The Art and Design of Genetic Screens: Drosophila Melanogaster

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THE ART AND DESIGN OF GENETIC SCREENS: DROSOPHILA MELANOGASTER

Daniel St Johnston The success of Drosophila melanogaster as a model organism is largely due to the power of forward genetic screens to identify the genes that are involved in a biological process. Traditional screens, such as the Nobel-prize-winning screen for embryonic-patterning mutants, can only identify the earliest phenotype of a mutation. This review describes the ingenious approaches that have been devised to circumvent this problem: modifier screens, for example, have been invaluable for elucidating signal-transduction pathways, whereas clonal screens now make it possible to screen for almost any phenotype in any cell at any stage of development.

POLYTENE CHROMOSOME The fruitfly Drosophila melanogaster has been one of An unfortunate feature of genetic model organisms A giant chromosome that is the favourite model organisms of geneticists, since is that the easier they are to work with, the worse they formed by many rounds of Thomas Hunt Morgan decided to use it to investigate are as models for the animal that most funding agencies replication of the DNA. The the chromosomal theory of inheritance at the begin- find most interesting, namely ourselves. In this respect, replicated DNA molecules 1 tightly align side-by-side in ning of the last century . Morgan chose Drosophila however, Drosophila provides a very happy compro- parallel register, which creates a because it is easy and cheap to rear in the laboratory, mise. A surprisingly large number of developmental non-mitotic chromosome that is has a ten-day generation time and produces many processes seem to be conserved between flies visible by light microscopy. progeny. However, he soon discovered that it has several and vertebrates, even though they diverged at the PROTOSTOME–DEUTEROSTOME PROTOSTOME–DEUTEROSTOME other advantages for genetic analyses. For example, split ~700 million years ago. To The two principal divisions of there is no meiotic recombination in males, and there cite two of the more famous examples: the dorsoventral animal phyla, based on how the are only four chromosomes, which can be directly visu- (D/V) axes of the Drosophila and vertebrate embryo are mouth forms in the embryo. alized in the giant POLYTENE CHROMOSOMES of the larval patterned by opposing gradients of Decapentaplegic salivary gland. Furthermore, its exoskeleton provides a (Dpp; BMP4 (bone morphogenetic protein 4) in verte- wealth of external features, such as bristles, wing veins brates) and Short gastrulation (Sog; CHRD (chordin) in and compound eyes, which can be affected by muta- vertebrates), even though the orientation of the axes is tions, and for which the resulting mutant phenotypes reversed; whereas Hedgehog (Hh) and its vertebrate can be scored simply by looking down the stereomicro- counterpart, sonic hedgehog (SHH), have remarkably scope. This early start has been built on by succeeding similar roles in limb patterning in both systems3,4.The generations of drosophilists, who have developed an sequencing of the Drosophila genome has now revealed Wellcome/CRC Institute 5 and Department ever-increasing repertoire of techniques that make the true extent of these similarities . Drosophila has only of Genetics, Drosophila one of the most tractable multicellular ~15,000 genes, which is fewer than has Caenorhabditis University of Cambridge, organisms for genetic analysis2. In fact, Drosophila has elegans, but twice as many of these have clear homo- Tennis Court Road, only one main drawback, which is that the stocks have logues in humans (E-value <10–50)6.Furthermore,197 Cambridge CB2 1QR, UK. to be continuously maintained in the laboratory out of 287 known human disease genes have Drosophila e-mail: [email protected] because it is not possible to freeze them (and success- homologues, and even those that do not can produce DOI: 10.1038/nrg751 fully revive them afterwards). very similar symptoms when expressed in flies7,8.So,

