Name: induction and diagnosis of tumors in drosophila imaginal disc epithelia
Uploaded: 2017-07-25T14:00:00.000+00:00
Duration: 8 min 14 s
Description: Watch this Scientific Journal Video about Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia at JoVE.com

We thank J. Vaughen for critical reading of the manuscript. This work was supported by grants from JSPS KAKENHI Grant Numbers 26891025, 15H01500 and The Takeda Science Foundation Research Grant to Y.T.

1. Fly Crosses and Clone Induction
<ol>
	<li>Remove all flies in the vial 12 h before collecting virgin flies.</li>
	<li>Anesthetize the flies in the vial by injecting CO2 gas and place flies onto a CO2 fly pad.</li>
	<li>Transfer 10 - 20 virgin females and 10 males from the CO2 fly pad into a fresh vial and incubate for 1 day at 25 &#176;C.</li>
	<li>Transfer these flies into a fresh vial and incubate for 12 h at 25 &#176;C. 
	NOTE: Discard the first vial as virgin females do not lay enough eggs in the first day.</li>
	<li>For enhancer-GAL4 lines, remove the adult flies and incubate the vial at 25 &#176;C until dissection.</li>
	<li>For induction of mosaic clones by flip-out-GAL4, remove the adult flies and incubate the vial for 2 - 4 days at 25 &#176;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.</li>
	<li>For induction of mosaic clones by flip-out-GAL4, heat shock the vial by immersion in a 37 &#176;C water bath for 10 - 45 min followed by incubation for 2 - 3 days at 25 &#176;C. 
	NOTE: Information of heat-shock time, timing, and incubation time in each experiment is shown in the figure legends.</li>
</ol>
2. Dissection of Larvae
<ol>
	<li>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).</li>
	<li>Wash the larvae in PBS by pipetting with 2 mL plastic transfer pipettes.</li>
	<li>Pinch the center of the larva with one forceps and tear the body in half with the other forceps.</li>
	<li>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.</li>
	<li>Remove unnecessary materials such as salivary glands or fat bodies with forceps.</li>
</ol>
3. Fixation and Antibody Staining
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#176;C.</li>
	<li>Remove PBTG and add primary antibody solution appropriately diluted with PBTG (see Materials Table) and nutate overnight at 4 &#176;C.</li>
	<li>Remove the primary antibody solution and wash the tissues with 1 mL PBT three times for 15 min each.</li>
	<li>Remove PBT and add secondary antibody solution appropriately diluted with PBTG (1:400). Nutate for 2 h at room temperature or overnight at 4 &#176;C.</li>
	<li>Remove secondary antibody solution and wash the tissues with 1 mL of PBT two times for 15 min each.</li>
	<li>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.</li>
	<li>To counterstain nuclear DNA, remove PBT and add DAPI (4&#39;, 6-diamidino-2-phenylindole) solution (0.5 &#181;g/mL of DAPI in PBS). Then nutate 10 min. 
	CAUTION: DAPI has carcinogenic potential. Wear protective gloves and clothing to prevent skin contact.</li>
	<li>Remove DAPI solution and wash the tissues with 1 mL of PBT two times for 15 min each.</li>
	<li>Rinse once in 1 mL of PBS for 10 min at room temperature.</li>
	<li>Remove PBS and add 500 &#181;L of 100 % glycerol as the pre-mounting medium.</li>
</ol>
4. Mounting onto Microscope Slides
<ol>
	<li>Place the stained tissues on a microscope slide using a 2 mL plastic transfer pipette.</li>
	<li>Transfer the tissues to drops of mounting medium on another microscope slide with forceps.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Gently cover the imaginal discs with a coverslip and seal with nail polish; store at 4 &#176;C.</li>
</ol>
5. Confocal Microscopy
<ol>
	<li>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.</li>
	<li>To capture single confocal sections of an entire wing imaginal disc, use a 20X objective lens.</li>
	<li>Acquire 3-dimensional images by z-stack scanning at step-size of 0.5 - 1.0 &#181;m using 40X or 60X lenses. 
	NOTE: To analyze cellular phenotypes in high resolution, an image size should be larger than 512 x 512 pixels.</li>
</ol>
6. Image Analysis Using ImageJ
<ol>
	<li>Use Fiji, an open-source ImageJ software focused on biological-image analysis (https://fiji.sc/) 25, to acquire and analyze confocal z-stack images.</li>
	<li>To acquire vertical sections, open the z-stack images in ImageJ and select the menu item &#34;Image/Stacks/Reslice.&#34; 
	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.</li>
</ol>
7. Diagnosis of Neoplastic Phenotypes
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Categorize tumor phenotypes according to the flowchart described in Figure 1.</li>
</ol>

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The authors have nothing to disclose.

