Name: dissection and mounting of drosophila pupal eye discs
Uploaded: 2014-11-09T15:00:00.000+00:00
Duration: 7 min 48 s
Description: Watch this Scientific Journal Video about Dissection and Mounting of Drosophila Pupal Eye Discs at JoVE.com

We appreciate and would like to thank the Howard Hughes Medical Institute for the HHMI Professor award to U.B. which made this project possible. We thank the college at the University of California, Los Angeles for providing facilities and teaching infrastructure support for this work. The work was also supported with funding from Midwestern University and a generous donation from the Charity Fidelity Gift Fund. We thank John VandenBrooks for comments on the manuscript and Krista Pearman for her technical assistance.

This procedure is a 2 day procedure.
Day 1 (2 hr + dissecting time)
1. Pupal Eye Disc Dissection
<ol>
	<li>Select a pupa for dissection. 
	NOTE: The age of the pupa to be dissected will be determined by the experimental needs. However, if examining cell morphology, this is frequently done at 42 hr after puparium formation (APF) at 25 &#176;C, which is the age of the pupae shown in the video. Collect white pupae (considered 0 hr APF) with a wetted paintbrush and arrange them in chronological order (based on collection time) in a new fly food vial maintained at 25 &#176;C. Dissect the aged pupae in this same chronological order to keep the time differences between pupae as close as possible.</li>
	<li>Transfer the pupa into a drop of phosphate-buffered saline (PBS, pH 7.4, see materials for recipe) on a silicone dissecting plate.</li>
	<li>Hold the top of the pupal case with forceps and pierce and open the lower portion of the pupal case with a pair of sharp forceps. Remove the pupa from the case in the newly formed hole.</li>
	<li>Make a diagonal cut with scissors, mid-thorax, in the pupa. Remove the debris or transfer the pupa head to a new drop of PBS. Clean using the pipette blower tube (see materials and Figure 1 for details) and add additional PBS as needed. Make sure to avoid dehydration.</li>
	<li>Securely grip the top of the pupal head cuticle with a pair of forceps. Using the tip of the blower tube, gently push on the pupal case to extrude the brain and other tissues, including the eye discs, out of the pupal case. Be sure to avoid grabbing any tissue within the cuticle.</li>
	<li>Using the blower tube filled with PBS, gently blow the fat body tissue and other debris out of the head case to clearly separate and identify the brain and eye discs. 
	NOTE: Keeping the brain attached to the pupal head case will allow the researcher to use the head case as a &#8220;handle&#8221; for future manipulation of the tissue without damaging the eye discs or brain. However, it is common to dislodge the brain and eye discs from the head case and this is not detrimental to the overall procedure and results. It often takes multiple attempts of blowing and pushing to successfully extract the eye discs and brain from within the pupal head case and so that they are clearly separate from surrounding tissue. The cleaner and more separate one can get the eye discs from the surrounding tissue will help prevent extraneous tissue from fixing to the eye discs.</li>
	<li>Following dissection, immediately transfer the tissue to a 3 well glass dish on ice with 0.5 ml of Fix solution (see materials). Float the tissue on the surface of the Fix solution for a few minutes (typically the time it takes to dissect the next set of eye discs). 
	NOTE: If this is not the first eye disc to be dissected in this round of dissection, completely submerge the previous eye disc into the Fix solution. If performing special staining procedures (e.g., reactive oxygen species, lysosomes, etc.) this must be done prior to fixation. In order to do this, store the dissected eye discs in ice-cold PBS prior to continuing with the appropriate staining protocol.</li>
	<li>Following dissection and submersion of all eye discs being dissected, move the 3 well glass dish to a rocker and rock at room temperature for 30 min.</li>
</ol>
2. Immunostaining
<ol>
