Name: dissection and immunostaining of imaginal discs from drosophila melanogaster
Uploaded: 2014-09-20T13:00:00.000+00:00
Duration: 10 min 10 s
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 the middle of the dissection plate.</li>
	<li>Using #5 forceps, transfer a single cleaned larva from the small pool to the larger pool of dissection buffer.</li>
</ol>
2. Coarse Dissection of Larvae
<ol>
	<li>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).</li>
	<li>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.</li>
	<li>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 &#8220;guts&#8221; of the larva to spill out. This allows for the imaginal discs to remain in their normal conformation and prevents them from being deformed.</li>
	<li>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.</li>
	<li>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&#160;3).</li>
	<li>Repeat steps 2.1&#8211;2.5 with additional larvae for 15&#8211;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).</li>
</ol>
3. Fixation and Staining of Tissue with Antibodies
<ol>
	<li>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 &#181;l 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.</li>
	<li>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 &#181;l to minimize dilution of the wash buffer. Note: all dissected tissues should be completely submerged.</li>
	<li>Transfer 20&#8211;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&#8211;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.</li>
	<li>Remove wash buffer and replace with 100 &#181;l of blocking solution: 10% normal goat serum in wash buffer. Incubate at RT with gentle rotation on a table top rotator for 10 min.</li>
	<li>Remove blocking solution and replace with 100 &#181;l of primary antibodies that have been appropriately diluted in 10% normal goat serum. Incubate at RT with gentle rotation for 16 hr.</li>
	<li>Remove primary antibodies and replace with 750 &#181;l 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 &#176;C) and reused later. Reusing antibodies multiple times can help in reducing non-specific binding.</li>
	<li>Allow heads to settle to the bottom of the tube, then remove wash buffer and add 100 &#181;l of secondary antibodies that have been appropriately diluted in 10% normal serum. Incubate at RT with gentle rotation for 2&#8211;4 hr.</li>
	<li>Remove secondary antibody solutions from tissue and replace with 500 &#181;l of wash buffer. Allow tissue to settle to the bottom of the tube.</li>
	<li>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.</li>
</ol>
4. Fine Dissection of Eye-Antennal Disc Complexes and Mounting onto Slides
<ol>
	<li>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&#160;3, red arrow) and swiftly pulling the brain away from the mouth hooks.</li>
	<li>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&#160;3, blue arrow). Note: the eye-antennal disc complexes should be free of all tissue (Figure&#160;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&#160;1B-E).</li>
	<li>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 &#8212; 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.</li>
	<li>Using a P-20 with an uncut tip, add 9 &#181;l of an anti-bleaching agent to the middle of the glass microscope slide. This prevents bleaching of the tissue when viewed with fluorescent light.</li>
	<li>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 &#181;l or less.</li>
	<li>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.</li>
	<li>Using a fine paintbrush gently lower a coverslip onto the specimen. This prevents the formation of air bubbles.</li>
	<li>Store slides at -20 &#176;C until ready to view the eye-antennal or other imaginal discs using light or fluorescent microscopy.</li>
</ol>

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The authors declare that they have no competing financial interests.

