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The art and design of genetic screens: zebrafish
Author: David Gems
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"956 | DECEMBER 2001 | VOLUME 2 www.nature.com/reviews/genetics REVIEWS Twenty years ago, George Streisinger?s pioneering research revealed the potential of the zebrafish as a ver- tebrate organism that was suitable for forward genetic screening 1 . A relatively small fish (3?4 cm long as an adult), zebrafish can be easily managed in large num- bers in the laboratory environment. The ability to com- bine embryological and genetic methodology has established the zebrafish as a powerful research tool. External development of transparent embryos allows fundamental vertebrate developmental processes ? from gastrulation to organogenesis ? to be visualized and studied; in addition, the heart beats and blood cir- culation of the embryo are readily observed. Touch, sight and behavioural responses can also be monitored in live embryos under the dissecting microscope. Several features, such as a short generation time of 3?4 months, mean that zebrafish are particularly suitable for genetic studies. In addition, mutations can be induced with high frequency in zebrafish, and recessive mutations can be recovered within two generations 2,3 . Large progeny sizes (females lay about 100?200 eggs) facilitate large-scale genetic screening and mutation analysis in a Mendelian fashion. Here, we review the methods of genetic screening in zebrafish, and include some examples of the resulting mutants and genes discovered from diploid, haploid and GYNOGENETIC diploid screens. We follow with a sam- pling of the recent, resourceful genetic screening tech- niques now carried out in zebrafish to show the poten- tial and versatility of the zebrafish genetic system. First large-scale vertebrate genetic screens Systematic genome-wide screens for mutations in worms, flies and plants have successfully identified many genes that define embryological pathways. Smaller collections of mammalian mutants provide valuable insights into developmental processes. However, identifying large numbers of mutations in the mammalian system is problematic because of intrauterine development and expensive supporting laboratory facilities. The remarkable characteristics of the zebrafish, along with the initial success of the first zebrafish genetic screens 4 , inspired two groups of scien- tists in Boston 5 and T�bingen 6 to undertake the first large-scale genetic screens in a vertebrate organism. The Boston and T�bingen screens identified mutant embryonic phenotypes in the F 3 generation (FIG. 1). Some of the ~2,000 mutated developmental genes that were identified in these two screens have been cloned, which assists in the dissection of the gene networks that control early development. For example, the genes that are mutated in the endoderm mutants casanova (cas), bonnie and clyde (bon), and faust (fau) can be assem- bled into a genetic pathway, and have been shown to encode transcription factors that are necessary for endoderm formation 7?12 . Embryo transparency, as well as conspicuous heart, blood and blood vessels in the zebrafish have also made it possible to identify cardio- vascular system mutants in a manner that is unprece- dented in other animal systems. As an example, the gene jekyll (jek) is necessary for formation of the THE ART AND DESIGN OF GENETIC SCREENS: ZEBRAFISH E. Elizabeth Patton and Leonard I. Zon Inventive genetic screens in zebrafish are revealing new genetic pathways that control vertebrate development, disease and behaviour. By exploiting the versatility of zebrafish, biological processes that had been previously obscured can be visualized and many of the responsible genes can be isolated. Coupled with gene knockdown and overexpression technologies, and small-molecule-induced phenotypes, genetic screens in zebrafish provide a powerful system by which to dissect vertebrate gene function and gene networks. GYNOGENESIS Development of an organism derived from the genetic material of the female gamete. Howard Hughes Medical Institute, Children?s Hospital of Boston, 300 Longwood Avenue, Enders 750, Boston, Massachusetts 02115, USA. Correspondence to L.I.Z. e-mails: epatton@enders. tch.harvard.edu; zon@enders.tch.harvard.edu � 2001 Macmillan Magazines Ltd NATURE REVIEWS | GENETICS VOLUME 2 | DECEMBER 2001 | 957 REVIEWS PARTHENOGENESIS Development of an organism derived from an unfertilized gamete. RHOMBOMERE Each of seven neuroepithelial segments found in the embryonic hindbrain that adopt distinct molecular and cellular properties, restrictions in cell mixing and ordered domains of gene expression. Ferrochelatase, which, when disrupted in humans, leads to erythropoietic protoporphyria owing to the accumu- lation of light-sensitive, toxic intermediates of the haem biosynthetic pathway 19 . The yqe gene encodes uropor- phyrinogen decarboxylase (UROD), and as in the fish, homozygous mutations in this enzyme lead to hepato- erythropoietic porphyria in humans 20 . The wealth of knowledge gained from just a sampling of the phenotyp- ic groups of mutants identified in the Boston and T�bingen screens illustrates the power and potential of zebrafish genetics, and validates the use of zebrafish as a model for vertebrate biological processes. Haploid and homozygous diploid screens. Phenotypic detection of a recessive allele within an F 3 generation requires many crosses in F 2 families (FIG. 1). Streisinger et al. attempted to eliminate this ?cumbersome? step by cre- ating haploid and PARTHENOGENETIC diploid animals that were suitable for use in genetic screens 1 (BOX 1).A geneti- cally convenient feature of zebrafish is that they can live up to 3 days post-fertilization as haploid organisms 1 . Recessive alleles in F 1 fish are therefore exposed within a generation, streamlining the screen for both size and time (BOX 1, figure panel a). Haploids have the same body plan as diploids, but are shorter, contain more and small- er cells than diploids, and can have other morphological defects that complicate screening (BOX 1, figure panel b). Nevertheless, molecular markers that are essentially nor- mal in wild-type, haploid embryos can be used success- fully in haploid screens to identify genes that are crucial for the early stages of zebrafish embryogenesis. For example, a haploid screen successfully uncovered genes that were involved in brain patterning 21 . Haploid embryos from F 1 females were screened simultaneously for the expression pattern of six genes in the brain by RNA in situ hybridization 21 .Three valentino (val) alleles had atypical krox20 expression, as well as abnormally smooth and unsegmented hindbrains. val is required for the subdivision and expansion of the hindbrain region that normally gives rise to RHOMBOMERES (r) 5 and 6, and is a homologue of the mouse segmentation-factor-encod- ing gene Mafb (kreisler) 22 . The lazarus (lzr) mutant is defective for krox20 expression in r3 and r5, but then recovers expression in r5 at 15 hours post-fertilization (hpf) 23 . lzr encodes a novel Pbx factor, which together with the Hox segmentation factors orchestrates the vertebrate body plan 23 . Manipulation of the normal egg cell cycle, with heat shock (HS) or early hydrostatic pressure (EP), contributes to the creation of gynogenetic diploid embryos 24 (BOX 1, figure panel c). EP has been used successfully in a screen for primary motor-neuron mutants, which can be visual- ized with anti-motor-neuron antibodies at 24 hpf (REFS 24?26). Primary motor neurons are the earliest developing spinal motor neurons, and have distinct axon projections and pathways. The mutant stumpy (sty) extends the Caudal Primary motor neuron (CaP) along common but not CaP-neuron-specific pathways 24,25 . EP-treated clutch- es from F 1 females that are heterozygous for sty show Mendelian inheritance (50% wild type, 50% sty) and, accordingly, the sty mutation maps near to the cardiac valve, which prevents retrograde blood flow between the atrium and ventricle 13 . The jek gene encodes the enzyme UDP(uridine 5?