Until 1980, most drosophilists were still preoccupied Box 1 | Mutagenesis in Drosophila with understanding the nature of the gene, and most Ethyl methane sulphonate mutagenesis studies were designed to discover new alle- This is the most commonly used mutagen in Drosophila because it is easy to administer les of existing genes or to find out how many genes there and causes the highest frequency of mutations. It mainly induces single-base changes were in a particular region of the genome. This all (point mutations), which disrupt gene function by causing missense or nonsense changed when Christiane Nüsslein-Volhard and Eric mutations, and the frequency at which a gene can be mutated therefore depends on the Wieschaus published their Nobel-prize-winning Nature size of the coding regions and the number of crucial amino acids that it contains. Using paper on mutations that affect the patterning of the the standard mutagenesis protocol with 25 mM ethyl methane sulphonate (EMS), the embryo10 (FIG. 1a). This work was revolutionary, because mutation rate for the average gene is ~1 in 1,000 (REF.93). This varies enormously, it was the first mutagenesis in any multicellular organ- however, and mutations in very large genes, such as dumpy (dp) are recovered at more ism that attempted to find most or all of the mutations than 20 times this rate. A disadvantage of EMS in the past has been that it was very that affect a given process, and because it was one of the difficult and laborious to map point mutations to specific genes. This problem has been first screens for phenotypes in the embryo rather than solved largely by the development of single nucleotide polymorphism (SNP) maps that the adult, which allowed them to identify null or strong allow the rapid meiotic mapping of mutations to regions of less than 50 kb, and SNP maps that are specifically designed for mapping mutants from FRT (Flp recombinase mutations in most of the essential patterning genes that 11 target) screens (see main text) are now available94,95. A second drawback of EMS is that are used throughout development (FIG. 2a,b).As Peter the progeny of mutagenized males are often mosaic (that is, some of the cells carry the Lawrence pointed out, half of the talks at the Drosophila meeting in Crete ten years later were about genes that mutation, whereas others do not), and mutants identified in F1 screens will therefore not be transmitted to the next generation, unless the germ cells are also mutant. This were identified in this screen, which gives some idea of mosaicism arises because an EMS-induced base change in one strand of the spermatid the impact of the paper12. Two features of Drosophila DNA segregates from the unmutated strand during the first zygotic division, if the development had a profound effect on the success of the screen. First, because Drosophila has an exoskeleton, the mismatch has not been repaired yet. To overcome this problem, F1 screens are often carried out using X-ray irradiation as the mutagen. This is about an order of magnitude larval cuticle provides an exquisite readout of the pat- less efficient than EMS, but induces mainly double-stranded DNA breaks that do not terning of the embryo. Second, Drosophila embryogene- cause mosaicism. Because many X-ray-induced mutations are chromosomal sis has evolved to occur as rapidly as possible, and the rearrangements or deletions, they can often be detected cytologically in larval polytene mother therefore loads the egg with most of the prod- chromosomes, which allows mutations to be mapped rapidly to a region and then ucts of genes that do not need to be transcribed in a pre- identified on Southern blots. cise pattern in the embryo13. This means that, in contrast P-transposable elements to other organisms, very few mutations block embry- Another popular strategy is to screen for mutations caused by P-element insertions, onic development at early stages, and most mutants in because the mutated gene can be rapidly and easily identified using the P-element as a housekeeping genes complete embryogenesis and tag. P-elements are very inefficient mutagens, however, so the most common approach is secrete a normal cuticle. The screen was therefore very to screen the large collection of P-element insertions that are available from the Berkeley efficient at identifying the transcription factors and sig- Drosophila Genome Project, rather than generating new insertions by mobilizing the nalling molecules that generate positional information P-element oneself. The existing collection contains insertions in about one-quarter of in the embryo. the essential genes89. Because most genes are predicted to be cold spots for P-element No genetic screen can find everything, and it is worth insertion, saturation screens cannot be carried out, but this type of screen does provide considering what sort of genes could not be identified in a very efficient way of identifying some of the genes that are involved in a process. the famous Heidelberg screen. The analysis of the zygotic genes that pattern the anteroposterior (A/P) and there are now more reasons than ever for taking advan- D/V axes of the larvae revealed that the genes at the top tage of the powerful genetics of Drosophila to investigate of the hierarchy (the gap genes, and the D/V genes, such the basic biological questions that are common to flies as dpp, zerknullt (zen), twist (twi) and snail (sna)) are and humans. One of the most important tools that already expressed in discrete domains, even though they Drosophila provides is the ability to carry out large-scale are among the first genes to be transcribed in the genetic screens for mutations that affect a given process embryo. This indicated that they must be regulated by or, to coin a term I recently heard from a mouse geneti- maternal determinants that are deposited in the egg. To cist,“forward functional genomics”.The advantage of find these factors, the Nüsslein-Volhard lab, and Eric this approach is that it provides an unbiased way to Wieschaus and Trudi Schüpbach, carried out saturation identify the genes that function in a particular process, mutageneses for maternal-effect mutations, which iden- whereas the mutants themselves are a very valuable tified many of the genes that are involved in generating resource for dissecting the function of the gene. the four maternal signals that define the two main axes of the embryo14,15 (FIG. 2c). Traditional genetic screens A second class of genes that were missed in the Although the early drosophilists isolated many visible screen comprises those that have specific roles in the mutations, these were all spontaneous alleles from patterning of internal structures, such as the nervous natural populations, and genetic screens only became system, because it is obviously impossible to identify possible once better ways to generate mutations were mutants that have no effect on the structure that is being developed. The most efficient method to do this is to screened. Indeed, several groups have subsequently used feed flies ethyl methane sulphonate (EMS), which a very similar approach to carry out large-scale screens induces point mutations, following the protocol for mutants that affect the organization of the central described by Lewis and Bacher in 1968 (REF.9) (BOX 1). nervous system, which led to the discovery of genes that

a EMS

Balancer

* F1 ×

* × * F2

25%50% 25%

* * F3 *

Balancer homozygotes: Balanced mutant stock: Homozygous for mutagenized die as larvae viable chromosome: screen embryos for phenotype

X-rays b sevB4 sevd2 sevd2 sevd2 ×

X2 3 X/Y 2 3

sevB4 sevd2 d2 sev ** * and F1 or

Screen for mutants with no R7 cells in the eye

Figure 1 | Outline of the crossing schemes used in the Heidelberg screen and the first screen for enhancers of sevenless. a | A typical crossing scheme for a screen for mutants on an autosome that produce a zygotic phenotype, based on those used in the BALANCER CHROMOSOME Heidelberg screens for mutants that affect the pattern of the larval cuticle. Male flies are fed ethyl methane sulphonate (EMS) to induce A chromosome with one or mutations and are crossed en masse to virgin females (indicated by the symbol ) that carry a BALANCER for the chromosome to be more inverted segments that screened (grey). As the mutations are induced in mature spermatids, each F male inherits a mutagenized chromosome (red) carrying suppress recombination. They 1 a different spectrum of mutations (asterisk). Single F males that carry a mutagenized chromosome in trans to the balancer are then are used as genetic tools because 1 backcrossed to balancer stock to generate F males and females that carry the same mutagenized chromosome. When these are they allow lethal mutations to be 2 crossed to each other, 25% of the F progeny will be homozygous for the mutagenized chromosome. As only one chromosome is maintained without selection. 3 screened at a time, the other chromosomes are not shown. In the Heidelberg screens, the lines were first screened for the absence

MATERNAL-EFFECT MUTATION of flies that were homozygous for the mutagenized chromosome, which indicated that it carried a zygotic lethal mutation, and cuticle Homozygous-viable mutation preparations were then done on the embryos from these crosses to see if 25% showed a phenotype. In the screens for MATERNAL- that causes little or no EFFECT MUTATIONS (see main text), the eggs laid by homozygous females from non-lethal lines were screened for phenotypes. phenotype in the mutant b | The crossing scheme for the original screen for enhancers of sev (REF. 32). Males that are hemizygous for a null allele of sev (sevd2) mothers, but leads to the were mutagenized with X-rays and crossed to sevd2 homozygous females that carry a temperature-sensitive allele of sev (sevB4) as a development of abnormal transgene inserted on the third chromosome balancer, TM3. The F1 TM3 flies were then screened for a reduction in the number of R7 offspring. photoreceptor cells in the eye.

