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.     

Coupled mutagenesis screens and genetic mapping in zebrafish

Forward genetic analysis is one of the principal advantages of the zebrafish model system. However, managing zebrafish mutant lines derived from mutagenesis screens and mapping the corresponding mutations and integrating them into the larger collection of mutations remain arduous tasks. To simplify and focus these endeavors, we developed an approach that facilitates the rapid mapping of new zebrafish mutations as they are generated through mutagenesis screens. We selected a minimal panel of 149 simple sequence length polymorphism markers for a first-pass genome scan in crosses involving C32 and SJD inbred lines. We also conducted a small chemical mutagenesis screen that identified several new mutations affecting zebrafish embryonic melanocyte development. Using our first-pass marker panel in bulked-segregant analysis, we were able to identify the genetic map positions of these mutations as they were isolated in our screen. Rapid mapping of the mutations facilitated stock management, helped direct allelism tests, and should accelerate identification of the affected genes. These results demonstrate the efficacy of coupling mutagenesis screens with genetic mapping.     

Simple Methods for Generating and Detecting Locus-Specific Mutations Induced with TALENs in the Zebrafish Genome

The zebrafish is a powerful experimental system for uncovering gene function in vertebrate organisms. Nevertheless, studies in the zebrafish have been limited by the approaches available for eliminating gene function. Here we present simple and efficient methods for inducing, detecting, and recovering mutations at virtually any locus in the zebrafish. Briefly, double-strand DNA breaks are induced at a locus of interest by synthetic nucleases, called TALENs. Subsequent host repair of the DNA lesions leads to the generation of insertion and deletion mutations at the targeted locus. To detect the induced DNA sequence alterations at targeted loci, genomes are examined using High Resolution Melt Analysis, an efficient and sensitive method for detecting the presence of newly arising sequence polymorphisms. As the DNA binding specificity of a TALEN is determined by a custom designed array of DNA recognition modules, each of which interacts with a single target nucleotide, TALENs with very high target sequence specificities can be easily generated. Using freely accessible reagents and Web-based software, and a very simple cloning strategy, a TALEN that uniquely recognizes a specific pre-determined locus in the zebrafish genome can be generated within days. Here we develop and test the activity of four TALENs directed at different target genes. Using the experimental approach described here, every embryo injected with RNA encoding a TALEN will acquire targeted mutations. Multiple independently arising mutations are produced in each growing embryo, and up to 50% of the host genomes may acquire a targeted mutation. Upon reaching adulthood, approximately 90% of these animals transmit targeted mutations to their progeny. Results presented here indicate the TALENs are highly sequence-specific and produce minimal off-target effects. In all, it takes about two weeks to create a target-specific TALEN and generate growing embryos that harbor an array of germ line mutations at a pre-specified locus.     

Acridine mutagenesis of zebrafish (Danio rerio)

Mutagenesis screening, in which heritable traits are isolated following damage to the genome, is a powerful approach for investigating gene function. Among vertebrate model organisms, the zebrafish (Danio rerio) is ideally suited to mutagenesis screens. The success of large-scale screens is dependent on the way in which changes are identified. The type of damage induced is also pivotal. Single base coding region deletions and insertions are suited to abolition of gene function whilst inducing a small physical alteration to the genome. Such mutations are not commonly found following mutagenesis schemes reported to date. Here, we show that an acridine mutagen, ICR191, which in other model organisms frequently induces single base deletions and insertions, is mutagenic in zebrafish. ICR191 induces hallmark phenotypes associated with genetic damage in treated embryos. Alterations are heritable. Offspring of mutagenised fish had mutations in a marker gene and were found to produce offspring with abnormal development. Using an adaptation of a molecular mutation detection method, fluorescent arbitrary primed PCR, we identified an induced alteration directly. The estimated frequency of induced mutations was sufficiently high to make it feasible to employ this approach for mutagenesis screening.     

Genetics of gamma-irradiation-induced mutations in Arabidopsis thaliana: large chromosomal deletions can be rescued through the fertilization of diploid eggs.