-diphosphate)- glucose dehydrogenase, which is required for the pro- duction of (among other products) glycosaminogly- cans 14 . Valve formation therefore seems to require Jekyll function in a new glycosaminoglycan-dependent signalling pathway. Several of the mutant phenotypes identified in the screens resemble human genetic disease conditions, and some of the genes cloned have homologues in other ani- mal systems. Anaemic mutants (scored by their low blood-cell counts) can be models for human blood dis- eases 15?17 . Positional cloning of the weissherbst (weh) mutant identified a mutation in a novel iron exporter, Ferroportin 1 (REF. 18; FIG. 2b). Blood mutants that are analogous to erythropoietic porphyria syndromes have also been found, such as the dracula (drc) and yquem (yqe) mutants, which have a rapid, light-dependent lysis of red blood cells 15,16 . The drc gene encodes +/+ x ENU-treated male P +/+ x +/m F 1 +/m x x x +/m F 2 Random sibling crosses Raise families (50% +/+, 50% +/m) 25% +/+ 50% +/m 25% m/m F 3 Figure 1 | Outline of large-scale F 2 genetic screens. In F 2 screens, a mutagen, such as ethylnitrosourea (ENU), is used to generate hundreds of point mutations in the male pre- meiotic germ cells (spermatogonia). ENU-treated males are crossed to wild-type females to produce the F 1 heterozygous progeny. F 1 fish are then crossed to siblings to create F 2 families, half of which are genotypically heterozygous for a specific mutation (m), whereas the other half are wild type. F 2 siblings are crossed, and the resulting F 3 progeny are 25% wild type (+/+), 50% heterozygous (+/m) and 25% homozygous (m/m) for a recessive mutation. Together, the Boston and T�bingen screens, starting from about 300 ENU founder males, involved raising more than 5,000 F 2 families, analysing more than 6,000 mutagenized genomes and selecting more than 2,000 new developmental mutants for characterization. � 2001 Macmillan Magazines Ltd 958 | DECEMBER 2001 | VOLUME 2 www.nature.com/reviews/genetics REVIEWS CHIASMA INTERFERENCE The inhibition of crossover events during meiosis such that there is generally only one crossover event per chromosome arm. tions that are mosaic for an estimated tenfold greater mutational load 28 . In theory, mosaic fish are able to carry this mutational burden because wild-type cells in all tis- sues compensate for the heterozygous mutant cells 28 .By screening F 2 haploids that are derived from mosaic F 1 mothers, numerous heart mutations have been success- fully identified, including many new mutations that affect cardiac induction and AP patterning 30 . This screen shows the value of mosaic screening; however, several mutations in haploid embryos and the unpredictable phenotypic representation within a clutch can compli- cate screening and allele recovery. Nonetheless, mosaic screening has proven to be a robust and rapid method to identify cardiac induction and AP-patterning mutations, a phenotypic class under-represented in the Boston and T�bingen screens 5,6,30 . Mutagen selection. The mutagen selected to generate the parental fish affects the number and types of muta- tion that are passed on to subsequent generations 31 . ENU induces mainly point mutations, and was effec- tively used in the Boston and T�bingen screens 2,3,5,6 . ENU efficiently generates mutations in most genes, although the mutation frequency can vary widely between loci. Ethyl methanesulphonate (EMS), although a potent mutagen in Drosophila, is much less potent in zebrafish 2,3 . Ionizing radiation, such as ?-rays and X-rays, also induce genetic alterations, which can centromere 25 (BOX 1). A second motor-neuron mutant was identified that also has defects in somite formation; its phenotype was caused by a new allele of the fused-somite deadly seven (des) mutation 26 . Other somite mutants also have neuronal hyperplasia, but nervous system abnor- malities had not previously been described for des 27 .By altering the search criteria from somite formation to motor-neuron projections, this new allele of des has cou- pled anterior?posterior (AP) patterning of the somites and the myotome with nervous system development 26 . So, although mutant representation varies in an EP-treat- ed clutch, EP screens provide an efficient way to uncover new mutant phenotypes in specific genetic pathways. Mosaic screens. Haploid and diploid screens can be altered to increase the frequency of mutation events of interest 28?30 . Standard ethylnitrosourea (ENU) mutagen- esis involves mutagenizing pre-meiotic germ cells in adult males, then breeding for several weeks to fix the mutation through several rounds of DNA replication before the development of mature sperm cells 2,3 . As ENU alkylates the bases on only one DNA strand, mutations in the ENU-treated post-meiotic germ cells become fixed only during cell divisions after fertilization 2,3 .The resulting F 1 generation is genetically mosaic for a specific mutation. To increase the efficiency of identifying specif- ic mutations, breeding the ENU-mutagenized males for a brief period after mutagenesis will generate F 1 genera- b wt weh wtwt hag oep wt oep ca Figure 2 | Examples of mutants identified in zebrafish large-scale screening efforts. a | Mutants for one-eyed pinhead (oep), which encodes a member of the Nodal signalling pathway, lack endoderm, prechordal plate and ventral neuroectoderm, which results in severe cyclopia (arrows denote lens position) among other defects. Lateral (top panels) and anterior?ventral (bottom panels) view of wild type (wt) and oep mutants. Reproduced with permission from REF. 78 � (1996) Company of Biologists Ltd. b | The recessive embryonic- lethal mutation weissherbst (weh) results in hypochromic blood with decreasing blood cell counts. Staining of 2-day-old embryos with O-dianisidine to visualize haemoglobin (arrows) shows reduced levels of haemoglobin in the weh mutant. Reproduced with permission from REF. 15 � (1996) Company of Biologists Ltd. c | A dominant mutation, hagoramo (hag), which results in a disrupted stripe pattern of adult fish and encodes a protein with a possible