a d ef

Naked Denticle cuticle belt bc ghi

Figure 2 | Examples of mutant phenotypes from standard F3 screens, and from a screen for enhancers and suppressors of a rough-eye phenotype. a–c | Cuticle preparations of first instar larvae. a | Wild type. The alternating denticle belts and naked cuticle along the anteroposterior axis (left–right) are indicated. b | A wingless mutant embryo, showing the loss of naked cuticle between the denticle belts in the ventral region of each segment. c | An embryo laid by a female that is homozygous for a null mutation in staufen (stau), which disrupts the localization of the anterior and posterior determinants. The embryo has a reduced head and no abdomen, but forms a normal thorax and telson (the posterior-most region of a Drosophila embryo). d–f | The central nervous system of the embryo stained with a marker for longitudinal and commissural axons. d | In the wild-type embryonic nervous system, each segment contains two commissures that cross between the longitudinal axonal tracts. e | In roundabout mutants, IPSILATERAL AXONS are no longer prevented from crossing the midline, and axons that should only cross once, recross multiple times. f | In embryos that are mutant for commissureless, which encodes a product that is required for axon guidance, most axons fail to cross the midline, and the commissures fail to form. g–i | Scanning electron micrographs of adult eyes. g | Wild type. h | A rough eye produced by overexpression of the Egfr (Epidermal growth factor receptor) inhibitor, Argos (GMR–argos/+). i | A mutation in sprouty (sty) acts as a dominant suppressor of the GMR–Argos rough-eye phenotype. (Panel b courtesy of Bénédicte Sanson, University of Cambridge, UK; panels d–f courtesy of Guy Tear, MRC Centre for Developmental Neurobiology, Guy’s Campus, London, UK, and reproduced with permission from REF.100 © (1998) Elsevier Science; panels g–i courtesy of Matthew Freeman, MRC–LMB, Cambridge, UK, and h, i reproduced with permission from REF.44 © (1999) Elsevier Science.)

control axon pathfinding, such as roundabout (robo) be found in the screens for maternal-effect mutations, and commissureless (comm)16–19 (FIG. 2d–f). As our ability because they need to be expressed zygotically to mediate to visualize the details of patterning has advanced, more later Wg signalling events and are therefore lethal. This sophisticated screens have become possible. For exam- problem applies to many essential proteins that are sup- ple, a recent screen used a Gal4 driver (BOX 2) to label the plied maternally to the embryo, as homozygous mutants dendrites of peripheral neurons with green fluorescent in these genes only show a zygotic phenotype when the protein (GFP), and this has allowed the discovery of maternal contribution runs out, and this often happens new genes that control dendritic morphology20–22. gradually, making the phenotype hard to analyse. Although the embryonic lethal screen identified The discussion above illustrates a general limitation mutants in most of the signalling molecules that are of genetic screens for homozygous lethal phenotypes, involved in patterning the embryo, such as Wingless which is that only the first essential function of a gene (Wg), Dpp, Hh, Spitz (Spi; an epidermal growth factor- can be analysed. Indeed, Eric Wieschaus and others like molecule) and Delta (Dl), they were much less have carried out an elegant series of experiments that successful at finding the other components of the signal- have taken this to its logical extreme. They looked at transduction pathways through which these ligands act. embryos that were homozygous for a series of large For example, the only components of the Wg pathway deletions (also called deficiencies) that remove hun- that give a clear SEGMENT-POLARITY phenotype in homozy- dreds of genes at the same time. Because most genes gous mutant embryos are wg itself, arrow (arr), are not required zygotically for early development, the armadillo (arm; also known as β-catenin) and pangolin first defects in the embryo reveal the phenotype of the (pan; also known as TCF/LEF1), which interact to form gene within the deficiency that has the earliest essential the transcriptional activator at the end of the path- function. By using a series of deletions that span most way23–26. Many of the remaining components, such as of the genome, they could therefore identify most or all IPSILATERAL AXON An axon that does not cross the the proteins that are required for the processing and of the regions that contain these early-acting genes, midline. secretion of Wg, the Frizzled (Fz) receptors, Dishevelled and this led to the surprising conclusion that develop- (Dsh) and those of the Adenomatous polyposis coli ment to the cellular blastoderm stage requires only SEGMENT-POLARITY GENE (Apc) complex, were not identified, because their mater- seven zygotic genes27,28. As deficiencies are equivalent to A gene that is required for nal contribution is sufficient for early Wg signalling. null mutations in all of the genes that are deleted, this anteroposterior patterning within each segment, such as Wingless signalling is used over and over again in devel- type of deficiency screen provides a rapid way to scan wingless, engrailed and hedgehog. opment, however, and mutants in these genes could not the genome for zygotically acting genes that are

Box 2 | The GAL4–UAS system for directed gene expression

The yeast transcriptional activator Gal4 can be used to Enhancer-trap GAL4 UAS–gene X regulate gene expression in Drosophila by inserting the upstream activating sequence (UAS) to which it binds next to a gene of interest (gene X)96. The GAL4 gene has been inserted at random positions in the Drosophila genome to generate ‘enhancer-trap’ lines that express GAL4 under the control of nearby genomic enhancers, and there is now a large collection × of lines that express GAL4 in a huge variety of cell-type and tissue-specific patterns97. Therefore, the expression of gene X can be driven in any of these GAL4 patterns by crossing the appropriate GAL4 enhancer- trap line to flies that carry the UAS–gene X transgene. This system has been adapted to carry out genetic Genomic enhancer GAL4 gene X screens for genes that give phenotypes when UAS misexpressed in a particular tissue (modular Tissue-specific expression Transcriptional activation misexpression screens)79. of GAL4 of gene X

involved in any early developmental process, but it can (FIG. 1b). First, the progeny of mutagenized flies can be

