A mouse model of Waardenburg syndrome type IV resulting from an ENU‐induced mutation in endothelin 3

A line of mutant mice (114‐CH19) exhibiting white spotting and preweaning lethality was identified during an N‐ethyl‐N‐nitrosourea (ENU) mutagenesis screen. The trait segregated as a semidominant bellyspot with reduced penetrance. Homozygous mutant mice showed preweaning lethality, and exhibited white spotting over the majority of the body surface, with pigmented patches remaining around the pinnae, eyes and tail. Linkage analysis localized 114‐CH19 on mouse chromosome 2, suggesting endothelin 3 (Edn3) as a candidate gene. Sequence analysis of Edn3 identified a G > A transversion that encodes an arginine to histidine substitution (R96H). This mutation is predicted to disrupt furin‐mediated proteolytic cleavage of pro‐endothelin that is necessary to form biologically active EDN3. This mutation is novel among human and mouse EDN3 mutants, is the first reported EDN3 ENU mutant, and is the second reported EDN3 point mutation. This study demonstrates the power of using ENU mutagenesis screens to generate new animal models of human disease, and expands the spectrum of EDN3 mutant alleles.     

Characterization of a novel missense mutation on murine Pax3 through ENU mutagenesis

N-ethyl-N-nitrosourea (ENU) mutagenesis has led to the elucidation of several regulator genes for melanocyte and skin development.Here we characterized a mutant from ENU mutagenesis with similar phenotype as that of Splotch mutant,including exencephaly,spina bifida and abnormal limbs in homozygotes as well as white belly spotting and occasionally loop-tail in heterozygotes.This novel mutant was named as SpxG..Through genome-wide linkage analysis in backcross progenies with microsatellite markers,the SpxG was confined to a region between D1MIT415 and D1M IT7 on chromosome 1,where notable Pax3 gene was located.Direct sequencing revealed that SpxG carried a nucleotide A894G missense transition in exon 6 of Pax3 gene that resulted in Asn to Asp substitution at amino acid 269 within the highly-conserved homeodomain (HD) DNA recognition module,which was the first point mutation found in this domain in mice.This N269D mutation impaired the transactivation capacity of Pax3 protein,but exerted no effect on Pax3 protein translation.The characterization of the new mutation expanded our understanding the transactivation and DNA-binding structure of Pax3 protein.     

Kit无义突变致W~(-3Bao)小鼠生殖腺异常及纯合子贫血死亡

W-3Bao是本研究组通过诱变获得、Kit无义突变的白斑小鼠.突变基因杂合子小鼠腹部、四肢肢端及尾尖白化,其部分精曲小管内无精原细胞.突变纯合子小鼠在胚胎后期色泽苍白、个体矮小,于出生前后死亡;血液学检查发现纯合子小鼠血色素极低且红细胞变大:18.5天胚胎的连续切片可见精曲小管轮廓欠清晰,精原细胞分散分布于睾丸间质,未迁入精曲小管:卵巢结构紊乱,无明显的原始卵泡结构;骨髓等器官组织未见显著异常.结论:Kit无义突变不仅导致了W-3Bao杂合子小鼠白斑形成及纯合子小鼠贫血死亡,同时影响生殖腺发育.     

Identification of a novel point mutation of mouse proto-oncogene c-kit through N-ethyl-N-nitrosourea mutagenesis

Manipulation of the mouse genome has emerged as an important approach for studying gene function and establishing human disease models. In this study, the mouse mutants were generated through N-ethyl-N-nitrosourea (ENU)-induced mutagenesis in C57BL/6J mice. The screening for dominant mutations yielded several mice with fur color abnormalities. One of them causes a phenotype similar to that shown by dominant-white spotting (W) allele mutants. This strain was named Wads because the homozygous mutant mice are white color, anemic, deaf, and sterile. The new mutation was mapped to 42 cM on chromosome five, where proto-oncogene c-kit resides. Sequence analysis of c-kit cDNA from Wads(m/m) revealed a unique T-to-C transition mutation that resulted in Phe-to-Ser substitution at amino acid 856 within a highly conserved tyrosine kinase domain. Compared with other c-kit mutants, Wads may present a novel loss-of-function or hypomorphic mutation. In addition to the examination of adult phenotypes in hearing loss, anemia, and mast cell deficiency, we also detected some early developmental defects during germ cell differentiation in the testis and ovary of neonatal Wads(m/m) mice. Therefore, the Wads mutant may serve as a new disease model of human piebaldism, anemia, deafness, sterility, and mast cell diseases.     

