Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

Summary

Mosaic clone analysis in Drosophila imaginal disc epithelia is a powerful model system to study the genetic and cellular mechanisms of tumorigenesis. Here we describe a protocol to induce tumors in Drosophila wing imaginal discs using the GAL4-UAS system, and introduce a diagnosis method to classify the tumor phenotypes.

Abstract

In the early stages of cancer, transformed mutant cells show cytological abnormalities, begin uncontrolled overgrowth, and progressively disrupt tissue organization. Drosophila melanogaster has emerged as a popular experimental model system in cancer biology to study the genetic and cellular mechanisms of tumorigenesis. In particular, genetic tools for Drosophila imaginal discs (developing epithelia in larvae) enable the creation of transformed pro-tumor cells within a normal epithelial tissue, a situation similar to the initial stages of human cancer. A recent study of tumorigenesis in Drosophila wing imaginal discs, however, showed that tumor initiation depends on the tissue-intrinsic cytoarchitecture and the local microenvironment, suggesting that it is important to consider the region-specific susceptibility to tumorigenic stimuli in evaluating tumor phenotypes in imaginal discs. To facilitate phenotypic analysis of tumor progression in imaginal discs, here we describe a protocol for genetic experiments using the GAL4-UAS system to induce neoplastic tumors in wing imaginal discs. We further introduce a diagnosis method to classify the phenotypes of clonal lesions induced in imaginal epithelia, as a clear classification method to discriminate various stages of tumor progression (such as hyperplasia, dysplasia, or neoplasia) had not been described before. These methods might be broadly applicable to the clonal analysis of tumor phenotypes in various organs in Drosophila.

Introduction

Epithelial tissues are highly organized systems that have the remarkable homeostatic ability to maintain their organization through development and cell turnover. This robust self-organizing system, however, is progressively disrupted during tumor development. At the beginning of tumor development, individual mutant cells arising from oncogene activation or tumor-suppressor gene inactivation emerge within an epithelial layer. When this transformed "pro-tumor cell" evades a suppressive environment, disrupts epithelial organization, and begins uncontrolled proliferation, tumorigenesis occurs ¹. During the past few decades, outstanding technological advances in genetics and molecular biology have made remarkable progresses on cancer research. In particular, recent studies using the genetically mosaic analysis tools in Drosophila melanogaster, such as FLP-FRT (flippase recombinase/flippase recombinase target) mitotic recombination ² and flip-out-GAL4-UAS (upstream activating sequence) systems ³, have greatly contributed to better understanding the genetic mechanisms involved in the formation and metastasis of tumors ^4,5,6.

Studies of a group of conserved Drosophila tumor-suppressor genes, lethal giant larvae (lgl), discs large (dlg), and scribble (scrib), highlighted the critical relationship between loss of epithelial organization and tumor development, as these genes play key roles in regulation of apical-basal cell polarity and cell proliferation in epithelial tissues ⁷. While Drosophila imaginal discs are normally monolayered epithelia, homozygous mutations in any of these three genes cause cells to lose structure and polarity, fail to differentiate, overproliferate, and ultimately form multilayered amorphous masses that fuse with adjacent tissues ⁷. Similarly, disruption of these genes in mammals is involved in the development of malignant tumors ^8,9. The neoplastic phenotypes exhibited by the mutant tissues have led to the classification of these three genes as conserved, neoplastic tumor-suppressor genes (nTSGs) ^7,8. However, when homozygous nTSG mutant cells are sporadically generated in developing wild-type imaginal discs using FLP-FRT-mediated mitotic recombination, mutant cells are eliminated from the tissue through c-Jun N-terminal kinase (JNK)-dependent apoptosis ^{10,11,12,13,14}, extrusion ^15,16, or engulfment and phagocytosis by neighbors ¹⁷. In this genetically mosaic epithelia, apoptosis is mostly detected in nTSG mutant cells located at the clone boundary, suggesting that adjacent normal cells trigger the apoptosis of nTSG mutant cells ^10,11,12,18. Recent studies in mammalian cells have confirmed that this cell competition-dependent elimination of pro-tumor cells is an evolutionarily conserved epithelial self-defense mechanism against cancer ^{19,20,21,22,23}.