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 epithelial cells. GAL4 driver lines regulated by enhancer sequences such as GAL4 enhancer-trap lines or Janelia GAL4 lines 27 have unique, spatiotemporally restricted expression patters. Therefore, these enhancer-GAL4 lines are useful to induce tumors consistently in restricted areas of imaginal discs. In addition to sd-GAL4 and upd-GAL4 shown in this study, we confirmed that lgl-knockdown induced by other enhancer-trap GAL4 lines including engrailed-GAL4 (en-GAL4), hedgehog-GAL4 (hh-GAL4), optomotor-blind-GAL4 (omb-GAL4), patched-GAL4 (ptc-GAL4) and spalt-major-GAL4 (salm-GAL4) can generate neoplastic tumors in the hinge regions of wing imaginal discs. If the enhancer-GAL4 expression pattern needs to be temporally controlled, a combination of GAL4 and a temperature-sensitive version of GAL80 (GAL80ts) is an option. GAL80ts represses GAL4 function at 18 &#176;C but not at 29 &#176;C, which permits UAS-transgenes expression to be switched on or off as necessary 28.
The flip-out-GAL4 mosaic system, which combines the FLP-FRT system and the GAL4-UAS system 3, generates random clones in imaginal discs. By combining heat-shock-inducible flippase (hsFLP) with flip-out GAL4, the timing of clone generation is controllable. Because the tumorigenic potential of imaginal disc cells may be dependent on the developmental stage, endogenous signaling events, and/or cellular differentiation 29,30, it is very important to examine the effect of altered heat-shock timing on tumorigenic phenotypes.
It has been previously shown that nTSG mutant clones undergo cell competition-dependent apoptosis when surrounded by wild-type cells 10,11,12,18, suggesting that multiple mutations are required for cell transformation during cancer development. In fact, overexpression of oncogenic Ras or Yki in lgl or scrib mutant clones transforms them into aggressive tumors 10,18,31 whereas misexpression of RasV12 or Yki3SA does not induce malignant neoplasm by themselves without nTSG mutations. As shown here and previously 16, pro-tumor nTSG-knockdown clones generated in wild-type wing discs also undergo apoptosis and do not show tumorigenesis in the wing pouch 16. At the same time, nTSG-knockdown clones located in hinge &#34;hotspots&#34; develop into neoplastic tumors without any additional oncogenic mutations. In hinge regions, nTSG-deficient cells hijack endogenous Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling to drive tumorigenesis 16. Therefore, hinge-specific GAL4s such as upd-GAL4 are useful tools for consistently inducing neoplastic tumor formation by RNAi-mediated knockdown of tumor suppresser genes. Although flip-out-GAL4-induced nTSG-knockdown mosaic clones also induce neoplastic tumors in hinge regions, tumorigenesis occurrence is dependent on initial clone size: large clones generated by a heat shock over 20 min usually induce tumorigenesis, whereas most small clones generated by heat shock shorter than 10 min are completely eliminated (K.M. and Y.T., unpublished data).
A number of recent studies on tumor development and cancer progression use Drosophila imaginal discs as a model system 32,33. Common to these studies include several markers of neoplastic tumors: deviant cell masses, deformed disc shapes, accumulation of F-actin, enhanced cell cycling (BrdU/EdU incorporation or PH3 expression), activation of JNK signaling and its downstream target MMP1, basement membrane disruption, and/or epithelial disorganization, including loss or subcellular delocalization of proteins involved in apical-basal cell polarity (Crumbs, Stardust, PatJ, aPKC, Bazooka/PAR-3 or PAR-6), septate junctions (Lgl, Scrib, Dlg or Coracle), or adherens junctions (E-Cadherin or Armadillo). These phenotypes are readily detectable by conventional antibody staining. Although it is difficult to determine if a clonal phenotype is hyperplasia, dysplasia, benign neoplasm or malignant neoplasm in the early stage of tumorigenesis, it should be simpler in the Drosophila imaginal disc epithelia composed of a single epithelial monolayer. In this study, we introduced a diagnosis method to classify tumorous clone phenotypes induced in the epithelial monolayer (Figure 1). This method requires careful observations of five criteria using z-stack confocal images; each diagnostic criterion can be examined by conventional antibody staining and confocal imaging. One of the new criteria we introduced in this method is &#34;4-cell-diameter criteria&#34; which discriminates between dysplasia and neoplasm in imaginal discs. Pro-tumor cells such as nTSG mutant clones are frequently extruded from epithelial layers through cell competition or spindle misorientation 15,16,34. In most situations, these extruded cells apoptose, and so they cannot proliferate. Although it might be possible that the pro-tumor cells undergo cell division once during extrusion and once or twice more after extrusion, this makes a cell mass of 2- to 3-cell diameter. Therefore, if a cell mass of pro-tumor cells deviated from epithelial layer is larger than 4-cell diameter, we can determine that they are surviving and proliferating as a neoplasm outside of the epithelial layer.
Although this diagnosis method is simple, it is critical to take a time course of phenotypes, as tumor development and malignancy progresses throughout time. For example, dysplasia is a cytologically abnormal tissue and a transitional state between completely benign growths and those that are premalignant. Therefore, even if an observed clone is defined as a dysplasia according to the flowchart (Figure 1), it is possible that the dysplasia subsequently develops into a neoplasm. Although the larval stage is limited in time (5 - 6 days at 25 &#176;C or 4 - 5 days at 29 &#176;C AED), tumor growth in imaginal discs prolongs the larval stage through an insulin-like peptide induced delay of pupariation 35,36. Therefore, neoplastic tumor formation in imaginal discs makes it possible to observe tumor clone phenotypic progression for several more days. Our simple classification method might be broadly applicable to the clonal analysis of tumor phenotypes in various organs in Drosophila.

