Genetic Analysis in Drosophila

Genetic Screens and Tools

A. Genetic analysis of development

Many genetic studies in Drosophila are intimately tied to experiments devised to understand or take advantage of the development of Drosophila, so it is important that you have a basic understanding of Drosophila development. Developmental Biologists seek to answer a basic question: How is a complex animal (or plant) formed from a single cell? In practice, this has meant learning a great deal about processes such as signal transduction, transcriptional regulation, and many aspects of cell biology. Because many aspects of Drosophila development are now so well understood, many times scientists can turn this around and take advantage of Drosophila phenotypes to analyze their favorite molecule or signal transduction pathway. A major feature of development is growth, and not surprisingly, many oncogenes and tumor suppressors turn out to have important roles during animal development. I. Oogenesis

Drosophila has two ovaries, which are composed of strings of developing oocytes in ovarioles. Within the germarium, there are 2-3 stem cells, which divide to form a cystoblast. This cell goes through four incomplete cell divisions to make a 16-cell cyst. All cells of the cysts are connected by ring canals. These connections allow for communication such that the cyst is polarized. This is facilitated by a microtubule network that runs through all the cells and is responsible for a gradient resulting in the accumulation of proteins and RNAs in one of the cells with four ring canals or connections. This gradient is required for oocyte differentiation. The figures below show how the polarity of the embryo (A-P and D-V axis) are established in the oocyte. The oocyte nucleus plays a key role. It first takes up a posterior position and signals the follicle cells (the somatic cells surrounding the oocyte) to take up a posterior fate. The nucleus then moves, based on a microtubule network, to the anteriordorsal surface and signals the follicle cells their to adopt a dorsal fate. We will discuss these signal transduction pathways later. At maturity, the Drosophila egg is about 400m long and about 160m in diameter. Its polarity that is morphologically (its asymmetric) and molecularly visible.

from (Riechmann and Ephrussi 2001).

II.

Early embryonic development

Fertilization occurs through the micropyle in the anterior tip of the egg, and after one round of replication the male and female pronuclei fuse. Early Drosophila embryos are a syncytium, in which a series of rapid nuclear divisions occur within a single large cell. There are 13 rapid nuclear divisions (termed cycles) before cellularization occurs. For the first seven cycles all the nuclei are in the central region of the embryo. The nuclei then begin to migrate towards the periphery (cortex) of the embryo, and they arrive there by cycle 10. The pole cells (the precursors of the germline cells) form after the 10th nuclear cycle. After cycle 13, there are about 6000 nuclei. These nuclei are then simultaneously separated into 6000 cells through the process of cellularization, which is a specialized, massive form of cytokinesis. This occurs at about three hours of development. The Drosophila embryo is loaded with a large endowment of maternally transcribed RNAs. As we will discuss later, this has important implications for the genetic analysis in Drosophila. All of early Drosophila embryogenesis is driven by maternally provided RNAs and proteins. Zygotic genes dont start to be transcribed until the cycles of DNA replication slow down. The early cycles are so rapid (about 8-10 min) that they dont allow much time for transcription. The earliest requirements for zygotic gene activity occur during the process of cellularization. Immediately after the cellular blastoderm. forms, the embryo starts to gastrulate. The main tissue layers of an organism (endoderm, mesoderm, and ectoderm) are established during gastrulation through cell movements. In Drosophila, gastrulation proceeds without cell division. The mesoderm is formed from an invagination of cells along the ventral side of the embryo, called the ventral furrow. The endoderm (gut) is formed from cells that invaginate through the anterior and posterior midgut invaginations, which eventually meet in the middle of the embryo to form a continuous tube. The region of the animal between the head and the tail is called the germband. As gastrulation begins in Drosophila, the germband elongates, and extends posterior and then wraps around the posterior end. The germband first becomes visibly segmented when it is fully extended. The segments are demarcated by invaginations that form between them. In the Drosophila embryo 3 head segments, three thoracic segments, and nine abdominal segments can be seen. The nervous system is formed from ectodermal cells that delaminate from the neuroepithelium. This process begins during germband extension. A couple hours after fully extending, the germband retracts. After it retracts the ectoderm seals over the amnioserosa in a process called dorsal closure.