Despite the demonstrated value of chromosomal deletions and deficiencies as tools in plant and animal genome research, in the genetic model plant species Arabidopsis thaliana, such mutations have not been extensively studied. For example, it is not known whether large deletions in different regions of the genome can be tolerated in diploid plants that are heterozygous for such mutations. Similarly the viability or inviability of monosomics has not been examined in detail. To investigate these questions, we have used gamma-irradiated haploid wild-type pollen to pollinate diploid and tetraploid multimarker lines of Arabidopsis. Examination of M1 progenies revealed that chromosome loss mutations and large deletions were induced in the irradiated pollen. Such mutations were eliminated in diploid M1 plants due to dominant lethality but could be rescued in triploid M1 progeny. The use of irradiated pollen and tetraploid marker lines of Arabidopsis is a convenient way of generating deletions and modified chromosomes and provides a genetic tool for deletion mapping and for analysis of chromosomal regions essential for chromosome maintenance.     

Heritable and Precise Zebrafish Genome Editing Using a CRISPR-Cas System

We have previously reported a simple and customizable CRISPR (clustered regularly interspaced short palindromic repeats) RNA-guided Cas9 nuclease (RGN) system that can be used to efficiently and robustly introduce somatic indel mutations in endogenous zebrafish genes. Here we demonstrate that RGN-induced mutations are heritable, with efficiencies of germline transmission reaching as high as 100%. In addition, we extend the power of the RGN system by showing that these nucleases can be used with single-stranded oligodeoxynucleotides (ssODNs) to create precise intended sequence modifications, including single nucleotide substitutions. Finally, we describe and validate simple strategies that improve the targeting range of RGNs from 1 in every 128 basepairs (bps) of random DNA sequence to 1 in every 8 bps. Together, these advances expand the utility of the CRISPR-Cas system in the zebrafish beyond somatic indel formation to heritable and precise genome modifications.     

基于高通量测序数据构建斑马鱼隐性遗传突变筛选系统

目的:针对斑马鱼高通量测序数据,通过一系列质量控制和突变过滤,构建一套有效隐性遗传突变筛选系统。方法:A)经过与斑马鱼参考基因组比对,利用GATK获得初始的VCF格式突变信息。B)使用perl语言编写本地脚本,对突变信息进行过滤,注释等。C)通过绘制纯合突变点分布图,找出突变富集区域。整合以上步骤,构建出一套针对斑马鱼高通量测序数据的遗传性突变筛选系统。结果:对获得的突变位点进行了一系列的质量控制,定位到具体染色体上的特定区域,并且对获得的突变进行了功能上的注释。结论:本过滤筛选系统能有效控制检测的假阳性率,对斑马鱼致病性的隐性突变位点筛查提供有力参考。     

Genome editing with RNA-guided Cas9 nuclease in Zebrafish embryos

Recent advances with the type II clustered regularly interspaced short palindromic repeats (CRISPR) system promise an improved approach to genome editing. However, the applicability and efficiency of this system in model organisms, such as zebrafish, are little studied. Here, we report that RNA-guided Cas9 nuclease efficiently facilitates genome editing in both mammalian cells and zebrafish embryos in a simple and robust manner. Over 35% of site-specific somatic mutations were found when specific Cas/gRNA was used to target either etsrp, gata4 or gata5 in zebrafish embryos in vivo. The Cas9/gRNA efficiently induced biallelic conversion of etsrp or gata5 in the resulting somatic cells, recapitulating their respective vessel phenotypes in etsrp^y11 mutant embryos or cardia bifida phenotypes in fau^tm236a mutant embryos. Finally, we successfully achieved site-specific insertion of mloxP sequence induced by Cas9/gRNA system in zebrafish embryos. These results demonstrate that the Cas9/gRNA system has the potential of becoming a simple, robust and efficient reverse genetic tool for zebrafish and other model organisms. Together with other genome-engineering technologies, the Cas9 system is promising for applications in biology, agriculture, environmental studies and medicine.     