role in proteolysis, was generated by insertional mutagenesis. Reproduced with permission from REF. 37 � (2000) Elsevier Science. � 2001 Macmillan Magazines Ltd NATURE REVIEWS | GENETICS VOLUME 2 | DECEMBER 2001 | 959 REVIEWS polygenic burden of radiation-induced deletions 34 . Positional cloning of mutations generated with the above mutagens has been highly successful, and com- pletion of the zebrafish genome in 2002 will greatly facilitate positional cloning efforts 35 . Nonetheless, it remains labour intensive and expensive. vary from point mutations through large genomic dele- tions to translocation events 32,33 . Large deletions and translocations can alter more than one gene and gener- ate several phenotypes in a clutch of embryos. The chemical mutagen trimethylpsoralen includes small deletions (100 bp to 15 kb), and might circumvent the Box 1 | Haploid and homozygous diploid screens To avoid the cumbersome step of screening thousands of progeny for recessive mutations in conventional F 2 screens (FIG. 1), methods have been devised to uncover recessive alleles in a single generation by exploiting the ability to create haploid or homozygous diploid embryos. Haploid screens Recessive mutations can be revealed more quickly in zebrafish by taking advantage of their ability to survive for several days as haploid organisms 77 .In a haploid screen (a), female F 1 fish (derived from the cross between a wild-type female and an ethylnitrosourea (ENU)-mutagenized male) are squeezed gently to release their eggs, which are then fertilized with ultraviolet (UV)-treated sperm to generate haploid embryos. UV treatment destroys the parental DNA, without affecting its ability to activate the egg. A haploid clutch derived from a heterozygous female will contain 50% mutant and 50% wild-type embryos. Panel b shows 3-day-old diploid (top) and haploid (bottom) zebrafish embryos. Note that diploid and haploid embryos share a similar overall morphology, but haploid embryos are visibly shorter with abnormal eye (arrow) and otic vesicle (arrowhead) development. Homozygous diploid screens: methods to induce gynogenesis Eggs extracted from a female have completed meiosis I (the separation of homologous chromosomes) during ovulation, and initiate meiosis II (the separation of sister chromatids) on fertilization 24 . Early pressure (EP) applied to embryos during the first few minutes post-fertilization breaks down the meiotic spindle, and the egg maintains both sister chromatids (c, left). Subsequently, eggs undergo their first mitosis as diploids, with two sets of maternal chromosomes. By contrast, heat-shock treatment (HS; c, right) inhibits the first mitotic division, and eggs activated with UV-treated sperm enter the first mitotic division as haploids, abort mitosis and directly enter the second mitotic division as diploids. In meiosis, recombination and CHIASMA INTERFERENCE occur between homologous chromosomes when aligned as tetrads, so that there is, on average, a single crossover event per chromosome arm. Therefore, embryos that are derived from EP treatment will be homozygous for loci that are proximal to the crossover event that occurred at meiosis I (allele ?a? in the figure) and heterozygous for loci that are distal to it. Similar to haploid clutches, a gynogenetic diploid clutch that is derived from a heterozygous female and generated by HS will contain 50% mutant and 50% wild-type embryos. As embryos generated by HS are homozygous at all loci, they would be preferable to embryos generated by EP for use in genetic screens, except for their reportedly poor viability (10?20%). +/+ x ENU-treated male bb aa +/m UV-treated sperm P F 1 F 2 50% + 50% m BB Metaphase oocyte AA bB aa bB Meiosis I UV-treated sperm EP Meiosis II Diploid oocytes Haploid oocytes Diploid embryos Diploid embryos Haploid embryos AA bB aa bB AA bB aa bB AA Meiosis II b a bb aa BB aa HS Mitosis I B a b A B A bB aa bB AA b A B A a c b � 2001 Macmillan Magazines Ltd 960 | DECEMBER 2001 | VOLUME 2 www.nature.com/reviews/genetics REVIEWS SWIM BLADDER An internal fish organ filled with gases, which are regulated to allow the fish to rise and fall. In some teleosts, the swim bladder can have a role in respiration and sound production and reception. SOMITE Paired cubical paraxial mesodermal segment, which is often used as a staging index during embryogenesis. INVERSE PCR A technique for amplifying DNA by PCR that uses primers that initiate replication in opposite directions to each other, as compared with standard PCR, which uses primers that initiate replication towards one another. by Nancy Hopkins and colleagues 36 (in Cambridge, Massachusetts, USA; FIG. 3). Similar to the Boston and T�bingen screens, the Cambridge screen has identified genetic mutations with both specific and non-specific phenotypes 36 . Non-specific defects generally cause death and are predicted to be mutations in genes that are necessary for cell survival or growth. Some exam- ples of highly specific mutations include those that affect the pigment, fin, brain, circulation, body size and SWIM BLADDER formation. Among these, the smoothened (smo) mutant has phenotypic characteristics that are similar to Hedgehog (Hh) pathway mutants, including U-shaped SOMITES, mild cyclopia and lack of pectoral fin outgrowth 38 . Analysis of smo shows that, as in Drosophila, it is required for Hh signalling, which pro- vides new insight into the conserved and divergent roles of smo in vertebrates 38 . Two dominant alleles of hagoramo (Japanese for a dress for a goddess, hag; FIG. 2c) were also identified by their disrupted body- stripe pattern in the adult F 1 generation 36,37 . The hag gene encodes an F-box/WD40 repeat protein, which are often involved in regulating ubiquitin-mediated proteolysis 37 . Although insertional mutagenesis is only one-ninth as effective at generating mutations as ENU, disrupted genes are rapidly and easily cloned by INVERSE PCR methods. Overall, the relatively facile cloning method of insertional mutagenesis compensates for its labour intensivity, making this a powerful screening instrument in zebrafish. Inventive screening techniques The optical clarity of the zebrafish reveals developmental processes that are obscured in other organisms, such as Drosophila 5,6 . Molecular markers that highlight processes of interest, including RNA or protein staining, fluores- cence and precise measurements of organ or tissue func- tion, have unveiled zebrafish mutations that are normal- ly not visible to the eye. Creative, behaviour-based assays have also been designed in zebrafish to begin dissecting the complex workings of animal behaviour. Fluorescent reporters. Similar to mammals, zebrafish larvae process lipids throughout the intestine and hepatobiliary system, and are sensitive to drugs that block cholesterol synthesis. Farber et al. exploited this homology to design a screen that would identify mutants that are unable to correctly process phospho- lipids and cholesterol 39 . As a reporter for lipid process- ing, an engineered, quenched fluorescent moiety was placed at a phospholipase A 2 (PLA 2 ) cleavage site that is normally targeted by a phospholipid (PED6), so that cleavage resulted in unquenching and visual fluo- rescence. Normal PLA 2 cleavage of the quenched lipid (and hence lipid metabolism) resulted in immediate fluorescence of the gall bladder and the intestinal lumen in living larvae. Direct testing of pancreatic and intestinal mutants ? slim jim (slj) and piebald (pie) ? had reduced gall-bladder fluorescence, which shows that the differences in fluorescence could be detected in mutants that are abnormal for digestive organ morphology 39 . As an alternative to chemical- or radiation-induced mutations, insertional mutagenesis with a retrovirus is mutagenic and allows for rapid cloning of the gene 36?38 . Large-scale screening for recessive developmental mutations with retroviral insertions is now being done P F 1 1,000?2,000-cell stage embryos Cross- founders Select multi- insert fish ? at least 3 unique inserts Inject virus +/m +/m F 2 Random sibling crosses Raise families *Fish crosses ** * * ** * * * * * 25% +/+ 50% +/m 25% m/m F 3 +/m x x x x +/+ (with mutations in other loci) x x x Figure 3 | Outline of insertional mutagenesis screen. The goal of the Cambridge screen is to identify ~1,000 genes involved in embryogenesis 36 . The protocol for large- scale insertional mutagenesis screening involves injecting virus into 250,000 embryos at the 1,000?2,000-cell stage 36 . The virus infects many cells, several times, among them the primordial germ cells. Approximately 36,000 embryos are raised (the founder fish; P), mated and several insertions transmitted to the F 1 generations. More than 10,000 F 1 families are raised, and multi-insert F 1 fish are selected by carrying out real-time PCR and Southern blot analysis of DNA isolated from tail biopsies. Multi-insert F 1 fish are crossed to each other and 10,000 F 2 families are raised. More than six sibling matings for each F 2 family are carried out to identify ~1,000 homozygous mutations in the F 3 generation. m, mutagenized chromosome. � 2001 Macmillan Magazines Ltd NATURE REVIEWS | GENETICS VOLUME 2 | DECEMBER 2001 | 961 REVIEWS tract, which indicates a possible defect in bile synthesis or secretion. Visualizing enzymatic activity in a genetic screen of live larvae therefore identified a mutant with normal organ morphology but with a specific digestive defect in bile synthesis or secretion. Similarity between fish and humans in lipid process- ing is further underscored by the finding that the drug atorvastatin ? an inhibitor of cholesterol synthesis in humans ? blocked PED6 labelling of the gall bladder in zebrafish 39 . Future screens for mutants that bypass or exacerbate drug function, or screens for drugs that alter mutant function, will help to probe the mechanistic basis of digestive physiology and possibly human diges- tive disorders. Other fluorescent reporters have been used for screening and to directly visualize specific processes. Fluorescent dyes injected into the eyes of day 5 larvae label the entire length of the retinal axons, which can be directly visualized through the skin. Using fluorescent dyes as a marker, a screen of F 3 embryos for defects in the retinotectal pathfinding of retinal ganglion cells (RGCs) recovered the viable, recessive mutant ast (ast) 40,41 . Eye transplantation experiments, coupled with fluorescent monitoring of retinal axons, showed ast activity to be required in the eye and not in the brain 42 . Interestingly, ast mutants show no visual-behaviour defects 43 (see below). The ast phenotype is caused by a mutation in the zebrafish roundabout (robo2) gene, which is expressed in the extending axons of RGCs 42 . Roundabout proteins belong to a family of axon-guid- ance receptors that bind specific ligands to guide axons to their destinations through a complex environment. So, the use of fluorescent dyes has uncovered an eye- autonomous gene that is required for RGC pathfinding and has provided new insight into the molecular basis of vertebrate visual projection. By linking green fluorescent protein (GFP) to genes or promoters of interest, it is possible to visual- ize normally obscured processes. Spatial and temporal expression patterns, and the development of embry- onic haematopoiesis, can be visualized in living zebrafish by the expression of GFP from the promoter of the haematopoietic transcription factor gata1 (REF. 44). Other tissue-specific promoters that have been linked to GFP include the skin-specific type II cytokeratin (CK) promoter and the muscle-specific muscle creatine kinase (mck) promoter 45 . These, and other fluorescent-based tools, provide important markers for monitoring the development of specific tissues and will be valuable reagents when screening for mutants that perturb normal gene expression, protein abundance and tissue development. Behaviour screens. Genetic screens in zebrafish can be designed to reveal mutations not only in developmental and physiological processes, but also in behavioural activities and movement. Normal 2?3-day-old larvae are not very active, but will swim rapidly away from a stimulus (such as a needle) applied to the tail. As part of the first, large-scale zebrafish screens, Granato et al. 46 selected embryos that were defective in the touch In a pilot screen, new mutants that were defective for digestive organ function were revealed using the fluores- cent lipid reporter 39 (FIG. 4).F 3 embryos that arose from crosses in F 2 families were bathed in PED6 and screened for fluorescence. One mutation, called fat free, has nor- mal digestive organ morphology, but with greatly reduced gall-bladder fluorescence (FIG. 4b). Although several other digestive mutants were found with reduced fluorescence, fat free was the only mutation that was defective for specific organ function but not mor- phology, and therefore would not have been identified in a screen based on organ morphology alone. Assays to measure the enzymatic activity of PLA 2 determined that, although there was less overall PLA 2 activity, PLA 2 remained active in fat free animals. Using another fluo- rescent lipid ? a cholesterol analogue that requires bil- iary emulsification for absorption ? fat free showed very weak fluorescence in the gall bladder and digestive Larvae ingest engineered phospholipid a bc 25% +/+ 25% m/m 50% +/m +/m x +/m fat-free wt fat-free wt Figure 4 | Fluorescent reporter screens. a | Fluorescent reporters can be used to screen for mutations in specific enzymatic processes in live embryos 39 . For example, larvae derived from crossing two fish that are heterozygous for a specific digestive tract mutation (for example, fat free, see part b) ingest phospholipids that are engineered to fluoresce during normal lipid processing. One-quarter of the larvae are homozygous for a recessive, digestive-tract mutation and lack gall-bladder fluorescence (arrow), which indicates a defect in normal lipid processing. b,c | The larval digestive mutant fat free is defective for lipid processing. Wild type (wt) and fat free mutants were bathed in a type of phospholipid (PED6) that was engineered to fluoresce when