During the remaining hours of embryonic development, hundreds of different cell types are specified and the different tissues of the animal established. Altogether embryogenesis in Drosophila takes about 24 hours at 25C. At the end of embryonic development, the first instar larvae hatches.

Take home message: dont memorize these developmental stages. The important thing is that they are reproducible in wild-type, and that (developmental) mutants can be isolated which effect a specific developmental event.

B. The Genetic analysis of embryonic development

In the late 1970s Eric Wieschaus & Christiane (Janni) Nsslein Volhard embarked on a massive genetic screen that has profoundly influenced biology ever since. They set out to identify, in a systematic way, all of the essential genes in Drosophila that could be mutated to give a phenotype in the cuticle of the animal at the end of embryogenesis. This was the first time that anyone had really ever set out to systematically identify genes that are important for development, and they were enormously successful. The genes they identified continue to be a focus of much research. As the genes they identified began to be cloned, scientists began to realize that they were homologous to genes that existed in humans and other animals. Many genes that play important roles in the development of humans and other animals were actually first identified in this screen, and then cloned as homologues of their Drosophila counterparts. The screen was particularly effective at finding genes involved in pattern formation - i.e., genes that tell cells where they are and what they are supposed to do. One reason the screen was so successful is that Eric & Janni made themselves experts on the larval cuticle, so that they could identify even subtle phenotypes.

C. Mutagenesis in Drosophila
A mutagen is an agent (chemical, radiation, transposable element) that induces changes in the sequence of the DNA. Chemical and irradiation Mutagens are not completely random, but most approximate it closely enough for practical use. For example, the most common chemical mutagen, Ethyl methane sulfonate (EMS) gives mostly G to A transitions. Usually males are treated with the mutagen, and the crossed with females to generate progeny, each of which can potentially have one or more induced mutations in their genome. Mutagenizing males is more efficient in terms of progeny production, since males produce a lot more sperm than females produce eggs. When a male is mutagenized (be feeding or injection of the mutagen) DNA base changes are made in his sperm. Thus, each of his sons or daughters inherits a different mutation(s). Since they also receive a maternal chromosome, these progeny are heterozygous for these new mutations. If you cant recognize the phenotype mutation in the next generation (in an F1 screen because there is a dominant phenotype, see below), then you need to do further crosses to see the effects of the mutation in a homozygote (F2 screen). I. Types of mutagens: chemical---e.g. EMS, generates point mutations at a reasonable rate. One lethal per chromosome arm, or one mutation per 1000 chromosomes for an average gene.

radiation----X-rays or gamma-rays, generates mutations at lower rates (~ 1/5000). They often generate deletions or other chromosome rearrangements. transposable elements-- Transposable elements are not a very efficient mutagen, and are less random that the other methods, but they have the great advantage of providing a molecular tag that can be used to clone a gene. The principle transposable element that is used for mutagenesis in Drosophila is the P element.

D. Screen for maternal effect mutants

Nsslein-Volhard, Wieschaus, Schpbach and others carried out similar screens to identify mutations that acted maternally to influence the patterning of the embryo. In these maternal effect mutations, the phenotype of an individual embryo is not determined by its own genotype, but that of its mother. Modified from (Schupbach and Wieschaus 1989; Schpbach and Wieschaus 1991) cn bw sp/ CyO, Cy cn bw (EMS treated)

cn bw / CyO, Cy

Fs(2)D/ CyO, Cy

cn bw / CyO, Cy cn bw / Fs(2)D Fs(2)D/ CyO, Cy

fertile females, Cy sterile females, Cy+ sterile females, Cy cross males to fertile females

cn bw / CyO, Cy

cn bw / CyO, Cy

cn bw / cn bw if alive, test for fertility keep cn bw / CyO, Cy for stock. Series of two papers I. Maternal effect mutations embryos made but do not develop II. Mutations blocking oogenesis or altering egg morphology no embryos.

I.

Analysis

I.1. Genetic mapping By meiotic recombination - determine the approximate location of the linkage map. By deficiency - determine a segment of the chromosome containing the gene. Remember that if the deficiency effects the gene of interest, than mat(2)a/Df(2) will have a mutant phenotype. Genetic mapping is an essential part of positional cloning and also required for advanced genetic studies. I.2. Establishing complementation groups - genetic definition of the gene.