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 &#34;pro-tumor cell&#34; 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 &#34;hinge&#34; region and never observed in the central &#34;pouch&#34; 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 &#34;tumor coldspot&#34; where pro-tumor cells do not show dysplastic overgrowth, whereas the peripheral hinge region behaves as a &#34;tumor hotspot&#34; 16. In &#34;coldspot&#34; pouch regions, nTSG-knockdown cells delaminate from the basal side and undergo apoptosis. In contrast, as &#34;hotspot&#34; 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).

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Wako</td><td>162-19321</td><td></td></tr><tr><td>TritonX-100</td><td>Wako</td><td>168-11805</td><td></td></tr><tr><td>Formaldehyde</td><td>Wako</td><td>064-00406</td><td></td></tr><tr><td>bovine serum albumin</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>normal goat serum</td><td>Sigma</td><td>G6767</td><td></td></tr><tr><td>mounting medium, Vectashield</td><td>Vector Laboratories</td><td>H-1000</td><td></td></tr><tr><td>DAPI</td><td>Sigma</td><td>D9542</td><td></td></tr><tr><td>mouse-anti-Dlg 4F3</td><td>Developmental Studies Hybridoma Bank</td><td>4F3 anti-discs large, RRID:AB_528203</td><td>dilute in PBTG, 1:40</td></tr><tr><td>mouse-anti-MMP1</td><td>Developmental Studies Hybridoma Bank</td><td>3A6B4, RRID:AB_579780</td><td>3 mixed 1:1:1 and dilute in PBTG, 1:40</td></tr><tr><td>mouse-anti-MMP1</td><td>Developmental Studies Hybridoma Bank</td><td>3B8D12, RRID:AB_579781</td><td>3 mixed 1:1:1 and dilute in PBTG, 1:40</td></tr><tr><td>mouse-anti-MMP1</td><td>Developmental Studies Hybridoma Bank</td><td>5H7B11, RRID:AB_579779</td><td>3 mixed 1:1:1 and dilute in PBTG, 1:40</td></tr><tr><td>mouse-anti-&lt;span class=&quot;font13&quot;&gt;a&lt;/span&gt;&lt;span class=&quot;font8&quot;&gt;tubulin&lt;/span&gt;</td><td>Developmental Studies Hybridoma Bank</td><td>AA4.3, RRID:AB_579793</td><td>dilute in PBTG, 1:100</td></tr><tr><td>Alexa Fluor 546 Phalloidin</td><td>Molecular probes</td><td>A22283</td><td>dilute in PBS, 1:40</td></tr><tr><td>goat anti-mouse IgG antibody, Alexa Fluor 546</td><td>Molecular probes</td><td>A11030</td><td>dilute in PBTG, 1:400</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Fly strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;sd-Gal4&lt;/em&gt;</td><td>Bloomington Drosophila Stock Center</td><td>#8609</td><td>recombined with &lt;em&gt;&lt;span class=&quot;font11&quot;&gt;UAS-EGFP&lt;/span&gt;&lt;/em&gt;</td></tr><tr><td>&lt;em&gt;upd-Gal4&lt;/em&gt;</td><td>Bloomington Drosophila Stock Center</td><td>#26796</td><td>recombined with &lt;em&gt;&lt;span class=&quot;font11&quot;&gt;UAS-EGFP&lt;/span&gt;&lt;/em&gt;</td></tr><tr><td>&lt;em&gt;UAS-lgl-RNAi&lt;/em&gt;</td><td>Vienna Drosophila RNAi center</td><td>#51247</td><td></td></tr><tr><td>&lt;em&gt;UAS-scrib-RNAi&lt;/em&gt;</td><td>Vienna Drosophila RNAi center</td><td>#105412</td><td></td></tr><tr><td>&lt;em&gt;UAS-Ras&lt;span class=&quot;font12&quot;&gt;&lt;sup&gt;V12&lt;/sup&gt;&lt;/span&gt;&lt;/em&gt;</td><td>Bloomington Drosophila Stock Center</td><td>#64196</td><td></td></tr><tr><td>&lt;em&gt;UAS-Yki&lt;span class=&quot;font12&quot;&gt;&lt;sup&gt;3SA&lt;/sup&gt;&lt;/span&gt;&lt;/em&gt;</td><td>Bloomington Drosophila Stock Center</td><td>#28817</td><td></td></tr><tr><td>&lt;em&gt;hsFLP&lt;/em&gt;</td><td>Bloomington Drosophila Stock Center</td><td>#6</td><td></td></tr><tr><td>&lt;em&gt;Act&gt;CD2&gt;GAL4 (flip-out GAL4)&lt;/em&gt;</td><td>Bloomington Drosophila Stock Center</td><td>#4780</td><td>recombined with &lt;em&gt;&lt;span class=&quot;font11&quot;&gt;UAS-EGFP&lt;/span&gt;&lt;/em&gt;</td></tr><tr><td>&lt;em&gt;UAS-EGFP&lt;/em&gt;</td><td>Bloomington Drosophila Stock Center</td><td>#5428</td><td>X chromosome</td></tr><tr><td>&lt;em&gt;UAS-EGFP&lt;/em&gt;</td><td>Bloomington Drosophila Stock Center</td><td>#6658</td><td>third chromosome</td></tr><tr><td>&lt;em&gt;UAS-Dicer2&lt;/em&gt;</td><td>Bloomington Drosophila Stock Center</td><td>#24650</td><td>second chromosome</td></tr><tr><td>&lt;em&gt;UAS-Dicer2&lt;/em&gt;</td><td>Bloomington Drosophila Stock Center</td><td>#24651</td><td>third chromosome</td></tr><tr><td>&lt;em&gt;vkg-GFP&lt;/em&gt;</td><td>Morin et al. 2001</td><td></td><td>GFP protein trap</td></tr></tbody></table>

