Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

For more than a century the fruit fly, Drosophila melanogaster, has been a premier system to study development, behavior and physiology. Development in the fly can be divided into two broad stages: embryonic and post-embryonic with much of the latter taking place within monolayer epithelia called imaginal discs ^1-3. Drawings of imaginal discs were first published in 1864 by August Weismann as part of his broad monograph on insect development ¹. These discs begin their development during embryogenesis, are patterned during the larval stages, survive the massive histolysis of the early pupal stages, and ultimately give rise to a high percentage of adult structures that are found within the adult fly ^1-14. During larval development each disc makes several critical decisions regarding fate, shape and size. Within the first and second larval instars, the discs are tasked with adopting a primary fate, establishing compartment boundaries, adopting the correct shape and generating the requisite number of cells ^15-16. During the third larval instar and early pre-pupal stage, the imaginal discs continue to divide and are patterned as cells adopt their terminal fates ¹⁶.

During the early history of Drosophila developmental biology, imaginal discs were studied nearly exclusively in the context of normal development and in the limited cases in which a loss or gain-of-function mutant was viable. The use of X-rays to induce mitotic recombination allowed for lethal mutations to be analyzed in cell clones within the larval and adult tissues. This method has been improved by the introduction of transgenic methods to analyze loss and gain-of-function mutations in both larval and adult tissues. The number of antibodies, transcriptional reporters and protein traps for describing the molecular landscape of wild type and mutant tissues is also constantly growing. Using these molecular markers to analyze loss and gain-of-function mutant cell clones has made it increasingly feasible to gain a real-time understanding of how mutant cells deviate from their wild type cousins during development. To properly take advantage of these tools and reagents it is critical to have high quality preparations of imaginal discs that can be viewed, photographed and analyzed. The goal of this manuscript is to provide an optimized protocol for the isolation and preparation of the eye-antennal disc complex (Figure 1A). It can also be successfully used to isolate a wide variety of additional discs including those that give rise to the wings, halteres, T1-T3 legs and the genitals (Figure 1B-E). This procedure, with minor modifications, has been used to isolate imaginal discs from Drosophila for nearly eighty years.

As described above, since most genes are expressed during multiple stages of development and in a multitude of tissues, it is often impossible to study the effects that null mutants have on the entire eye as the animal dies well before the third instar larval stage. Four methods have made the study of more developed tissues such as the retina significantly more tractable. The first is the Flippase (FLP)/Flippase Recombination Target (FRT) method of generating mutant cell clones within an otherwise wild type tissue ^17-19. In this instance the mutant tissue is identified by the absence of a visual marker such as Green Fluorescent Protein (GFP) and can be compared to the surrounding wild type tissue in which GFP is present (Figure 2D). The second is the “flp-out” method in which a transgene is expressed in a population of cells ²⁰. In this instance the cell clones are identified by the presence of GFP and compared to the surrounding wild type tissue that lacks the GFP reporter (Figure 2E). The third is the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique, which combines elements of the FLP/FRT mutant clone and flp-out expression systems ²¹. With this method a transgene can be expressed within a population of cells that are simultaneously mutant for an individual genetic locus. Like flp-out clones, MARCM clones are identified by the presence of GFP and compared to the surrounding wild type tissue that lacks the GFP marker (Figure 2F). And lastly, the genes and RNAi constructs can be expressed within imaginal tissues under the control of specific promoter-GAL4 constructs. These four methods have increased the interest in studying imaginal discs since mutant or over-expression clones or patterns can be directly compared to adjacent wild type tissue. The method described in this procedure has been developed so that researchers who study the post-embryonic development of adult tissues in Drosophila, particularly those derived from the eye-antennal disc, will be able to obtain high quality tissue for analysis. Although individual researchers have made slight modifications, the core of this procedure (which we describe here) has remained largely unaltered. Since obtaining high quality tissue is critical to the study of the imaginal discs we hope this written protocol and accompanying video will serve as a valuable teaching resource.

Protokół

1. Preparation of Larvae

1. Fill a 35 mm Petri dish with dissection buffer.
2. Place larvae in Petri dish and allow them to swim around for a few minutes (self-cleaning step).
3. Transfer larvae to a pool of dissection buffer on a silicone-based dissection plate. This pool should be at one edge of the plate. The dissection plate consists of a silicone solution that has been poured and hardened within a glass Petri dish.
4. Using a pasteur-pipet, place a larger pool of dissection buffer in the middle of the dissection plate.
5. Using #5 forceps, transfer a single cleaned larva from the small pool to the larger pool of dissection buffer.

