. In total, 1,961 F1s were screened. Outliers were identified by running means and standard deviations. Of 70 mice showing consistent abnormalities in plasma biochemistry, 29 were entered into inheritance testing. Of these, 9 phenotypes were confirmed as inherited, 10 found not to be inherited, and 10 are still being tested. Inherited mutant phenotypes include abnormal lipid profiles (low total and HDL cholesterol, high triglycerides); abnormalities in bone and liver metabolism (low ALP, high ALP, high ALT, and AST); abnormal plasma electrolyte levels (high sodium and chloride); as well as phenotypes of interest for the study of diabetes (high glucose). The gene loci bearing the mutations are currently being mapped and further characterized. Our results have validated our biochemical screen, which is applicable to other mutagenesis projects, and we have produced a new set of mutants with defined metabolic phenotypes.     

Induction of recessive lethal and specific locus mutations in the zebrafish with ethyl nitrosourea.

Recessive lethal mutations and mutations at the gol-1 locus were induced in the zebrafish by exposure of mature sperm to the alkylating agent ethyl nitrosourea (ENU). Embryonic lethal phenotypes were recognized among the parthenogenetic progeny of mutagenized animals or among the progeny of daughters of mutagenized animals. Novel specific locus mutations were identified by the failure of mutagenized chromosomes to complement pre-existing mutant alleles at the gol-1 locus. Each mutagenized individual harboured approximately 10 embryonic lethal mutations in its germ line and about 1 in 500 mutagenized animals harboured a new mutation at the gol-1 locus. Three lines of evidence indicate that the majority of mutations that were recovered following treatment of mature sperm with ENU were probably point mutations. First, the soma and germ lines of mutagenized animals were mosaic, as expected following simple alkylation of sperm DNA. Second, mutations induced by ENU at the gol-1 locus affected pigmentation but not viability, unlike the majority of mutations induced at this locus with gamma-irradiation. Third, the ratio of specific locus:recessive lethal mutations induced by ENU was approximately 50-fold lower than the ratio observed following mutagenesis with gamma-rays. Comparison of the incidence with which embryonic recessive lethal mutations were induced with the incidence with which specific locus mutations arose indicates that there are greater than 5000 genes essential to the development and viability of the zebrafish embryo.     

The identification of zebrafish mutants showing alterations in senescence-associated biomarkers

There is an interesting overlap of function in a wide range of organisms between genes that modulate the stress responses and those that regulate aging phenotypes and, in some cases, lifespan. We have therefore screened mutagenized zebrafish embryos for the altered expression of a stress biomarker, senescence-associated beta-galactosidase (SA-beta-gal) in our current study. We validated the use of embryonic SA-beta-gal production as a screening tool by analyzing a collection of retrovirus-insertional mutants. From a pool of 306 such mutants, we identified 11 candidates that showed higher embryonic SA-beta-gal activity, two of which were selected for further study. One of these mutants is null for a homologue of Drosophila spinster, a gene known to regulate lifespan in flies, whereas the other harbors a mutation in a homologue of the human telomeric repeat binding factor 2 (terf2) gene, which plays roles in telomere protection and telomere-length regulation. Although the homozygous spinster and terf2 mutants are embryonic lethal, heterozygous adult fish are viable and show an accelerated appearance of aging symptoms including lipofuscin accumulation, which is another biomarker, and shorter lifespan. We next used the same SA-beta-gal assay to screen chemically mutagenized zebrafish, each of which was heterozygous for lesions in multiple genes, under the sensitizing conditions of oxidative stress. We obtained eight additional mutants from this screen that, when bred to homozygosity, showed enhanced SA-beta-gal activity even in the absence of stress, and further displayed embryonic neural and muscular degenerative phenotypes. Adult fish that are heterozygous for these mutations also showed the premature expression of aging biomarkers and the accelerated onset of aging phenotypes. Our current strategy of mutant screening for a senescence-associated biomarker in zebrafish embryos may thus prove to be a useful new tool for the genetic dissection of vertebrate stress response and senescence mechanisms.     