cleaved by the phospholipase (PLA 2 ) enzyme (for details, see REF. 39). Wild- type day 5 larvae show intense gall-bladder fluorescence (arrowhead in b), whereas fat free day 5 larvae show severely reduced gall-bladder fluorescence. Note that under normal lighting conditions (c), the digestive tract appears normal in fat free mutant larvae. m, mutation. Panel b is reproduced with permission from REF. 39 � (2000) American Association for the Advancement of Science. � 2001 Macmillan Magazines Ltd 962 | DECEMBER 2001 | VOLUME 2 www.nature.com/reviews/genetics REVIEWS TECTUM The dorsal portion of the midbrain (mesencephalon) that mediates reflexive responses to visual and auditory stimuli. DOPAMINERGIC INTERPLEXIFORM CELL (DI-IPC). Residing in the inner nuclear layer of the retina, this type of cell releases dopamine to regulate light adaptation in the retina. the OMR test by not accumulating at one end of the channel, were re-examined by electroretinography and by histology 43,48 . These secondary assays allowed the authors to distinguish between sight-specific and sight- non-specific mutations that might cause a fish to fail one or both of the tests 43 (such as a locomotive defect). By using previously identified developmental mutants, this screen questioned if the genes that are required for development might overlap with those required for visual-behaviour responses. At least 25 developmental mutants were found to have defects in the normal visual-behaviour response to stimuli 43 . Interestingly, mutants that failed the OKR and OMR tests were not restricted to defects in a specific develop- mental process. Small- or large-eye mutants, microps (mic) and blowout (blw) respectively, passed the OKR and OMR tests, indicating that eye morphology might be controlled by a different set of genes than visual per- formance 43 . Interestingly, darkly pigmented fish fre- quently failed the visual-behaviour response tests. In a light environment, melanin in the melanophore aggre- gates and the animal appears pale, whereas in darker surroundings melanin is dispersed and the animal dark- ens. Thirteen mutants with dispersed pigment were also defective for both the OKR and OMR, probably because they were unable to visually process their surrounding environment 43 . As this adaptation response is controlled by a projection from the retina to the hypothalamus and is a neuroendocrine process, the dark fish might be black as a secondary response to being blind. For exam- ple, the dark fish lakritz (lak) (German for liquorice) failed both the OKR and OMR, has a reduced number of RGCs and has a markedly smaller optic nerve 43 .Some of the zebrafish mutants resemble human disease phe- notypes and 13 of the sight mutants involved retinal degeneration ? the most common cause of hereditary blindness in humans. Fish might share genetic similarity with humans in eye development and prove a valuable model for studying human eye disorders. Using a behavioural assay based on the zebrafish escape response (FIG. 6) in an F 1 screen for dominant defects in visual sensitivity to light, revealed an unusual mutation called night blindness b (nbb) 50 . Subsequent experiments showed that an increased visual threshold in nbb +/? fish is related to a reduction of DOPAMINERGIC INTERPLEXIFORM CELLS (DA-IPCs) in the inner retina 50 . Supporting this conclusion, infusion of the dopamine inhibitor 6-hydroxydopamine (6-OHDA), which kills DA-IPCs, phenocopies the nbb +/? phenotype 50,51 . Importantly, nbb +/? animals carry an unexpected defect in the neural retina connections with the centrifugal fibres originating from the terminal nerves of the olfac- tory bulb 50 . Underscoring the biological significance of this neuronal connection defect, the nbb +/? phenotype can be mimicked by excising the olfactory epithelium and bulb. Innervation of the retina with olfactory cen- trifugal fibre connections might therefore be an impor- tant DA-IPC regulatory mechanism, and therefore con- trol dark-adapted visual sensitivity. So, a subtle assay for visual adaptation mutants has shown unexpected con- nections between distinct neurological pathways. response. One mutant, nevermind (nev), rotates around its long body axis and displays a corkscrew swimming pattern. Although the muscle morphology is normal, the dorsal retinotectal axon projections in nev are abnormal, terminating on both the dorsal and ventral side of the TECTUM 40,46 . Another mutant, space cadet (spc) shows specific abnormalities in the fast-turning escape response, and will also spontaneously undergo escape- response turning while swimming 46,47 (FIG. 5). The spc gene is required for the axonal pathfinding of a subset of commissural axon trajectories that control locomotive behaviour 47 . So, a simple touch test has identified com- plex mutant phenotypes that link locomotion with neu- ronal development defects. More recently, screens have been developed to moni- tor behaviour in response to a visual stimulus, revealing genes that control vertebrate visual-behaviour respons- es. Neuhauss et al. conducted a ?shelf screen? and re- examined more than 400 zebrafish developmental mutants from the T�bingen screen for abnormal behav- ioural responses to visual stimuli 43,48 . To monitor their optokinetic response (OKR), a black and white drum was rotated slowly around zebrafish larvae that had been partially immobilized in a Petri dish, and their eye movements were recorded 43,48 . Normal fish eyes will smoothly scan in the direction of the stripes (clockwise or counter-clockwise) and, then, in a rapid eye move- ment, reset to the midline before following the stripes again 48 . The optomotor response (OMR) was measured by placing day 5 larvae at one end of transparent, long, thin chambers, that were placed on top of, and at right angles to, black and white stripes that move across the computer screen 43 . Wild-type fish swim in the direction of the motion and accumulate at the opposite end of the channel. Mutants with an abnormal OKR, or who failed A1 0 ms A2 3 ms A3 5 ms A4 9 ms A5 14 ms A6 20 ms B1 0 ms B2 9 ms B3 12 ms B4 18 ms B5 35 ms B6 43 ms B7 54 ms B8 62 ms B9 74 ms B10 79 ms B11 87 ms B12 98 ms Figure 5 | The space cadet locomotion mutant. The space cadet mutant shows abnormal swimming behaviours during a stimulus-induced escape response. High-speed camera images capture the C-shaped bend of a wild-type larva as it rapidly moves its head away from the stimulus source (A1?A4; the timing of each frame is given, in milliseconds (ms)). The larva then makes a less powerful counterturn (A5, A6), before rapidly swimming away from the source (not shown). In response to a stimulus, the space cadet larva makes a C-shaped bend away from the stimulus in a similar manner to wild-type larvae (B1?B4), but has a poor counterturn (B5, B6). It then initiates a second turn towards the same side (B7?B9), before swimming away using a series of fast, bilateral tail flexures. A movie of the swimming defects of space cadet can be viewed at http://dev.biologists.org/cgi/content/full/128/11/2131/DC1 and http://www.nature.com/nsu/010607/010607-1.html. Modified