Development and Genetics Of Drosophila BSc Module PDF

Development and Genetics of Drosophila Großhans laboratory BSc module “Function and Development I” Genetics and cell biology of development LV-17-026-271 WS 2024/25 Prof. J. Großhans, Dr. D. Kong Important notes on the...

Development and Genetics of Drosophila Großhans laboratory BSc module “Function and Development I” Genetics and cell biology of development LV-17-026-271 WS 2024/25 Prof. J. Großhans, Dr. D. Kong Important notes on the report: You will prepare a report which contains the theory of the experiment (where necessary divided into theory of the biological system/organism plus theory of the experiment), implementation, result, evaluation and discussion for each course day/course section/experiment. The procedure should be summarized briefly and concisely in your own words. Evaluation means, for example, that embryos are shown correctly oriented on images, i.e. anterior to the left and ventral to the bottom, and relevant patterns or tissues are marked with arrows and explained in the figure legend. In addition, the protocol includes: a cover sheet, a list of contents, sources and figures as well as page numbers. The transcript should be written on a computer, be justified and not exceed 15 pages (including cover sheet etc.) + appendix. Drawings made during the practical course are added to the appendix. These drawings can be added to the transcript either in the original or as a scan if submitted electronically. Deadline for submission of the report: Sunday January 12th, 2025 Ilias folder Name of the report: YourSurname-FirstName.pdf 2 Programme Week 1 1. Expression of the primary pair rule gene even-skipped in seven stripes. You will analysed embryos stained for Eve protein by immune-histochemistry. You will recognize developmental stages of embryos and relate to the dynamics of the expression pattern in wild type and patterning mutants. In addition you will analyse cuticles of the same mutants. 2. Expression of segmentation genes by lacZ reporter genes. You will stain embryos expressing lacZ reporter constructs driven by the promoter of segmentation genes. You will fix embryos and conduct X-gal stainings. You will recognize developmental stages and relate to the dynamics of the expression pattern. Group 1 Group 2 Experiment Tuesday, 10.12 Thursday, 12.12 Exp 2: Expression pattern of segmentation genes by lacZ reporter transgenes. X-gal staining of embryos Wednesday Friday 13.12 Exp. 1: Segmentation mutants: Cuticles and expression 11.12 pattern of Even-skipped (prepared slides) Exercises about sequence analysis and gene structure Week 2 3. Dpp expression at the compartment boundary in imaginal discs. You will prepare imaginal discs from living third instar larvae and stain them for dpp expression by X-gal. The larvae contain transgenes with part of the dpp promoter driving GAL4 and with a UAS-lacZ reporter. You will recognized leg discs and describe the expression pattern. 4. Eyeless induces eye development. You will analyse flies for additional eyes or eye rudiments. In these flies the gene eyeless was ectopically expressed in all imaginal discs in addition to its endogenous expression in the eye-head disc by the GAL4-UAS system. You will prepare the flies and describe the morphology of the extra eyes. 5. Four-winged diptera due to a homeotic haltere-wing transformation. You analyse mosaic flies, which are mutant for the homeotic gene Ubx only in the haltere disc. The Ubx knockout is induced by expression of CAS12 endonuclease only in larval haltere discs by the GAL4xUAS system together with a ubiquitous expression of Ubx specific guide RNAs. Group 1 Group 2 Experiment Tuesday, 17.12 Thursday 19.12 Exp.3: Preparation of larval imaginal discs, Expression pattern of dpp (dpp-GAL4 x UAS-lacZ) Wednesday 18.12 Friday 20.12 Exp. 4: Ectopic expression of eyeless in leg discs. Analysis of adult morphology for ectopic eyes Wednesday 18.12 Friday 20.12 Exp. 5: Four-winged diptera. Tissue specific gene knock- out by CrispR. Homeotic transformation of halteres to wings in Ubx mutants Exercises about sequence analysis and gene structure 6. Computational sequence and gene analysis. You will practice the analysis of nucleotide and amino acid sequences. You will learn how to retrieve sequences from data bases and subject 3 them to computational analysis such as finding open reading frames, translating nucleotide sequences into peptide sequences, finding evolutionary conserved genes and protein domains by sequence comparison with BLAST. 