Name: dissection and immunostaining of imaginal discs from drosophila melanogaster
Uploaded: 2014-09-20T13:00:00
Description: Watch this Scientific Journal Video about Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster at JoVE.com

We would like to thank Donald Ready and Kevin Moses for teaching JPK the original imaginal disc dissection procedure. We also thank Bonnie Weasner for the genital disc in Figure&#160;1B and the eye disc in Figure&#160;2A, Brandon Weasner for Figure&#160;3, the Bloomington Drosophila Stock Center for fly stains and the Developmental Studies Hybridoma Bank for antibodies. CMS has been supported by a stipend from the National Institutes of Health (NIH) GCMS Training Grant (T32-GM007757), the Frank W. Putnam Research Fellowship, and the Robert Briggs Research Fellowship. JPK is supported by a grant from the National Eye Institute (R01 EY014863)

1. Preparation of Larvae
<ol>
	<li>Fill a 35 mm Petri dish with dissection buffer.</li>
	<li>Place larvae in Petri dish and allow them to swim around for a few minutes (self-cleaning step).</li>
	<li>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.</li>
	<li>Using a pasteur-pipet, place a larger pool of dissection buffer in th...

<ol>
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F., Hanson, T. E., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+Drosophila+retina,+a+neurocrystalline+lattice.">Development of the Drosophila retina, a neurocrystalline lattice.</a> Dev. Biol. 53, 217-240 (1976).</li><li>Wolff, T., Ready, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beginning+of+pattern+formation+in+the+Drosophila+compound+eye:+the+morphogenetic+furrow+and+the+second+mitotic+wave.">The beginning of pattern formation in the Drosophila compound eye: the morphogenetic furrow and the second mitotic wave.</a> Development. 113, 841-850 (1991).</li><li>Heberlein, U., Wolff, T., Rubin, G. 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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&#160;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...

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&#160;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&#160;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&#160;2D). The second is the &#8220;flp-out&#8221; 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&#160;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&#160;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.

<table border="0" cellpadding="0" cellspacing="0" width="915">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Name of Material/Equipment</td>
			<td style="width:161px;">Company</td>
			<td style="width:165px;">Catalog Number</td>
			<td style="width:360px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td>Dow Corning</td>
			<td>184 SIL ELAST KIT 0.5KG</td>
			<td>Used to create base for dissection plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pryex Glass Petri Dish 150x20mm</td>
			<td>Dow Corning</td>
			<td>3160-152CO</td>
			<td>Use the cover for dissection plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">#5 Dissecting Forceps</td>
			<td>Ted Pella</td>
			<td>525</td>
			<td>Forceps must be kept very sharp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">9 well watch glass</td>
			<td>Vairous Vendors</td>
			<td>N/A</td>
			<td>Used for fixation of imaginal disc complexes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50ml Erlenmeyer Flask</td>
			<td>Various Vendors</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small Stir Bar</td>
			<td>Various Vendors</td>
			<td>N/A</td>
			<td>Small enough to fit into Erlenmeyer Flask</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50ml Conical Tubes</td>
			<td>Various Vendors</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5ml Microfuge Tubes</td>
			<td>Various Vendors</td>
			<td>N/A</td>
			<td>Clear or Dark depending upon application</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microfuge Rack</td>
			<td>Various Vendors</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benchtop Rotator</td>
			<td>Various Vendors</td>
			<td>N/A</td>
			<td>100ul volume should not splatter at low setting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Macron Chemicals</td>
			<td>2-26555-1</td>
			<td>Serves as fixative</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Phosphate Monobasic</td>
			<td>Sigma Chemical Co</td>
			<td>S-3139</td>
			<td>Used to make dissection and wash buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Phosphate Dibasic</td>
			<td>Sigma Chemical Co</td>
			<td>71636</td>
			<td>Used to make dissection and wash buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lysine</td>
			<td>Acros Organics</td>
			<td>125221000</td>
			<td>Used in the fixative solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Periodate</td>
			<td>Sigma Chemical Co</td>
			<td>S-1878</td>
			<td>Used in the fixative solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>EMD Chemicals</td>
			<td>MTX1568-1</td>
			<td>Used to perforate imaginal discs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Hydroxide</td>
			<td>EM Science</td>
			<td>SX0593-3</td>
			<td>Used to dissolve paraformaldehyde</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100% Normal Goat Serum`</td>
			<td>Jackson Laboratories</td>
			<td>005-000-121</td>
			<td>Serves as a blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primary Antibodies</td>
			<td>Various Vendors</td>
			<td>N/A</td>
			<td>Dilute in 10% goat serum as directed by manufacturer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Seondary Antibodies</td>
			<td>Various Vendors</td>
			<td>N/A</td>
			<td>Dilute in 10% goat serum as directed by manufacturer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectashield</td>
			<td>Molecular Probes</td>
			<td>H-1000</td>
			<td>Prevents bleaching of samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope Slides</td>
			<td>Fischer Scientific</td>
			<td>48312-003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass Cover Slips 18x18mm</td>
			<td>Fischer Scientific</td>
			<td>12-542A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kimwipe Tissue</td>
			<td>Various Vendors</td>
			<td>NA</td>
			<td>Prevents Glass slides from adhering to silicone base</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Panit Brush 000</td>
			<td>Various Vendors</td>
			<td>NA</td>
			<td>Use to gently lower coverslip on to samples</td>
		</tr>
	</tbody>
</table>

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.

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, ...

Watch this Scientific Journal Video about Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster at JoVE.com

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.

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 ...

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