Loss of Function of Endothelin-2 Leads to Reduced Ovulation and CL Formation

Endothelin-2 (EDN2), a potent vasoconstrictive peptide, is transiently produced by periovulatory follicles at the time of ovulation when corpus luteum (CL) formation begins. EDN2 induces contraction of ovarian smooth muscles ex vivo via an endothelin receptor A-mediated pathway. In this study, we aimed to determine if EDN2 is required for normal ovulation and subsequent CL formation in?vivo. In the ovaries of a mouse model that globally lacks the Edn2 gene (Edn2 knockout mouse; Edn2KO), histology showed that post-pubertal Edn2KO mice possess follicles of all developmental stages, but no corpora lutea. When exogenous gonadotropins were injected to induce super-ovulation, Edn2KO mice exhibited significantly impaired ovulation and CL formation compared to control littermates. Edn2KO ovaries that did ovulate in response to gonadotropins did not contain histologically and functionally identifiable CL. Intra-ovarian injection of EDN2 peptide results suggest partial induction of ovulation in Edn2KO mice. Endothelin receptor antagonism in wild type mice similarly disrupted ovulation, CL formation, and progesterone secretion. Overall, this study suggests that EDN2 is necessary for normal ovulation and CL formation.     

乙酰基亚硝基脲诱导小鼠突变的初步研究

目的　探索乙酰基亚硝基脲 (ENU)诱导小鼠突变的效率 ,筛查并获得能显性遗传的突变型小鼠。方法　采用 8～ 10周龄的雄性C57BL 6小鼠 33只、DBA 2小鼠 18只 ,腹腔注射ENU10 0mg kg ,每周一次共三次 ,与同品系母鼠配种 ,在后代小鼠中针对可见表型筛查突变个体。结果　处理雄鼠有 9至 13周的不育期 ;在已经筛查的12 4 1只小鼠中得到眼睛异常、多趾、少趾及腹部白斑、矮小等突变个体 6 1只 ,突变率约 5 % ;获得单基因显性遗传的突变品系 2种。结论　ENU为小鼠的强诱突变剂 ;通过诱变可以得到遗传突变小鼠 ,为建立人类疾病动物模型提供条件 ;大规模诱变实验对小鼠功能基因组的研究有重要意义     

An ENU-induced mutation in the Ankrd11 gene results in an osteopenia-like phenotype in the mouse mutant Yoda.

The mechanisms that regulate bone mass are important in a variety of complex diseases such as osteopenia and osteoporosis. Regulation of bone mass is a polygenic trait and is also influenced by various environmental and lifestyle factors, making analysis of the genetic basis difficult. As an effort toward identifying novel genes involved in regulation of bone mass, N-ethyl-N-nitrosourea (ENU) mutagenesis in mice has been utilized. Here we describe a mouse mutant termed Yoda that was identified in an ENU mutagenesis screen for dominantly acting mutations. Mice heterozygous for the Yoda mutation exhibit craniofacial abnormalities: shortened snouts, wider skulls, and deformed nasal bones, underlined by altered morphology of frontonasal sutures and failure of interfrontal suture to close. A major feature of the mutant is reduced bone mineral density. Homozygosity for the mutation results in embryonic lethality. Positional cloning of the locus identified a missense mutation in a highly conserved region of the ankyrin repeat domain 11 gene (Ankrd11). This gene has not been previously associated with bone metabolism and, thus, identifies a novel genetic regulator of bone homeostasis.     

The induction of forward and reverse specific-locus mutations and dominant cataract mutations in spermatogonia of treated strain DBA/2 mice by ethylnitrosourea

The mutagenic effectiveness of ethylnitrosurea (ENU) was assessed in treated spermatogonia of DBA/2 mice. In a total of 17,515 offspring examined following 160 mg ENU/kg body weight treatment of parental males, 26 forward specific-locus mutations, 2 reverse specific-locus mutations and 9 dominant cataract mutations were recovered. ENU increased the mutation rate to all 3 genetic endpoints. However, ENU was less effective in treated DBA/2 mice than in the standard experimental protocol employing treated hybrid (102 X C3H)F1 male mice. This observed difference for a direct-acting mutagen such as ENU may result from differences in the detoxification of ENU or from differences in the DNA-repair capabilities of strain DBA/2. The first documented reverse mutation of the b allele is reported. The reversion was shown to be due to an AT to GC transition. To date, in addition to the reverse mutation of the b allele, 5 independent ENU-induced mutations recovered in germ cells of the mouse have been molecularly characterized and all have been shown to be base substitutions at an AT site. This is in contrast to the expected mechanism of ENU mutation induction due to O6-ethylguanine adduct formation which results in a GC to AT base-pair substitution and emphasizes the complexities of mutagenesis in germ cells of mammals.     