4 Introduction You will find a detailed introduction to Drosophila as part of the materials to the course “Drosophila-Intro.pdf”. Please thoroughly read this introduction. In the first part the life cycle is described. The genetic part is rather specific in part, which requires more practice for full understanding. Experiment 1 Analysis of patterning mutants One aim of the genetic cascade for patterning of the anterior-posterior axis is formation of the fourteen segments. While the gap genes define the positions of each seven stripes of the primary pair-rule genes even-skipped, runt and paired, the segment polarity genes double that number to 14. The seven stripes of eve (similar for runt or paired) are controlled by the upstream acting maternal genes such as bicoid and gap genes such as hunchback or Krüppel but not by genes acting downstream of the genetic cascade such as the segment polarity genes including wingless, or hedgehog. Therefore the seven eve stripes will be affected in a characteristic manner in hunchback mutants but not in wingless mutants, for example (Figure 1). Please keep in mind that the embryos are generated by a cross of heterozygous parent flies resulting in only one quarter of mutant embryos in case of recessive mutations. The slides will contain a mixture of homo- and heterozygous embryos with corresponding mutant and wild type phenotypes. Please also keep in mind that the proportion of mutant embryos is very different for maternal mutations, in which only the maternal genotype matters. Bicoid is an example of a maternal mutation. In case of maternal mutations, all embryos are generated by homozygous females and thus show the mutant phenotype. Figure 1: Eve expression pattern and cuticle phenotype of knirps mutants. (A) Cuticle phenotype and (B) pattern of eve mRNA distribution in a knirps mutant background. Knirps is a gap gene acting upstream of eve. Eve RNA and Eve protein expression pattern largely correspond to each other except for a short delay by several minutes. You will receive slides marked with numbers containing cuticle preparations of mutants from the segmentation cascade similar to what you analysed in the BM3 last spring to remind you about their classification in gap genes, pair-rule genes and segment polarity genes. You will also receive slides with embryos stained for the pair-rule gene even-skipped. Eve protein was visualized by histochemical staining after incubation with an anti-Eve antibody. To analyze the wild type pattern of eve as a reference, you will also receive slides with embryos of the wild type strain called Oregon R, also labeled with anti-Eve. 5 Procedure 1. Analyse wild type embryos. Select two representative wild type embryos at two different times (earlier and later) of Eve expression. Draw the embryos with the pattern of Eve staining. 2. Using the 40x objective, assess the exact width of the stripes (e.g. 3 cells, 4 cells). Eve protein is a transcription factor and thus labels nuclei. Draw what you observe. Indicate the width of the stripe. 3. Analyse the mutant embryos. Draw the corresponding perturbations in the expression pattern of the Eve staining using the two different mutants. 4. Analyse the corresponding cuticle preparations. Draw a representative wild-type embryo laterally and ventrally (showing the dentical belts) and a representative embryo of each of the four different mutants. 5. Record photographs with your handy camera of the nicest embryos/cuticles. Report 1.1 Four drawings of wild type at low magnification, one drawing at high magnification (40x objective) 1.2 Drawings of Eve staining in four mutants 1.3 Drawings of cuticles 1.4 Handy photographs 1.5 Assign the class of mutant (gap, pair rule or segment polarity) to the preparations? Questions 1.6 What is the difference between primary and secondary pair-rule genes? Name examples! 1.7 How were the fixed embryos stained for Eve protein? Briefly describe the steps of the procedure! 1.8 Is it possible to detect eve RNA? What is the name of the procedure? Would the result be similar? 