2. Coarse Dissection of Larvae

1. While the larva is still within the large pool of dissection buffer clasp the larva with the forceps. One pair of forceps should be used to grab the mouth hooks while the other pair of forceps is used to hold the animal still (grab the larva gently at 1/3 body length).
2. Hold steady the pair of forceps containing the cuticle near the mouth hooks while quickly pulling the rest of the body away with the second pair of forceps.
3. When the larva begins to tear apart you will feel a slight release in tension. Release the larva from the forceps and allow for the “guts” of the larva to spill out. This allows for the imaginal discs to remain in their normal conformation and prevents them from being deformed.
4. With one pair of forceps grasp the mouth hooks again to hold the front end of the larva in place. Using the other pair of forceps remove the lower 2/3 of the larvae including the inner guts. Note: a complex containing the mouth hooks, the eye-antennal discs, brain hemispheres, ventral ganglion, the salivary glands, some leg discs, and the overlying cuticle will remain.
5. With a pair of forceps gently remove overlying cuticle, salivary glands, leg discs and other tissue. Note: only tissue that should remain is the mouth hooks, eye-antennal discs, brain hemispheres, and ventral ganglion (Figure 3).
6. Repeat steps 2.1–2.5 with additional larvae for 15–20 min. Note: imaginal discs that remain in dissection buffer for longer periods of time tend to degrade and ultimately will appear as less than ideal specimens to be photographed. Thus, after a maximum of 20 min all dissected tissues should be transferred to the PLP fixative (see below).

3. Fixation and Staining of Tissue with Antibodies

1. Using a P-200 pipetman, transfer the dissected tissues to a watch glass containing cold Paraformaldehyde-Lysine-Periodate (PLP) fixative. Limit volume of transferred dissection buffer to 50 µl to minimize dilution of PLP. Be sure to cut the tip with a razor blade so that the tip opening is large enough to accommodate the dissected tissues. Using a pair of forceps or a tungsten needle ensure that the dissected tissues are completely submerged in order for proper fixation to occur. Incubate dissected tissues in cold PLP fixative for 45 min. This incubation can take place at RT without agitation.
2. Using a P-200 pipetman, transfer the dissected tissues to wash buffer (RT) using another cut yellow tip for 45 min. Limit volume of transferred PLP to 50 µl to minimize dilution of the wash buffer. Note: all dissected tissues should be completely submerged.
3. Transfer 20–30 sets of dissected tissue to 1.5 ml microfuge tube. Note: if larger numbers of eye-antennal disc complexes are combined within the tube, there is a possibility that the tissue at the bottom of the tube will not be exposed properly to the antibodies. For optimal results no more than 20–30 eye-antennal disc complexes should be present within a single tube. The dissected tissue will settle to the bottom of the microfuge tube. Therefore, in subsequent steps use a pipetman to remove and replace wash buffer, blocking solution, and antibodies.
4. Remove wash buffer and replace with 100 µl of blocking solution: 10% normal goat serum in wash buffer. Incubate at RT with gentle rotation on a table top rotator for 10 min.
5. Remove blocking solution and replace with 100 µl of primary antibodies that have been appropriately diluted in 10% normal goat serum. Incubate at RT with gentle rotation for 16 hr.
6. Remove primary antibodies and replace with 750 µl of wash buffer. Place tubes on a nutator and allow to nutate at RT for 10 min. Note: The primary antibody can be saved (store at 4 °C) and reused later. Reusing antibodies multiple times can help in reducing non-specific binding.
7. Allow heads to settle to the bottom of the tube, then remove wash buffer and add 100 µl of secondary antibodies that have been appropriately diluted in 10% normal serum. Incubate at RT with gentle rotation for 2–4 hr.
8. Remove secondary antibody solutions from tissue and replace with 500 µl of wash buffer. Allow tissue to settle to the bottom of the tube.
9. Using a P-200 and a cut yellow tip, transfer all dissected tissues to a pool of wash buffer that has been placed on the dissecting dish. While proceeding with the next step of fine dissection, the tissue will incubate in the wash buffer. This helps to remove excess secondary antibodies.