Dissecting hematopoiesis and disease using the zebrafish

The study of blood has often defined paradigms that are relevant to the biology of other vertebrate organ systems. As examples, stem cell physiology and the structure of the membrane cytoskeleton were first described in hematopoietic cells. Much of the reason for these successes resides in the ease with which blood cells can be isolated and manipulated in vitro. The cell biology of hematopoiesis can also be illuminated by the study of human disease states such as anemia, immunodeficiency, and leukemia. The sequential development of the blood system in vertebrates is characterized by ventral mesoderm induction, hematopoietic stem cell specification, and subsequent cell lineage differentiation. Some of the key regulatory steps in this process have been uncovered by studies in mouse, chicken, and Xenopus. More recently, the genetics of the zebrafish (Danio rerio) have been employed to define novel points of regulation of the hematopoietic program. In this review, we describe the advantages of the zebrafish system for the study of blood cell development and the initial success of the system in this pursuit. The striking similarity of zebrafish mutant phenotypes and human diseases emphasizes the utility of this model system for elucidating pathophysiologic mechanisms. New screens for lineage-specific mutations are beginning, and the availability of transgenics promises a better understanding of lineage-specific gene expression. The infrastructure of the zebrafish system is growing with an NIH-directed genome initiative, providing a detailed map of the zebrafish genome and an increasing number of candidate genes for the mutations. The zebrafish is poised to contribute greatly to our understanding of normal and disease-related hematopoiesis.     

The emerging use of zebrafish to model metabolic disease

The zebrafish research community is celebrating! The zebrafish genome has recently been sequenced, the Zebrafish Mutation Project (launched by the Wellcome Trust Sanger Institute) has published the results of its first large-scale ethylnitrosourea (ENU) mutagenesis screen, and a host of new techniques, such as the genome editing technologies TALEN and CRISPR-Cas, are enabling specific mutations to be created in model organisms and investigated in vivo. The zebrafish truly seems to be coming of age. These powerful resources invoke the question of whether zebrafish can be increasingly used to model human disease, particularly common, chronic diseases of metabolism such as obesity and type 2 diabetes. In recent years, there has been considerable success, mainly from genomic approaches, in identifying genetic variants that are associated with these conditions in humans; however, mechanistic insights into the role of implicated disease loci are lacking. In this Review, we highlight some of the advantages and disadvantages of zebrafish to address the organism’s utility as a model system for human metabolic diseases.     

Pentachlorophenol treatment in vivo elevates point mutation rate in zebrafish p53 gene

Pentachlorophenol (PCP), a probable human carcinogen, has been heavily used as an aseptic and a biocide throughout the world, and is widely present in the environment. Recent survey in Germany revealed that the average PCP amount in the urine of general German populations was 1.04 microg/L while the peak concentration could reach up to 19.1 microg/L. PCP was reported to cause DNA damage, but whether it can be involved in inducing point mutations in genome is unknown. To determine the genotoxicity of PCP on vertebrate, we first performed acute toxicity test on zebrafish for the effect of PCP exposure. The LC50 values of zebrafish exposed to PCP at 24, 48, 72 and 96 h were determined to be 0.196, 0.130, 0.130 and 0.130 mg/L, respectively. We then treated zebrafish with PCP for 10 days at 0 (control), 0.5, 5 and 50 microg/L, respectively, to determine whether PCP could be involved in inducing point mutations. Employing denaturing high-performance liquid chromatography analysis and DNA sequencing, we demonstrated that exposure of PCP to zebrafish at a concentration as low as 5 microg/L for 10 days elevates point mutation rate in p53 gene in liver cells. This is the first direct evidence revealing that PCP can elevate point mutation rate in the vertebrate genomes. The result implies PCP might be involved in carcinogenesis by elevating point mutation rate in the somatic genomes.     

A meta-analysis of kidney microarray datasets: investigation of cytokine gene detection and correlation with rt-PCR and detection thresholds

Background

Large-scale mutagenesis screens in the zebrafish employing the mutagen ENU have isolated several hundred mutant loci that represent putative developmental control genes. In order to realize the potential of such screens, systematic genetic mapping of the mutations is necessary. Here we report on a large-scale effort to map the mutations generated in mutagenesis screening at the Max Planck Institute for Developmental Biology by genome scanning with microsatellite markers.