induction and diagnosis of tumors in drosophila imaginal disc epithelia

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.

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 in subdomains of the hinge, including strong expression in two domains of the dorsal medial fold, mild expression in one anterior-lateral domain, and weak expression in the ventral-posterior domain (Figure 2 and Figure 3C); and (3) heat-shock-induced flip-out GAL4, which generates random mosaic clones expressing UAS-genes (Figure 4 and Figure 5). In all of the experiments, UAS-EGFP was co-expressed with UAS-RNAi to mark the knockdown clones.
First, sd-GAL4-driven lgl-RNAi was used to induce knockdown of lgl in the wing pouch and hinge regions. In control third instar wing discs without UAS-lgl-RNAi, no morphological alteration occurred (Figure 3A). In contrast, in third instar wing discs expressing lgl-RNAi, epithelial deformations and tumorigenic overgrowths were clearly observed in certain parts of the hinge (Figure 3B). In the wing pouch region, however, dysplastic overgrowth was not observed. Strong MMP1 (matrix metalloprotease-1) expression was observed in the tumorous hinge lesions but not in the wing pouch region (Figure 3B). Since MMP1 expression is involved in degradation of basement membranes, it is possible that these hinge tumors have a metastatic capability (Figure 3B). Next, upd-GAL4 was used to induce lgl knockdown in the hinge region. In control third instar wing discs without UAS-lgl-RNAi, no morphological alterations occurred (Figure 3C). However, after 5 days AED, third instar wing discs with upd-GAL4 and lgl-RNAi displayed prominent epithelial disorganization, with F-actin accumulation and clone displacement from the epithelial layer (Figure 3D). At 6 days AED, neoplastic tumors expressing MMP1 overgrew in the hinge region (Figure 3G), albeit with variation in tumor size between samples (Figure 3E and F). A vertical section of the hinge region confirmed that the neoplastic tumor underwent deviating outgrowth at the apical side of epithelial layer (Figure 3H).
To monitor the progression of tumor growth induced by lgl knockdown, sporadic clones expressing lgl-RNAi were generated in wing discs using the heat-shock-induced flip-out GAL4 mosaic system. Two days after heat-shock to second instar larvae (2 days AED), some lgl-knockdown clones located in the hinge region showed deviation away from the apical side of the epithelial layer (Figure 4A). Four days after heat-shock induction, dysplastic lesions in the hinge region became clear, suggesting that lgl-knockdown clones displaced away from the apical side proliferate in the lumen (Figure 4B). Seven days after heat-shock induction, tumorigenic overgrowths dominated the hinge regions (Figure 4C).
To demonstrate the classification of clonal phenotypes with our diagnosis method, sporadic clones expressing scrib-RNAi were generated in wing discs using the heat-shock-induced flip-out GAL4 mosaic system. Five days after heat-shock to second instar larvae (2 days AED), certain GFP-expressing scrib-knockdown clones showed tumorigenic phenotypes (Figure 5A). We analyzed four clones (B, C, D and E in Figure 5A) in vertical sections of z-stack confocal images utilizing ImageJ, because it is difficult to acquire the detailed cytological and structural phenotypes in the 2D confocal images (Figure 4 and Figure 5A). Clones B and C were classified as dysplasia as septate-junction protein Discs large (Dlg) was mislocalized (Figure 5B and C). In clone B, the size of the cell mass displaced from the epithelium was not greater than 4-cells in diameter (Figure 5B), while clone C did not deviate from the epithelium (Figure 5C); therefore, clones B and C were not considered neoplastic. However, clones D and E, which also showed mislocalization of Dlg, formed tumorous cell masses outside of the epithelium (Figure 5D and 5E). As the size of these displaced tumorous clones was more than 4-cells in diameter, the clones were classified as neoplastic.