Complementation tests genetic definition of the gene mat(2)a/ CyO crossed to mat(2)b/ CyO what is the phenotype of mat(2)a/mat(2)b If sterile, than mutations in the same gene, if fertile, than mutations in different genes. I.3. Types of mutations identified The mutants were divided into general classes based on their phenotypes. The reason for this should become clear, but it is a first attempt to classify the genes based on functions. This is where it is very important to have an intimate knowledge of the organism and its biology.

Mature stage 14 oocyte, the oocyte (red) and dorsal appendage are shown. Results divided into a series of two papers: I. Maternal effect mutations Class 1: Presyncytial arrest early, subito, bientot

Class 2: Syncytial blastoderm arrest grauzone, cortex, presto Class 3: Irregular cellularization at blastoderm stage cribble, sieve, valois Class 4: Abnormal cell behavior at onset of gastrulation cactus, dorsal, tudor, vasa (only one allele) II Mutations blocking oogenesis or altering egg morphology Class 1A: No or few egg chambers Class 1B: Abnormal number of nurse cells and tumorous egg chambers BicD, egl Class 1C: Early degeneration Class 2A: Degeneration of egg chambers in mid oogenesis Class 2B: abnormalitites in follicle cell migration, abnormalities in dorsal-ventral patterning of follicle cells or dorsal appendage formation cappuccino, gurken, torpedo, aubergine, gourd, okra, zucchini, squash, vasa It is possible that the genes in a class function in a common pathway. For example, gurken, aubergine, okra, zucchini, and vasa function in a pathway which regulates oocyte polarity (for example, vasa translationally regulates gurken, more below). It is also possible that hypomorphic alleles of a gene will be in a different class than null alleles. This would be expected to be in a class with a phenotype manifested later in development. I.4. Saturation of the screen

how many alleles per locus, then compare to predictions of the Poisson distribution if many genes with one allele, not saturated why would one not concentrate on genes with only single alleles? one reason, weak alleles of zygotic lethals. A maternal effect mutation must survive as a homozygote until reproductive age. Why would one not concentrate on genes with only single alleles? one reason, weak alleles of zygotic lethals. A maternal effect mutation must survive as a homozygote until reproductive age.

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E. The sensitized screen

Traditional genetic screens can miss genes that are important for a process of interest because of problems of pleiotropy or redundancy. In addition, traditional screens are time consuming because they hunt for recessive mutants (generations required to make a mutation homozygous One trick to get around these problems is to conduct a sensitized screen, which is also known as a dominant modifier screen. The fundamental concept behind a sensitized screen is to create a genetic condition whereby a mutation that would ordinarily be recessive can be isolated as a dominant enhancer or suppressor of another mutation. Simon et al (Simon et al. 1991) paper presents a classic example of this. In this study, the authors first created a temperature sensitive allele of the sevenless gene, such that they could create flies that just barely had enough sevenless function for normal eye development. They reasoned that in this condition, even a two fold decrease in the levels of activity of other genes in the sevenless pathway might yield a dominant phenotype (ie, in the background of the sevenless mutation, they would effectively have been rendered haploinsufficient). I. i. the Drosophila eye The Drosophila compound eye is composed of about 800 ommatidia. Each ommatidia includes 8 photoreceptor cells and a dozen support cells. ii. The ommatidia are formed progressively as a wave of differentiation, demarcated by the morphogenetic furrow, sweeps across the eye. The formation of each ommatidia involves the progressive recruitment and specification of cells. Because the eye is not essential for viablity, it has proved to be a good model system for a wide variety of genetic studies.

iii.

II.

Enhancer of sevenless screen

Sevenless encodes a transmembrane protein tyrosine kinase (similar to mammalian c-ros). photoreceptors (R7). In mutants, the R7 cell become a nonneuronal cell. Thus, the sev gene is required for the R7 cell to receive a signal to become a photoreceptor. That is all it does.

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However, other genes in the signal transduction pathway might be required in other tissues, and therefore, might be lethal when mutated.