also be used to look for dominant effects in deficiency screened directly (an F1 screen), because the mutations heterozygotes, which is a quick means of assessing the do not need to be made homozygous, which means number of potential target genes in an enhancer or that an order of magnitude more flies can be screened 29 suppressor screen (see below) . To facilitate deficiency than in an F3 screen. Second, lethal mutations in essen- screens, the Bloomington stock center has assembled a tial genes that function at many stages of development collection of deficiencies that provide maximal cover- can be isolated, because the sensitized background only age of the genome in the minimum number of stocks, affects the eye, which is not required for viability. Third, which is often referred to as the ‘deficiency kit’ . the whole genome can be screened at once, because there is no need to use balancers to make particular Enhancer and suppressor screens chromosomes homozygous. At the time this screen was One way to find the missing components of a develop- carried out, biochemical experiments had identified mental pathway that cannot be found in traditional several RTK substrates, but the pathway through screens is to carry out a screen for dominant enhancers which they signal to the nucleus was still unclear. The or suppressors. Loss-of-function mutations in almost mutants from the screen led to the demonstration that all genes are recessive, which indicates that 50% of the Sev, and other Drosophila RTKs, such as the Epidermal wild-type level of a protein is sufficient for normal growth factor receptor (Egfr) and Torso (Tor), signal development. When a particular process is already par- through Son of Sevenless (Sos), Downstream of recep- tially disrupted by another mutation, however, this tor kinase (Drk; an SH3/SH2 adaptor protein) and amount might no longer suffice, and mutations in the Corkscrew (Csw; a protein tyrosine phosphatase), to genes that are involved in the pathway can therefore be activate Ras31–34. Two other types of screen for modi- identified as dominant enhancers or suppressors in this fiers of sev identified partially overlapping sets of sensitized genetic background. Some of the most suc- genes, and illustrate the different ways that this type of cessful screens of this type were carried out to find the screen can be designed. Rogge et al.35 used a stronger components in the signal-transduction pathway down- sev hypomorphic mutation and screened for domi- stream of Sevenless (Sev), which is a receptor tyrosine nant suppressors that increase the number of R7 cells. kinase (RTK) that controls cell-fate choice between one This led to the identification of the opposite types of of the eight photoreceptors in the eye, the R7 cell, and mutant to the enhancer screen, such as a gain-of-func- non-neuronal cone cells30. A weak allele of sev provides tion mutant in Sos. By contrast, a screen for dominant just enough signalling activity for most of the R7 cells to suppressors of a constitutively active form of the Sev form, but these cells are transformed to cone cells if receptor identified a loss-of-function allele of drk 36. there is any further reduction in the efficiency of signal Although these screens were remarkably effective, transduction. Under these conditions, halving the dose they did not find the genes that function downstream of of some of the downstream components in the pathway ras, presumably because these act too far downstream causes most of the R7 cells to become cone cells, with- of sev for a reduction in their levels to have a significant out affecting signalling through the other RTKs in the effect in the sensitized backgrounds. Because ras is an fly. This sensitized genetic background therefore essential gene that is required throughout development, allowed Simon et al.30 to identify mutants in compo- none of the alleles gave a viable phenotype that was suit- nents of the RTK signalling pathway as dominant able for modifier screens; however, this problem could enhancers of a visible eye phenotype. This approach has be circumvented by making a transgenic construct in several important advantages over traditional screens which the sev enhancer drives the expression of an

38 OMMATIDIA activated form of Ras in the eye. The eye-specific activa- et al. , who screened for modifiers of an activated form The compound eye of tion of the RTK pathway by this transgene transforms of Raf and identified many of the same genes. This is not Drosophila is formed from 800 cone cells into R7 cells to produce a rough-eye pheno- the end of the story, however, because new screens for ommatidia, each of which type (in which the surface of the eye loses its smooth modifiers of other components of the pathway continue contains eight photoreceptor cells, surrounded by four cone appearance; see FIG. 2g,h). A genetic screen for dominant to identify new genes that are important in this cells that secrete the lens, and enhancers and suppressors of this phenotype resulted in process39–42. seven pigment cells. the identification of mutants in the downstream compo- The screens for mutants in the Sev pathway illustrate nents, such as Raf (a serine/threonine kinase), mitogen- several important features for designing successful mod- activated protein (MAP) kinase kinase, MAP kinase ifier screens. Although I have mentioned some of the (encoded by rolled (rl)), and Phyllopod (Phyl; a nuclear interacting genes that proved to have a direct role in the protein)37. A similar approach was taken by Dickson pathway, many mutants were also isolated from these screens that modify the eye phenotype for less obvious reasons, and it is therefore crucial to have simple assays Box 3 | Using the Flp/FRT system to generate mitotic clones to sort out the different classes of interactors. For example, some mutants might modify the phenotype by Flp recombinase mediates site-specific recombination between FRT (Flp recombinase interfering with unrelated processes that occur in the target) sites during replication of the yeast 2µ plasmid, and works very efficiently when expressed in Drosophila49. Flp-mediated recombination can be used to generate mitotic same cells, whereas others might do so because they alter clones by creating flies with transgenic FRT sites at identical positions on homologous the expression of the allele or construct used to generate chromosomes98. If the site-specific recombination between homologues occurs after the sensitized background. This latter class can be DNA replication, and the daughter chromatids segregate appropriately, the region of the screened out because they should also suppress con- chromosome arm that lies distal to the FRT site will be made homozygous, with each structs that are under the same transcriptional regula- daughter cell inheriting two copies of this region from one of the parental chromosomes tion but produce the opposite phenotype, whereas bona (see figure). This site-specific recombination event can be used to make a mutagenized fide components of the pathway should have the con- chromosome arm (red) homozygous in clones of cells, which can then be screened for a verse effect. Second, not all components of a pathway