Genetic determinants of hepatic steatosis in man

Hepatic steatosis is one of the most common liver disorders in the general population. The main cause of hepatic steatosis is nonalcoholic fatty liver disease (NAFLD), representing the hepatic component of the metabolic syndrome, which is characterized by type 2 diabetes, obesity, and dyslipidemia. Insulin resistance and excess adiposity are considered to play key roles in the pathogenesis of NAFLD. Although the risk factors for NAFLD are well established, the genetic basis of hepatic steatosis is largely unknown. Here we review recent progress on genomic variants and their association with hepatic steatosis and discuss the potential impact of these genetic studies on clinical practice. Identifying the genetic determinants of hepatic steatosis will lead to a better understanding of the pathogenesis and progression of NAFLD.     

Comet assay as a tool to screen for mouse models with inherited radiation sensitivity

Recent in vivo and in vitro data of patients analyzed for genetic susceptibility to radiation during cancer therapy have shown structural changes in the chromosomes to be prevalent both in the patients being treated and in their immediate family members. As structural changes in chromosomes frequently lead to activation of proto-oncogenes and elimination of tumor-suppressor genes, they represent important mechanisms for the initiation of DNA repair processes and tumorigenesis. With the exception of rare genetic syndromes such as AT (Ataxia telangiectasia) or NBS (Nijmegen Breakage Syndrome), the background for the inheritance of genetic susceptibility to radiation is unknown. Recently, a large-scale genetic screen of mouse mutants has been established within the German Human Genome Project (Hrabè de Angelis and Balling 1998). The goal of this ENU (ENU: ethylnitrosourea) mutagenesis screen is the generation of mutant mice that will serve as animal models for human diseases and genetic susceptibility. In order to fully utilize the potential of a genetic screen of this magnitude, in which exploration for genes responsible for genomic instability and radiation sensitivity is to occur, it is necessary to establish a simple assay system that is amenable to automation. Hence, we are using the single-cell gel electrophoresis (comet assay) to detect mouse mutants that display a genetic susceptibility to ionizing radiation. We have established the analysis parameters in the comet assay which are currently used to detect radiation-sensitive mouse mutants and to control the variance within the mouse population in the ENU screen. The assay can be used to isolate genes that are responsible for DNA repair and radiation sensitivity in mouse and human. Received: 16 December 1999 / Accepted: 17 December 1999     

Efficient Recovery of Enu-Induced Mutations from the Zebrafish Germline

We studied the efficiency with which two chemical mutagens, ethyl methanesulfonate (EMS) and N-ethyl-N-nitrosourea (ENU) can induce mutations at different stages of spermatogenesis in zebrafish (Brachydanio rerio). Both EMS and ENU induced mutations at high rates in post-meiotic germ cells, as indicated by the incidence of F(1) progeny mosaic for the albino mutation. For pre-meiotic germ cells, however, only ENU was found to be an effective mutagen, as indicated by the frequencies of non-mosaic mutant progeny at four different pigmentation loci. Several mutagenic regimens that varied in either the number of treatments or the concentration of ENU were studied to achieve an optimal ratio between the mutagenicity and toxicity. For the two most mutagenic regimens: 4 X 1 hr in 3 mM ENU and 6 X 1 hr in 3 mM ENU, the minimum estimate of frequencies of independent mutations per locus per gamete was 0.9-1.3 X 10(-3). We demonstrate that embryonic lethal mutations induced with ENU were transmitted to offspring and that they could be recovered in an F(2) screen. An average frequency of specific-locus mutations of 1.1 X 10(-3) corresponded to approximately 1.7 embryonic lethal mutations per single mutagenized genome. The high rates of mutations achievable with ENU allow for rapid identification of large numbers of genes involved in a variety of aspects of zebrafish development.     

pyewacket, a new zebrafish fin pigment pattern mutant

Many mutants that disrupt zebrafish embryonic pigment pattern have been isolated, and subsequent cloning of the mutated genes causing these phenotypes has contributed to our understanding of pigment cell development. However, few mutants have been identified that specifically affect development of the adult pigment pattern. Through a mutant screen for adult pigment pattern phenotypes, we identified pyewacket (pye), a novel zebrafish mutant in which development of the adult caudal fin pigment pattern is aberrant. Specifically, pye mutants have fin melanocyte pigment pattern defects and fewer xanthophores than wild-type fins. We mapped pye to an interval where a single gene, the zebrafish ortholog of the human gene DHRSX, is present. pye will be an informative mutant for understanding how xanthophores and melanocytes interact to form the pigment pattern of the adult zebrafish fin.     