This screen is effective because it is an F1 screen, and the mutants can be spotted by a simple visual inspection (importance simplicity). Because of this screen, genes were isolated that have a role in other developmental stages. Thus, while SEV is specific to eye development, it interacts with a signal transduction pathway that is involved in many cell types. Interesting genes were isolated, including ras and CDC25, thus implicating the ras pathway in eye development. The objective of genetic screens: These genetic screens are a method to identify the components of a signal transduction pathway or other biological process (such as a biochemical pathway). A combination of genetic screens is often performed with the goal of identifying most or all of the genes that function in a given process. Once the genes are identified, more advanced

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studies are performed to analyze the mutant phenotype and molecularly and biochemically characterize the gene product.

F. The RNAi screen and cell culture

Expressing double stranded RNA in Drosophila works effectively to reduce or eliminate gene function. This can be used in a reverse genetics approach to test the mutant phenotype of a specific gene. It can also be used to screen for genes affecting a specific function. Using a library of a library of double-stranded RNAs (dsRNAs) directed against all predicted open reading frames, the whole genome can be screened for a mutant phenotype. An example of this is at http://flyrnai.org/. Due to current technical limitations on transformation technology, this can only be performed in cell culture.

G. Working with Pleiotropic mutations

Mutations affect more than one distinct phenotype. Pleiotropy can be a complication for genetic analysis, because the requirement for a gene activity during one process may preclude your ability to assess its requirements during other processes. Drosophila geneticists have been able to overcome the problem of pleiotropy between zygotic and maternal requirements for gene function by combining three techniques: I. The production of germline clones. By inducing mitotic recombination, it is possible to generate cells that are homozygous mutant for a gene of interest in an animal that is otherwise heterozygous. II. The use of FLP-FRT mediated recombination, so that clones can be generated at a high enough frequency to be practical for screening. This techniques creates a group of mutant cells surrounded by wild-type cells. III. The use of dominant female sterile mutations or other markers like GFP. By deploying dominant female sterile mutations, one does not have to search for mutant phenotypes against a background of phenotypically wild-type eggs.

H. Important cytological methods

1)RNA in situ hybridization - when and where is the RNA expressed - often using single stranded RNA probes 2) antibody staining - when and where is the protein expressed - antibodies against protein from gene of interest - or use epitope tagging 3) live analysis using GFP tagged proteins

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I. Applications in Drosophila molecular biology modifying the genome for benefit.

Modern genetic analysis in Drosophila is always combined with molecular analysis. e.g. rather than trying to isolate a gain-of-function allele, now a days we will just use the cloned gene to create one. I. Insertional mutagenesis

Around the time of the development of transformation techniques (see below), it also became clear that P elements could be used to mutate existing genes. The appeal of this approach is that the P element provides a molecular tag that facilitates identification and cloning of the mutated gene. For example, inverse PCR can be used to identify the sequences adjacent to the P element insertion site. However, this approach is not the most efficient form of mutagenesis. First, there is a nonrandom insertion of P elements: certain sites are hot spots, while others appear to be cold spots. Also, the overall rate is lower than for chemical mutagenesis. Nonetheless, one goal of the Drosophila genome project is to get P element insertions in most or all of the genes in Drosophila (one insertion every 10 kb) - it is estimated that this will involve analyzing 100,000 to 200,00 independent insertions. Due to the insertion preference, 30% of all new insertions occur in previously mutated genes. However, the remaining 70% are more randomly inserted. With the genome sequence complete, new insertions can be readily mapped by inverse PCR and DNA sequencing. Another important feature of P element mobilization is the phenomenon of local hopping. P elements tend to reinsert near (within 200 kb of) their original location. One can take advantage of this in reverse genetic approaches where a P element resides in a gene that is near a gene of interest. By starting with a nearby P element, the chances that a new insertion will occur in the desired gene are greatly increased. For example, assume a P-element is found to be inserted near a gene of interest and the original insertion does not affect expression of the gene. This can be used to make deletion alleles. w; P(2)[w+] /CyO w/y+Y; CyO/+; +/ Sb D2-3 w/Y; P(2)[w+] /CyO; + /Sb D2-3 (dysgenic male) pick Cy Gla+ flies with white eyes w /Y; P(2)[w-]/ SM6, Cy

w; Gla/SM6, Cy

w; Gla/SM6, Cy

The excision of the P-element results in loss of the marker gene and a white eye phenotype. Each chromosome that lost the P-element is tested for a mutant phenotype. After two generations can make the P(2)[w-] chromosome homozygous. That is, cross P(2)[w-]/ SM6, Cy 14

males and females. If lethal, no Cy+ progeny will be observed. Otherwise, the Cy+ flies can be examined other phenotypes.