	<li>Carefully remove the Fix solution and add 0.5 ml of PBS with 0.3% Triton X-100 (0.3% PBT, see materials) and wash the tissue on a rocker for 10 min. Repeat this step three more times for a total of 4 washes (10 min each). 
	NOTE: Careful removal of the solution from the well can be performed under a dissecting microscope to prevent tissue damage or loss. The solution can be aspirated carefully with a pipet; we prefer a fine-tipped, disposable transfer pipet that has had the opening flattened to reduce the chance of aspirating tissue.</li>
	<li>At the end of the 4th wash, remove the wash and add 0.5 ml block solution (see materials) to the well and incubate for 30 min. 
	NOTE: This time frame can be extended according to your needs.</li>
	<li>At the end of the 30 min block time, pipet 10 &#956;l of Primary Antibody Solution (diluted in Block solution) into the appropriate number of wells in a microwell plate. 
	NOTE: One well of a microwell plate will accommodate about 4 pairs of eye discs attached to the pupal case or 8 isolated pairs of eye discs with brains attached. The primary antibodies used in this protocol for the representative data are anti-phosphotyrosine (1:500 dilution) and anti-Cut (1:200 dilution).</li>
	<li>Transfer the dissected tissue into the microwell plate wells filled with the Primary Antibody Solution from the 3 well glass dish.</li>
	<li>Place the microwell plate on a wetted paper towel (to prevent dehydration) in an empty pipet tip box (or other similar container) and store at 4 &#176;C overnight.</li>
</ol>
Day 2 (4 hr + mounting time)&#160;
<ol start="6">
	<li>Transfer the tissue from the microwell plate to a new 3 well glass dish with 0.3% PBT (&#8776;0.5 ml) and wash the tissue for 10 min on the rocker. Repeat this step three more times for a total of 4 washes (10 min each)</li>
	<li>Remove the last wash and add 0.5 ml of Block solution to the well. Block for 30 min. 
	NOTE: This time frame can be extended according to your needs.</li>
	<li>Remove the Block solution and add 0.5 ml of the Secondary Antibody Solution (made from fluorescently labeled antibodies appropriate to your primary antibody and fluorescent wavelength, diluted in Block solution) to the well and protect the 3 well glass dish from light with a cover or aluminum foil and incubate for at least 2 hr at room temperature. 
	NOTE: All subsequent steps should be similarly protected from light exposure. This incubation can be extended to overnight at 4 &#176;C, in a setup similar to the Primary Antibody incubation, but it can also be performed in a 3 well glass dish covered with laboratory film, or a microwell plate if antibody conservation is desired.</li>
	<li>At end of the antibody incubation, remove the Secondary Antibody Solution and add 0.5 ml of 0.3% PBT to the well and wash on the rocker for 10 min. Repeat this step for a total of 4 washes (10 min each). 
	NOTE: If DAPI staining is desired, before starting the first wash, add 0.5 &#956;l of DAPI stock solution (see materials) to the 500 &#956;l of PBT that will be used for the first wash (1:1,000 dilution).</li>
</ol>
3. Mounting&#160;
<ol>
	<li>At the end of the last wash, mount the antennal/eye discs on a slide using 70% glycerol or PBS as a medium to remove the brain and other unwanted tissues in a pool on one side of the slide.</li>
	<li>Carefully move the tissue using hydrostatic pressure between the forceps, or very gently with a forceps tip, and line up the eye discs in a column towards the center of the slide.</li>
	<li>Remove all extraneous glycerol or PBS from around the tissue (using wicking action of a laboratory wipe or the forceps work for this).</li>
	<li>Place a small amount (&#8776;10 &#956;l) of mounting medium along one side of the eye disc column and place a coverslip on the tissue so as to spread the mounting medium onto the eye discs.</li>
	<li>Let slide sit for 1 to 2 min to allow the tissue/mounting medium to spread out completely.</li>
	<li>Wipe any extra mounting medium off from around the edges of the coverslip and seal it with clear nail polish.</li>
	<li>Capture fluorescent images of the eye discs using confocal microscopy.</li>
</ol>