Genetic modifiers of non-alcoholic fatty liver disease progression

Non-alcoholic fatty liver disease (NAFLD) is now recognised as the most common cause of liver dysfunction worldwide. However, whilst the majority of individuals who exhibit features of the metabolic syndrome including obesity and insulin resistance will develop steatosis, only a minority progress to steatohepatitis, fibrosis and cirrhosis. Subtle inter-patient genetic variations and environment interact to determine disease phenotype and influence progression. A decade after the sequencing of the human genome, the comprehensive study of genomic variation offers new insights into the modifier genes, pathogenic mechanisms and is beginning to suggest novel therapeutic targets. We review the current status of the field with particular focus on advances from recent genome-wide association studies.     

Phenotype-based identification of mouse chromosome instability mutants

There is increasing evidence that defects in DNA double-strand-break (DSB) repair can cause chromosome instability, which may result in cancer. To identify novel DSB repair genes in mice, we performed a phenotype-driven mutagenesis screen for chromosome instability mutants using a flow cytometric peripheral blood micronucleus assay. Micronucleus levels were used as a quantitative indicator of chromosome damage in vivo. Among offspring derived from males mutagenized with the germline mutagen N-ethyl-N-nitrosourea (ENU), we identified a recessive mutation conferring elevated levels of spontaneous and radiation- or mitomycin C-induced micronuclei. This mutation, named chaos1 (chromosome aberration occurring spontaneously 1), was genetically mapped to a 1.3-Mb interval on chromosome 16 containing Polq, encoding DNA polymerase theta. We identified a nonconservative mutation in the ENU-derived allele, making it a strong candidate for chaos1. POLQ is homologous to Drosophila MUS308, which is essential for normal DNA interstrand crosslink repair and is unique in that it contains both a helicase and a DNA polymerase domain. While cancer susceptibility of chaos1 mutant mice is still under investigation, these data provide a practical paradigm for using a forward genetic approach to discover new potential cancer susceptibility genes using the surrogate biomarker of chromosome instability as a screen.     

The ubiquitin ligase Phr1 regulates axon outgrowth through modulation of microtubule dynamics

To discover new genes involved in axon navigation, we conducted a forward genetic screen for recessive alleles affecting motor neuron pathfinding in GFP reporter mice mutagenized with ENU. In Magellan mutant embryos, motor axons were error prone and wandered inefficiently at choice points within embryos, but paradoxically responded to guidance cues with normal sensitivity in vitro. We mapped the Magellan mutation to the Phr1 gene encoding a large multidomain E3 ubiquitin ligase. Phr1 is associated with the microtubule cytoskeleton within neurons and selectively localizes to axons but is excluded from growth cones. Motor and sensory neurons from Magellan mutants display abnormal morphologies due to a breakdown in the polarized distribution of components that segregate between axons and growth cones. The Magellan phenotype can be reversed by stabilizing microtubules with taxol or inhibiting p38MAPK activity. Thus, efficacious pathfinding requires Phr1 activity for coordinating the cytoskeletal organization that distinguishes axons from growth cones.     