II.

Germline transformation

P elements are naturally occurring transposable elements. Spradling and Rubin took advantage of their property of mobility in the presence of transposase to develop a system for doing germline transformations (Spradling and Rubin 1982). Basically, the gene of interest is cloned into a vector that contains intact P element terminal repeats, but lacks the transposase gene. This plasmid is injected into embryos together with another plasmid, the helper which has an intact transposase gene but lacks normal P element ends. The DNA is injected during early, syncytial stages of embryogenesis, before the pole cells have formed. If some of this DNA is incorporated into the pole cells, then transposase can mediate the insertion of the P-element vector into germline chromosomes. Since, the transposase plasmid cannot insert into the genome, it will eventually be lost, after which the inserted construct will be stably maintained. In order to identify individuals into which a DNA construct has been successfully transformed, P element vectors also contain wild-type copies of genes that can be used as markers. Commonly used markers are two genes that affect eye color, white and rosy, and one that effects body color, yellow. DNA is injected into embryos that are mutant for one of these genes.

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Since P element transposition is involved with DNA transformation in flies, the size of the construct is inversely proportional to the efficiency of transformation. For example, cosmids transform at much lower rates than small plasmids. III. The Gal4-UAS system

The ability to mis-express (ectopic expression) or over-express a gene is a powerful technique for studying its function. Ectopic expression allows us to ask what a gene is sufficient to do when expressed outside of its normal context, as opposed to what it is necessary for where it is normally expressed. Mis-expression effectively creates dominant, gain-of-function alleles that can be used for genetic studies (e.g. genetic screens, epistasis tests).Traditionally this could be done by cloning a gene of interest under the control of a heterologous promoter that may be inducible (e.g. heat shock promoter), or expressed in a specific pattern. Several years ago, Brand and Perrimon (Brand and Perrimon 1993) developed the UAS-Gal4 system for driving the expression of cloned genes under the control of heterologous promoters. Their approach is a two component system that employs a transcriptional activator from yeast, the GAL4 protein. In yeast, GAL4 binds to its UAS (Upstream Activation Sequence) and drives the transcription of adjacent promoters. GAL4 does not normally exist in Drosophila, nor do flies have any genes that are regulated by the GAL4 UAS. Thus expression of GAL4 alone, or the presence of a transgene under adjacent to UAS sequences, has no effect in the fly. However, in animals containing both a GAL4 expressing transgene and a gene of interest under UAS-control, the gene of interest will be expressed in whatever pattern GAL4 is expressed in. This is done by crossing flies containing separate UAS and Gal4 transgenes. The Gal4-expressing lines can either be constructed as fusions between a desired enhancer and the Gal4 ORF, or they can be generated by the technique of enhancer trapping (see below). Driving ectopic expression using a 2-component system such as this has three important advantages: Can bypass problems of lethality Its modular - by generating one UAS transgene, hundreds of different enhancers become available It facilitates co-expression experiments

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IV.