<ol>
	<li>Ready, D. 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 (2), 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 (3), 841-850 (1991).</li><li>Carthew, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+formation+in+the+Drosophila+eye.">Pattern formation in the Drosophila eye.</a> Curr Opin Genet Dev. 17 (4), 309-313 (2007).</li><li>Bao, S., Cagan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+adhesion+mediated+by+Hibris+and+Roughest+regulates+morphogenesis+and+patterning+in+the+Drosophila+eye.">Preferential adhesion mediated by Hibris and Roughest regulates morphogenesis and patterning in the Drosophila eye.</a> Dev cell. 8 (6), 925-935 (2005).</li><li>Grzeschik, N. A., Knust, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IrreC/rst-mediated+cell+sorting+during+Drosophila+pupal+eye+development+depends+on+proper+localisation+of+DE-cadherin.">IrreC/rst-mediated cell sorting during Drosophila pupal eye development depends on proper localisation of DE-cadherin.</a> Development. 132 (9), 2035-2045 (2005).</li><li>Sprecher, S. G., Desplan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Switch+of+rhodopsin+expression+in+terminally+differentiated+Drosophila+sensory+neurons.">Switch of rhodopsin expression in terminally differentiated Drosophila sensory neurons.</a> Nature. 454 (7203), 533-537 (2008).</li><li>Zuker, C. S., Cowman, A. F., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+structure+of+a+rhodopsin+gene+from+D.+melanogaster.">Isolation and structure of a rhodopsin gene from D. melanogaster.</a> Cell. 40 (4), 851-858 (1985).</li><li>Voas, M. G., Rebay, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+integration+during+development:+insights+from+the+Drosophila+eye.">Signal integration during development: insights from the Drosophila eye.</a> Dev Dyn. 229 (1), 162-175 (2004).</li><li>Hsiao, H. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+and+immunohistochemistry+of+larval,+pupal+and+adult+Drosophila+retinas.">Dissection and immunohistochemistry of larval, pupal and adult Drosophila retinas.</a> J Vis Exp. , 4347 (2012).</li><li>Call, G. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomewide+clonal+analysis+of+lethal+mutations+in+the+Drosophila+melanogaster+eye:+comparison+of+the+X+chromosome+and+autosomes.">Genomewide clonal analysis of lethal mutations in the Drosophila melanogaster eye: comparison of the X chromosome and autosomes.</a> Genetics. 177 (2), 689-697 (2007).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery-based+science+education:+functional+genomic+dissection+in+Drosophila+by+undergraduate+researchers.">Discovery-based science education: functional genomic dissection in Drosophila by undergraduate researchers.</a> PLoS Biol. 3 (2), 59 (2005).</li><li>Wolff, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+techniques+for+pupal+and+larval+Drosophila+eyes.">Dissection techniques for pupal and larval Drosophila eyes.</a> CSH Protoc. 2007, (2007).</li><li>Williamson, W. R., Hiesinger, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+developing+and+adult+Drosophila+brains+and+retinae+for+live+imaging.">Preparation of developing and adult Drosophila brains and retinae for live imaging.</a> J Vis Exp. 37, 1936 (2010).</li><li>Xu, T., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+genetic+mosaics+in+developing+and+adult+Drosophila+tissues.">Analysis of genetic mosaics in developing and adult Drosophila tissues.</a> Development. 117 (4), 1223-1237 (1993).</li><li>Duffy, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GAL4+system+in+Drosophila:+a+fly+geneticist's+Swiss+army+knife.">GAL4 system in Drosophila: a fly geneticist's Swiss army knife.</a> Genesis. 34 (1-2), 1-15 (2002).</li></ol>

The authors have nothing to disclose.

While it appears that the process is simple and easy to perform, in reality, this technique requires a great deal of practice to master. Routinely, we start students off by learning to dissect and mount third instar eye discs12, which are much easier to work with. This practice helps to develop an appropriate dissection position of the arms, hands and fingers13 so that manipulation of the forceps under the dissecting microscope is stable, easy and experienced. In essence, the practice period should achieve the goal of making the forceps into a natural extension of the fingers and thumb, allowing for the discrete and subtle movements required in the dissection. 
If using PBS to mount in, always have extra PBS available, as the small amount of PBS quickly evaporates in the mounting process. Another common problem is the folding of the eye discs in the Fix solution, thus fixing the eye disc in that orientation. This can be avoided by floating the eye disc on the surface of the Fix solution for some time (typically the length of the next eye disc dissection) before submerging it completely in the Fix solution.
While other published techniques focus on the dissection of the pupal eye disc at the midpupal stage9, when dissection is easily accomplished, this technique provides a method that can be applied to any pupal developmental time, except in the case of the pharate adult or early white pupa. We feel our technique provides an alternative and complementary approach to already published methods for pupal eye disc dissection. 
This technique, accompanied with common and useful genetic manipulations like FLP-FRT analysis14, UAS-GAL expression of transgenes15 and others, will allow researchers to study the eye disc in further detail during any pupal time frame.