Characterization of seed-specific benzoyloxyglucosinolate mutations in Arabidopsis thaliana

Glucosinolates are secondary metabolites involved in pathogen and insect defense of cruciferous plants. Although seeds and vegetative tissue often have very different glucosinolate profiles, few genetic factors that determine seed glucosinolate accumulation have been identified. An HPLC-based screen of 5500 mutagenized Arabidopsis thaliana lines produced 33 glucosinolate mutants, of which 21 have seed-specific changes. Five of these mutant lines, representing three genetic loci, are compromised in the biosynthesis of benzoyloxyglucosinolates, which are only found in seeds and young seedlings of A. thaliana. Genetic mapping and analysis of T-DNA insertions in candidate genes identified BZO1 (At1g65880), which encodes an enzyme with benzoyl-CoA ligase activity, as being required for the accumulation of benzoyloxyglucosinolates. Long-chain aliphatic glucosinolates are elevated in bzo1 mutants, suggesting substrate competition for the common short-chain aliphatic glucosinolate precursors. Whereas bzo1 mutations have seed-specific effects on benzoyloxyglucosinolate accumulation, the relative abundance of 3-benzoyloxypropyl- and 4-benzoyloxybutylglucosinolates depends on the maternal genotype.     

Insertional mutagenesis in zebrafish: genes for development, genes for disease.

In order to rapidly identify a substantial fraction of the genes with a unique and essential role in vertebrate development, the laboratory of Nancy Hopkins at MIT has performed a large insertional mutagenesis screen in zebrafish using a pseudotyped retroviral vector as the mutagen. We have recovered mutations in about one-quarter of the embryonic essential genes in this organism, and have identified the mutated genes in nearly all of these (333). As the ease of gene identification allowed us to clone the mutated genes for nearly all of the mutants rather than prioritizing based upon the initially observed phenotypes, this has provided an unbiased view of the diversity of genes required for vertebrate development as well as a large collection of mutants to be screened for more specific phenotypes. In collaboration with other labs, we have screened the insertional mutant for the development of a variety of organs and cell types, as well as phenotypes that could represent disease models, such as cystic kidney and hepatomegaly. Furthermore, while all of these mutants are embryonic lethal in their homozygous state, we are investigating the heterozygous adults for additional phenotypes, such as cancer predisposition.     

Forward genetic analysis of visual behavior in zebrafish

The visual system converts the distribution and wavelengths of photons entering the eye into patterns of neuronal activity, which then drive motor and endocrine behavioral responses. The gene products important for visual processing by a living and behaving vertebrate animal have not been identified in an unbiased fashion. Likewise, the genes that affect development of the nervous system to shape visual function later in life are largely unknown. Here we have set out to close this gap in our understanding by using a forward genetic approach in zebrafish. Moving stimuli evoke two innate reflexes in zebrafish larvae, the optomotor and the optokinetic response, providing two rapid and quantitative tests to assess visual function in wild-type (WT) and mutant animals. These behavioral assays were used in a high-throughput screen, encompassing over half a million fish. In almost 2,000 F2 families mutagenized with ethylnitrosourea, we discovered 53 recessive mutations in 41 genes. These new mutations have generated a broad spectrum of phenotypes, which vary in specificity and severity, but can be placed into only a handful of classes. Developmental phenotypes include complete absence or abnormal morphogenesis of photoreceptors, and deficits in ganglion cell differentiation or axon targeting. Other mutations evidently leave neuronal circuits intact, but disrupt phototransduction, light adaptation, or behavior-specific responses. Almost all of the mutants are morphologically indistinguishable from WT, and many survive to adulthood. Genetic linkage mapping and initial molecular analyses show that our approach was effective in identifying genes with functions specific to the visual system. This collection of zebrafish behavioral mutants provides a novel resource for the study of normal vision and its genetic disorders.     