phenotype. The principal advantages of this approach are: first, F1 screens can be carried will necessarily be dosage sensitive. Only one allele of out for recessive loss-of-function phenotypes, as there is no longer a need to go through Raf was isolated in all of these screens, whereas hun- two additional generations to make the mutagenized chromosomes homozygous, as is dreds of mutations were recovered in rl (MAP kinase), the case in a traditional genetic screen; and second, by controlling where and when the which indicates that the levels of the former are proba- recombination occurs, only the cells of interest are made homozygous. The tissue- bly not crucial for the efficiency of signal transduction specific phenotypes of mutations in essential genes can therefore be identified, in the eye. Third, each screen isolated a different spec- regardless of their other functions in development. One disadvantage is that Flp/FRT trum of mutations, and it might be necessary to carry screens cannot detect mutations that lie proximal to the FRT site. However, FRT out screens in various different sensitized backgrounds insertions have been recovered close to the centromere on all of the main chromosome to find most of the components in a pathway. Finally, arms, and it is now possible to screen ~95% of the euchromatin, although this requires many of the mutations turned out to be homozygous carrying out separate screens for each of the five arms56,99. See animation of mitotic viable and have no phenotype when removed from the recombination online. sensitized background. Suppression or enhancement of the phenotype of interest is therefore no guarantee that FRT a gene has an essential role in the process. * Sister Many other enhancer and suppressor screens have * chromatids G2 Flp recombinase been carried out in Drosophila, and they continue to provide a very effective means of finding new components in almost any process29,43–45. Some idea of their impact comes from the hundreds of genes listed in FlyBase, whose names indicate that they were identified as either enhancers or suppressors, even though they represent only a subset of the genes found in this way * (for example, Enhancer of split (E(spl)), Suppressor of Hairless (Su(H)) and Suppressor of deltex (Su(dx)) in the Notch pathway). The eye is by far the most popular Mitosis place in which to conduct such screens, because it is not * essential for viability or fertility, and is easy to score. Furthermore, it is particularly suited to modifier screens because it is composed of ~800 regularly packed OMMATIDIA, and numerous defects in cell-fate determination and differentiation give a rough-eye phenotype, the severity of which reflects the number of * ommatidia affected. It is even possible to carry out * screens for modifiers of genes the functions of which have not been characterized in the eye, if the eye-spe-

Homozygous mutant clone Wild-type twin-spot clone cific expression of a wild-type or mutant construct of the gene produces a rough-eye phenotype46.

IMAGINAL DISC Clonal screens mitotic recombination between homologous chromo- Sac-like infolding of the Another extremely powerful way to get around the somes, and this technique was used successfully to carry epithelium in the larva. They problem of only being able to analyse the first pheno- out genetic screens for X-linked mutations that give give rise to most of the external type of a mutation is to carry out screens in mitotic phenotypes in germ-line clones47,48.However,a much structures of the adult. Imaginal disc cells are set aside in the clones, in which only the cells of interest are homo- more efficient way to generate clones is to use the Flp embryo and continue to divide zygous for the mutagenized chromosome, whereas the recombinase from the yeast 2µ plasmid, which mediates until pupation, when they rest of the organism is heterozygous. The old-fashioned efficient site-specific recombination between its target differentiate. way of generating clones was to use X-rays to induce sites, called FRT sites, when these are integrated into the Drosophila genome49. The Flp recombinase works sur- a b prisingly well when the transgenic FRT sites are present at identical positions on homologous chromosomes, and this is probably because homologues are paired in mitotic cells in Drosophila, in contrast to most other organisms. This site-specific recombination event can, therefore, be used to make a mutagenized chromosome arm homozygous in clones of cells, which can then be screened for a phenotype (BOX 3). By expressing Flp in larvae under the control of a heat-inducible promoter, any embryonic phenotype can be bypassed by specifically inducing clones in the IMAGINAL DISCS. The first Flp/FRT screens used this c d approach to identify mutants that affect the growth and patterning of the discs on the basis of their visible adult phenotypes, and show the immense power of this tech- re nique. Xu et al.50 searched for mutations that produce tumorous outgrowths in the discs, and identified the tumour-suppressor gene warts (wts; also known as lats), and a negative regulator of the Dpp and Wg signalling pathways, supernumerary limbs (slmb; also known as la me slimb). Jiang and Struhl also identified slmb, and isolated an allele of Protein kinase A, which was found to be a negative regulator of the Hh pathway51,52 (FIG. 3a). Similar screens for mutants that produce bubbles in the wing identified 14 loci that are required for adhesion between the dorsal and ventral compartments, and led to the identification of novel components of the inte- R7 grin adhesion complexes that hold the two surfaces R8 together53–55 (FIG. 3b). e Germ-line clones. An ingenious refinement to the Flp/FRT system, called the dominant female sterile (DFS) technique, has been developed by Chou and Perrimon to select for mutant clones in the germ line56,57. The DFS method uses the dominant ovoD mutation to kill the non-recombinant germ cells, so that females lay only eggs that derive from homozygous mutant germ-line clones. This approach provides a Figure 3 | Examples of mutant phenotypes from Flp/FRT screens. a | A NOTUM containing a powerful way to screen for the maternal-effect pheno- homozygous lats/warts mutant clone, which has overgrown to form a large tumerous outgrowth types of lethal genes (BOX 4) and has allowed the identifi- (arrow). b | An adult fly with bubbles in both wings produced by clones of a mutant in piopio (pio), cation of many of the essential genes that were missed in which disrupts adhesion between the dorsal and ventral surfaces of the wing. c,d | Section previous screens because their products are required for through an adult head showing the projections of retinal axons into the lamina (la) and medulla 58 (me) of the optic lobe. ‘re’ marks the position of the retina. c | Wild type. The R7 and R8 axons embryogenesis but are supplied maternally . For exam- project to two distinct layers in the medulla. d | The R7 and R8 axons terminate in the same region ple, these screens have led to the discovery of several of the medulla in Leukocyte-antigen-related-like (Lar) mutant clones, generated using eye–FLP new genes that are involved in signal-transduction path- with the Minute technique. In panels a–d, anterior is to the left. e | Scanning electron micrographs ways in the embryo, such as tout velu (ttv), which is of the head and thorax of a wild-type fly (centre), and flies from the ‘pinhead’ screen with either a required for the diffusion of Hh protein and encodes smaller (left) or larger (right) than normal head. (Panel a courtesy of Tain Xu, Yale University, USA, the homologue of the human disease gene exostoses and reproduced with permission from © (1995) The Company of Biologists, Ltd; panel b REF.50 (EXT), and sugarless (sgl ) and sulfateless (sfl ), which are courtesy of Nick Brown and Christian Boekel, Wellcome/CRC Institute, Cambridge, UK; panels c, d courtesy of Barry Dickson, Institute for Molecular Pathology, Vienna, and reproduced with both enzymes involved in the synthesis of heparan sul- permission from REF.72 © (2001) Elsevier Science; panel e courtesy of Ernst Hafen, University of phate glycosoaminoglycans, which are required for Wg Zürich, Switzerland.) and fibroblast growth factor (Fgf) signalling59–62.