Results

We have selected a set of microsatellite markers and developed methods and scoring criteria suitable for efficient, high-throughput genome scanning. We have used these methods to successfully obtain a rough map position for 319 mutant loci from the Tübingen I mutagenesis screen and subsequent screening of the mutant collection. For 277 of these the corresponding gene is not yet identified. Mapping was successful for 80 % of the tested loci. By comparing 21 mutation and gene positions of cloned mutations we have validated the correctness of our linkage group assignments and estimated the standard error of our map positions to be approximately 6 cM.

Conclusion

By obtaining rough map positions for over 300 zebrafish loci with developmental phenotypes, we have generated a dataset that will be useful not only for cloning of the affected genes, but also to suggest allelism of mutations with similar phenotypes that will be identified in future screens. Furthermore this work validates the usefulness of our methodology for rapid, systematic and inexpensive microsatellite mapping of zebrafish mutations.     

Efficient mutation identification in zebrafish by microarray capturing and next generation sequencing

Fish models like medaka, stickleback or zebrafish provide a valuable resource to study vertebrate genes. However, finding genetic variants e.g. mutations in the genome is still arduous. Here we used a combination of microarray capturing and next generation sequencing to identify the affected gene in the mozartkugel^p11cv (mzl^p11cv) mutant zebrafish. We discovered a 31-bp deletion in macf1 demonstrating the potential of this technique to efficiently isolate mutations in a vertebrate genome.     

Zebrafish as a genomics model for human neurological and polygenic disorders

Whole exome sequencing and, to a lesser extent, genome-wide association studies, have provided unprecedented advances in identifying genes and candidate genomic regions involved in the development of human disease. Further progress will come from sequencing the entire genome of multiple patients and normal controls to evaluate overall mutational burden and disease risk. A major challenge will be the interpretation of the resulting data and distinguishing true pathogenic mutations from rare benign variants.While in model organisms such as the zebrafish,mutants are sought that disrupt the function of individual genes, human mutations that cause, or are associated with, the development of disease, are often not acting in a Mendelian fashion, are frequently of small effect size, are late onset, and may reside in noncoding parts of the genome. The zebrafish model is uniquely poised for understanding human coding- and noncoding variants because of its sequenced genome, a large body of knowledge on gene expression and function, rapid generation time, and easy access to embryos. A critical advantage is the ease of zebrafish transgenesis, both for the testing of human regulatory DNA driving expression of fluorescent reporter proteins, and the expression of mutated disease-associated human proteins in specific neurons to rapidly model aspects of neurological disorders. The zebrafish affords progress both through its model genome and it is rapidly developing transparent model vertebrate embryo.     

Efficient mapping and cloning of mutations in zebrafish by low-coverage whole-genome sequencing

The generation and analysis of mutants in zebrafish has been instrumental in defining the genetic regulation of vertebrate development, physiology, and disease. However, identifying the genetic changes that underlie mutant phenotypes remains a significant bottleneck in the analysis of mutants. Whole-genome sequencing has recently emerged as a fast and efficient approach for identifying mutations in nonvertebrate model organisms. However, this approach has not been applied to zebrafish due to the complicating factors of having a large genome and lack of fully inbred lines. Here we provide a method for efficiently mapping and detecting mutations in zebrafish using these new parallel sequencing technologies. This method utilizes an extensive reference SNP database to define regions of homozygosity-by-descent by low coverage, whole-genome sequencing of pooled DNA from only a limited number of mutant F(2) fish. With this approach we mapped each of the five different zebrafish mutants we sequenced and identified likely causative nonsense mutations in two and candidate mutations in the remainder. Furthermore, we provide evidence that one of the identified mutations, a nonsense mutation in bmp1a, underlies the welded mutant phenotype.     