Mutations in alpha-tubulin cause abnormal neuronal migration in mice and lissencephaly in humans

The development of the mammalian brain is dependent on extensive neuronal migration. Mutations in mice and humans that affect neuronal migration result in abnormal lamination of brain structures with associated behavioral deficits. Here, we report the identification of a hyperactive N-ethyl-N-nitrosourea (ENU)-induced mouse mutant with abnormalities in the laminar architecture of the hippocampus and cortex, accompanied by impaired neuronal migration. We show that the causative mutation lies in the guanosine triphosphate (GTP) binding pocket of alpha-1 tubulin (Tuba1) and affects tubulin heterodimer formation. Phenotypic similarity with existing mouse models of lissencephaly led us to screen a cohort of patients with developmental brain anomalies. We identified two patients with de novo mutations in TUBA3, the human homolog of Tuba1. This study demonstrates the utility of ENU mutagenesis in the mouse as a means to discover the basis of human neurodevelopmental disorders.     

An XXY mouse, the result of a rearrangement between one X and a Y chromosome

A male mouse with irregular white spotting, typical of piebald, s, arose during an experiment designed to search for mutations induced in spermatogonial cells by ethylnitrosourea (ENU). On being examined cytologically it was found to carry 40 chromosomes but was effectively XXY since one of the two X chromosomes present was distally fused to a Y chromosome. In common with the previously described XXY mice, all of which carried 41 chromosomes, the mouse was sterile with a total absence of germ cells. Because of this, it was not possible to determine if the white spotting was inherited. The spotting could not be related to any observable abnormality of chromosomes known to carry spotting genes, nor could it be linked in any way with the X and Y fusion. It was concluded from the cytological considerations and the time interval (6 months) that had elapsed between mutagen treatment and birth of the offspring, that whereas the spotting was probably the result of ENU damage in a spermatogonial stem cell, the XY fusion was probably a later and spontaneous event.     

A new mouse mutant for the LDL receptor identified using ENU mutagenesis

In an effort to discover new mouse models of cardiovascular disease using N-ethyl-N-nitrosourea (ENU) mutagenesis followed by high-throughput phenotyping, we have identified a new mouse mutation, C699Y, in the LDL receptor (Ldlr), named wicked high cholesterol (WHC). When WHC was compared with the widely used Ldlr knockout (KO) mouse, notable phenotypic differences between strains were observed, such as accelerated atherosclerotic lesion formation and reduced hepatosteatosis in the ENU mutant after a short exposure to an atherogenic diet. This loss-of-function mouse model carries a single base mutation in the Ldlr gene on an otherwise pure C57BL/6J (B6) genetic background, making it a useful new tool for understanding the pathophysiology of atherosclerosis and for evaluating additional genetic modifiers regulating hyperlipidemia and atherogenesis. Further investigation of genomic differences between the ENU mutant and KO strains may reveal previously unappreciated sequence functionality.     

Interactions between Sox10, Edn3 and Ednrb during enteric nervous system and melanocyte development

The requirement for SOX10 and endothelin-3/EDNRB signalling pathway during enteric nervous system (ENS) and melanocyte development, as well as their alterations in Waardenburg-Hirschsprung disease (hypopigmentation, deafness and absence of enteric ganglia) are well established. Here, we analysed the genetic interactions between these genes during ENS and melanocyte development. Through phenotype analysis of Sox10;Ednrb and Sox10;Edn3 double mutants, we show that a coordinate and balanced interaction between these molecules is required for normal ENS and melanocyte development. Indeed, double mutants present with a severe increase in white spotting, absence of melanocytes within the inner ear, and in the stria vascularis in particular, and more severe ENS defects. Moreover, we show that partial loss of Ednrb in Sox10 heterozygous mice impairs colonisation of the gut by enteric crest cells at all stages observed. However, compared to single mutants, we detected no apoptosis, cell proliferation or overall neuronal or glial differentiation defects in neural crest cells within the stomach of double mutants, but apoptosis was increased in vagal neural crest cells outside of the gut. These data will contribute to the understanding of the molecular basis of ENS, pigmentation and hearing defects observed in mouse mutants and patients carrying SOX10, EDN3 and EDNRB mutations.     

ENU Mutagenesis Reveals a Novel Phenotype of Reduced Limb Strength in Mice Lacking Fibrillin 2

Background

Fibrillins 1 (FBN1) and 2 (FBN2) are components of microfibrils, microfilaments that are present in many connective tissues, either alone or in association with elastin. Marfan''s syndrome and congenital contractural arachnodactyly (CCA) result from dominant mutations in the genes FBN1 and FBN2 respectively. Patients with both conditions often present with specific muscle atrophy or weakness, yet this has not been reported in the mouse models. In the case of Fbn1, this is due to perinatal lethality of the homozygous null mice making measurements of strength difficult. In the case of Fbn2, four different mutant alleles have been described in the mouse and in all cases syndactyly was reported as the defining phenotypic feature of homozygotes.