Because the ovoD selection means that all of the progeny clones can therefore be selected by the absence of mater- of mutant females show the phenotype, it is not possible nal GFP and screened for phenotypes, and the muta- to recover the mutant chromosomes from the females, tions can be recovered from the eggs that are derived and these screens must, therefore, be carried out in the from non-recombinant germ cells, which express GFP.

F2 generation, so that the mutant can be isolated from Flp/FRT screens can be targeted to particular tis- the sibling males. More recently, Luschnig et al.63 have sues by putting the Flp recombinase under the control

adapted this approach to carry out F1 screens for lethal of a tissue-specific promoter, which removes any risk mutations with maternal-effect phenotypes, by marking of losing mutants because of the effects of clones in the non-mutagenized FRT chromosome with GFP.The other tissues. Duffy et al. have generated upstream embryos from the homozygous mutant germ-line activating sequence (UAS)–FLP lines that allow the

Box 4 | The dominant female sterile technique for selecting homozygous germ-line clones Dominant female sterile (DFS) mutations in the ovo gene (ovoD mutants) cause female germ cells to die during early oogenesis, and therefore completely block egg production. These mutants can be used to eliminate non-recombinant cells in Flp/FRT screens for mutations that give phenotypes in germ-line clones, by placing them on the FRT chromosome arm that is to be selected against. In the absence of recombination, the ovoD transgene kills all of the germ cells, so the only egg chambers to survive are those that have lost ovoD and are homozygous for the other FRT chromosome arm (see figure). By mutagenizing this chromosome, genetic screens can be carried out for lethal mutations that give maternal-effect phenotypes, in which 100% of the eggs that are laid derive from homozygous mutant germ-line clones, and this provides a very powerful way to find mutants in maternally supplied components that are essential for embryonic development58. Although the ovo gene is on the X chromosome, Chou and Perrimon have generated a set of transgenic lines that carry the mutant form of the gene and a proximal FRT site on each of the four major autosomal chromosome arms55. This makes it possible to use the DFS technique to do germ-line clone screens of most of the genome. hs, heat shock; m, mutation; w, white gene.

P[ovoD] FRT m FRT ×

Balancer Balancer

P[ovoD]

m w hs–FLP; m FRT/P[ovoD]FRT

+ Flp

P[ovoD] P[ovoD] Ovaries

m/ovoD D Flp recombinase m/m P[ovo ] m m m

P[ovoD] m

P[ovoD] m NOTUM The dorsal or upper surface of the thoracic segment of any Homozygous mutant Twin-spot clone Non-recombinant insect. germ-line clone DEAD DEAD

FOLLICLE STEM CELL targeted expression of Flp with any Gal4 driver, and The most sophisticated Flp/FRT screens combine Each ovariole (chambers in the have shown that a driver that is expressed in the tissue-specific expression of the recombinase with a ovary through which the egg FOLLICLE STEM CELLS can make up to 30% of the follicle selection for the homozygous mutant clones, and, passes during development) cells homozygous for a particular chromosome arm perhaps not surprisingly, these have been devised for contains 2–3 follicle stem cells, which produce the somatic (that is, 60% of the cells have undergone recombina- carrying out screens in the eye (BOX 4). Flp has been follicle cells that surround the tion)64. This has made it possible to carry out clonal expressed specifically in the developing eye disc chambers. The follicle cells then screens for mutants that affect several aspects of folli- either by using an eyeless (ey)–GAL4 driver and differentiate into several cell cle cell behaviour. For example, Liu and Montell have UAS–FLP, or by fusing the recombinase directly to types, including the border cells, which migrate from the anterior screened for mutants that disrupt the migration of the the ey regulatory region (ey–FLP). In addition, sev- of the egg chamber towards the border follicle cells towards the anterior of the oocyte eral different tricks have been used to select against oocyte, where they contribute to and found two new transcription factors that regulate the non-recombinant cells and the twin-spot clones the formation of the micropyle. this movement, whereas Pai et al. identified a new (the reciprocal product of the recombination event, At the end of oogenesis, the dorsalizing mutation that proved to be an allele of which inherits two copies of the non-mutagenized follicle cells secrete the eggshell 69,70 69 and undergo apoptosis. Cbl, which targets the activated Egf receptor for chromosome arm) . Newsome et al. used FRT degradation65–68. chromosomes that carry Minute (M) mutations,

+ Adult eye a Genotype: w eye–FLP; FRT P[w ] /FRT* P[w+]

+ Twin-spot P[w ] P[w+] clone Eye discs FRT expressing Dark red eye–FLP

Flp P[w+] * * * Homozygous clone * White

P[w+]

Non- recombinant * Red

+ Adult eye b Genotype: w eye–FLP; FRT P[w ] M /FRT* P[w+] M

+ Twin-spot P[w ] P[w+] clone Eye discs FRT M expressing M Dead eye–FLP M P[w+] * * * Homozygous clone * White