To induce hyperplastic tumors or cyst formations in the wing imaginal discs, sporadic clones expressing oncogeneic Ras (RasV12) or constitutively active form of Yorkie, a transcriptional coactivator of the Hippo pathway, (Yki3SA) were generated using the heat-shock-induced flip-out GAL4 mosaic system. Four days after heat shock to second instar larvae (2 days AED), RasV12-expressing clones labeled by GFP became round cell masses in the imaginal discs (Figure 6A). A vertical section of the RasV12-expressing clone (clone B in Figure 6A) revealed that the cell mass dissociated from the epithelial layer has proper localization of Dlg, suggesting that the cell mass maintains normal epithelial structures outside the epithelial layer (Figure 6B). MMP1 expression was not observed in these RasV12-expressing clones (Figure 6C). Therefore, the clone B was classified as cyst formation. Four days after heat shock to second instar larvae (2 days AED), Yki3SA-expressing clones labeled by GFP formed big cell masses in the imaginal discs (Figure 7A). We focused on two clones (B and C in Figure 7A). Clone B showed integration in the epithelial layer and proper localization of Dlg in the sub-apical membranes of the epithelial cells, suggesting that the Yki3SA-expressing clone B maintained epithelial structure. Therefore, clone B was classified as hyperplasia (Figure 7B). Clone C located in the hinge region showed mislocalization of Dlg and formed multi-layered structure at the basal side of the epithelial layer. MMP1 expression was not observed in the Yki3SA-expressing clones. Taken together, clone C was classified as benign neoplasm (Figure 7C and D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55901/55901fig1.jpg" /> 
Figure 1: Flowchart for Classification of Tumor Phenotypes. This flowchart represents a diagnosis method to classify clonal lesions in imaginal epithelia. The classification is based on five criteria: (1) deviation of clones from the epithelial layer, (2) subcellular mislocalization of junctional proteins, (3) cellular proliferation, (4) clone size and (5) MMP1 expression. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55901/55901fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55901/55901fig2.jpg" /> 
Figure 2: (A) Wild-type wing imaginal disc stained for Dlg (green) and nuclei (DAPI, magenta). (B) Schematic representation of Drosophila wing imaginal discs showing wing pouch (blue), hinge (orange) and notum (green) regions. (C) Normal wing imaginal disc stained for F-actin (red) and microtubules (green). Basement membranes are labeled with Collagen IV/Vkg-GFP (blue). (D) Vertical section of the site marked with white dotted line in (C). (E) Line drawings trace the apical (red) and basal (blue) sides of the epithelial layer in (D). Dotted lines trace the peripodial membrane. Distal (D.f.), medial (M.f.) and proximal (P.f.) folds of the dorsal hinge are indicated. 
Detailed genotypes: A, w- C-D, Vkg-GFP <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55901/55901fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55901/55901fig3.jpg" /> 
Figure 3: Site-specific Tumorigenesis Induced by Enhancer-GAL4s. (A, B) Confocal images show wing imaginal discs dissected from third instar larvae of indicated genotypes stained for MMP1 (red). The sd-GAL4-expressing regions were labeled by GFP expression (green). (C-G) Confocal images show wing imaginal discs dissected from third instar larvae of indicated genotypes at the indicated time point AED. F-actin was stained with Phalloidin (red) in (C-F). MMP1 expression is stained with anti-MMP1 antibodies (red) in (G). The upd-GAL4-expressing regions were labeled by GFP expression (green). Bottom panels of (C-F) show magnified views of regions indicated in upper panels. White arrows indicate dysplastic lesions induced by lgl-knockdown clones. (H) Vertical section of the site marked with white dotted line in the bottom panel of (E). White dotted lines mark the boundaries between wing pouch and hinge regions in (A) and (B). Nuclei were labeled with DAPI (blue). Scale bars = 100 &#181;m in (A-G) and 50 &#181;m in (H). Detailed genotypes: A, w-, sd-GAL4, UAS-EGFP; UAS-dicer2; B, w-, sd-GAL4, UAS-EGFP; UAS-dicer2; UAS-lgl-RNAi; C, w-, upd-GAL4, UAS-EGFP; UAS-dicer2; D-H, w-, upd-GAL4, UAS-EGFP; UAS-dicer2; UAS-lgl-RNAi <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55901/55901fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55901/55901fig4.jpg" /> 
Figure 4: Tumor Phenotypes Induced by Random Mosaic Clones. (A-C) Confocal images show mosaic wing imaginal discs of third instar larvae with clones co-expressing lgl-RNAi and GFP (green) at the indicated time point after heat-shock induction. Nuclei were labeled by DAPI (blue). The middle panels show magnified views of the white square regions marked in left panels. The right panels show schematic line drawings of the apical side of the epithelial layers (blue) and the GFP-expressing clones (green). Mosaic clones located within the epithelium are depicted in light green while neoplastic tumors deviating from the epithelium are shown in magenta. White dotted lines in the left panels mark the boundaries between wing pouch and hinge regions. White arrows in the middle panels indicate dysplastic lesions induced by lgl-knockdown clones. Scale bars = 100 &#181;m. Detailed genotypes: A-C, hsFLP; UAS-dicer2; act&#62;CD2&#62;GAL4, UAS-EGFP/UAS-lgl-RNAi (heat-shocked 30 min at 2 days AED, incubate 2 days at 25 &#176;C in A, incubate 4 days at 25 &#176;C in B, incubate 7 days at 25 &#176;C in C after heat shock). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55901/55901fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55901/55901fig5.jpg" /> 