A recent study in Drosophila imaginal discs, however, showed that mosaic nTSG-knockdown clones induces neoplastic tumors in specific regions of wing imaginal discs ¹⁶. Initial tumor formation was always found in the peripheral "hinge" region and never observed in the central "pouch" region of the wing disc epithelium, suggesting that the tumorigenic potential of nTSG-knockdown cells depends on the local environment. The central pouch region functions as a "tumor coldspot" where pro-tumor cells do not show dysplastic overgrowth, whereas the peripheral hinge region behaves as a "tumor hotspot" ¹⁶. In "coldspot" pouch regions, nTSG-knockdown cells delaminate from the basal side and undergo apoptosis. In contrast, as "hotspot" hinge cells possess a network of robust cytoskeletal structures on their basal sides, nTSG-knockdown cells delaminate from the apical side of the epithelium and initiate tumorigenic overgrowth ¹⁶. Therefore, analysis of tumor phenotypes in imaginal discs requires careful consideration of the region-specific susceptibility to tumorigenic stimuli.

Here, we describe a protocol to induce neoplastic tumor formation in the Drosophila wing imaginal discs utilizing the GAL4-UAS-RNAi system by which nTSG-knockdown cells are generated in normal wing disc epithelia. Although these experimental systems are useful to study the early stages of cancer, a clear classification method to evaluate the stages of tumor progression in imaginal disc epithelia has not been clearly described before. Therefore, we also propose a diagnosis method to classify pro-tumor clonal phenotypes induced in the wing disc epithelia into three categories: hyperplasia (accumulation of an excessive number of normal-appearing cells with increased proliferation), dysplasia (premalignant tissue composed of abnormally appearing cells), and neoplasia (benign or malignant tumor composed of cells having an abnormal appearance and abnormal proliferation pattern).

Protocol

1. Fly Crosses and Clone Induction

1. Remove all flies in the vial 12 h before collecting virgin flies.
2. Anesthetize the flies in the vial by injecting CO₂ gas and place flies onto a CO₂ fly pad.
3. Transfer 10 - 20 virgin females and 10 males from the CO₂ fly pad into a fresh vial and incubate for 1 day at 25 °C.
4. Transfer these flies into a fresh vial and incubate for 12 h at 25 °C.
   NOTE: Discard the first vial as virgin females do not lay enough eggs in the first day.
5. For enhancer-GAL4 lines, remove the adult flies and incubate the vial at 25 °C until dissection.
6. For induction of mosaic clones by flip-out-GAL4, remove the adult flies and incubate the vial for 2 - 4 days at 25 °C. The incubation time before heat shock is dependent on the experimental purpose. A heat shock 2 days after egg deposition (AED) generates mosaic clones in immature second instar imaginal epithelia. A heat shock after 3 or 4 days AED generates mosaic clones in differentiated early- to mid-third instar imaginal epithelia.
   NOTE: It is important to examine the effect of altered heat-shock timing on tumorigenic phenotypes.
7. For induction of mosaic clones by flip-out-GAL4, heat shock the vial by immersion in a 37 °C water bath for 10 - 45 min followed by incubation for 2 - 3 days at 25 °C.
   NOTE: Information of heat-shock time, timing, and incubation time in each experiment is shown in the figure legends.

2. Dissection of Larvae

1. Collect wandering third instar larvae with a wooden stick or blunt forceps, genotype them by appropriate fluorescent markers (e.g., EGFP) under a fluorescence stereoscopic microscope and place them in a dissection dish with PBS (phosphate buffered saline).
2. Wash the larvae in PBS by pipetting with 2 mL plastic transfer pipettes.
3. Pinch the center of the larva with one forceps and tear the body in half with the other forceps.
4. Pinch the mouth hook of the anterior half with one forceps and push the mouth towards the body with the other forceps to turn the body inside out.
5. Remove unnecessary materials such as salivary glands or fat bodies with forceps.