P[w+] M Non- recombinant; slow-growing * Red

Figure 4 | The eye–FLP technique for targeting clones to the eye. By placing the FLP recombinase gene under the control of the eyeless enhancer (which drives expression specifically in the eye–antennal imaginal disc), Flp/FRT-mediated recombination can be targeted to this disc to generate homozygous mutant clones in the eye in flies that are otherwise heterozygous. a | The non-mutant chromosome (the asterisk indicates a mutation) is marked by a mini-white transgene, but there is no selection against the twin-spot clones or non-recombinant cells, and both the mutant clones (white) and the twin-spot clones (darker red, because they carry two copies of white+) are relatively small. b | The effects of incorporating a Minute mutation (M) onto the non-mutant FRT chromosome. The mutant clones now occupy almost all of the eye, because they outcompete the slow-growing non-recombinant cells (which are M/+), whereas the twin-spot clones die. (Photographs courtesy of Barry Dickson, Institute of Molecular Pathology, Vienna, and reproduced with permission from REF.69 © (2000) The Company of Biologists, Ltd.)

which are mutants in essential components of the mutants that disrupt size regulation, by using the translation apparatus, such as ribosomal proteins, ey–FLP system with a selection against the twin spots that are recessive lethal and cause a dominant inhibi- to generate flies in which all of the regions of the head tion of cell growth71. The twin-spot cells that are that derive from the eye–antennal imaginal disc are homozygous for the M mutation therefore die, mutant, whereas the rest of the body is wild type, and whereas the non-recombinant cells grow more slowly by screening for flies with small heads (FIG. 3e) (S. and are largely eliminated through competition with Oldham, H. Stocker and E. Hafen, personal commu- the faster growing M+ clones (FIG. 4). Stowers and nication). Because flies naturally come in different Schwarz went even further, and completely elimi- sizes depending on their nutritional state, it is much nated the non-recombinant and twin-spot cells by easier to spot a mutant that affects size control when creating an FRT with a dominant eye-specific cell- only the head is affected, and the rest of the body acts lethal construct, in which another eye-specific pro- as an internal control. moter, GMR (glass multiple reporter), drives the The efficiency of the Flp/FRT system for generat- expression of head involution defective (hid; also ing clones has made Drosophila the only multicellular known as wrinkled) — a gene that triggers organism in which it is now possible to carry out apoptosis70. It turns out, however, that ey–FLP is so screens in almost any cell at any stage of develop- efficient that nearly all of the cells undergo recombi- ment, without having to worry about the pleiotropic nation, so there is no need to eliminate the non- effects of mutations. Only a few examples of mutants recombinant cells. The twin-spot cells can simply be that have been isolated in such clonal screens have removed by placing a recessive cell-lethal mutation been published so far, but they already illustrate the on the other FRT chromosome69. These reagents enormous potential of this approach, and the range make it possible to generate flies in which almost all of such screens can be further enhanced by combin- eye cells are mutant, but the rest of the animal is wild ing them with sophisticated ways of visualizing par-

type, and allow simple F1 or F2 screens for the eye- ticular attributes of a cell. For example, mutations specific phenotypes of all genes. that affect the expression or localization of a specific One of the first screens to take advantage of the protein can be screened for, by generating the appro- ey–FLP approach used a lacZ line that was expressed priate transgenic GFP reporter constructs78. Given the in the photoreceptor cells to screen for defects in their versatility of this technique, it seems very likely that axonal projections into the optic lobe of the brain. many more Flp/FRT screens will be carried out in This approach identified 210 mutant lines that specif- future to address a host of biological questions. ically disrupt axon pathfinding, without affecting photoreceptor cell differentiation69. Although most of Misexpression screens these mutants remain to be characterized, two of the All of the screens described so far can be carried out complementation groups correspond to the mutants on a sufficiently large scale that, in theory, it should in the receptor tyrosine phosphatases, PTP69 and be possible to identify all of the genes in which loss- Leukocyte-antigen-related-like (Lar; also known as of-function mutations give the phenotype of interest. Dlar)72 (FIG. 3c–d). Similar mutants have also been Not all genes can be found in this way, however, and

recovered in an elegant F1 screen, which used a behav- one class that might be missed comprises genes that ioural assay to select mutant flies that do not detect have redundant functions. A useful complement to motion73,74. Finally, there are some types of screen these screening strategies is to use the Gal4 system that can be conceived only as clonal screens, and par- (BOX 2) to carry out a targeted misexpression screen. ticularly ingenious examples are screens for mutants Rørth et al. have generated a P-element vector called that cause cells to divide more rapidly than normal, the EP element that carries UAS sites at one end, so without disrupting patterning or causing a tumorous that any gene that it inserts next to can be activated by overgrowth phenotype. These take advantage of the Gal4 (REF. 79). This makes it possible to carry out a fact that a mutant clone and a twin-spot clone are gain-of-function screen by simply crossing a Gal4 generated at the same cell division, and therefore nor- driver that is expressed in the appropriate tissue to a mally grow to be about the same size. Mutant and large number of EP insertion lines, and screening for MINI-WHITE GENE twin-spot clones in the eye that were marked by the phenotypes caused by the mis- or overexpression of A truncated version of the white presence of either no copies or two copies of the MINI- the adjacent genes80. The effectiveness of this type of gene that is commonly used as the selectable marker in WHITE GENE were generated using ey–FLP, and these ‘modular misexpression screen’ in identifying func- transformation constructs. One were screened for cases in which the eye was normally tions that would be missed in loss-of-function copy of the transgene usually proportioned, but the mutant clone was much larger screens is exemplified by a screen for genes that produces yellow or orange eyes than the twin spot. Mutants from these screens fall impair border-cell migration when overexpressed in in a white mutant background, whereas two copies give more into more than 20 complementation groups, and either the border cells or the germ cells through 81,82 complete rescue and produce include alleles of the Drosophila homologues of the which the border cells migrate . Migration was dis- darker eye colours. This allows human tuberous sclerosis complex 1 and 2 genes, rupted by 2 out of 8,500 EP insertions tested, and more than one transgene to be Pten and archipelago (ago), which regulates cyclin E these led to the demonstration that EGF and followed at a time, and flies that degradation and is the homologue of the human PDGF/VEGF (platelet-derived growth factor/vascular are either heterozygous or homozygous for a particular tumour-suppressor gene HCDC4 (REFS 75–77).The endothelial growth factor) homologues function as insertion to be distinguished. Hafen lab is carrying out a related ‘pinhead’ screen for redundant signalling molecules that direct migration