4. Fine Dissection of Eye-Antennal Disc Complexes and Mounting onto Slides

1. Use one pair of forceps to clasp the cuticle by the mouth hooks with the ventral side of the complex facing downwards. With a second pair of forceps remove the two brain lobes by closing the second forceps in the space between the brain and eye discs (Figure 3, red arrow) and swiftly pulling the brain away from the mouth hooks.
2. While continuing to hold onto the mouth hooks, pinch off the tissue as close as possible to the connection between the antennal section of the eye-antennal disc and the mouth hooks (Figure 3, blue arrow). Note: the eye-antennal disc complexes should be free of all tissue (Figure 1A). Continue until all desired tissue desired that can be placed onto slides has been dissected. This protocol can be adapted to isolate wing, haltere, leg and genital imaginal discs (Figure 1B-E).
3. Add two small pieces of tissue paper (size of coverslips) approximately 3 in. apart on the dissecting dish. Place a glass slide onto the dish — the two pieces of tissue paper should be under the ends of the slide. This will prevent the slide from sticking to the silicone base of the dissecting dish.
4. Using a P-20 with an uncut tip, add 9 µl of an anti-bleaching agent to the middle of the glass microscope slide. This prevents bleaching of the tissue when viewed with fluorescent light.
5. Using the same uncut tip, gather all eye-antennal discs and add them to the drop of anti-bleaching reagent. Minimize the amount of wash buffer that is carried over. Try to limit the amount of wash buffer to 10 µl or less.
6. Use a pair of forceps to separate and spread out the eye-antennal discs within the drop of anti-bleaching reagent. Allow discs to incubate in anti-bleaching reagent for several minutes. Note: discs will turn clear as the tissue absorbs the anti-bleaching reagent.
7. Using a fine paintbrush gently lower a coverslip onto the specimen. This prevents the formation of air bubbles.
8. Store slides at -20 °C until ready to view the eye-antennal or other imaginal discs using light or fluorescent microscopy.

Wyniki

The method that is described above reliably produces high quality material for analysis with in situ probes, transcriptional reporters, protein traps and antibodies. In Figure 1 we display eye-antenna, genital, wing, haltere and leg discs that are routinely recovered with this method. These discs have been treated with a phalloidin-conjugated fluorophore, which binds to F-actin and therefore outlines each cell. If the tissue has been fixed properly then the morphogenetic furrow of the eye disc, ...

Dyskusje

Although this procedure has largely focused on the isolation and subsequent treatment of eye-antennal discs, it is amenable to being used to isolate and analyze the wing, haltere, leg and genital discs (Figure 4). The only required modification of the protocol for isolating these discs (as opposed to the eye-antennal disc) is the method of coarse dissection (section 2 of the protocol). The first thoracic leg (T1) pair is found at the anterior of the larva and can be recovered by following the protoc...

Materiały

Name	Company	Catalog Number	Comments
Name of Material/Equipment	Company	Catalog Number	Comments
Sylgard 184 Silicone Elastomer Kit	Dow Corning	184 SIL ELAST KIT 0.5KG	Used to create base for dissection plate
Pryex Glass Petri Dish 150x20mm	Dow Corning	3160-152CO	Use the cover for dissection plate
#5 Dissecting Forceps	Ted Pella	525	Forceps must be kept very sharp
9 well watch glass	Vairous Vendors	N/A	Used for fixation of imaginal disc complexes
50ml Erlenmeyer Flask	Various Vendors	N/A
Small Stir Bar	Various Vendors	N/A	Small enough to fit into Erlenmeyer Flask
50ml Conical Tubes	Various Vendors	N/A
1.5ml Microfuge Tubes	Various Vendors	N/A	Clear or Dark depending upon application
Microfuge Rack	Various Vendors	N/A
Benchtop Rotator	Various Vendors	N/A	100ul volume should not splatter at low setting
Paraformaldehyde	Macron Chemicals	2-26555-1	Serves as fixative
Sodium Phosphate Monobasic	Sigma Chemical Co	S-3139	Used to make dissection and wash buffers
Sodium Phosphate Dibasic	Sigma Chemical Co	71636	Used to make dissection and wash buffers
Lysine	Acros Organics	125221000	Used in the fixative solution
Sodium Periodate	Sigma Chemical Co	S-1878	Used in the fixative solution
Triton X-100	EMD Chemicals	MTX1568-1	Used to perforate imaginal discs
Sodium Hydroxide	EM Science	SX0593-3	Used to dissolve paraformaldehyde
100% Normal Goat Serum`	Jackson Laboratories	005-000-121	Serves as a blocking solution
Primary Antibodies	Various Vendors	N/A	Dilute in 10% goat serum as directed by manufacturer
Seondary Antibodies	Various Vendors	N/A	Dilute in 10% goat serum as directed by manufacturer
Vectashield	Molecular Probes	H-1000	Prevents bleaching of samples
Microscope Slides	Fischer Scientific	48312-003
Glass Cover Slips 18x18mm	Fischer Scientific	12-542A
Kimwipe Tissue	Various Vendors	NA	Prevents Glass slides from adhering to silicone base
Panit Brush 000	Various Vendors	NA	Use to gently lower coverslip on to samples