Figure 5: Diagnostic Examination of Dysplastic Clonal Phenotypes. (A) The left panel shows a mosaic wing imaginal disc of third instar larva with clones co-expressing scrib-RNAi and GFP (green) 5 days after heat-shock induction, stained for Dlg (red). The right panel shows a schematic line drawing of the apical side of the epithelial layers (blue) and the GFP-expressing clones (green). Mosaic clones in the epithelial layers are shown in light green while neoplastic tumors displaced from the epithelium are shown in magenta. (B-E) The confocal images show vertical sections of the scrib-knockdown clones indicated in the right panel of (A). The second panels from the left show the Dlg localization patterns (red). The third panels from the left show schematic line drawings of the apical (red) and basal (blue) sides of epithelial layers and the GFP-expressing scrib-knockdown clones (green). Dysplastic clones in the epithelial layers are shown in light green (B and C) while neoplastic clones displaced from the epithelium are shown in magenta (D and E). White arrows indicate scrib-knockdown clones showing tumorigenic phenotype. Nuclei were labeled with DAPI (blue). Scale bar = 50 &#181;m. The tumor phenotype of each clone was classified according to the flowchart shown in Figure 1. Detailed genotypes: A-E, hsFLP; UAS-scrib-RNAi; act&#62;CD2&#62;GAL4, UAS-EGFP (heat-shocked 30 min at 2 days AED, incubate 4 days at 25 &#730;C after heat shock). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55901/55901fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/55901/55901fig6.jpg" /> 
Figure 6: Diagnostic Examination of RasV12-expressing Clonal Phenotypes. (A) The left panel shows a mosaic wing imaginal disc of third instar larva with clones co-expressing oncogenic RasV12 and GFP (green) 4 days after heat-shock induction, stained for Dlg (red). The right panel shows a schematic line drawing of the apical side of the epithelial layers (blue) and the GFP-expressing clones (green). Scale bar represents 50 &#181;m. (B) Vertical sections of the RasV12 expressing clones indicated in the right panel of (A). The second panels from the left show the Dlg localization patterns (red). The third panels from the left show schematic line drawings of the apical (red) and basal (blue) sides of epithelial layers and the RasV12 and GFP expressing clones (green). Scale bar = 25 &#181;m. (C) Mosaic wing imaginal disc of third instar larva with clones co-expressing RasV12 and GFP (green) 4 days after heat-shock induction, stained for MMP1 (red). Scale bar = 100 &#181;m. Detailed genotypes: A-C, hsFLP; UAS-RasV12; act&#62;CD2&#62;GAL4, UAS-EGFP (heat-shocked 45 min at 2 days AED, incubate 4 days at 25 &#176;C after heat shock). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55901/55901fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/55901/55901fig7.jpg" /> 
Figure 7: Diagnostic Examination of Yki3SA-expressing Clonal Phenotypes. (A) The left panel shows a mosaic wing imaginal disc of third instar larva with clones co-expressing constitutively active Yki3SA and GFP (green) 4 days after heat-shock induction, stained for Dlg (red). The right panel shows a schematic line drawing of the apical side of the epithelial layers (blue) and the GFP-expressing clones (green). Scale bar = 50 &#181;m. (B-C) Vertical sections of the Yki3SA expressing clones indicated in the right panel of (A). The second panels from the left show the Dlg localization patterns (red). The third panels from the left show schematic line drawings of the apical (red) and basal (blue) sides of epithelial layers and the Yki and GFP expressing clones (green). Scale bar = 25 &#181;m. (D) Mosaic wing imaginal disc of third instar larva with clones co-expressing Yki3SA and GFP (green) 4 days after heat-shock induction, stained for MMP1 (red). Scale bar = 100 &#181;m. Detailed genotypes: A-D, hsFLP;; UAS-Yki3SA/ act&#62;CD2&#62;GAL4, UAS-EGFP (heat-shocked 30 min at 2 days AED, incubate 4 days at 25 &#176;C). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55901/55901fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia at JoVE.com

Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

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.

Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

In the early stages of cancer, transformed mutant cells show cytological abnormalities, begin uncontrolled overgrowth, and progressively disrupt tissue ...

induction-diagnosis-tumors-drosophila-imaginal-disc

Nanyang Technological University

Research

JoVE Journal

Cancer Research

14.8K Views.  SOKENDAI. 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.

Video: Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific molecular profiles of a tumor may predict responsiveness to therapeutic agents or provide knowledge about prognosis. At our institution a tumor genotyping program was established as part of routine clinical care, screening both hematologic and solid tumors for a wide spectrum of mutations using two next-generation sequencing (NGS) panels: a custom, 33 gene hematological malignancies panel for use with peripheral blood and bone marrow, and a commercially produced solid tumor panel for use with formalin-fixed paraffin-embedded tissue that targets 47 genes commonly mutated in cancer. Our workflow includes a pathologist review of the biopsy to ensure there is adequate amount of tumor for the assay followed by customized DNA extraction is performed on the specimen. Quality control of the specimen includes steps for quantity, quality and integrity and only after the extracted DNA passes these metrics an amplicon library is generated and sequenced. The resulting data is analyzed through an in-house bioinformatics pipeline and the variants are reviewed and interpreted for pathogenicity. Here we provide a snapshot of the utility of each panel using two clinical cases to provide insight into how a well-designed NGS workflow can contribute to optimizing clinical outcomes.

This manuscript describes clinical protocols for two next-generation sequencing panels. One panel interrogates hematologic malignancies while the other panel targets genes commonly mutated in solid tumors. Molecular classification of driver mutations in human malignancies offers valuable prognostic and predictive information.

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific ...

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.

Biology

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more than 50 histological subtypes of these rare solid tumors have been identified. Thus, due to their extraordinary diversity and low incidence, our understanding of the biology of these tumors is still limited. Patient-derived cultures represent the ideal platform to study STS pathophysiology and pharmacology. We thus developed a human preclinical model of STS starting from tumor specimens harvested from patients undergoing surgical resection. Patient-derived STS cell cultures were obtained from the surgical specimens by collagenase digestion and isolated by filtration. Cells were counted, seeded, and left for 14 days in standard monolayer cultures and then processed by downstream analysis. Before performing molecular or pharmaceutical analyses, the establishment of STS primary cultures was confirmed through the evaluation of cytomorphologic features and, when available, immunohistochemical markers. This method represents a useful tool 1) to study the natural history of these poorly explored malignancies and 2) to test the effects of different drugs in an effort to learn more about their mechanisms of action.

The following protocol focuses on the establishment of a primary culture of patient-derived soft tissue sarcoma (STS). This model could help us to better understand the molecular background and pharmacological profile of these rare malignancies and could represent a starting point for further research aimed at improving STS management.