with permission from REF. 47 � (2001) Company of Biologists Ltd. � 2001 Macmillan Magazines Ltd NATURE REVIEWS | GENETICS VOLUME 2 | DECEMBER 2001 | 963 REVIEWS TELEOST Ray-finned bony fishes. TURBIDOMETRY A way to measure the solution turbidity, this can be used to assay for the formation of fibrin in the form of visible clotting in plasma. Biochemical blood screens. In mammals, coagulation requires the activation of factor VII to initiate a cascade of proteases that eventually lead to the cleavage of fib- rinogen and to fibrin clotting. Similarities between mammals and TELEOSTS have recently been reported to extend to the blood coagulation pathway with the iden- tification of factor VII in zebrafish 54 . Using new bio- chemical assays, adult F 2 -generation EP-generated diploids were screened for clotting defects in zebrafish, by clipping the tail of individual fish (the tail regenerates within a few weeks), collecting small amounts of blood and testing for the conversion of human fibrinogen to fibrin by TURBIDOMETRY 55 . Eight mutants were found that had prolonged clot formation. To further characterize which part of the coagulation pathway was defective, reconstitution assays for the extrinsic, intrinsic and common pathways were developed 55 . It is expected that these biochemical advances will provide a fruitful genet- ic means by which to explore the homology between zebrafish and human coagulation pathways. Screening at high magnification. During retinal devel- opment, unspecified neuroepithelium progresses to a highly organized, laminated structure of differentiated retinal cells. The transition from multipotent precursor cells to fully mature retinal cells requires cells first to withdraw from the cell cycle and undergo specification (marked by migration to the appropriate laminar posi- tion and distinct gene expression), followed by differ- entiation and maturation (marked by morphological and biochemical changes). Lamination, a hallmark of normal retinal development, can be clearly viewed at high resolution. Using a magnification of �200, differ- ential interference contrast (DIC) microscopy was used to screen F 3 -generation day 4 embryos derived from ENU-mutagenized founders for defects in retinal lami- nation 56,57 .Two young (yng) alleles were identified that were blocked for the transition from specification to differentiation 56 . Two mutants, perplexed (plx) and con- fused (cfs), were unable to exit the cell cycle and to become post-mitotic retinal cells 57 . Although yng, plx and cfs are seen to have slightly smaller eyes later in development, screening with microscopy for retinal development defects has identified subtle mutants that are defective for key transitions in retinal histogenesis. Finally, high-resolution DIC time-lapse video has also been reportedly used to visualize details of nuclear movement in live embryos 58 , which is suggestive of a new screening tool in zebrafish. Dissecting genetic pathways in zebrafish Altering an initial phenotype, through genetic or synthet- ic means, can provide information about gene function and gene order in pathways. Genetic screens for sec- ondary mutations that suppress or enhance a phenotype are an exciting next step in zebrafish genetics, and some are already underway. A serendipitous recessive enhancer of cyclops (cyc), called squint (sqt), was identified in specif- ic genetic backgrounds that enhanced the severity of mesoendoderm formation 59 . sqt, like cyc, encodes a Nodal-related signalling protein 59 . Enhancer mutations in Zebrafish show many other behavioural responses that are amenable to genetic screening, including complex social behaviours, such as schooling and ter- ritorial responses (see REF. 52 and references therein). In response to low levels of alcohol, aggressive and locomotive behaviours are increased, whereas higher levels of alcohol have inhibitory effects; screening is already underway for mutations that alter the behav- ioural effects of alcohol on zebrafish 52 . Cocaine also alters zebrafish behaviour and zebrafish will show a preference for a chamber supplemented with cocaine over an adjoining chamber without cocaine 53 (called conditioned place preference, CPP). The genetics of cocaine addiction have been probed in an F 2 -genera- tion screen for zebrafish with altered, cocaine- induced CPP. Three dominant mutants, dumbfish (dum), jumpy (jpy) and goody-two-shoes (gts), are insensitive to cocaine and could provide insight into the normal dopaminergic signalling in the brain and addiction-related behaviours 53 . P +/M +/+ F 1 +/m +/+ x ENU-treated male Figure 6 | Behaviour screens: visual adaptation mutants. A behavioural test can be a measure of visual sensitivity and be used to screen for subtle, eye-specific mutations in adult zebrafish. Adult F 1 -generation fish derived from ethylnitrosourea (ENU)-mutagenized founders are placed in a transparent container that is surrounded by a rotating drum marked with a black square that represents a threatening object 49,50 . After initial dark adaptation, normal zebrafish rapidly ?escape? the threatening object in light above (but not below) their visual threshold (right drum). By contrast, fish that are heterozygous for the mutation night blindness b show the escape response at a visual threshold that is 2?3 log units above the average 50 . M, dominant mutation on an ENU- mutagenized chromosome. � 2001 Macmillan Magazines Ltd 964 | DECEMBER 2001 | VOLUME 2 www.nature.com/reviews/genetics REVIEWS HOLOPROSENCEPHALY Failure of the forebrain (prosencephalon) to divide into hemispheres or lobes, often accompanied by a deficit in midline facial development. RNA CAGING RNA inactivation through the covalent attachment of a photo- removable synthetic compound called the caging group. RNA is reactivated by photo- illumination with a specific light wavelength. GAL4/UAS SYSTEM A genetic system for controlling the induction of gene expression. An activator line that expresses the yeast transcriptional activator GAL4 gene under the control of the heat-shock 70 promoter (hsp70) or a tissue-specific promoter is crossed to an effector line that carries the DNA-binding motif of Gal4 (UAS) fused to the gene of interest. As a result, the progeny of this cross expresses the gene of interest in an activator- specific manner. severe embryonic defects and death, indicating that non-conditional alleles of reg6 might have defects in normal embryogenesis 61 . Zebrafish geneticists have a range of tools that can be used in conjunction with genetic mutations to confirm and explore gene function, to study maternal versus zygotic gene usage and to link genetic path- ways. Morpholino (MOs) gene-knockdown technol- ogy has been recently developed in zebrafish 62 . MOs are antisense, chemically modified oligonucleotides that inhibit translation in a specific manner, and have been used to show genetic interactions 62 . For exam- ple, sonic hedgehog (shh) mutations cause HOLOPROS- ENCEPHALY in humans 63 , but zebrafish shh does not have dorsal?ventral patterning defects 64 . MOs of both shh and