Enhancer trapping

About 15 years ago, another important technique for identifying genes was developed in Drosophila, the enhancer trap method. The basis for this approach is that when a transgene is inserted into a chromosome, its expression can come under the influence of the enhancers that regulate the expression of the gene into which it has inserted. Enhancer trapping was discovered accidentally, when scientists who were studying transcriptional control regions took putative regulatory sequences and used them to drive the expression of a reporter gene like lacZ (galactosidase). Subsequently, vectors were developed that have a minimal promoter driving lacZ expression. Enhancer trapping allows the cloning of genes based on their expression pattern, rather than on their mutant phenotypes. This has both advantages and disadvantages over a traditional genetic screen that is based on looking for mutant phenotypes - hence it facilitated the identification of a number of interesting genes that would not otherwise have been identified. Since the enhancer trap is a P element insertion, in some cases it will disrupt the gene whose expression it reports, and produce a mutant phenotype. For reasons that are not well understood, but presumably have to do with chromatin structure, P elements preferentially insert 5 to transcription units or introns. Another advantage of enhancer trapping as a method for identifying new genes of interest is that the genes are molecularly tagged, which facilitates cloning. The simplest way to generate new enhancer trap insertions is to use a strain containing an insertion, P[ry+ 2-3], which makes P element transposase but cannot move itself. These flies are crossed to another strain that contains an inserted enhancer trap construct. By using markers to follow the linkage of P insertions, one can score for new transpositions only and stain for new expression patterns of interest. A strategy for this was described in previous notes. The enhancer trap insertion presents a starting point for genetic analysis of the gene. Even if the original insertion is not a mutation in the gene, the phenomenon of imprecise excision makes it relatively straightforward to obtain mutations (as described for subito). When P elements excise, sometimes the excision is imprecise - presumably because the free ends of the DNA were attacked by nucleases, and this damage was not fully repaired. Since imprecise excision results in the deletion of sequences flanking the original P element insertion, and P elements often insert close to the start of transcription, imprecise excision can usually be used to efficiently generate null mutations. In addition to lacZ, enhancer trapping has also been done with other reporters such as GFP. One very important variation is the Gal4 enhancer trap. V. P-element used in genetic mapping

Fine genetic mapping: Another important use of P elements is as markers for positional cloning. Since all P elements are marked genetically, and their position can be unambiguosuly determined molecularly, the order and distance between a P element and a mutant of interest as determined

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by genetic recombination can be used to help define the molecular location of that gene. This can be done either using standard recombination or P-induced male recombination VI. FLP-FRT

An alternative method for generating ectopic expression is the FLP-FRT or Flip-out method. In this system, a gene of interest is expressed under the control of a constitutive, ubiquitously expressed promoter, such as an actin promoter.
constitutive promoter FRT marker transcription terminator FRT My Gene

A transcription terminator is cloned in between the promoter and the gene of interest, such that the gene is not expressed. Flanking the transcription terminator are FRTs - flipase recombination target sites. In the presence of the flipase enzyme, recombination between the two FRT sequences can occur, resulting in excision of the transcription terminator and expression of the gene of interest. Low level, transient expression of flipase is induced (using a heat shock promoter), such that recombination will only occur in a few cells. As these cells proliferate, they will give rise to clones of cells (Flip-out clones) in which a gene of interest is ectopically expressed.

rk ma

er

tran s term criptio inat n or

constitutive promoter

FRT FRT

My Gene

ma

rke

tran s term criptio inat n or

FRT constitutive promoter

+
FRT My Gene

Typically, a marker gene is also included within the DNA that will be excised by flipasemediated recombination, so that cells in which this recombination has occurred can be unambiguously identified. In the FLP-out system, the pattern in which a gene is ectopically expressed is lineage-based, rather than position based as in the UAS-Gal4 system. Since lineage in most Drosophila tissues is indeterminate, this makes it much more versatile in terms of the possible patterns in which a gene can be expressed. Having a lineage-based system with a constitutive promoter also reduces the chances of generating artifacts in genetic experiments that might derive from influences on the expression of the specific promoter being used. An especially powerful and versatile combination is the Flp-out Gal4 method, in which the gene expressed by Flp-out is Gal4. This allows one to take advantage of all the pre-existing UAS lines, but still conduct Flip-out experiments. It also facilitates easy co-expression in Flip-out clones (e.g. epistasis experiments, markers). It is important to realize that as ectopic expression techniques both the UAS-GAL4 and Flip-out methods have the potential to generate misleading information about gene function. That is, what 18

a gene can do when it is ectopically expressed is not always an accurate reflection of its normal role. The clearest understandings of gene function usually come when both loss-of-function and gain-of-function techniques are applied. VII. FRT based screens

In a standard F2 genetic screen, a recessive mutation (*) induced in the germline of the parent is identified in homozygous flies of individual lines that are generated from three generations of crosses.