6 Experiment 2: Expression analysis of patterning genes by lacZ reporters Several methods are available to reveal gene expression patterns within a tissue or embryo. A method that was an important step forward in genetic research due to its simplicity and ease are lacZ reporters and enhancer traps. For reporter genes the known or assumed promoter region including upstream regulatory sequences (enhancer, silencer) are put together with the cDNA of reporters genes such as lacZ, encoding ß-galactosidase from E. coli. For enhancer traps a transposon with a minimal reporter gene are inserted randomly in the genome and screened for specific expression pattern. Nowadays, visible reporters such as GFP and variants are more commonly used. In Experiment 2 you will analyse early embryos in blastoderm stage and gastrulation for expression of patterning genes. The embryos contain transgenic reporter genes, in which the regulatory sequences are fused to a minimal promoter and the lacZ coding sequence. Thus lacZ will be expressed in the same pattern as the endogenous gene. Your task will be to analyse and describe the expression pattern. The difficulty is that the expression pattern changes with development. You need to recognize the developmental stage and the expression pattern. Detection of ß-gal activity in situ Fixation of a tissue by chemical crosslinking as is caused by treatment with formaldehyde or glutaraldehyde does not completely destroy the enzymatic activity. It is therefore possible to conduct the enzymatic reaction in the fixed tissue. In contrast to bacterial or yeast genetics, where the reaction is conducted with colonies of living cells, it is important to fix the tissue as color development may take several hours in which the embryos would further develop. In the first step ß-galactosidase catalyzes the hydrolysis of X-gal to galactose and a indoxyl derivative. In the second, spontaneous, step two indoxyl molecules dimerize and are oxidized to the blue dye indigo. The oxidation substrate may be oxygen from air. To promote oxidation, the staining solution contains hexacyanoferrate (Figure 2). Figure 2: Hydrolysis and oxidation of X-gal to indigo. X-gal is a galactose derivate, whose hydrolysis is catalysed by ß- galactosidase to galactose and an indoxyl derivate. The blue dye indigo is formed by dimerization of the indoxyl followed by oxidation. You will receive apple juice plates which contain with an age of embryos in the age of zero to four after fertilization/egg laying. The plates have been stored in the cold room to pause development. 7 The petri dishes are coded with numbers. You will prepare and fix the embryos. Following staining you will analyse, describe and document the expression pattern of the lacZ reporter gene. Buffers and solutions Dechorionisation solution: 50% (v/v) household Klorix, what contains the oxidizing agent hypochlorite, ClO– Fixative: 25% glutaraldehyde in 100 mM Na-phosphate [pH 7.4]. Dilute 2.7 ml 25% glutardialdehyde with 0.3 ml 1 M phosphate buffer [pH7.4], 15 ml n-heptane. Shake vigorously allowing to dissolve some of the fixative into the organic phase. For caregivers: Shake vigorously immediately before use and only remove and use the upper heptane phase! Staining solution: 10 mM Na-phosphate buffer [pH 7.2], 150 mM NaCl, 1 mM MgCl2, 3.3 mM K4[Fe(II)(CN)6], 3.3 mM K3[Fe(III)(CN)6], 0.1% X-gal PBT wash buffer: PBS, 0.1 % Tween Mounting medium: 25% glycerol in PBT Procedure Preparation (under the fume cupboard!) 1. Place 2 ml of the fixation solution in a 12-well plate under the fume cupboard and mark your well. 2. Spread Klorix onto the apple juice plates with the embryos. Gently swirl to ensure that all embryos are covered with Klorix. 3. Incubate for three minutes, at least. Gently swirl the plate and observe whether they are detached from the surface. 4. Pour the Klorix solution into the nets to collect the embryos. You may do this over the sink. 5. Wash the embryos in the net several times in a block bowl filled with water and dab the net with the dechorionized embryos briefly on paper towel to remove excess Klorix solution. Fixing (in the hood) 6. P l a c e the net with the dechorionized, dried embryos in the fixation solution of the 12- well plate. 7. Place the lid on the 12-well plate and fix the embryos on the shaker for approx. 10 min with constant mixing. 8. In the meantime, prepare a block dish with 2 ml of staining solution. Please do not forget to label it! 9. After 10 min of fixation, dab the net with the fixed embryos briefly on paper towel to remove any of the heptane. 10. Collect the waste (fixation solution) in the liquid waste container in the hood. Staining (on your lab bench) 11. Quickly place the net with the fixed embryos into the staining solution and leave to stain at 37°C for approx. 45 min. 8  12. Check the intensity of the staining from time to time. If the staining is weak, extend the dyeing process by a further 15 minutes, at least. 