Methods for reverse genetic screening in zebrafish by resequencing and TILLING

Animal models provide an in vivo system to study gene function by transgenic and knockout approaches. Targeted knockout approaches have been very successful in mice, but are currently not feasible in zebrafish due to the inability to grow embryonic stem cells. As an alternative, a reverse genetic approach that utilizes screening by resequencing and/or TILLING (Targeting Induced Local Lesions INGenomes) of mutagenized genomes has recently gained popularity in the zebrafish field. Spermatogonia of healthy males are mutagenized using ENU (N-ethyl-N-nitrosourea) and F1 progeny is collected by breeding treated males with healthy wild type females. Sperm and DNA banks are generated from F1 males. DNA is screened for ENU-induced mutations by sequencing or TILLING. These mutations can then be studied by in vitro fertilization (IVF) from the cryopreserved sperm of the corresponding F1 male followed by breeding to homozygosity. A high-throughput method of screening for rare heterozygotes and efficient recovery of mutant lines are important in identification of a large number of mutations using this approach. This article provides optimized protocols for resequencing and TILLING based on our experiences. We performed a pilot screen on 1235 F1 males by resequencing 54 exons from 17 genes and analyzed the sequencing data using multiple programs to maximize the mutation detection with minimal false positive detection. As an alternative to sequencing, we developed the protocols for TILLING by capillary electrophoresis using an ABI Genetic analyzer 3100 platform followed by fragment analysis using GeneScan and Genotyper softwares. PCR products generated by fluorescently labeled universal primers and tailed exon-specific primers were pooled 4-fold prior to heteroduplex formation. Overall, our pilot screen shows that a combination of TILLING and sequencing is optimal for achieving cost-effective, high-throughput screening of a large number of samples. Amplicons with fewer common SNPs are ideal for TILLING whereas amplicons with multiple SNPs and in/del polymorphisms are best suited for sequencing followed by analysis with SNPdetector.     

A double‐mutant collection targeting MAP kinase related genes in Arabidopsis for studying genetic interactions

Mitogen‐activated protein kinase cascades are conserved in all eukaryotes. In Arabidopsis thaliana there are approximately 80 genes encoding MAP kinase kinase kinases (MAP3K), 10 genes encoding MAP kinase kinases (MAP2K), and 20 genes encoding MAP kinases (MAPK). Reverse genetic analysis has failed to reveal abnormal phenotypes for a majority of these genes. One strategy for uncovering gene function when single‐mutant lines do not produce an informative phenotype is to perform a systematic genetic interaction screen whereby double‐mutants are created from a large library of single‐mutant lines. Here we describe a new collection of 275 double‐mutant lines derived from a library of single‐mutants targeting genes related to MAP kinase signaling. To facilitate this study, we developed a high‐throughput double‐mutant generating pipeline using a system for growing Arabidopsis seedlings in 96‐well plates. A quantitative root growth assay was used to screen for evidence of genetic interactions in this double‐mutant collection. Our screen revealed four genetic interactions, all of which caused synthetic enhancement of the root growth defects observed in a MAP kinase 4 (MPK4) single‐mutant line. Seeds for this double‐mutant collection are publicly available through the Arabidopsis Biological Resource Center. Scientists interested in diverse biological processes can now screen this double‐mutant collection under a wide range of growth conditions in order to search for additional genetic interactions that may provide new insights into MAP kinase signaling.     

Multi-SNP Analysis of GWAS Data Identifies Pathways Associated with Nonalcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) is a common liver disease; the histological spectrum of which ranges from steatosis to steatohepatitis. Nonalcoholic steatohepatitis (NASH) often leads to cirrhosis and development of hepatocellular carcinoma. To better understand pathogenesis of NAFLD, we performed the pathway of distinction analysis (PoDA) on a genome-wide association study dataset of 250 non-Hispanic white female adult patients with NAFLD, who were enrolled in the NASH Clinical Research Network (CRN) Database Study, to investigate whether biologic process variation measured through genomic variation of genes within these pathways was related to the development of steatohepatitis or cirrhosis. Pathways such as Recycling of eIF2:GDP, biosynthesis of steroids, Terpenoid biosynthesis and Cholesterol biosynthesis were found to be significantly associated with NASH. SNP variants in Terpenoid synthesis, Cholesterol biosynthesis and biosynthesis of steroids were associated with lobular inflammation and cytologic ballooning while those in Terpenoid synthesis were also associated with fibrosis and cirrhosis. These were also related to the NAFLD activity score (NAS) which is derived from the histological severity of steatosis, inflammation and ballooning degeneration. Eukaryotic protein translation and recycling of eIF2:GDP related SNP variants were associated with ballooning, steatohepatitis and cirrhosis. Il2 signaling events mediated by PI3K, Mitotic metaphase/anaphase transition, and Prostanoid ligand receptors were also significantly associated with cirrhosis. Taken together, the results provide evidence for additional ways, beyond the effects of single SNPs, by which genetic factors might contribute to the susceptibility to develop a particular phenotype of NAFLD and then progress to cirrhosis. Further studies are warranted to explain potential important genetic roles of these biological processes in NAFLD.     

