We would like to thank Dr. Sarala Pradhan for working on an earlier version of this protocol. We would like to thank NSF-IOS 1354282 for funding while we developed this protocol. We would like to thank NIH-NIDCR RO1DE025311 for funding our lab currently.

1. Collecting Eggs to Generate Larvae for Dissection
<ol>
	<li>Cross 30-40 virgin female Dpp-GAL4/TM6 Tb Hu flies to 10-15 male Sp/CyO-GFP; UAS-Dpp-GFP/TM6 Tb Hu. 
	NOTE: Any two genotypes containing Dpp-GAL4 and UAS-Dpp-GFP may be used as long as the balancers have larval markers allowing for selection of the appropriate progeny during the larval stage.</li>
	<li>To collect eggs, flip the crossed flies into a fresh vial of food and allow them to lay for 3&#8211;4 h before removing them from the vial. To encourage egg laying, a small amount of yeast paste made of granulated yeast mixed with a small amount of water can be added to the top of the food before the flies are introduced to the vial. The same set of crossed flies can be kept and used for up to 7 days of egg collections. 
	NOTE: Any standard Drosophila cornmeal food recipe should be acceptable. Used here is the food recipe described by Hazegh and Reis20.</li>
	<li>Keep the egg collection vials at 25 &#176;C for approximately 144 h until larvae are at the third instar (wandering) stage.</li>
	<li>To select the appropriate larvae for dissection (those expressing both Dpp-GAL4 and UAS-Dpp-GFP) first choose larvae that are non-Tb. These larvae will be normal length rather than short and fat.</li>
	<li>Among the non-Tb larvae there will be larvae expressing CyO-GFP and larvae expressing Dpp-GFP. To select the Dpp-GFP expressing larvae, view them under a fluorescence stereomicroscope. While both Dpp-GFP and CyO-GFP expressing larve will have some GFP fluorescence, the Dpp-GFP expressing larvae can be distinguished in that the GFP is restricted to the wing discs and the fluorescence is much less bright than the CyO-GFP expressing larvae. Select these less bright larvae for dissection.</li>
</ol>
2. Preparing Solution for Larval Imaging and Dissection
<ol>
	<li>For live imaging of wing discs, prepare modified HL3 media21 (70 mM NaCl, 5 mM KCl, 1.5 mM CaCl2 dyhydrate, 4 mM MgCl2, 10 mM NaHCO3, 5 mM trehalose dyhydrate, 115 mM sucrose, 5 mM HEPES). This media allows the wing disc to be kept alive for imaging21.</li>
	<li>Adjust pH to 7.1 using HEPES and filter with a 0.22 &#956;m filter. Use this media for both the wing disc dissections and as the mounting media for live imaging.</li>
</ol>
3. Preparing Slides for Wing Disc Mounting
<ol>
	<li>Place two pieces of colorless double-sided tape widthwise across a microscope slide, leaving a space of about 5 mm between them. The pieces of tape should be long enough that the ends of the tape lie flush with the edges of the microscope slide.</li>
	<li>Use a flat edge (such as the handle end of a pair of dissecting forceps) to press the tape firmly to the slide so that no media can seep underneath it.</li>
	<li>Place 25 &#956;L of modified HL3 media between the two pieces of tape. The wing disc will be mounted in this drop of media between pieces of tape so that the tape prevents the coverslip from crushing the disc (Figure 1A).</li>
</ol>
4. Dissecting and Mounting Wing Discs for Imaging
<ol>
	<li>Dissect larvae in the prepared modified HL3 media26.</li>
	<li>Gently tear larvae in half using two pairs of forceps and discard the posterior half of the larva.</li>
	<li>Grasp the middle of the anterior half of the larva with one pair of forceps and use another pair of closed forceps to push the mouth hooks back into the larva until the larva is completely inverted.</li>
	<li>The wing discs can be most easily found by looking for the sharp bends in the two darker colored primary branches of the tracheae that run down both sides of the larvae. The wing discs lie directly under these bends in the tracheae in an inverted larva.</li>
	<li>Gently remove the wing discs and transfer them to the 25 &#956;L of HL3 media on the prepared microscope slide. It is important to make sure that no pieces of trachea are left attached to the dissected wing discs as this can cause the discs to float, making imaging difficult.</li>
	<li>Arrange the wing disc so that the peripodial side of the wing pouch is facing upward away from the microscope slide with the disc lying flat (see Figure 1A,B).</li>
	<li>Cover the wing disc and tape with a coverslip and seal the coverslip using nail polish. Once the polish is dry, immediately proceed to the imaging steps below.</li>
</ol>
5. Imaging Dpp Release
<ol>
	<li>Image the mounted wing disc using a confocal laser scanning microscope with a 40x oil objective and 488 nm laser. The Dpp-GFP should appear as a stripe of GFP fluorescence down the center of the wing disc with small puncta of Dpp-GFP being released from this center stripe of cells.</li>
	<li>Collect images at a speed of 2 Hz for 2 min to visualize Dpp release. For best results, set the laser at a setting just strong enough to get a clear image of the Dpp-releasing cells to avoid over-bleaching during image collection. The images can be collected as a time series which can then be exported as AVI files (no compression, 3 frames/s) to obtain video files of the Dpp release.</li>
</ol>