13. As soon as the staining is clearly visible, carefully pipette off the staining solution in the block dish and wash the net with 1 ml PBT in the block dish. 14. Dab the net with the stained embryos briefly on paper towel and transfer the embryos with a brush to a slide on which one drop of 25% glycerin/PBT has been placed. 15. Carefully distribute the embryos and carefully place a 24 x 36 mm coverslip on top. 16. Observe your preparation under the microscope and analyze it. 17. Prepare drawings of three embryos, at least. Prepare a drawing with a 10x objective. Prepare a second drawing of the same embryo with a 40x objective. In total you will prepare six drawings, at least. 18. Record a photograph with your hand camera of a representative embryo. Safety instructions and protective measures Klorix contains the strong oxidizing agent hypochlorite (ClO – ), which bleaches and decolors textiles. Avoid spilling Klorix on the bench. n-heptane and glutardialdehyde (fixing solution) can irritate the respiratory tract and cause skin reactions and eye damage. Work under the fume cupboard during handling! Make sure to keep the fume cupboard closed as far as possible. A separate liquid waste container is provided under the fume cupboard for the disposal of the fixing solution! The staining solution contains 0.1% X-Gal, which may cause skin irritation. A lab coat, protective gloves and eye protection must be worn during practical work. Report 2.1 Drawing of three embryos (each at 10x and 40x) 2.2 Photographs from Handy 2.3 Describe the expression pattern in words. Be remined that the expression changes with developmental stage. Based on the expression pattern, what class of patterning gene is it (gap gene, pair rule gene, segment polarity gene)? Name representative genes of this class! Questions 2.4 What is the function of n-heptane during fixation of the embryos? 2.5 How does Glutharaldehyde fix cells/embryos/tissue? What is the underlying chemical reation? 2.6 Briefly describe the procedure how the lacZ coding sequence was introduced into the Drosophila genome? How is the transcription of lacZ controlled in the embryos from these stocks? 9 Experiment 3: Gene expression pattern of the dpp in larval imaginal discs The GAL4-UAS system is a binary genetic system for controlled gene expression independent of the endogenous expression pattern and levels (Figure 3). The expression pattern is determined by the spatio-temproal expression pattern of the transcription factor GAL4 from yeast. GAL4 is a prototypic transcription factor from yeast, which binds to a specific DNA sequence (UAS, upstream activating sequence) and will activate transcription at a nearby promoter. GAL4 is pretty neutral in Drosophila cells and induces dominant effects by its own only at high expression levels. The second part is a cDNA under control of a minimal promoter and UAS sites. Transgenes are available with a UAS sequence upstream of the cDNA of a gene of interest for most genes. Again this UAS transgene is neutral and not or little expressed by itself. However in the presence of GAL4, transcription is activated by GAL4 bound to the UAS sites. The binary system allows the expression of any gene in any selected expression pattern even if it is toxic or lethal. Figure 3. The GAL4/UAS system in Drosophila. Females homozygous for the GAL transgene are crossed with males homozygous for the UAS transgene or vice versa. Progeny from the cross, embryos, larvae, pupae or adults contain each one copy of the GAL4 and UAS transgene. Corresponding to the expression of GAL4, the gene behind the UAS will be expressed in a similar spatial and temporal pattern. from Duffy, Genetics 34 (2002) 1–15. (C) Expression pattern of a gene driven by a dpp-GAL4 in a larval wing disc. Dpp is expressed at the dorsal-ventral border. Here you will visualize the expression pattern of the dpp gene (decapentaplegic) with a UAS-lacZ reporter. For this purpose, you will receive fly tubes in which dpp-Gal4 and UAS-lacZ flies have already been crossed with each other. In Experiment 4 will use the dpp-GAL4 line to induce ectopic expression of the gene eyeless in imaginal discs. In contrast to the previous experiments with embryos, you will analyse larval tissues (Figure 4). Beside the differential tissues, the larvae contain undifferentiated so-called imaginal discs, which will build the adult tissues during pupation while most larval tissues are degraded. During larval stages the imaginal discs proliferate with about a cell cycle every ten hours. Only during pupation, the imaginal discs differentiate and build the adult specializations and morphology. You will learn how to prepare larval discs by manual dissection. 