tiggy-winkle hedgehog (twhh), a shh homo- logue that has no MO phenotype, act synergistically to produce strong dorsal?ventral patterning defects, indicating that shh and twhh in zebrafish might func- tion together to carry out the function of shh in mammals 62 . The strength of this technique is under- scored by the number of genes that have been subject to MOs, including blood and pigment genes, and genes that are involved in embryonic patterning, angiogenesis and brain development 65 (FIG. 7). Ectopic gene-expression methods have been used to place genes in genetic pathways. Notably, forced expres- sion (by mRNA injection) of the haematopoietic stem- cell transcription factor gene scl, or the haematopoieti- cally expressed homeobox gene hhex, can partially rescue the blood and endothelial deficiencies of cloche (clo), placing scl and hhex downstream of clo 66,67 . Other overexpression techniques include RNA CAGING and the GAL4/UAS SYSTEM, which allow for specific control over the time and tissue in which a gene is expressed 68?70 .Control of gene expression in individual cells has also been achieved by exploiting the optical clarity of the embryo to focus a laser microbeam or beam of light onto specif- ic cells, thereby activating expression of a transgene or uncaging RNA molecules, respectively 68,71 . Another way to identify and clone genes, and to place them into functional pathways, relies on looking for genes with informative expression patterns. This technique, called whole-mount in situ hybridization is described in BOX 2. Integration of Tc3, a member of the Tc1/mariner/ Sleeping Beauty family of transposons is another advancing technology that provides the basis for trans- poson-mediated genetic transformation and inducible removal of DNA from integrated constructs 72 . In addi- tion, the recent production of germ-line chimaeras from zebrafish embryo cell cultures is progress towards achieving targeted gene inactivation in the zebrafish 73 . Among the many useful applications of such technolo- gy is a reverse genetic approach to screening. Small molecules can also generate specific pheno- types in zebrafish. The zebrafish CHORION is permeable to small molecules, and is amenable to high-through- put screening for chemicals that specifically inhibit developmental processes and mimic genetic muta- tions. Unlike gene disruptions, chemicals can be zebrafish can therefore identify genes that are involved in the same genetic process, and this example validates the use of zebrafish for suppressor/enhancer-based screens. Allele specificity can also assist in dissecting a genet- ic pathway. Stronger or weaker alleles can be generated by crossing an F 1 generation from an ENU-treated founder with a heterozygous mutant, and by screening for non-complementing alleles. This has been success- fully done to generate stronger alleles of sonic you (syu), you-too (yot), fused somites (fss) and unplugged (unp), simultaneously. Van Eeden et al. 60 crossed fish that were heterozygous for syu, yot, fss and unp to the F 1 genera- tion of ENU-mutagenized fish. To ensure that the F 1 ENU fish were not mixed up with the heterozygous fish, the F 1 ENU fish carried homozygous leopard (leo) mutations that cause spotted pigmentation, and a dominant long fin (lof) mutation that causes long fins, thereby allowing easy separation of fish after mating 60 . One application of this technique might be to generate non-lethal hypomorphic alleles of embryonic-lethal mutants that could be subsequently used in suppres- sor/enhancer screens. Temperature-sensitive (ts) alleles can be used to reveal stage-specific gene function. For example, an EP screen for adult fish with ts defects in fin regeneration identified several regeneration (reg) mutants, including reg6 ? a mutant that is defective in the early outgrowth stage of regeneration 61 . At the permissive temperature, reg6 mutants regenerate fins normally, in contrast to the non-permissive temperature, at which reg6 mutants develop misshapen fin rays and dysmorphic blood- filled growths on the regenerating fins. reg6 embryos raised at the non-permissive temperature result in a Control c Control b urod-MO d urod-MO Figure 7 | Morpholino-induced antisense phenotypes. An example of morpholino (MO) targeting of the uroprophyrinogen decarboxylase (urod) gene phenocopies the urod loss-of- function mutant, called yquem (yqe). b, d | As in the yqe mutant, the urod-MO causes porphyria phenotypes similar to those seen in humans, including red-blood-cell autofluorescence in ultraviolet light (b), and rapid lysis when exposed to light (d). Panels a and c show sibling embryos to those shown in (b, d), injected with a control MO. Reproduced with permission from REF. 62 � (2000) Macmillan Magazines Ltd. � 2001 Macmillan Magazines Ltd NATURE REVIEWS | GENETICS VOLUME 2 | DECEMBER 2001 | 965 REVIEWS CHORION An extraembryonic membrane that surrounds the zebrafish embryo during the first 2 days of development. OTOLITH One of the small particles of calcium carbonate in the sacculus of the inner ear. Pressure of the otoliths on the hair cells of the macula (the most sensitive area of the ear) provide sensory inputs about acceleration and gravity. Practical considerations and conclusion Designing a genetic screen in zebrafish involves balanc- ing the screen parameters that best allow mutant detec- tion with those that are manageable in individual labo- ratories. Zebrafish genetic screens are also labour intensive, requiring many hours of fish husbandry, preparation, screen processing and mutant re-identifi- cation. Owing to the large space and time requirements that are necessary for large-scale screening of F 3 diploids, our laboratory has carried out large-scale screening for blood mutants in collaboration with the N�sslein-Volhard lab. By comparison, in our own fish facility, we have carried out several small-scale haploid and EP screens simultaneously, which are managed by individuals or small teams of investigators. The Boston and T�bingen screens discovered thousands of mutants, including alleles of previously identified mutants, but even so, did not reach saturation. From the first large-scale screen, members from our lab have contributed to finding 32 blood mutants, which formed 17 complementation groups 15 , whereas in our small-scale screens (covering ~600 genomes), we recover up to ten mutants, most being single alleles. We generally invest 1 year for our small-scale screens: about 6 weeks to 2 months to generate ENU-mutagenized males (founders), ~3?5 months for F 1 ENU females to be primed for egg extraction, and at least 3 months to initiate several sibling crosses to re-identify mutants in the F 2 generations. With the high homology between zebrafish and human genes, and conserved gene order (synteny), genetic screens in zebrafish provide a powerful means to elucidate the complexities of development and dis- ease. Genetic and radiation hybrid maps, and the soon- to-be-completed zebrafish genome sequence, are con- tinually advancing the ease of cloning mutated genes of interest. Although practical considerations shape screen design, as the screens described here show, imaginative screens can, and will continue to divulge gene functions and illuminate the intricacies of gene networks. applied during specific windows of time in develop- ment, bypassing effects in early development that might lead to gross abnormalities or death. Chemicals can also potentially interfere with one or more targets, which circumvents problems with genetic redundancy in a particular pathway. One molecule, called 31N3, disrupts OTOLITH formation in the ear in a temporally specific manner 74 . 