3. Fixation and Antibody Staining

1. Transfer the dissected anterior half of the larval body (including imaginal wing discs) to a 1.5 mL plastic tube and fix in 1 mL of Fix solution (4% Formaldehyde in PBS) for 10 min at room temperature in the dark with gentle rotation.
   CAUTION: Formaldehyde is toxic and has carcinogenic potential. Wear protective gloves and clothing to prevent skin contact.
   NOTE: In this part, all steps take place on a nutator at room temperature in the dark unless otherwise noted.
2. Remove the Fix solution and discard. Wash the tissues with 1 mL of PBT (0.3% Triton X-100 in PBS) three times for 15 min each.
3. Remove the PBT and add 1 mL of PBTG (0.2% bovine serum albumin and 5% normal goat serum in PBT) for blocking and nutate 1 h at room temperature or overnight at 4 °C.
4. Remove PBTG and add primary antibody solution appropriately diluted with PBTG (see Materials Table) and nutate overnight at 4 °C.
5. Remove the primary antibody solution and wash the tissues with 1 mL PBT three times for 15 min each.
6. Remove PBT and add secondary antibody solution appropriately diluted with PBTG (1:400). Nutate for 2 h at room temperature or overnight at 4 °C.
7. Remove secondary antibody solution and wash the tissues with 1 mL of PBT two times for 15 min each.
8. To stain F-actin, remove PBT and add Phalloidin solution appropriately diluted in PBS (1:40). Then nutate for 20 min. Remove the Phalloidin solution and wash the tissues with 1 mL of PBT two times for 15 min each.
9. To counterstain nuclear DNA, remove PBT and add DAPI (4', 6-diamidino-2-phenylindole) solution (0.5 µg/mL of DAPI in PBS). Then nutate 10 min.
   CAUTION: DAPI has carcinogenic potential. Wear protective gloves and clothing to prevent skin contact.
10. Remove DAPI solution and wash the tissues with 1 mL of PBT two times for 15 min each.
11. Rinse once in 1 mL of PBS for 10 min at room temperature.
12. Remove PBS and add 500 µL of 100 % glycerol as the pre-mounting medium.

4. Mounting onto Microscope Slides

1. Place the stained tissues on a microscope slide using a 2 mL plastic transfer pipette.
2. Transfer the tissues to drops of mounting medium on another microscope slide with forceps.
3. Hold down the end of dissected tissue with one forceps and pull away brain and eye antennal discs with the other forceps.
   NOTE: Keep the dissected brains to place them near the wing imaginal discs. The brains act as a platform preventing the coverslip from crushing the wing imaginal discs.
4. To isolate the wing imaginal discs hold down the end of dissected tissue with one forceps and gently scratch the body wall and tear off the discs with the other forceps.
   NOTE: If it is difficult to find wing imaginal discs, peel the trachea from the posterior to the anterior side. Wing imaginal discs stick to the trachea.
5. Gently cover the imaginal discs with a coverslip and seal with nail polish; store at 4 °C.

5. Confocal Microscopy

1. To acquire confocal images using a confocal microscope set image acquisition parameters including range of emission wavelength, laser intensity, gain, offset, scanning speed and image size ²⁴.
   NOTE: For detailed procedure of image acquisition settings, refer to the instruction manual supplied by each microscope manufacturer.
2. To capture single confocal sections of an entire wing imaginal disc, use a 20X objective lens.
3. Acquire 3-dimensional images by z-stack scanning at step-size of 0.5 - 1.0 µm using 40X or 60X lenses.
   NOTE: To analyze cellular phenotypes in high resolution, an image size should be larger than 512 x 512 pixels.

6. Image Analysis Using ImageJ

1. Use Fiji, an open-source ImageJ software focused on biological-image analysis (https://fiji.sc/) ²⁵, to acquire and analyze confocal z-stack images.
2. To acquire vertical sections, open the z-stack images in ImageJ and select the menu item "Image/Stacks/Reslice."
   NOTE: Either X-Z axis or Y-Z axis are selectable in the Reslice menu. Vertical section can be obtained also in an arbitrary direction by drawing a straight line or rectangle onto the z-stack image.