Methodology/Principal Findings

As part of a large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis screen, we identified a mouse mutant, Mariusz, which exhibited muscle weakness along with hindlimb syndactyly. We identified an amber nonsense mutation in Fbn2 in this mouse mutant. Examination of a previously characterised Fbn2-null mutant, Fbn2^fp, identified a similar muscle weakness phenotype. The two Fbn2 mutant alleles complement each other confirming that the weakness is the result of a lack of Fbn2 activity. Skeletal muscle from mutants proved to be abnormal with higher than average numbers of fibres with centrally placed nuclei, an indicator that there are some regenerating muscle fibres. Physiological tests indicated that the mutant muscle produces significantly less maximal force, possibly as a result of the muscles being relatively smaller in Mariusz mice.

Conclusions

These findings indicate that Fbn2 is involved in integrity of structures required for strength in limb movement. As human patients with mutations in the fibrillin genes FBN1 and FBN2 often present with muscle weakness and atrophy as a symptom, Fbn2-null mice will be a useful model for examining this aspect of the disease process further.     

Endothelin-2-Mediated Protection of Mutant Photoreceptors in Inherited Photoreceptor Degeneration

Expression of the Endothelin-2 (Edn2) mRNA is greatly increased in the photoreceptors (PRs) of mouse models of inherited PR degeneration (IPD). To examine the role of Edn2 in mutant PR survival, we generated Edn2^−/− mice carrying homozygous Pde6b^rd1 alleles or the Tg(RHO P347S) transgene. In the Edn2^−/− background, PR survival increased 110% in Pde6b^rd1/rd1 mice at post-natal (PN) day 15, and 60% in Tg(RHO P347S) mice at PN40. In contrast, PR survival was not increased in retinal explants of Pde6b^rd1/rd1; Edn2^−/− mice. This finding, together with systemic abnormalities in Edn2^−/− mice, suggested that the increased survival of mutant PRs in the Edn2^−/− background resulted at least partly from the systemic EDN2 loss of function. To examine directly the role of EDN2 in mutant PRs, we used a scAAV5-Edn2 cDNA vector to restore Edn2 expression in Pde6b^rd1/rd1; Edn2^−/− PRs and observed an 18% increase in PR survival at PN14. Importantly, PR survival was also increased after injection of scAAV5-Edn2 into Pde6b^rd1/rd1 retinas, by 31% at PN15. Together, these findings suggest that increased Edn2 expression is protective to mutant PRs. To begin to elucidate Edn2-mediated mechanisms that contribute to PR survival, we used microarray analysis and identified a cohort of 20 genes with >4-fold increased expression in Tg(RHO P347S) retinas, including Fgf2. Notably, increased expression of the FGF2 protein in Tg(RHO P347S) PRs was ablated in Tg(RHO P347S); Edn2^−/− retinas. Our findings indicate that the increased expression of PR Edn2 increases PR survival, and suggest that the Edn2-dependent increase in PR expression of FGF2 may contribute to the augmented survival.     

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.     

The Mouse MC13 Mutant Is a Novel ENU Mutation in Collagen Type II,Alpha 1

Phenotype-driven mutagenesis experiments are a powerful approach to identifying novel alleles in a variety of contexts. The traditional disadvantage of this approach has been the subsequent task of identifying the affected locus in the mutants of interest. Recent advances in bioinformatics and sequencing have reduced the burden of cloning these ENU mutants. Here we report our experience with an ENU mutagenesis experiment and the rapid identification of a mutation in a previously known gene. A combination of mapping the mutation with a high-density SNP panel and a candidate gene approach has identified a mutation in collagen type II, alpha I (Col2a1). Col2a1 has previously been studied in the mouse and our mutant phenotype closely resembles mutations made in the Col2a1 locus.     

Random mutagenesis of the mouse genome: a strategy for discovering gene function and the molecular basis of disease

Mutagenesis of mice with N-ethyl-N-nitrosourea (ENU) is a phenotype-driven approach to unravel gene function and discover new biological pathways. Phenotype-driven approaches have the advantage of making no assumptions about the function of genes and their products and have been successfully applied to the discovery of novel gene-phenotype relationships in many physiological systems. ENU mutagenesis of mice is used in many large-scale and more focused projects to generate and identify novel mouse models for the study of gene functions and human disease. This review examines the strategies and tools used in ENU mutagenesis screens to efficiently generate and identify functional mutations.