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The Art and Design of Genetic Screens: Zebrafish

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The Art and Design of Genetic Screens: Zebrafish REVIEWS 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 Twenty years ago, George Streisinger’s pioneering First large-scale vertebrate genetic screens Development of an organism research revealed the potential of the zebrafish as a ver- Systematic genome-wide screens for mutations in derived from the genetic tebrate organism that was suitable for forward genetic worms, flies and plants have successfully identified material of the female gamete. screening1. A relatively small fish (3–4 cm long as an many genes that define embryological pathways. adult), zebrafish can be easily managed in large num- Smaller collections of mammalian mutants provide bers in the laboratory environment. The ability to com- valuable insights into developmental processes. bine embryological and genetic methodology has However, identifying large numbers of mutations in established the zebrafish as a powerful research tool. the mammalian system is problematic because of External development of transparent embryos allows intrauterine development and expensive supporting fundamental vertebrate developmental processes — laboratory facilities. The remarkable characteristics of from gastrulation to organogenesis — to be visualized the zebrafish, along with the initial success of the first and studied; in addition, the heart beats and blood cir- zebrafish genetic screens4, inspired two groups of scien- culation of the embryo are readily observed. Touch, tists in Boston5 and Tübingen6 to undertake the first sight and behavioural responses can also be monitored large-scale genetic screens in a vertebrate organism. in live embryos under the dissecting microscope. The Boston and Tübingen screens identified mutant Several features, such as a short generation time of 3–4 embryonic phenotypes in the F3 generation (FIG. 1). months, mean that zebrafish are particularly suitable Some of the ~2,000 mutated developmental genes that for genetic studies. In addition, mutations can be were identified in these two screens have been cloned, induced with high frequency in zebrafish, and recessive which assists in the dissection of the gene networks that mutations can be recovered within two generations2,3. control early development. For example, the genes that Large progeny sizes (females lay about 100–200 eggs) are mutated in the endoderm mutants casanova (cas), facilitate large-scale genetic screening and mutation bonnie and clyde (bon), and faust (fau) can be assem- Howard Hughes Medical analysis in a Mendelian fashion. bled into a genetic pathway, and have been shown to Institute, Children’s Hospital of Boston, Here, we review the methods of genetic screening in encode transcription factors that are necessary for 300 Longwood Avenue, zebrafish, and include some examples of the resulting endoderm formation7–12. Embryo transparency, as well Enders 750, Boston, mutants and genes discovered from diploid, haploid as conspicuous heart, blood and blood vessels in the Massachusetts 02115, USA. and GYNOGENETIC diploid screens. We follow with a sam- zebrafish have also made it possible to identify cardio- Correspondence to L.I.Z. e-mails: epatton@enders. pling of the recent, resourceful genetic screening tech- vascular system mutants in a manner that is unprece- tch.harvard.edu; niques now carried out in zebrafish to show the poten- dented in other animal systems. As an example, the [email protected] tial and versatility of the zebrafish genetic system. gene jekyll (jek) is necessary for formation of the 956 | DECEMBER 2001 | VOLUME 2 www.nature.com/reviews/genetics © 2001 Macmillan Magazines Ltd REVIEWS P Ferrochelatase, which, when disrupted in humans, leads x to erythropoietic protoporphyria owing to the accumu- lation of light-sensitive, toxic intermediates of the haem +/+ ENU-treated male biosynthetic pathway19. The yqe gene encodes uropor- phyrinogen decarboxylase (UROD), and as in the fish, F1 homozygous mutations in this enzyme lead to hepato- x erythropoietic porphyria in humans20. The wealth of knowledge gained from just a sampling of the phenotyp- +/+ +/m ic groups of mutants identified in the Boston and F2 Tübingen screens illustrates the power and potential of x zebrafish genetics, and validates the use of zebrafish as a model for vertebrate biological processes. Raise x families (50% +/+, Haploid and homozygous diploid screens. Phenotypic 