31N3 is potent even at very low doses, and related compounds with only minor chemi- cal modifications have no effect on otolith formation 74 . Another molecule, called concentramide, mimics a heart and soul (has) mutant that is defective in heart- chamber morphogenesis 75 . Both the has mutation and concentramide allow for chamber formation, but the ventricle is abnormally formed in the atrium. Interestingly, has mutations and concentramide seem to operate in different molecular pathways: has encodes PKC? (protein kinase C?), which is required for cell polarity, epithelial layer integrity and spindle orientation 75,76 , and concentramide disrupts AP pat- terning in the heart 75 . So, a combined genetic and chemical approach has identified a pivotal, previously uncharacterized, developmental step in heart develop- ment. Future chemical-based screens could identify other compounds that might suppress, enhance or otherwise alter specific mutant phenotypes, laying the foundation for future drug design. Box 2 | Complementing the genetic screen: in situ-based screens Whole-mount in situ hybridization (WISH)-based screens reveal expression patterns for new genes and known genes and can be a complement to the genetic screen (C. Thisse and B. Thisse, personal communication). Using cDNA libraries from organ- or stage-specific embryos, WISH reveals genes with similar expression patterns that can be grouped as potentially working in the same genetic pathway. Genes with interesting expression patterns are directly sequenced, and mapped, and their map position compared with the locations of ethylnitrosourea-generated mutants. This technique has been used to clone casanova 7 , and chardonnay (cdy)a haematopoietic divalent metal iron transporter (DMT1; A. Donovan, L.Z., C. Thisse and B. Thisse, personal communication). 1. Streisinger, G. et al. Production of clones of homozygous diploid zebrafish (Brachydanio rerio). Nature 291, 293?296 (1981). A landmark paper in the zebrafish field reveals that zebrafish are suitable for genetic analysis and screening. 2. Solnica-Krezel, L. et al. Efficient recovery of ENU-induced mutations from the zebrafish germline. Genetics 136, 1401?1420 (1994). 3. Mullins, M. C. et al. 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Alexander, J. et al. Screening mosaic F1 females for mutations affecting zebrafish heart induction and patterning. Dev. Genet. 22, 288?299 (1998). 31. Knapik, E. W. et al. ENU mutagenesis in zebrafish?from genes to complex diseases. Mamm. Genome 11, 511?519 (2000). 32. Chakrabarti, S. et al. Frequency of ?-ray induced specific locus and recessive lethal mutations in mature germ cells of the zebrafish, Brachydanio rerio. Genetics 103, 109?123 (1983). 33. Walker, C. & Streisinger, G. Induction of mutations by ?-rays in pregonial germ cells of zebrafish embryos. Genetics 103, 125?136 (1983). 34. Ando, H. et al. Efficient mutagenesis of zebrafish by a DNA cross-linking agent. Neurosci. Lett. 244, 81?84 (1998). 35. Talbot, W. S. & Schier, A. F. Positional cloning of mutated zebrafish genes. Methods Cell Biol. 60, 259?286 (1999). 36. Amsterdam, A. et al. A large-scale insertional mutagenesis screen in zebrafish. Genes Dev. 13, 2713?2724 (1999). 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Behavioral screening for cocaine sensitivity in mutagenized zebrafish. Proc. Natl Acad. Sci. USA 98, 11691?11696 (2001). 54. Sheehan, J. et al. Demonstration of the extrinsic coagulation pathway in Teleostei: identification of zebrafish coagulation factor. Proc. Natl Acad. Sci. USA 98, 8768?8773 (2001). 55. Jagadeeswaran, P. et al. Haemostatic screening and identification of zebrafish mutants with coagulation pathway defects: an approach to identifying novel haemostatic genes in man. Br. J. Haematol. 110, 946?956 (2000). 56. Link, B. A. et al. The zebrafish young mutation acts non- cell-autonomously to uncouple differentiation from specification for all retinal cells. Development 127, 2177?2188 (2000). 57. Link, B. A. et al. The perplexed and confused mutations affect distinct stages during the transition from proliferating to post-mitotic cells within the zebrafish retina. Dev. Biol. 236, 436?453 (2001). 58. Herbomel, P. Spinning nuclei in the brain of the zebrafish embryo. Curr. 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Positional cloning of heart and soul reveals multiple roles for PKC? in zebrafish organogenesis. Curr. Biol. 11, 1492?1502 (2001). 77. Walker, C. et al. Haploid screens and ?-ray mutagenesis. Methods Cell Biol. 60, 43?70 (1999). 78. Schier, A. F. et al. Mutations affecting the development of the embryonic zebrafish brain. Development 123, 165?178 (1996). Acknowledgements We thank members of the Zon laboratory for helpful discussions and critical reading of the manuscript. E.E.P. is funded by a long- term postdoctoral fellowship from the Human Frontier Science Program. L.I.Z. is funded by the Howard Hughes Medical Institute and by a National Institutes of Health grant. Online links DATABASES The following terms in this article are linked online to: LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/ CK | factor VII | Ferrochelatase | Ferroportin 1 | fibrinogen | gata1 | Hh | Mafb | mck | Nodal | shh | twhh | UDP-glucose dehydrogenase | UROD Medscape Drug Info: http://promini.medscape.com/drugdb/search.asp atorvastatin OMIM: http://www.ncbi.nlm.nih.gov/Omim/ erythropoietic protoporphyria | hepatoerythropoietic porphyria ZFIN: http://zfin.org/cgi-bin/ZFIN_jump?record=JUMPTOGENE ast | blw | bon | cas | cfs | cdy | clo | cyc | des | drc | fau | fss | hag | has | jek | krox20 | lak | leo | lof | mic | nbb | nev | oep | pie | plx | reg | reg6 | slj | smo | spc | sqt | sty | syu | unp | val | weh | yot | yng | yqe FURTHER INFORMATION Encyclopedia of Life Sciences: http://www.els.net Zebrafish as an experimental organism Zebrafish anatomy guide: http://zebrafish.mgh.harvard.edu/anatomy.html ZFIN (Zebrafish Information Network): http://zfin.org Zon lab: http://genetics.med.harvard.edu/~zonlab Access to this interactive links box is free online. 966 | DECEMBER 2001 | VOLUME 2 www.nature.com/reviews/genetics REVIEWS � 2001 Macmillan Magazines Ltd "
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Genetics
Gene Inheritance and Transmission
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Cell Origins and Metabolism
Proteins and Gene Expression
Subcellular Compartments
Cell Communication
Cell Cycle and Cell Division
Scientific Communication
Career Planning
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