10 Genotypes of the fly strains dpp-Gal4 (T041) B-#1553 w[*]; wg[Sp-1]/CyO; P{w[+mW.hs]=GAL4-dpp.blk1}40C.6/TM6B, Tb UAS-LacZ (T039) B-#1777: w[*]; P{w[+mC]=UAS-lacZ.B}Bg4-2-4b Simplified crossing scheme: Sp/Cy0; dpp-Gal4/TM6B, Tb x +/+; UAS-lacZ/UAS-lacZ Buffer/solutions: Wash buffer (PBT): Phosphate-buffered saline (PBS) + 0.1% Tween Fixation solution: 4% formaldehyde in PBT + 1% Triton-X 100 Staining solution: 10 mM Na-phosphate [pH 7.2], 150 mM NaCl, 1 mM MgCl2 , 3.3 mM K4 [Fe(II)(CN)6 ], 3.3 mM K3 [Fe(II)(CN)6 ], 0.1% X-Gal Mounting medium: 25% glycerine/PBT Procedure 1. The crossbreeding scheme results in different genotypes in the offspring. Create a Punett's square based on the genotypes. 2. What percentage of the offspring have both the dpp-Gal4 driver line and the UAS-lacZ reporter gene? 3. Why is it essential for the experiment to distinguish between the genotypes that occur? 4. Collect at least five larvae of the third and thus the largest larval stage from the fly tubes provided. Make sure you have the correct phenotype! The ddp-Gal4 line is heterozygous and is maintained via the balancer TM6B with the marker Tb (tubby). This marker results phenotypically in shorter larvae and a stocky appearance. Preparation of discs 5. Transferred the larvae into a block dish with 1 ml PBT and briefly wash them once. 6. Fix a larva at the rear end with tweezers, grasp the head skeleton at the front end with a second, very fine pair of tweezers (Dumont #5) and carefully tear apart the larva. The larvae will be opened and release to the outside the imaginal appendages as well as the salivary glands and the CNS. Fixation 7. Carefully peel off the solution and exchange with 2 ml of the fixative (formaldehyde in PBT). Incubate for approx. 20 min at room temperature. 8. Remove the fixation solution (caution: do not aspirate the tissue!) and add approx. 1 ml PBT. 9. Repeat this washing step twice with 1 ml PBT each time Staining reaction 10. Add 2 ml of staining solution to the prepared tissue in the block dish. 11. Then cover the block dish with a lid and incubate for at least 60 min at 37°C in the incubator. 11  12. Check the dyeing intensity after one hour and, if necessary, continue dyeing until a visible coloration appears. 13. Remove the staining solution and wash the tissue twice with 1 ml PBT each time. Mounting and documentation 14. Then transfer the tissue in a drop (≈200 µl) of 25% glycerol/PBT to a microscope slide, expose the imaginal discs and cover with a 24x36 mm coverslip. 15. Select three imaginal discs (eye, leg and wing/holder) with a clear staining. Manually draw three discs with the staining, at least. Label your drawings (anterior-posterior, type of imaginal disc, microscope objective used). 16. Record images of representative examples with your handy camera. Important: The fabric must not dry out at any time during the test Report 3.1 Drawing of three types of imaginal discs with proper labeling 3.2 Photographs of imaginal discs Questions 3.3 Why did you prepare imaginal discs from third instar larvae instead of first or second instar larvae? Would you expect a similar pattern in the younger discs 3.4 Can transcription factors other than GAL4 be used for a binary expression system? What are the prerequisites for functionality in Drosophila? 3.5 What is the function of dpp in imaginal discs? Brief answer, few words only 3.6 Why did we use X-gal staining for detection instead of antibody staining as you did in experiment 1? Which antibody would you need for staining? Figure 4: Anatomy of the larvae. (A) Schematic drawing of a larva. Tissues, imaginal discs and organs are indicated. (B) Exemplary imaginal discs from 3rd larvae, stained with X-gal for a LacZ reporter that stains all imaginal discs. 12 Experiment 4: Eyes in the leg by ectopic expression of eyeless A classical experiment to assess the function of a gene is ectopic expression or activation. Typically mutants reveal a loss of function phenotype, i. e. they indicate whether a gene is required for eye development, for example. Certainly, all sorts of genes are required including general house-hold genes. Thus, a plain requirement is not necessarily informative. In a second step the sufficiency, for example, for eye development needs to be assessed. For genes with spatially or temporally restricted expression, this is achieved by ectopic expression. Such an experiment allow to reveal the activity of a gene outside its normal situation. You will analyse flies, in which the gene eyeless was expressed in