The fields of developmental and cell biology have been greatly impacted by the model organism: Drosophila melanogaster. Within this model, studies of the eye disc have contributed a great deal of knowledge concerning signaling, cell biology and other areas. The late third larval instar eye disc has been studied extensively and is a powerful model to utilize, as it gives a snapshot of a series of developmental periods, each with its own unique signaling molecules and processes, as the morphogenetic furrow progresses across the eye disc8. However, there is a need to further expand our understanding of developmental processes into the pupal phase of development. While there have been studies on the pupal eye disc3-7, our knowledge does not approach the breadth of work that has been performed on the third instar eye disc. This is due, in part, to the greater difficulty in dissecting the pupal eye disc. Therefore, a presentation of the proper method of dissection could greatly expand research in this area.
While there are stages within pupal eye disc development that are easily dissected, particularly around the mid-pupal period, other time periods are much more challenging to dissect. This protocol represents one method for dissecting pupal eye discs that can be universally used for all pupal developmental time frames. This protocol can be used as an alternative to another protocol9 that shows an easier and faster method to dissect eye discs from the midpupal time points. This protocol was originally filmed and developed for training advanced undergraduate students in the UCLA Undergraduate Research Consortium in Functional Genomics (URCFG)10,11 in the technique of pupal eye dissection. Many undergraduate students were able to utilize this video and method to learn this challenging technique.

<table><tbody><tr><td>Phosphate-buffered saline (PBS, pH 7.4)</td><td></td><td></td><td>80 g NaCl, 2g&amp;nbsp; KCl, 14.4 g Na&lt;sub&gt;2&lt;/sub&gt;HPO4, 2.4 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, Bring volume to 1 &amp;#956;l, adjust the pH to 7.4, autoclave or filter sterilize, dilute to 1x PBS with autoclaved ddH&lt;sub&gt;2&lt;/sub&gt;O before using.</td></tr><tr><td>Triton X-100</td><td>Promega</td><td>H5142</td><td>Caution: Irritant! Wear gloves.</td></tr><tr><td>0.3% PBT</td><td></td><td></td><td>1.5 ml of Triton X-100, 500 ml 1X PBS.</td></tr><tr><td>37% formaldehyde solution</td><td>Fisher Scientific</td><td>F75P1GAL</td><td>Caution: Toxic, probable human carcinogen! Wear gloves.&amp;nbsp;</td></tr><tr><td>Fix Solution</td><td></td><td></td><td>(&amp;asymp;4% Formaldehyde in PBS) 50 &amp;mu;l of 37% Formaldehyde, 450 &amp;mu;l 1x PBS, make fresh before use</td></tr><tr><td>Normal goat serum</td><td>Rockland antibodies &amp;amp; assays</td><td>B304</td><td>Aliquot in 1 ml volumes and store at -80 &amp;#176;C</td></tr><tr><td>Block Solution</td><td></td><td></td><td>10% NGS in PBT.&amp;nbsp; This can be made and stored at 4 &amp;deg;C for a few days prior to use.</td></tr><tr><td>DAPI stock solution</td><td>Life Technologies</td><td>D3571</td><td>For coutnerstaining nuclei. Prepare a 1 mg/ml solution with ddH&lt;sub&gt;2&lt;/sub&gt;O.</td></tr><tr><td>VectaShield Mounting Medium</td><td>Vector Labs</td><td>H-1000</td><td>Mounting medium</td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516</td><td>For mounting. Prepare 70% dilution with ddH&lt;sub&gt;2&lt;/sub&gt;O.</td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>Nutating mixer</td><td>VWR</td><td>82007-202</td><td>Used to rock tissue in 3 well glass dish</td></tr><tr><td>SylGard 182 Silicone Elastomer Kit</td><td>Krayden</td><td>NC9897184</td><td>Used to make silicone dissection dish</td></tr><tr><td>Silicone dissecting dish</td><td></td><td></td><td>Mix Sylgard elastomer kit (above) according to directions gently (to avoid bubbles). Pour mixture into Petri dish (any size). Allow SylGard to cure overnight in 37 &amp;deg;C incubator.</td></tr><tr><td>3 well glass dish</td><td>Corning</td><td>7220-85</td><td>The 3 well variety of these are no longer available, this is the 9 well product.</td></tr><tr><td>72 well microwell minitray</td><td>Nunc</td><td>438733</td><td></td></tr><tr><td>Sharp forceps (Dumont #55)</td><td>Fine Science Tools</td><td>11255-20</td><td></td></tr><tr><td>Vannas-type Micro Scissors, Straight, 5mm blade</td><td>Ted Pella</td><td>1346</td><td></td></tr><tr><td>100 mm Borosilicate glass capillaries</td><td>World Precision Instruments</td><td>1B100-4</td><td>Pull with needle puller to make fine point tip that allows a small stream of PBS to flow.</td></tr><tr><td>Disposable Transfer Pipets, Fine Tip</td><td>Samco Scientific</td><td>231</td><td></td></tr><tr><td>Tubing dimensions given are inner diameter (ID) x outer diameter (OD) x wall thickness in inches</td><td></td><td></td><td></td></tr><tr><td>PVC tubing (1/8 x 3/16 x 1/32)</td><td>Nalgene</td><td>8000-0010</td><td>Use these with pulled needle to assemble the blower tube as shown in Figure 2.</td></tr><tr><td>Tygon Silicone tubing (3/32 x 5/32 x 1/32)</td><td>Saint Gobain Performance Plastics</td><td>ABW00004</td><td></td></tr><tr><td>Tygon Silicone tubing (1/32 x 3/32 x 1/32)</td><td>Saint Gobain Performance Plastics</td><td>ABW00001</td><td></td></tr></tbody></table>