of this approach at the moment is that it cannot be Box 5 | What type of screen to do? carried out by simply feeding flies double-stranded Although a successful screen can generate enough mutants to keep a lab busy for RNA (as in worms), and it has to be injected into the many years, doing the screen and characterizing the mutants from it involve a huge embryo. This means that RNAi screens will have the amount of work, and it is therefore very important to design an efficient and same limitations as traditional screens for homozy- effective screening strategy. The most appropriate solution will be different in each gous mutations, in that they will only be able to case, but there are several factors to bear in mind. detect the first zygotic function of each gene.

•F1 screens for viable phenotypes are much less work than F3 screens, but it is Although the problem could be overcome by gener- always more work to map the mutations and to organize them into ating transgenic constructs that express double- complementation groups than to do the screen itself. stranded RNA for each transcript, this would be very • Because characterizing the mutants requires so much effort, it is important to laborious. In addition, there are several types of minimize the background of mutations that do not affect the process of interest. experiment that can be done with a mutant that are For this reason, it is sometimes better to do a more laborious but more specific not possible with RNAi. For example, a common way screen than a faster screen with a higher background, and the best way to do to dissect the functions of a protein is to determine this is to screen for a phenotype that is directly related to the process of interest. whether transgenic constructs that express truncated A good example of this approach is the screen for retinal axon-guidance mutants or altered forms of the protein rescue a null muta- 69 by Newsome et al. , which is very labour intensive because it requires the tion, and this is difficult to do using RNAi, because staining of dissected heads, but unambiguously identifies the mutants that this exogenous RNA will also target the transgenic affect axon guidance, thereby reducing the frustration of mapping irrelevant gene products. mutations. A disadvantage of this approach, however, is that not as many flies A second reason why forward genetic screens can be screened, and many mutations might not fall into complementation might become obsolete is that there is only a finite groups. In other cases, it is more efficient to carry out a less specific but easier number of genes in the genome and, at some point in screen, such as an F1 suppressor or enhancer screen, provided that simple re-screening assays are available that distinguish the interesting mutants from the future, there might be mutations in all of them. the noise at an early stage. At the moment this seems a long way off, because there are reported mutations in only a few thousand • It is often quite difficult to predict how well a particular screening strategy will of the ~15,000 predicted genes, but the Berkeley work and what types of background mutation it will isolate, and it is therefore a Drosophila Genome Project is rapidly increasing this good idea to do a small pilot screen first. coverage by screening through tens of thousands of • It is very important to ISOGENIZE the starting stocks beforehand, to ensure that P-element insertions89. Although this approach alone there are no lethal mutations present in the background. Otherwise, there is a risk will not saturate the genome, it might be possible to of isolating the same pre-existing mutation over and over again, or of identifying generate a null mutation in every gene by using a spurious lethal complementation groups, because two independent mutations combination of transposable elements. Nevertheless, carry the same background lethal mutation. I believe that there will still be good reasons for doing genetic screens, even when the ‘gene knockout kit’ is available. As screens have become more sophisti- towards the oocyte. Other screens of this type have cated, they have used more and more complex led to the identification of the cell-cycle inhibitor genetic backgrounds, which often contain several dif- Tribbles (Trbl), a large number of genes that disrupt ferent transgenes; it is much more work to cross axon guidance or synapse formation when expressed ~15,000 mutations individually into this background in motor neurons and a similar number that perturb than to generate new mutations in the appropriate the development of the adult sensory organs when stocks. More importantly, forward genetic screens expressed in the sensory organ precursor cells83–85. can generate a variety of alleles of a gene, ranging Although this type of screen can never be a saturating from amorphs (null mutations) to weak hypomorphs screen, because P-elements do not insert into every (partial loss-of-function mutations), and the latter gene, large collections of EP insertions are available in are often invaluable for elucidating its functions. For the public stock centres. example, most mutations that are identified in behavioural screens turn out to be hypomorphic ISOGENESIS Prospects mutations in essential genes, and these phenotypes A way of homogenizing the I hope that this review has given a flavour of the would probably not be detected using null genetic background of a line that types of genetic screen that are possible in mutations90. Many proteins carry out more than one is used for mutagenesis. In an isogenic stock, the two Drosophila, and will provide ideas for new screens function in a cell, and this is often revealed by having homologous chromosomes of (BOX 5). It is worth considering, however, whether the a series of alleles of differing strengths. To take each pair are identical, which time will come when forward genetic screens are no another example from our own work, the key func- ensures that no recessive lethal longer used. One reason that this might occur is if tions of par-1 and mago nashi (mago) would not have allele is present. there are faster ways of knocking out gene function, been noticed without having hypomorphic muta- RNA INTERFERENCE and RNA INTERFERENCE (RNAi) now provides an alterna- tions, as the null mutants block oogenesis before (RNAi). A process by which tive strategy for doing this86. Because the genome these later functions become apparent91,92. So, for- double-stranded RNA sequence can be used to design double-stranded ward genetic screens are likely to be an important specifically silences the RNAs against every predicted gene, it should be pos- tool in Drosophila for many years to come, particu- expression of homologous genes through degradation of their sible to carry out genome-wide screens by RNAi, as is larly if people continue to be so inventive in coming cognate mRNA. now being done in C. elegans 87,88. One disadvantage up with better ways to do them.

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