the leg imaginal discs during larval development. Normally eyeless is expressed in the eye/head disc but not other imaginal discs. Technically expression of eyeless is induced with the GAL4xUAS system. The flies contain two genetic elements, a transgene with an eyeless cDNA under the control of a UAS promoter and a transgene with the GAL4 sequence under the control of a promoter activated only in parts of the leg imaginal discs. Specifically, GAL4 will be expressed under control of the dpp promoter at the boundary of anterior and posterior compartment. You have already analysed the expression pattern of this transgene in Experiment 3. You will receive vials with progenies of a cross of these two stocks, thus containing each one copy of the transgenes. Full genotypes of the fly strains B-#1553: w ; Sp / CyO ; P{GAL4-dpp.blk1, w+}40C.6 / TM6B, Tb B-#6294: y1 w ; P{ UAS-ey.H, w+}UE11 ; +/+ Simplified crossing scheme: (handed out ready for evaluation) Sp / CyO ; dpp-GAL4{w+} / TM6B,Tb X UAS-eyeless{w+} / UAS-eyeless{w+} ; +/+ ---→ Four different genotypes, which are visible by the dominant markers Sp and TM6b, Tb as well as the w+ markers of the transgenes. Procedure 1. Anesthetize the flies from the tubes with ether under the hood and collect the animals in a large Petri dish, also take the fly tube back to its place! 2. If the flies awaken prematurely during the experiment, a lid can be placed on the Petri dish and the flies can be anaesthetized again. 3. Screen the flies according to the following criteria and make a note of them! 4. Eye color and shape as well as appearance and possible disorders of the antennae, legs and wings. 5. Draw two flies, at least, with ectopic eye structures! Note It may be necessary to dissect flies from the pupal case. Flies with disturbed morphology often 13 have problems to ecclose from the pupal case. Dumont tweezers are available for this purpose. Is this a special genotype? Instructions and protective measures Diethyl ether is extremely flammable and may cause drowsiness and dizziness. Work under the fume cupboard during handling. Ensure that the fume cupboard is closed as far as possible. Avoid potential sources of ignition. A lab coat and protective gloves must be worn during practical work. Report 4.1 Photographs/drawings of abnormal flies 4.2 What disorders did you observe in the flies? 4.3 Generate Punett's square using the genotypes and try to assign and classify the phenotypes that can be derived from them. Questions 4.4 How could you test whether the ectopic eyes are functional? 4.5 Briefly describe the difference of insect complex eyes with human eyes! 14 Experiment 5: Four-winged diptera In this experiment you will analyse flies with a homeotic transformation of haltere to wings resulting in a second pair of wings instead of haltere in the third segment of the thorax (Figure 5). Following segmentation by the patterning genes of gap genes, pair-rule genes and segment polarity genes, the information for the fate of the tissue is conferred to the Hox genes. Soon after, during gastrulation stages, the patterning genes stop to be expressed and the Hox genes, so-called segment identity genes, maintain the identity of the segments and tissues throughout the life cycle in larvae, pupae and adult flies. Figure 5: Four-winged diptera. Normally, the third thoracic segment forms oscillating bulbs (red arrow), which act as a kind of gyrometer to ensure flight stability. In the Ubx mutant, the third thoracic segment develops like a second thoracic segment, resulting in a second pair of wings and absence of the swinging flasks. Ubx is one of the Hox genes in Drosophila. Ubx is expressed initially in early embryos posterior to the hunchback expression domain in the posterior half of the embryo. Further on, Ubx expression becomes restricted to the region of the third thorax segment. All three thoracic segments form each a pair of legs. In addition the second segment forms the pair of wings, the third segment, a pair of halteres (“Schwingkölbchen” in German), which are important for keeping the balance during flight acting as gyrometers. Loss of Ubx function leads to a change in identity of the third segment to that of the second segment, i. e. formation of wings instead of halteres. Figure 6: Mutations caused by Cas/CrispR. (A) The endonuclease Cas12a is targeted by guide RNAs to a specific location on the chromosome that is determined by the sequence of the guide RNA. At this point, Cas9/12a cuts the DNA double helix and thus triggers DNA repair. Since the repair, non-homologous