The overall goal of this protocol is to demonstrate how to dissect the drosophila eye-antennal imaginal discs and brains, carrying genetically defined fluorescently-marked clones and how to process them to visualize and quantify gene expression changes and cell invasiveness. The described procedures can help to provide novel insights into the role of genes and epithelial development, homeostasis or disease and to dissect mechanisms underlying their competition, compensatory proliferation, or interclonal corporation. The main advantage of the study is that we provide an entire suit of protocols tailored for in-depth quantitative and qualitative analysis of the drosophila genetic mosaics.
Generally individuals new working with the drosophila model struggle because they cannot recognize the eye-antennal imaginal disc from the other imaginal disc. To ease the recognition of eye-antennal imaginal disc bearing florescently-marked clones within the larva body it is recommended to perform the initial dissections under a fluorescent microscope. Mosaic analysis with a repressible cell marker or MARCM enables the generation of genetically defined clonal patches in an otherwise phenotypically wild-type context.
A FLP driven by an eyeless promoter catalyzes recombination between two FRT elements flanking a stop cassette marked with a yellow gene. After DNA replication FLP catalyzes the exchange of the non-sister chromatids between the homologous chromosomes generating sister chromatids. One carrying the wild-type allele and Gal80 repressor and the other the mutant.
In mitosis these segregate to produce two daughter cells, one being a homozygous mutant and the other wild type. Loss of the Gal80 repressor allows GFP expression in the mutant whereas the wild-type cells stays GFP negative. To begin the experiment harvest mosaic larvae by squirting PBS into the fly bottle to cover the surface of the food.
Then soften the top layer with a spatula and pour the food slurry containing larvae into a petri dish. Gently pick the larvae and transfer them into an embryo dish filled with PBS. Collect at least 20 larvae for immunostaining and 80 larvae for quantification of tumor invasiveness.
Wash the larvae with PBS to remove all residual food. Using a fluorescent stereo microscope at 8:16x magnification identify and discard all leopard larvae which are those displaying random GFP positive spots throughout the body. Place the dish containing the remaining larvae in PBS on ice until dissection.
To dissect the eye-antennal imaginal disc under the stereo microscope use one pair of forceps and gently grab the larva at approximately 2/3 of the body length posterior to the head. With the second pair of forceps grab the larval mouth hooks and pull them away from the body. Using forceps disentangle the mouth hooks from the overlaying cuticle and extraneous tissue such as the salivary glands and fat body.
Alternatively to prepare the eye-antennal disc brain complex keeping the ventral nerve cord intact use forceps to cut a larva in the middle of the body. Discard the posterior half. Hold the anterior half of the larval body with the tips of one pair of forceps then flip the larva inside out by pushing the mouth hooks inward with the tip of the second pair and rolling the cuticle over it with the first forceps pair.
Carefully remove all extraneous tissue including the gut, the fat body, and the salivary glands. Gently pull the mouth hooks away from the cuticle. Release the eye-antennal disc brain complex attached to the mouth hooks by severing the axonal projections extending from the ventral nerve cord to the muscles and epidermis.
To transfer the tissue first coat a P20 micropipette tip by pipetting the remaining body carcasses up and down several times and then use the same tip to transfer the dissected tissue. To transfer larger eye-antennal disc brain complexes pre-cut the P20 tip to enlarge the opening. Fix the samples in 400 micro-liters of 4%PFA fixative for 25 minutes at room temperature while nutating.
Then remove the fixative and wash the samples with PBST three times for 10 minutes with nutation. Next replace the PBST with 500 micro-liters of DAPI staining solution and nutate for 15 minutes in the dark. Wash the tissue once for 10 minutes with PBST.
To complete the dissection use a one milliliter pipette to transfer the tissue back into a PBS-filled glass dish. Using two pairs of forceps separate the brains to be used for quantification of invasiveness from the eye-antennal discs by cutting the optic stalks. Free the eye-antennal discs by clipping them off the mouth hooks.
Place a drop of mounting medium onto an abductive slide and using forceps distribute the liquid into a thin layer. To quantify invasiveness place at least 80 fixed intact brains for each genotype onto a single slide. Straighten and position the tissue as desired.
Gently touch one edge of a coverslip to the medium and lower it slowly with the help of forceps to avoid creating bubbles. Use a laboratory wipe to absorb excess mounting medium at the edges. The tissue can now be examined using confocal imaging.
Ask a neutral party to anonymize the slides so that scoring is unbiased. And have slides evaluated independently by at least two researchers. Disclose genotypes only after scoring is complete.
Evaluate the degree of malignancy under a fluorescent stereo microscope equipped with a GFP filter set. Using a marker label brains that have already been viewed to avoid double counting. Fluorescent confocal images show brains dissected from larvae bearing malignant RAS V12 scribble mutant GFP-labeled clonal tumors stained with DAPI.
The panels represent the four different grades of tumor invasiveness. Unbiased quantification revealed that inhibition of JNK signaling and RAS V12 scribbled one clones suppressed tumor invasiveness. In control eye-antennal discs the JNK-sensitive TRE-DsRED reporter labels only a narrow stripe of cells running from the antennal to the eye part.
In contrast TRE-DsRED activity was markedly enhanced in the GFP-positive malignant clonal tumors. By day 8 the tumor cells overgrew the eye-antennal discs and spread to the ventral nerve cord. The invading tumor cells also showed increased JNK activation compared to the surrounding tissue.
While blocking JNK clearly reduced the TRE-DsRED activity in the tumor clones, increasing the laser intensity revealed an enhancement in epithelial cells neighboring the clones. Additionally a hemolectin delta DsRED reporter showed that in contrast to a few hemocyte clusters found in the indentations of the control eye-antennal epithelium hemocytes accumulated in the eye-antennal and brain tissue comprised of malignant tumors. Upon inhibition of JNK the number of associated immune cells dramatically decreased.
The enhanced tumor invasiveness, JNK activity, and hemocyte infiltration were accompanied by significant up-regulation of MMP1 mNRA as determined by QRTPCR using total RNA isolated from dissected mosaic eye-antennal discs. Once mastered over 60 eye disc pairs and brains can be dissected within 30 minutes. While attempting this procedure it's important to remember to generate a sufficient number of larvae of the desired genotypes.
Be quick but precise while dissecting to keep samples intended for RNA isolation RNS free. Following dissection procedure western blot can be used as an alternative to immunostaining to assess changes in protein expression between different genotypes. Creating genetic mosaics and analyzing them with the described procedures enables research on otherwise lethal mutations and on mechanisms of oncogene corporation and After watching this video you should have good understanding of how to dissect mosaic tissues of the eye-antennal disc and brains from third instar larvae and how to process them for immunostaining, visualization of transgenic and port activity, RNA extraction, and the quantification of invasiveness.
Be reminded that PFA as well as phenol-chloroform and DAPI are highly toxic and that precautions such as wearing protective gloves and clothing should always be taken while working with these agents.