Maternal control of vertebrate development before the midblastula transition: mutants from the zebrafish I

Maternal factors control development prior to the activation of the embryonic genome. In vertebrates, little is known about the molecular mechanisms by which maternal factors regulate embryonic development. To understand the processes controlled by maternal factors and identify key genes involved, we embarked on a maternal-effect mutant screen in the zebrafish. We identified 68 maternal-effect mutants. Here we describe 15 mutations in genes controlling processes prior to the midblastula transition, including egg development, blastodisc formation, embryonic polarity, initiation of cell cleavage, and cell division. These mutants exhibit phenotypes not previously observed in zygotic mutant screens. This collection of maternal-effect mutants provides the basis for a molecular genetic analysis of the maternal control of embryogenesis in vertebrates.     

Increase of hepatic fat accumulation by liver specific expression of Hepatitis B virus X protein in zebrafish

The pathogenesis of fatty liver disease remains largely unknown. Here, we assessed the importance of hepatic fat accumulation on the progression of hepatitis in zebrafish by liver specific expression of Hepatitis B virus X protein (HBx). Transgenic zebrafish lines, GBXs, which selectively express the GBx transgene (GFP-fused HBx gene) in liver, were established. GBX Liver phenotypes were evaluated by histopathology and molecular analysis of fatty acid (FA) metabolism-related genes expression. Most GBXs (66–81%) displayed obvious emaciation starting at 4 months old. Over 99% of the emaciated GBXs developed hepatic steatosis or steatohepatitis, which in turn led to liver hypoplasia. The liver histology of GBXs displayed steatosis, lobular inflammation, and balloon degeneration, similar to non-alcoholic steatohepatitis (NASH). Oil red O stain detected the accumulation of fatty droplets in GBXs. RT-PCR and Q-rt-PCR analysis revealed that GBx induced hepatic steatosis had significant increases in the expression of lipogenic genes, C/EBP-α, SREBP1, ChREBP and PPAR-γ, which then activate key enzymes of the de novo FA synthesis, ACC1, FAS, SCD1, AGAPT, PAP and DGAT2. In addition, the steatohepatitic GBX liver progressed to liver degeneration and exhibited significant differential gene expression in apoptosis and stress. The GBX models exhibited both the genetic and functional factors involved in lipid accumulation and steatosis-associated liver injury. In addition, GBXs with transmissible NASH-like phenotypes provide a promising model for studying liver disease.     

Diabetes models by screen for hyperglycemia in phenotype-driven ENU mouse mutagenesis projects

More than 150 million people suffer from diabetes mellitus worldwide, and this number is expected to rise substantially within the next decades. Despite its high prevalence, the pathogenesis of diabetes mellitus is not completely understood. Therefore, appropriate experimental models are essential tools to gain more insight into the genetics and pathogenesis of the disease. Here, we describe the current efforts to establish novel diabetes models derived from unbiased, phenotype-driven, large-scale N-ethyl-N-nitrosourea (ENU) mouse mutagenesis projects started a decade ago using hyperglycemia as a high-throughput screen parameter. Mouse lines were established according to their hyperglycemia phenotype over several generations, thereby revealing a mutation as cause for the aberrant phenotype. Chromosomal assignment of the causative mutation and subsequent candidate gene analysis led to the detection of the mutations that resulted in novel alleles of genes already known to be involved in glucose homeostasis, like glucokinase, insulin 2, and insulin receptor. Additional ENU-induced hyperglycemia lines are under genetic analysis. Improvements in screen for diabetic animals are implemented to detect more subtle phenotypes. Moreover, diet challenge assays are being employed to uncover interactions between genetic and environmental factors in the pathogenesis of diabetes mellitus. The new mouse mutants recovered in phenotype-driven ENU mouse mutagenesis projects complement the available models generated by targeted mutagenesis of candidate genes, all together providing the large resource of models required for a systematic dissection of the pathogenesis of diabetes mellitus.