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><tbody><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Dow Corning</td><td>184 SIL ELAST KIT 0.5 KG</td><td>Used to create base for dissection plate</td></tr><tr><td>Pryex Glass Petri Dish 150 x 20 mm</td><td>Dow Corning</td><td>3160-152CO</td><td>Use the cover for dissection plate</td></tr><tr><td>#5 Dissecting Forceps</td><td>Ted Pella</td><td>525</td><td>Forceps must be kept very sharp</td></tr><tr><td>9 well watch glass</td><td>Vairous Vendors</td><td></td><td>Used for fixation of imaginal disc complexes</td></tr><tr><td>50 ml Erlenmeyer Flask</td><td>Various Vendors</td><td></td><td></td></tr><tr><td>Small Stir Bar</td><td>Various Vendors</td><td></td><td>Small enough to fit into Erlenmeyer Flask</td></tr><tr><td>50 ml Conical Tubes</td><td>Various Vendors</td><td></td><td></td></tr><tr><td>1.5 ml Microfuge Tubes</td><td>Various Vendors</td><td></td><td>Clear or Dark depending upon application</td></tr><tr><td>Microfuge Rack</td><td>Various Vendors</td><td></td><td></td></tr><tr><td>Benchtop Rotator</td><td>Various Vendors</td><td></td><td>100 &amp;#956;l volume should not splatter at low setting</td></tr><tr><td>Paraformaldehyde</td><td>Macron Chemicals</td><td>2-26555-1</td><td>Serves as fixative</td></tr><tr><td>Sodium Phosphate Monobasic</td><td>Sigma Chemical Co</td><td>S-3139</td><td>Used to make dissection and wash buffers</td></tr><tr><td>Sodium Phosphate Dibasic</td><td>Sigma Chemical Co</td><td>71636</td><td>Used to make dissection and wash buffers</td></tr><tr><td>Lysine</td><td>Acros Organics</td><td>125221000</td><td>Used in the fixative solution</td></tr><tr><td>Sodium Periodate</td><td>Sigma Chemical Co</td><td>S-1878</td><td>Used in the fixative solution</td></tr><tr><td>Triton X-100</td><td>EMD Chemicals</td><td>MTX1568-1</td><td>Used to perforate imaginal discs</td></tr><tr><td>Sodium Hydroxide</td><td>EM Science</td><td>SX0593-3</td><td>Used to dissolve paraformaldehyde</td></tr><tr><td>100% Normal Goat Serum</td><td>Jackson Laboratories</td><td>005-000-121</td><td>Serves as a blocking solution</td></tr><tr><td>Primary Antibodies</td><td>Various Vendors</td><td></td><td>Dilute in 10% goat serum as directed by manufacturer</td></tr><tr><td>Secondary Antibodies</td><td>Various Vendors</td><td></td><td>Dilute in 10% goat serum as directed by manufacturer</td></tr><tr><td>Vectashield</td><td>Molecular Probes</td><td>H-1000</td><td>Prevents bleaching of samples</td></tr><tr><td>Microscope Slides</td><td>Fischer Scientific</td><td>48312-003</td><td></td></tr><tr><td>Glass Cover Slips 18 x 18 mm</td><td>Fischer Scientific</td><td>12-542A</td><td></td></tr><tr><td>Kimwipe Tissue</td><td>Various Vendors</td><td></td><td>Prevents Glass slides from adhering to silicone base</td></tr><tr><td>Panit Brush 000</td><td>Various Vendors</td><td></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 ...

dissection-immunostaining-imaginal-discs-from-drosophila

Research

JoVE Journal

Biology

26.6K Views.  Indiana University. 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.

Video: Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

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.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

Neuroscience

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Developmental Biology

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 2. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 3 4. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes5: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s 6 7 8.
Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc9 and initiates the assembly of undifferentiated cells into ommatidia10-11. The 'founder cell' R8 is specified first and recruits R1-6 and then R7 12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes 15, including rhabdomere formation, synaptogenesis and eventually rh expression.
In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries made using this system have taken advantage of the spatiotemporal nature of photoreceptor differentiation in the developing eye imaginal disc. To use this system it is first necessary for the researcher to learn to identify and dissect the eye disc. We describe a novel RFP reporter to aid in the identification of the eye disc and the visualization of specific cell types in the developing eye. We detail a methodology for dissection of the eye imaginal disc from third instar larvae and describe how the eye-RFP reporter can aid in this dissection. This eye-RFP reporter is only expressed in the eye and can be visualized using fluorescence microscopy either in live tissue or after fixation without the need for signal amplification. We also show how this reporter can be used to identify specific cells types within the eye disc. This protocol and the use of the eye-RFP reporter will aid researchers using the Drosophila eye to address fundamentally important biological questions.

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

We describe a novel red fluorescent protein (RFP) reporter that is expressed specifically in the Drosophila eye. We detail a methodology for dissection of the eye imaginal disc and how this reporter can be used to aid in the dissection and identification of specific cell types in the developing eye.

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries ...

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the developmental mechanisms that generate neural diversity and drive circuit assembly. Given its complex three-dimensional organization, analysis of the optic lobe requires that one understand how its adult neuropils and larval progenitors are positioned relative to each other and the central brain. Here, we describe a protocol for the dissection, immunostaining and mounting of larval and adult brains for optic lobe imaging. Special emphasis is placed on the relationship between mounting orientation and the spatial organization of the optic lobe. We describe three mounting strategies in the larva (anterior, posterior and lateral) and three in the adult (anterior, posterior and horizontal), each of which provide an ideal imaging angle for a distinct optic lobe structure.

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

This protocol describes three steps to prepare larval and adult Drosophila optic lobes for imaging: 1) brain dissections, 2) immunohistochemistry and 3) mounting. Emphasis is placed on step 3, as distinct mounting orientations are required to visualize specific optic lobe structures.&#160;

The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the ...

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 Mounting of Drosophila Pupal Eye Discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

Dissection and Mounting of Drosophila Pupal Eye Discs

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

Summary

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Chapters in this video

Dissection of Larval CNS in Drosophila Melanogaster

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

Drosophila Pupal Abdomen Immunohistochemistry

Dissection and Mounting of Drosophila Pupal Eye Discs