50% +/m) detection of a recessive allele within an F3 generation x requires many crosses in F2 families (FIG. 1). Streisinger et al. attempted to eliminate this ‘cumbersome’step by cre- +/m +/m ating haploid and PARTHENOGENETIC diploid animals that Random sibling crosses were suitable for use in genetic screens1 (BOX 1).A geneti- cally convenient feature of zebrafish is that they can live up to 3 days post-fertilization as haploid organisms1. F Recessive alleles in F1 fish are therefore exposed within a 3 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- 25% +/+ 50% +/m 25% m/m er cells than diploids, and can have other morphological Figure 1 | Outline of large-scale F genetic screens. In F 2 2 defects that complicate screening (BOX 1, figure panel b). screens, a mutagen, such as ethylnitrosourea (ENU), is used to generate hundreds of point mutations in the male pre- Nevertheless, molecular markers that are essentially nor- meiotic germ cells (spermatogonia). ENU-treated males are mal in wild-type, haploid embryos can be used success- fully in haploid screens to identify genes that are crucial crossed to wild-type females to produce the F1 heterozygous progeny. F1 fish are then crossed to siblings to create F2 for the early stages of zebrafish embryogenesis. For families, half of which are genotypically heterozygous for a example, a haploid screen successfully uncovered genes specific mutation (m), whereas the other half are wild type. that were involved in brain patterning21. Haploid F siblings are crossed, and the resulting F progeny are 25% 2 3 embryos from F females were screened simultaneously wild type (+/+), 50% heterozygous (+/m) and 25% 1 homozygous (m/m) for a recessive mutation. Together, the for the expression pattern of six genes in the brain by 21 Boston and Tübingen screens, starting from about 300 ENU RNA in situ hybridization . Three valentino (val) alleles founder males, involved raising more than 5,000 F2 families, had atypical krox20 expression, as well as abnormally analysing more than 6,000 mutagenized genomes and smooth and unsegmented hindbrains.val is required for selecting more than 2,000 new developmental mutants for the subdivision and expansion of the hindbrain region characterization. that normally gives rise to RHOMBOMERES (r) 5 and 6,and is a homologue of the mouse segmentation-factor-encod- cardiac valve, which prevents retrograde blood flow ing gene Mafb (kreisler)22. The lazarus (lzr) mutant is between the atrium and ventricle13. The jek gene defective for krox20 expression in r3 and r5, but then encodes the enzyme UDP(uridine 5′-diphosphate)- recovers expression in r5 at 15 hours post-fertilization glucose dehydrogenase, which is required for the pro- (hpf)23. lzr encodes a novel Pbx factor, which together duction of (among other products) glycosaminogly- with the Hox segmentation factors orchestrates the cans14.Valve formation therefore seems to require Jekyll vertebrate body plan23. function in a new glycosaminoglycan-dependent Manipulation of the normal egg cell cycle, with heat signalling pathway. shock (HS) or early hydrostatic pressure (EP),contributes Several of the mutant phenotypes identified in the to the creation of gynogenetic diploid embryos24 (BOX 1, PARTHENOGENESIS screens resemble human genetic disease conditions, and figure panel c). EP has been used successfully in a screen Development of an organism some of the genes cloned have homologues in other ani- for primary motor-neuron mutants,which can be visual- derived from an unfertilized mal systems. Anaemic mutants (scored by their low ized with anti-motor-neuron antibodies at 24 hpf (REFS gamete. blood-cell counts) can be models for human blood dis- 24–26).Primary motor neurons are the earliest developing 15–17 RHOMBOMERE eases . Positional cloning of the weissherbst (weh) spinal motor neurons,and have distinct axon projections Each of seven neuroepithelial mutant identified a mutation in a novel iron exporter, and pathways. The mutant stumpy (sty) extends the segments found in the Ferroportin 1 (REF. 18; FIG. 2b). Blood mutants that are Caudal Primary motor neuron (CaP) along common but embryonic hindbrain that analogous to erythropoietic porphyria syndromes have not CaP-neuron-specific pathways24,25.EP-treated clutch- adopt distinct molecular and also been found, such as the dracula (drc) and yquem es from F females that are heterozygous for sty show cellular properties, restrictions 1 in cell mixing and ordered (yqe) mutants, which have
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