Using a strain having a P[FRT] element inserted near the centromere of a chromosome arm, induced recessive mutations on that arm can be identified in F1 heterozygous individuals by producing and examining somatic clones of cells that are homozygous for the mutagenized chromosome arm. In addition to being faster than a traditional screen, this approach bypasses problems of pleiotropy, and can also identify phenotypes that only occur in genetic mosaics.

J. Genetic mosaics
One of the most powerful and important techniques in the Drosophilists bag of tricks is the ease with which genetic mosaics can be created & analyzed. Gene expression in multicellular organisms poses many new and interesting questions the practioners of genetics in single celled organisms do not have to deal with. Where is a gene required? Does it act autonomously? What are the functions of an essential gene at later stages of life? These questions can be addressed by creating genetic mosaics. This is accomplished by altering the genetic content of cells that were identical at earlier stages. Genetic content can be altered by causing chromosomal loss, inducing mitotic recombination between heterozygous homologs, or by excision of a DNA segment.

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Genetic loss of the X chromosome is one of the oldest methods for creating genetic mosaics. Loss of an X chromosome in a female produces a gynandromorph, part male and part female. I. Mitotic recombination Loss of an allele can be induced by recombination between heterozygous homologs during mitosis. This occurs naturally at a very low rate, but Drosophila geneticists discovered long ago that it could be induced by X-rays. In this process, a cell heterozygous for a gene is made homozygous mutant in one daughter cell and homozygous wild-type in the other. As the daughter cells continue to proliferate, they will produce a clone of cells that are genetically alike, and distinct from other cells in the animal . The clones are marked using visible mutations that act cell-autonomously (i.e. genotype & phenotype are coincident). Two major drawbacks of generating mitotic recombination clones by X-rays are: 1) The process is still not that efficient, since if you make the X-ray dose too high you kill your subjects 2) Recombination does not occur in a discrete location, but can occur anywhere along the chromosome.

II. FLP-FRT mediated mitotic recombination In 1991 Golic & Lundquist introduced a new way to generate mitotic clones that solved the major limitations of X-ray mediated recombination. This process is so efficient that it can be used for genetic screens (see below). The process uses a site-specific recombinase from the yeast 2 circle, called Flipase (FLP). The target sequences for the FLP enzyme, called FRTs (Flip-recombination Target) are engineered into the Drosophila genome (by P-element based transformation see below). FLP expression is under the control of a heat shock promoter. Heat shock is used to transiently express the recombinase. This allows for its induction at virtually any time of development (except pre-blastoderm embryos). FRT sites have now been engineered into the bases of all chromosome arms (why is this important?). An important advance for the analysis of developing tissues by mitotic recombination has been

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the creation of molecular markers, such as the -Myc protein or GFP. -Myc is a non-functional protein that has had a Myc epitope tag added to it. Antibodies to the Myc epitope are commercially available, and expression of -Myc can be used as a cell autonomous marker in mosaic experiments where one is analyzing molecular (ie gene expression) phenotypes (see Figure: Example of clones in the Drosophila wing imaginal disc.

III.

A special type of mosaic - germ-line clones

Recall that for any gene required for zygotic development, mutants will not survive until adulthood. Thus, one cannot determine the effects of lacking this gene on an adult. There is a variation of mosaic analysis referred to as the dominant female sterile technique (Chou and Perrimon 1996). A dominant mutation (ovoD) prevents oogensis. Only if a mitotic recombination event occurs will there be cells lacking the mutation, and therefore, oogenesis can proceed. This recombination event also makes the other chromosome (with a gene of interest on it) homozygous.

In this way, the effects of a zygotic lethal mutation on oogenesis and early embryonic development can be determined. Whereas the homozygotes are lethal as embryos or larvae, in this method, the heterozygotes are grown up as heterozygotes. Then, using FLP/FRT, mitotic recombination is stimulated in the germline, producing homozygous mutants cells in an otherwise heterozygous animal.