<ol>
	<li>Ferguson, E. L., Anderson, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decapentaplegic+acts+as+a+morphogen+to+organize+dorsal-ventral+pattern+in+the+Drosophila+embryo.">Decapentaplegic acts as a morphogen to organize dorsal-ventral pattern in the Drosophila embryo.</a> Cell. 71 (3), 451-461 (1992).</li><li>Ferguson, E. L., Anderson, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+enhancement+and+repression+of+the+activity+of+the+TGF-beta+family+member,+decapentaplegic,+is+necessary+for+dorsal-ventral+pattern+formation+in+the+Drosophila+embryo.">Localized enhancement and repression of the activity of the TGF-beta family member, decapentaplegic, is necessary for dorsal-ventral pattern formation in the Drosophila embryo.</a> Development. 114 (3), 583-597 (1992).</li><li>Wharton, K. A., Ray, R. P., Gelbart, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+activity+gradient+of+decapentaplegic+is+necessary+for+the+specification+of+dorsal+pattern+elements+in+the+Drosophila+embryo.">An activity gradient of decapentaplegic is necessary for the specification of dorsal pattern elements in the Drosophila embryo.</a> Development. 117 (2), 807-822 (1993).</li><li>Raftery, L. A., Twombly, V., Wharton, K., Gelbart, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+screens+to+identify+elements+of+the+decapentaplegic+signaling+pathway+in+Drosophila.">Genetic screens to identify elements of the decapentaplegic signaling pathway in Drosophila.</a> Genetics. 139 (1), 241-254 (1995).</li><li>Raftery, L. A., Sanicola, M., Blackman, R. K., Gelbart, W. 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R., 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=An+inwardly+rectifying+K++channel+is+required+for+patterning.">An inwardly rectifying K+ channel is required for patterning.</a> Development. 139 (19), 3653-3664 (2012).</li><li>George, L. F., 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=Ion+Channel+Contributions+to+Wing+Development+in+Drosophila+melanogaster.">Ion Channel Contributions to Wing Development in Drosophila melanogaster.</a> G3. 9 (4), 999-1008 (2019).</li><li>Huang, H., Liu, S., Kornberg, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+signaling+at+cytoneme+synapses.">Glutamate signaling at cytoneme synapses.</a> Science. 363 (6430), 948-955 (2019).</li><li>Teleman, A. A., Cohen, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dpp+gradient+formation+in+the+Drosophila+wing+imaginal+disc.">Dpp gradient formation in the Drosophila wing imaginal disc.</a> Cell. 103 (6), 971-980 (2000).</li><li>Miesenbock, G., De Angelis, D. A., Rothman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+secretion+and+synaptic+transmission+with+pH-sensitive+green+fluorescent+proteins.">Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.</a> Nature. 394 (6689), 192-195 (1998).</li><li>Dahal, G. R., Pradhan, S. J., Bates, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inwardly+rectifying+potassium+channels+influence+Drosophila+wing+morphogenesis+by+regulating+Dpp+release.">Inwardly rectifying potassium channels influence Drosophila wing morphogenesis by regulating Dpp release.</a> Development. 144 (15), 2771-2783 (2017).</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><li>Hazegh, K. 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B., Roy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytonemes+as+specialized+signaling+filopodia.">Cytonemes as specialized signaling filopodia.</a> Development. 141 (4), 729-736 (2014).</li><li>Roy, S., Huang, H., Liu, S., Kornberg, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoneme-mediated+contact-dependent+transport+of+the+Drosophila+decapentaplegic+signaling+protein.">Cytoneme-mediated contact-dependent transport of the Drosophila decapentaplegic signaling protein.</a> Science. 343 (6173), 1244624 (2014).</li><li>Kornberg, T. 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The authors have nothing to disclose.