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.

As an example of the use of this protocol, results illustrating midpupal (42 hrs APF at 25 &#176;C) eye discs immunostained with different antibodies are presented in Figure 2. By using an antibody directed against phosphotyrosine residues, the membrane of the cells can be observed (Figure 2A). This can be used to identify the regular arrangement of ommatidial cells in the pupal eye following the final patterning processes that occur prior to the midpupal stage. Another representative image depicts the nuclei of cone cells can are identified by using an antibody against the Cut transcription factor (Figure 2B).
<img alt="Figure 1" fo:content-width="4.5in" src="/files/ftp_upload/52315/52315fig1highres.jpg" width="450" /> 
Figure 1: Assembly diagram of the blower tube used in the dissection procedure. See the materials section for details on the separate components of the blower tube.
<img alt="Figure 2" src="/files/ftp_upload/52315/52315fig2highres.jpg" /> 
Figure 2: Immunostaining of midpupal eye discs. Representative immunostaining demonstrating the use of this protocol to visualize A) the apical morphology of ommatidia (phosphotyrosine) and B) cone cell nuclei (Cut) from pupal eye discs at 42 hrs APF. Scale bars = 50 &#956;m.

Watch this Scientific Journal Video about Dissection and Mounting of Drosophila Pupal Eye Discs at JoVE.com

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.

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 ...

dissection-and-mounting-of-drosophila-pupal-eye-discs

Nanyang Technological University

Research

JoVE Journal

Biology

12.1K Views.  University of California at Los Angeles (UCLA). 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.

Video: Dissection and Mounting of Drosophila Pupal Eye Discs

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

Developmental Biology

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.

Long-term Live Imaging of Drosophila Eye Disc

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have been used to study many biological processes, such as cell proliferation, differentiation, growth, migration, apoptosis, competition, cell-cell signaling, and compartmental boundary formation. However, methods for the long-term ex vivo culture and live imaging of the imaginal discs have not been satisfactory, despite many efforts. Recently, we developed a method for the long-term ex vivo culture and live imaging of imaginal discs for up to 18 h. In addition to using a high insulin concentration in the culture medium, a low-melting agarose was also used to embed the disc to prevent it from drifting during the imaging period. This report uses the eye-antennal discs as an example. Photoreceptor R3/4-specific m&#948;0.5-Ga4 expression was followed to demonstrate that photoreceptor differentiation and ommatidial rotation can be observed during a 10 h live imaging period. This is a detailed protocol describing this simple method.