end joining) is highly error-prone errors, small deletions are introduced leading to frame shifts in the ORF. (B) The expression of Cas in our experiment is controlled by the transcription factor GAL4. GAL4 is activated only in haltere imaginal discs (pdm2-GAL4). The guide RNAs are expressed in all cells throughout development. Transcription occurs via an RNA polymerase III promoter (CFD244). Ubx mutations are embryonic lethal. Only partial loss of function alleles (hypomorphic) develop to adult flies. As such flies have difficulties to ecclose from the pupal case, the yield of four-wing flies is low. You will analyse mosaic flies, which are mutant for Ubx only in the haltere disc. We 15 will generate such genetic mosaics by inducing a Ubx knockout only in the haltere disc. CAS12 will be expressed in the larval haltere disc by the UASxGAL4 system (Figure 6). The Ubx specific guide RNAs are ubiquitously expressed with a RNA polymerase III promoter (U6 promoter). The efficiency of the CrispR system is so good that most cells in the haltere disc are mutant for Ubx by the end of larval stage. Stämme: Kreuzung: Cas (pFP1131) und Gal4 Treiber w UAS-Cas12a pdm2-GAL4{w+} w ; + ; U6:3>4xsgRNA für Halteren-Imaginalscheibe w ; UAS-Cas12a ; pdm2-GAL4{w+} X Y + U6:3>4xsgRNA w ; UASt-Cas12a{w+} ; pdm2-GAL4{w+} Transgene mit 4 sgRNAs HD12aCFD244 w; +/+ ; U6:3 >> 4xsgRNA w or w UAS-Cas12a ; U6:3>4xsgRNA Y w ; + pdm2-GAL4{w+} Figure 7: Crossing scheme and genotype of stocks for inducing Ubx knockout in haltere The three genetic elements needed for the experiment, CAS12a, guideRNA and GAL4, are transgenes stably introduced into the genome (Figure 7). One of the stocks contains two elements, the CAS12a under control of a UAS promoter (pFP1131) and a GAL4 under control of a haltere specific promoter (pdm2-GAL4, Obata et al. 2014).). In all flies of this stock, CAS12a is expressed in the haltere discs. As long as no guide RNAs are present, CAS12a does not cut any DNA. Similar to CAS9, CAS12a is an RNA-guided endonuclease, but is smaller, easier to handle and produces sticky rather than blunt ends (Port et al. PNAS 2020).The second stock (HD12a CFD244, F. Port, Heidelberg) contains a transgene with a total of four guide RNAs matching CAS12a. The four gRNAs target four different sites in the Ubx gene. The gRNAs are expressed under the control of the ubiquitous U6:3 promoter. U6 encodes a small RNA for ribosome biogenesis and is transcribed by RNA polymerase III (Figure 8). Figure 8: Induction of somatic mutations with sgRNA arrays. (Port et al. PNAS 2020).(A) The individual sgRNA (yellow, red, blue) are excised from the primary transcript by post-transcriptional processing. The RNA-dependent endonuclease CAS12a together with the guide RNAs cleaves sequence-specific genomic DNA and triggers its repair. (B) The combination of CAS12a and sgRNA array transgenes induces gene editing in the transheterozygous progeny. The activity of CAS12a increases with higher temperature. To produce the sgRNA arrays, the guide RNA sequences in the plasmid vector pCFD8 are cloned between tRNA genes (hairpin, red). The expression is carried out by a ubiquitous U6:3 promoter with RNA polymerase III. Procedure 1. You will receive fly tubes of the cross mentioned above. 16  2. Anesthetize the flies under the hood using ether and collect the animals in a large Petri dish, also take the fly tube back to its place! 3. Search for mutant flies 4. Count both mutant (four-winged) and wild type flies (two-winged) 5. Calculate the proportion of flies with complete and partial four-winged phenotype. 6. Describe the phenotypic variance in words (spectrum of phenotypes). 7. Take images of the most striking cases with you Handy camera. You may use a stereomicroscope for magnification. Insert images into your report. Safety instructions and protective measures Diethyl ether is flammable and may cause drowsiness and dizziness. Work under the fume cupboard during handling. Ensure that the fume cupboard is closed as far as possible. Avoid potential sources of ignition. A lab coat and protective gloves must be worn during practical work. Literature Port et al. PNAS 2020 Obata et al. 2014 Report 5.1 Scoring of wild type and mutant flies 5.2 Description of phenotypic variation 5.3 Photographs/drawings of mutant flies Questions 5.4 What is the common feature of Hox genes? 5.5 What are the classical Hox genes in Drosophila? 5.6 What are potential application of the CrispR system in medical therapy? 5.7 How can potential off-target effects of CrispR system be tested? 17