BMPs such as Dpp make their significant impact when they bind a complex of membrane bound receptors to cause a cascade of intracellular signaling in neighboring or apparently distant cells. Dr. Thomas Kornberg&#8217;s lab has shown that cells that produce the Dpp signal contact cells that receive the signal using actin based thin filapodia-like structures called cytonemes15,24,25. These data suggest that developmental cell-cell ...

Bone Morphogenetic Proteins (BMPs) are essential for early embryogenesis and patterning of adult structures. BMPs are produced and secreted to affect transcription of target genes needed for growth and cell differentiation in responding cells. Decapentaplegic (Dpp) is a Drosophila homolog of BMP4 that is important for development of embryonic and adult structures like the wing1,2,3,4. Several groups have focused on the role of Dpp in patterning adult fly wings because 1) the wings are comprised of two transparent epithelia sheets with a consistent venation pattern that can be easily assessed; 2) the wing discs are also reasonably flat, can be cultured outside of the larva, and are simple to image and quantify differences in pattern; and 3) the wing pattern development is sensitive to Dpp such that small perturbations in the pathway will impact wing venation pattern.
Dpp is produced in cells located in the anterior/posterior boundary of the wing disc5,6,7,8. Dpp binds to a complex of type 1 and type 2 serine/threonine kinase receptors9,10. Upon Dpp binding, the type 2 receptor phosphorylates the type 1 receptor which then phosphorylates Mothers against Dpp (Mad), a Smad 1/5/8 homolog. Phosphorylated SMAD recruits an additional co-Smad (Medea), which enables it enter to the nucleus where it regulates target genes, leading to downstream effects such as proliferation or differentiation4,11.
Recently, the Bates Lab has shown that the improper release of Dpp within the wing disc can lead to a decrease in Mad phosphorylation, reduction in target gene expression, and wing patterning defects12,13. Several ion channels impact development of the Drosophila wing and associated structures14,15. These ion channels could also be involved in Dpp release. In determining the mechanism of morphogen release, it is important that there is a method to visualize release events.
Drs. Aurelio Teleman and Stephen Cohen created a Dpp-GFP fusion protein which is able to rescue loss of Dpp, meaning that it is biologically active and is released in a biologically relevant manner16. Here, we describe how we visualize Dpp release events using this Dpp-GFP. This fusion protein is particularly useful because GFP is pH sensitive such that when it is in acidic vesicles, the fluorescence is quenched17. Therefore, when a protein tagged with GFP is released from a vesicle into the more neutral extracellular environment, GFP fluorescence intensity increases17. We took advantage of the pH sensitivity of GFP to determine if Dpp-GFP resides in acidic vesicles. We imaged wing discs expressing Dpp-GFP before and after the addition of ammonium chloride, which neutralizes intracellular compartments vesicles18. We found a significant increase in fluorescence of puncta after the addition of ammonium chloride, suggesting that intracellular Dpp-GFP is quenched before the addition of ammonium chloride18. We conclude that intracellular Dpp-GFP resides in acidic membrane-bound compartments, such as vesicles, and is unquenched upon addition of ammonium chloride to neutralize the pH of intracellular compartments18. This makes live imaging of Dpp-GFP a useful technique to visualize the dynamics of Dpp in the Drosophila wing disc as it is released from acidic compartments into the extracellular environment.
Here, we describe the method we use to visualize Dpp release events using Dpp-GFP. Dpp-GFP can be expressed in its native pattern in Drosophila wing discs using the UAS-GAL4 system19. This is the method that was used to determine that Irk channels impact Dpp release18. We validated the method by live-imaging z-stacks. We do not see Dpp-GFP puncta moving within the plane of focus in time series if we acquired in one plane of focus. We also do not see movement of Dpp-GFP puncta if we imaged in a z-stack. We conclude that the Dpp-GFP puncta seen using this method are release events rather than movement of vesicles intracellularly. This method of live-imaging of Dpp-GFP could potentially be used to test other putative modifiers of Dpp release for their impact on Dpp dynamics or could be modified to look at the dynamics of other ligands