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K. Gene targeting
A new approach for reverse genetics has become available in Drosophila, gene targeting by homologous recombination (Rong and Golic 2000; Rong and Golic 2001). The scheme involves creating a donor molecule homologous to the genomic sequences to be replaced, but with an introduced mutation. The donor sequence is cloned in between two FRT sites. In addition, a target site for a very rare cutting restriction enodonuclease, I-SceI (which has no sites in the Drosophila genome) is introduced in the middle of the donor DNA. This construct is then introduced into flies by P element transformation. By inducing the expression of the flipase enzyme and then I-SceI enzyme (expressed under heat shock control from different transgenes), the DNA in between the FRT sites is excised as a circle, and then cut by I-SceI. The resulting DNA, with a double strand break in the middle of the region of homology, is recombinogenic, presumably because the intact, wild-type allele can be used as a template for repairing the break, and in the course of this repair the donor DNA can invade and replace DNA at the endogenous locus. People had tried to establish homologous recombination for many years in Drosophila and failed. Although it is not certain why Rong & Golic succeeded, one hypothesis is that a chromosomally-derived donor molecule is more efficient at targeting. When excised, this DNA retains some important properties of chromosome structure.

Using the ends-in method, tandem copies of the gene are generated. This method is more complex but can be used to generate point mutations. The alternative, called endsout, results in gene replacement. This is a simpler methods since but is not used to introduce point mutations. The marker (functional yellow or white gene) is inserted in the gene, which also provides the inactivating mutation.

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In this example, a gene truncated at both ends was targeted. This generates two copies of the gene, with corresponding deletions. This only works if the gene is large enough, because at least 3-4kb is required for efficient targeting. Mutations can be engineered into both halves of the gene to generate mutations in both copies of the gene following targeting. If only one mutation is present (and therefore, targeting leaves one functional gene), then a reduction technique can be applied. Beside the white marker gene is a CreI cut site. Crossing the targeted flies to one expressing the enzyme CreI enzyme will result in a double strand break at the CreI site. This leads to a repair event in which the two copies of the gene essentially anneal to each other and leave a single copy (it is called single strand annealing). In about half the cases one expects the single gene to carry the introduced mutation. There is some excitement about this method since it might be applicable to a variety of organisms (the patentable idea is the excision from the chromosome being important).

I.

Sample cross: w; P{target::gene-x}/+ or balancer or homo. heat shock progeny as larvae to cause targeting in the germline pick Cy+ females. w; 70FLP/70FLP constitutive source of FLP

w; 70FLP 70I-SceI / S2 CyO souce of FLP and SceI enzymes

w; 70FLP 70I-SceI/ P{target::gene-x}

1. Screen for non-mosaic progeny (if moved, FLP no longer excises) 2. Cross to remove 70FLP, 70I-SceI and make stock w; P{target::gene-x}/+; 70FLP/+ test for linkage to appropriate chromosome. These are potential mutants.

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L. References
Brand, A.H. and N. Perrimon. 1993. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118: 401-415. Chou, T. and N. Perrimon. 1996. The autosomal FLP-DFS technique for generating germline mosaics in Drosophila melanogaster. Genetics 144: 1673-1679. Riechmann, V. and A. Ephrussi. 2001. Axis formation during Drosophila oogenesis. Curr. Opin. Genet. Dev. 11: 374-383. Rong, Y. and K.G. Golic. 2001. A targeted gene knockout in Drosophila. Genetics 157: 1307-1312. Rong, Y.S. and K.G. Golic. 2000. Gene targeting by homologous recombination in Drosophila. Science 288: 2013-8. Schupbach, T. and E. Wieschaus. 1989. Female sterile mutations on the second chromosome of Drosophila melanogaster. I. Maternal effect mutations. Genetics 121: 101-117. Schpbach, T. and E. Wieschaus. 1991. Female sterile mutations on the second chromosome of Drosophila melanogaster. II. Mutations blocking oogenesis or altering egg morphology. Genetics 129: 1119-1136. Simon, M.A., D.D. Bowtell, G.S. Dodson, T.R. Laverty, and G.M. Rubin. 1991. Ras1 and a putative guanine nucleotide exchange factor perform crucial steps in signaling by the sevenless protein tyrosine kinase. Cell 67: 701-16. Spradling, A.C. and G.M. Rubin. 1982. Transposition of cloned P elements into Drosophila germ line chromosomes. Science 218: 341-347.

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