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Drosophila has illuminated our understanding of the genetic basis of normal development and disease for the past several decades and today it continues to contribute immensely to our understanding of complex diseases 1-7. Progression of tumors from a benign to a metastatic state is a complex event 8 and has been modeled in Drosophila to help us better understand the genetic basis of this disease 9. Here I present a simple protocol to genetically induce, observe and then analyze the progression of tumors in Drosophila larvae. The tumor induction technique is based on the MARCM system 10 and exploits the cooperation between an activated oncogene, RasV12 and loss of cell polarity genes (scribbled, discs large and lethal giant larvae) to generate invasive tumors 9. I demonstrate how these tumors can be visualized in the intact larvae and then how these can be dissected out for further analysis. The simplified protocol presented here should make it possible for this technique to be utilized by investigators interested in understanding the role of a gene in tumor invasion.

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Cooperation between an activated oncogene like RASV12 and mutations in cell polarity genes like scribbled, result in tumor growth in Drosophila where tumor cells also display invasive behaviors. Here a simple protocol for the induction and observation of the benign and invasive tumors is presented.

Drosophila has illuminated our  understanding of the genetic basis of normal development and disease for the  past several decades and today it continues ...

Medicine

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A remarkable example is given by sensory epithelia, where cells of the same or distinct identities are brought together via cell-cell adhesion showing highly organized planar patterns. Cells align to one another in the same direction and display equivalent polarity over large distances. This organization of the mature epithelia is established over the course of morphogenesis. To understand how the planar arrangement of the mature epithelia is achieved, it is crucial to track cell orientation and growth dynamics with high spatiotemporal fidelity during development in vivo. Robust analytical tools are also essential to identify and characterize local-to-global transitions. The Drosophila pupa is an ideal system to evaluate oriented cell shape changes underlying epithelial morphogenesis. The pupal developing epithelium constitutes the external surface of the immobile body, allowing long-term imaging of intact animals. The protocol described here is designed to image and analyze cell behaviors at both global and local levels in the pupal abdominal epidermis as it grows. The methodology described can be easily adapted to the imaging of cell behaviors at other developmental stages, tissues, subcellular structures, or model organisms.

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

This protocol is designed for the imaging and analysis of the dynamics of cell orientation and tissue growth in the Drosophila abdominal epithelia as the fruit fly undergoes metamorphosis. The methodology described here can be applied to the study of different developmental stages, tissues, and subcellular structures in Drosophila or other model organisms.

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A ...

Developmental Biology

Drosophila Pupal Abdomen Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic morphologies 1-3. During pupation, which spans approximately 96 hours (at 25 &deg;C), proliferating populations of imaginal cells replace the larval epidermis to generate the adult abdominal segments. These imaginal cells, born during embryogenesis, exist as lateral pairs of histoblast nests in each abdominal segment of the larvae. Four pairs of histoblast nests give rise to the adult dorsal cuticle (anterior and posterior dorsal nests), the ventral cuticle (ventral nests) and the spiracles associated with each segment (spiracle nests) 4. Upon puparation, these diploid cells (distinguishable by size from the larger polyploid larval epidermal cells- LECs) begin a stereotypical process of proliferation, migration and replacement of the LECs. Various molecular and genetic tools can be employed to investigate the contributions of genetic pathways involved in morphogenesis of the adult abdomen. Ultimate adult phenotypes are typically analyzed following dissection of adult abdominal cuticles. However, investigation of the underlying molecular processes requires immunohistochemical analyses of the pupal epithelium, which present unique challenges. Temporally dynamic morphogenesis and the interactions of two distinct epithelial populations (larval and imaginal) generate a fragile tissue prone to excessive cell loss during dissection and subsequent processing. We have developed methods of dissection, fixation, mounting and imaging of the Drosophila pupal abdominem epithelium for immunohistochemical studies that generate consistent high quality samples suitable for confocal or standard fluorescent microscopy.

Drosophila Pupal Abdomen Immunohistochemistry

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...

Drosophila Larva Imaginal Disc Dissection: A Method to Observe Developing Epithelia

Source: Morimoto, K. and Tamori, Y. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/55901/" target="_blank">Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia</a>. J. Vis. Exp. (2017).
When staining Drosophila imaginal discs, often a rough dissection is performed where the discs are exposed but remain attached to the body. Once stained, the discs of interest are isolated and mounted. The example protocol demonstrates a procedure for wing imaginal disc dissection and mounting.

Source: Morimoto, K. and Tamori, Y. Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia. J. Vis. Exp. (2017).
When staining Drosophila ...

Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

Summary

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Chapters in this video

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

Drosophila Pupal Abdomen Immunohistochemistry

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

Drosophila Larva Imaginal Disc Dissection: A Method to Observe Developing Epithelia