<table><tbody><tr><td>Baker&#039;s yeast</td><td>Red Star</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; dyhydrate</td><td>Fisher Scientific</td><td>C79-500</td><td></td></tr><tr><td>Coverslips</td><td>VWR</td><td>484-457</td><td></td></tr><tr><td>Double-sided tape</td><td>Scotch</td><td></td><td></td></tr><tr><td>Drosophila Agar Type II</td><td>Apex</td><td>66-104</td><td></td></tr><tr><td>&lt;em&gt;Drosophila melanogaster:&lt;/em&gt; Dpp-GAL4/TM6 Tb Hu</td><td></td><td></td><td>This stock will soon be made available at Bloomington Drosophila Stock Center</td></tr><tr><td>&lt;em&gt;Drosophila melanogaster:&lt;/em&gt; Sp/CyO-GFP; UAS-Dpp-GFP/TM6 Tb Hu</td><td></td><td></td><td>This stock will soon be made available at Bloomington Drosophila Stock Center</td></tr><tr><td>Dumont Tweezers #5</td><td>World Precision Instruments</td><td>500233</td><td>Forceps for dissecting</td></tr><tr><td>HEPES</td><td>Sigma Aldrich</td><td>H3375</td><td></td></tr><tr><td>KCl</td><td>Fisher Scientific</td><td>AC193780010</td><td></td></tr><tr><td>Light Corn Syrup</td><td>Karo</td><td></td><td></td></tr><tr><td>Malt Extract</td><td>Breiss</td><td></td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher Scientific</td><td>AC223210010</td><td></td></tr><tr><td>Microscope slides</td><td>Sigma Aldrich</td><td>S8400</td><td></td></tr><tr><td>NaCl</td><td>Fisher Scientific</td><td>S271-500</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>RPI</td><td>S22060-1000.0</td><td></td></tr><tr><td>Nail polish</td><td>Electron Micsroscopy Sciences</td><td>72180</td><td></td></tr><tr><td>Propionic Acid</td><td>VWR</td><td>U330-09</td><td></td></tr><tr><td>Soy Flour</td><td>ADM Specialty Ingredients</td><td>062-100</td><td></td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>S5-3</td><td></td></tr><tr><td>Sucrose</td><td>Fisher</td><td>S512</td><td></td></tr><tr><td>Tegosept</td><td>Genesee Scientific</td><td>20-259</td><td></td></tr><tr><td>Trehalose dyhydrate</td><td>Chem-Impex International, Inc.</td><td>00766</td><td></td></tr><tr><td>Yellow Corn Meal</td><td>Quaker</td><td></td><td></td></tr><tr><td>Zeiss LSM 780 confocal microscope</td><td>Zeiss</td><td></td><td>Microscope for live imaging</td></tr><tr><td>Zeiss SteREO Discovery.V8 microscope</td><td>Zeiss</td><td></td><td>Microscope for dissections</td></tr></tbody></table>

imaging dpp release from a drosophila wing disc

The transforming Growth Factor-beta (TGF-&#946;) superfamily is essential for early embryonic patterning and development of adult structures in multicellular organisms. The TGF-&#946; superfamily includes TGF-&#946;, bone morphogenetic protein (BMPs), Activins, Growth and Differentiation Factors, and Nodals. It has long been known that the amount of ligand exposed to cells is important for its effects. It was thought that long-range concentration gradients set up embryonic pattern. However, recently it has become clear that the timing of exposure to these ligands is also important for their downstream transcriptional consequences. A TGF-&#946; superfamily ligand cannot have a developmental consequence until it is released from the cell in which it was produced. Until recently, it was difficult to determine when these ligands were released from cells. Here we show how to measure the release of a Drosophila BMP called Decapentaplegic (Dpp) from the cells of the wing primordium or wing disc. This method could be modified for other systems or signaling ligands.

Figure 2 shows representative live-imaging results of this protocol. When the protocol is successful, Dpp-GFP can be seen as a stripe down the center of the wing disc with nuclei visible as non-fluorescent circles within the Dpp-GFP region (Figure 2). Dpp-GFP release is visible as fluorescent puncta that appear and disappear. We have observed Dpp-GFP fluorescence appearing and disappearing in the cell bodies and far from the cell bodies. Dpp signaling is depende...

Watch this Scientific Journal Video about Imaging Dpp Release from a Drosophila Wing Disc at JoVE.com

Imaging Dpp Release from a Drosophila Wing Disc

The timing of exposure to ligands may impact their developmental consequences. Here we show how to image release of a Drosophila bone morphogenetic protein (BMP) called Dpp from cells of the wing disc.