Long-term Live Imaging of Drosophila Eye Disc

This protocol describes a detailed method for the long-term ex vivo culture and live imaging of a Drosophila imaginal disc. It demonstrates photoreceptor differentiation and ommatidial rotation within the 10 h period of live imaging of the eye disc. The protocol is simple and does not require expensive setup.

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have ...

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 ...

Neuroscience

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a reliable protocol for the dissection of the delicate Drosophila pupal retina. Our surgical approach utilizes readily-available microdissection tools to open pupae and precisely extract eye-brain complexes. These can be fixed, subjected to immunohistochemistry, and retinas then mounted onto microscope slides and imaged if the goal is to detect cellular or subcellular structures. Alternatively, unfixed retinas can be isolated from brain tissue, lysed in appropriate buffers and utilized for protein gel electrophoresis or mRNA extraction (to assess protein or gene expression, respectively). Significant practice and patience may be required to master the microdissection protocol described, but once mastered, the protocol enables relatively quick isolation of mainly undamaged retinas.

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and RNA-extraction.&#160;

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...

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 ...

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a plethora of mutant strains and technical tricks have been developed to allow sophisticated questions to be asked and answered in a reasonable amount of time. Fundamental insight into the interplay of components of all known major signaling pathways has been obtained in forward and reverse genetic Drosophila studies. The fly eye has proven to be exceptionally well suited for mutational analysis, since, under laboratory conditions, flies can survive without functional eyes. Furthermore, the surface of the insect eye is composed of some 800 individual unit eyes (facets or ommatidia) that form a regular, smooth surface when looked at under a dissecting microscope. Thus, it is easy to see whether a mutation might affect eye development or growth by externally looking for the loss of the smooth surface ('rough eye' phenotype; Fig. 1) or overall eye size, respectively (for examples of screens based on external eye morphology see e.g.1). Subsequent detailed analyses of eye phenotypes require fixation, plastic embedding and thin-sectioning of adult eyes.
The Drosophila eye develops from the so-called eye imaginal disc, a bag of epithelial cells that proliferate and differentiate during larval and pupal stages (for review see e.g. 2). Each ommatidium consists of 20 cells, including eight photoreceptors (PR or R-cells; Fig. 2), four lens-secreting cone cells, pigment cells ('hexagon' around R-cell cluster) and a bristle. The photoreceptors of each ommatidium, most easily identified by their light sensitive organelles, the rhabdomeres, are organized in a trapezoid made up of the six "outer" (R1-6) and two "inner" photoreceptors (R7/8; R8 [Fig. 2] is underneath R7 and thus only seen in sections from deeper areas of the eye). The trapezoid of each facet is precisely aligned with those of its neighbors and the overall anteroposterior and dorsoventral axes of the eye (Fig. 3A). In particular, the ommatidia of the dorsal and ventral (black and red arrows, respectively) halves of the eye are mirror images of each other and correspond to two chiral forms established during planar cell polarity signaling (for review see e.g. 3).
The method to generate semi-thin eye sections (such as those presented in Fig. 3) described here is slightly modified from the one originally described by Tomlinson and Ready4. It allows the morphological analysis of all cells except for the transparent cone cells. In addition, the pigment of R-cells (blue arrowheads in Fig. 2 and 3) can be used as a cell-autonomous marker for the genotype of a R-cell, thus genetic requirements of genes in a subset of R-cells can readily be determined5,6.

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

A standard approach to prepare adult Drosophila eyes for semi-thin sectioning and light microscopic analysis is presented here. The protocol can be used for gross morphological analysis of eye defects, or with the indicated adjustments can be used to determine genetic requirements of genes in specific cell types of the eye (e.g. clonal analysis of photoreceptors) or for electron microscopic analysis.

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a ...

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/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 ...

Dissection and Mounting of Drosophila Pupal Eye Discs

Summary

Explore More Videos

Chapters in this video

Live-imaging of the Drosophila Pupal Eye

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

Long-term Live Imaging of Drosophila Eye Disc

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

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

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

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

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