7. Diagnosis of Neoplastic Phenotypes

1. Open the vertical sections (X-Z or Y-Z axis) obtained from one set of z-stack images and analyze morphological phenotypes and antibody staining.
2. For the diagnosis of tumor phenotypes, mainly focus on the following three points: (1) if a cell mass, including knockdown clones, deviates from the main epithelial layer, (2) if the subcellular localization of junctional proteins is altered in this cell mass, and (3) if the diameter of this cell mass is larger than 4 cells.
3. Categorize tumor phenotypes according to the flowchart described in Figure 1.

Results

To demonstrate neoplastic tumor formation experimentally induced by RNAi-mediated nTSG-knockdown in Drosophila wing imaginal discs, three different GAL4 drivers were used to express UAS-RNAi for lgl or scrib: (1) sd-GAL4, which drives strong UAS expression in the wing pouch and mild expression in the hinge regions (Figure 2 and Figure 3A); (2) upd-GAL4, which drives i...

Discussion

The GAL4-UAS system is one of the most powerful genetic tools for targeted gene expression in Drosophila ²⁶ and greatly facilitates tumor cell induction and analysis in vivo ⁴. This system enables the generation of clones bearing knockdown of tumor-suppressor genes or overexpression of oncogenes within wild-type epithelial tissue, a situation highly similar to the initial stages of human cancer where transformed pro-tumor cells are surrounded by normal epi...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
Phosphate buffered saline (PBS)	Wako	162-19321
TritonX-100	Wako	168-11805
Formaldehyde	Wako	064-00406
bovine serum albumin	Sigma	A7906
normal goat serum	Sigma	G6767
mounting medium, Vectashield	Vector Laboratories	H-1000
DAPI	Sigma	D9542
mouse-anti-Dlg 4F3	Developmental Studies Hybridoma Bank	4F3 anti-discs large, RRID:AB_528203	dilute in PBTG, 1:40
mouse-anti-MMP1	Developmental Studies Hybridoma Bank	3A6B4, RRID:AB_579780	3 mixed 1:1:1 and dilute in PBTG, 1:40
mouse-anti-MMP1	Developmental Studies Hybridoma Bank	3B8D12, RRID:AB_579781	3 mixed 1:1:1 and dilute in PBTG, 1:40
mouse-anti-MMP1	Developmental Studies Hybridoma Bank	5H7B11, RRID:AB_579779	3 mixed 1:1:1 and dilute in PBTG, 1:40
mouse-anti-atubulin	Developmental Studies Hybridoma Bank	AA4.3, RRID:AB_579793	dilute in PBTG, 1:100
Alexa Fluor 546 Phalloidin	Molecular probes	A22283	dilute in PBS, 1:40
goat anti-mouse IgG antibody, Alexa Fluor 546	Molecular probes	A11030	dilute in PBTG, 1:400
Name	Company	Catalog Number	Comments
Fly strains
sd-Gal4	Bloomington Drosophila Stock Center	#8609	recombined with UAS-EGFP
upd-Gal4	Bloomington Drosophila Stock Center	#26796	recombined with UAS-EGFP
UAS-lgl-RNAi	Vienna Drosophila RNAi center	#51247
UAS-scrib-RNAi	Vienna Drosophila RNAi center	#105412
UAS-RasV12	Bloomington Drosophila Stock Center	#64196
UAS-Yki3SA	Bloomington Drosophila Stock Center	#28817
hsFLP	Bloomington Drosophila Stock Center	#6
Act>CD2>GAL4 (flip-out GAL4)	Bloomington Drosophila Stock Center	#4780	recombined with UAS-EGFP
UAS-EGFP	Bloomington Drosophila Stock Center	#5428	X chromosome
UAS-EGFP	Bloomington Drosophila Stock Center	#6658	third chromosome
UAS-Dicer2	Bloomington Drosophila Stock Center	#24650	second chromosome
UAS-Dicer2	Bloomington Drosophila Stock Center	#24651	third chromosome
vkg-GFP	Morin et al. 2001		GFP protein trap