Imaging Dpp Release from a Drosophila Wing Disc

The transforming Growth Factor-beta (TGF-&#946;) superfamily is essential for early embryonic patterning and development of adult structures in ...

imaging-dpp-release-from-a-drosophila-wing-disc

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Developmental Biology

5.6K Views.  University of Colorado School of Medicine. DPP beïnvloedt cellot door receptoren te binden en een signaal intracellularly aan affect transcriptie te verzenden. Als DPP wordt geproduceerd, maar niet vrijgegeven, heeft het geen toegang tot receptoren en kan het daarom zijn rollen niet uitvoeren. Deze techniek maakt het mogelijk om DPP release te visualiseren waardoor onderzoekers kunnen onderzoeken welke genetische en omgevingsfactoren DPP release dynamics veranderen.De BMP signaleringsroute wordt bewaard van vliegen naar mensen...

Video: Imaging Dpp Release from a Drosophila Wing Disc

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 ...

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 ...

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells named wing imaginal discs formed during embryonic development. In the larval stages the imaginal discs grow, increasing its number of cells and forming monolayered epithelial&#160;structures. Inside the pupal case, the imaginal discs bud out and fold into bilayers along a line that becomes the future margin of the wing. During this process, the longitudinal primodia veins originate vein cells on the prospective dorsal and ventral surfaces of the wing. During the pupal stage the stripes of vein cells of each surface communicate in order to generate tight tubes; at the same time, the cross-veins begin their formation.
With the help of appropriate molecular markers, it is possible to identify the major elements composing the wing during its development. For this reason, the ability to accurately detect transcripts or proteins in this structure is critical for studying their abundance and localization related to the development process of the wing.
The procedure described here focuses on manipulating pupal wings, providing detailed instructions on how to dissect the wing during the pupal stage. The dissection of pupal tissue is more difficult to perform than their counterparts in third instar larvae. This is why this approach was developed, to obtain rapid and efficient high quality samples. Details of how to immunostain and mount these wing samples, to allow the visualization of proteins or cell components, are provided in the protocol. With little expertise it is possible to collect 8-10 high quality pupal wings in a short amount of time.

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

The ability to accurately detect transcripts or proteins in Drosophila tissues is critical for studying their abundance and localization related to the development process. Here is the description of a straightforward procedure to dissect pupal wings. These wings can be used as samples in several techniques (immunohistochemistry, PCR assay, etc.).

Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

Biology

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 ...

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 ...

Extended Live Imaging of Drosophila Pupal Leg Morphogenesis Without Puparium

Long-Term Live Imaging of Drosophila Pupal Leg Development After Puparium Removal

Over the past decades, significant progress has been made in understanding the mechanisms of cell fate determination. However, the process by which fate-determined cells form three-dimensional organismal shapes remains unclear. Recent advances in confocal microscopy have facilitated efforts to observe cell dynamics during development through live imaging. The Drosophila melanogaster pupa is ideal for live imaging due to its immobility, transparent pupal cuticle, and the availability of fluorescent reporter lines. A primary challenge for imaging is the puparium, the cuticle surrounding the pupa, which obstructs optical imaging. While previous methods involved either partial or complete removal of the puparium, maintaining pupal viability for extended periods after this procedure has remained challenging. Here, a simple method is presented for days-long live imaging of the Drosophila leg during the pupal stage, involving complete puparium removal. The method includes removing the puparium from a pupa adhered to double-sided tape, followed by assembling a small chamber on a glass-bottom dish to enclose the pupa and a drop of water. This setup is straightforward, reliable, and supports extended pupal survival by preventing desiccation. Long-term live imaging of the Drosophila pupa has significantly contributed to capturing how the adult leg undergoes dramatic three-dimensional structural changes over 2-3 days. These changes include the transient formation of an intriguing structure (the Parthenon-like structure) by epithelial cells, rapid tissue narrowing, joint formation, and bristle elongation. This method is applicable to the observation of various tissues and can potentially be combined with other techniques, such as optical gene induction, to advance the understanding of cell dynamics during the final shape formation of tissues in the pupal stage.

Long-Term Live Imaging of Drosophila Pupal Leg Development After Puparium Removal

Here, a protocol is presented for long-term live imaging of the Drosophila leg during the pupal stage, involving complete removal of the puparium. This protocol can also be applied to other tissues.

Over the past decades, significant progress has been made in understanding the mechanisms of cell fate determination. However, the process by which ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

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 ...

Imaging Dpp Release from a Drosophila Wing Disc

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