Medaka and zebrafish, an evolutionary twin study

Comparison of two related species is one of the most successful approaches to decipher general genetic principles in eukaryotes. This is best illustrated in yeast, where the model systems Saccharomyyces. cervisiae and Schizosaccharomyces. pombe have been examined. Powerful forward genetics in both species, species-specific differences in biological features and the phylogenetic distance between the two species, make them well suited for a comparative approach. Recent whole genome sequencing has also facilitated comparative genomics of these simple eukaryotes. It is now possible to go a step further using higher eukaryotes. A duplication of the genome at the base of the teleost radiation, facilitated evolution of almost 25,000 fish species, more than half of all vertebrate species together. Two teleost genetic model systems have emerged in the past few decades: zebrafish, in which large-scale mutagenesis has been successfully performed, and Medaka, a Japanese killifish with a century of history in genetics and now, as reported in this issue, many induced mutations. In this review we will illustrate how comparison of these two model species, Medaka and zebrafish, can reveal conserved and species-specific genetic and molecular mechanisms underlying vertebrate development.     

Knockdown of duplicated zebrafish hoxb1 genes reveals distinct roles in hindbrain patterning and a novel mechanism of duplicate gene retention

We have used a morpholino-based knockdown approach to investigate the functions of a pair of zebrafish Hox gene duplicates, hoxb1a and hoxb1b, which are expressed during development of the hindbrain. We find that the zebrafish hoxb1 duplicates have equivalent functions to mouse Hoxb1 and its paralogue Hoxa1. Thus, we have revealed a 'function shuffling' among genes of paralogue group 1 during the evolution of vertebrates. Like mouse Hoxb1, zebrafish hoxb1a is required for migration of the VIIth cranial nerve branchiomotor neurons from their point of origin in hindbrain rhombomere 4 towards the posterior. By contrast, zebrafish hoxb1b, like mouse Hoxa1, is required for proper segmental organization of rhombomere 4 and the posterior hindbrain. Double knockdown experiments demonstrate that the zebrafish hoxb1 duplicates have partially redundant functions. However, using an RNA rescue approach, we reveal that these duplicated genes do not have interchangeable biochemical functions: only hoxb1a can properly pattern the VIIth cranial nerve. Despite this difference in protein function, we provide evidence that the hoxb1 duplicate genes were initially maintained in the genome because of complementary degenerative mutations in defined cis-regulatory elements.     

Characterization of zebrafish Rad52 and replication protein A for oligonucleotide-mediated mutagenesis

Zebrafish has become a favorite model organism not only in genetics and developmental biology, but also for the study of cancer, neuroscience and metabolism. However, strategies for reverse genetics in zebrafish are mostly limited to the use of antisense oligonucleotides, and therefore the development of other targeting methods is highly desirable. Here, we report an approach to gene targeting in this system in which single-stranded oligonucleotides and zebrafish Rad52 protein are employed. It has been proposed that a single-stranded oligonucleotide containing a mutation can be incorporated into the genome by annealing to the single-stranded region of the lagging strand of the replication fork. Rad52 is expected to accelerate the annealing step. In vitro experiments using purified truncated Rad52 proteins and replication protein A (RPA) showed that annealing of oligonucleotides is accelerated by Rad52 in the presence of RPA. We developed a simple and sensitive PCR-based method to detect point mutations in the genome. In exploratory experiments, we found that microinjection of single-stranded oligonucleotide targeted to a specific gene together with truncated Rad52 into zebrafish embryos resulted in a low level of recombinant copies in 3 of the 80 embryos tested under these conditions.     

Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases

We describe the use of zinc-finger nucleases (ZFNs) for somatic and germline disruption of genes in zebrafish (Danio rerio), in which targeted mutagenesis was previously intractable. ZFNs induce a targeted double-strand break in the genome that is repaired to generate small insertions and deletions. We designed ZFNs targeting the zebrafish golden and no tail/Brachyury (ntl) genes and developed a budding yeast-based assay to identify the most active ZFNs for use in vivo. Injection of ZFN-encoding mRNA into one-cell embryos yielded a high percentage of animals carrying distinct mutations at the ZFN-specified position and exhibiting expected loss-of-function phenotypes. Over half the ZFN mRNA-injected founder animals transmitted disrupted ntl alleles at frequencies averaging 20%. The frequency and precision of gene-disruption events observed suggest that this approach should be applicable to any loci in zebrafish or in other organisms that allow mRNA delivery into the fertilized egg.