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 v...

<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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+of+decapentaplegic+and+engrailed+expression+in+Drosophila+imaginal+disks:+do+these+genes+mark+the+anterior-posterior+compartment+boundary.">The relationship of decapentaplegic and engrailed expression in Drosophila imaginal disks: do these genes mark the anterior-posterior compartment boundary.</a> Development. 113 (1), 27-33 (1991).</li><li>Blackman, R. K., Sanicola, M., Raftery, L. A., Gillevet, T., 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+extensive+3'+cis-regulatory+region+directs+the+imaginal+disk+expression+of+decapentaplegic,+a+member+of+the+TGF-beta+family+in+Drosophila.">An extensive 3' cis-regulatory region directs the imaginal disk expression of decapentaplegic, a member of the TGF-beta family in Drosophila.</a> Development. 111 (3), 657-666 (1991).</li><li>de Celis, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+function+of+decapentaplegic+and+thick+veins+during+the+differentiation+of+the+veins+in+the+Drosophila+wing.">Expression and function of decapentaplegic and thick veins during the differentiation of the veins in the Drosophila wing.</a> Development. 124 (5), 1007-1018 (1997).</li><li>De Celis, J. F. <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+wing:+The+development+of+the+veins.">Pattern formation in the Drosophila wing: The development of the veins.</a> Bioessays. 25 (5), 443-451 (2003).</li><li>Letsou, A., 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=Drosophila+Dpp+signaling+is+mediated+by+the+punt+gene+product:+a+dual+ligand-binding+type+II+receptor+of+the+TGF+beta+receptor+family.">Drosophila Dpp signaling is mediated by the punt gene product: a dual ligand-binding type II receptor of the TGF beta receptor family.</a> Cell. 80 (6), 899-908 (1995).</li><li>Nellen, D., Affolter, M., Basler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+serine/threonine+kinases+implicated+in+the+control+of+Drosophila+body+pattern+by+decapentaplegic.">Receptor serine/threonine kinases implicated in the control of Drosophila body pattern by decapentaplegic.</a> Cell. 78 (2), 225-237 (1994).</li><li>Raftery, L. A., Sutherland, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+family+signal+transduction+in+Drosophila+development:+from+Mad+to+Smads.">TGF-beta family signal transduction in Drosophila development: from Mad to Smads.</a> Developmental Biology. 210 (2), 251-268 (1999).</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+regulate+Dpp+release+in+the+Drosophila+wing+disc.">Inwardly rectifying potassium channels regulate Dpp release in the Drosophila wing disc.</a> Development. 144 (15), 2771-2783 (2017).</li><li>Dahal, G. 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. E., Reis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Buoyancy-based+Method+of+Determining+Fat+Levels+in+Drosophila.">A Buoyancy-based Method of Determining Fat Levels in Drosophila.</a> Journal of Visualized Experiments. (117), e54744 (2016).</li><li>Feng, Y., Ueda, A., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+minimal+hemolymph-like+solution,+HL3.1,+for+physiological+recordings+at+the+neuromuscular+junctions+of+normal+and+mutant+Drosophila+larvae.">A modified minimal hemolymph-like solution, HL3.1, for physiological recordings at the neuromuscular junctions of normal and mutant Drosophila larvae.</a> Journal of Neurogenetics. 18 (2), 377-402 (2004).</li><li>Hsiung, F., Ramirez-Weber, F. A., Iwaki, D. D., 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=Dependence+of+Drosophila+wing+imaginal+disc+cytonemes+on+Decapentaplegic.">Dependence of Drosophila wing imaginal disc cytonemes on Decapentaplegic.</a> Nature. 437 (7058), 560-563 (2005).</li><li>Roy, S., Hsiung, F., 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=Specificity+of+Drosophila+cytonemes+for+distinct+signaling+pathways.">Specificity of Drosophila cytonemes for distinct signaling pathways.</a> Science. 332 (6027), 354-358 (2011).</li><li>Kornberg, T. 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. 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=Communicating+by+touch--neurons+are+not+alone.">Communicating by touch--neurons are not alone.</a> Trends in Cell Biology. 24 (6), 370-376 (2014).</li></ol>

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&#39;s yeast</td>
			<td>Red Star</td>
			<td>&#160;</td>
			<td>&#160;</td>
		</tr>
		<tr>
			<td>CaCl2 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>Drosophila melanogaster: 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>Drosophila melanogaster: 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>MgCl2</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>NaHCO3</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 ...

DPP affect cell fate by binding receptors and sending a signal intracellularly to affect transcription. If DPP is produced but not released, it doesn't have access to receptors and therefore cannot perform its roles. This technique makes it possible to visualize DPP release enabling researchers to examine which genetic and environmental factors change DPP release dynamics.The BMP signaling pathway is conserved from flies to humans so we expect that the mechanism that dictates DPP release will also be relevant for human development and regeneration. Begin by crossing 30 to 40 virgin female DPP GAL4 flies with 10 to 15 male UAS DPP-GFP flies. Encourage egg laying by adding a small amount of yeast paste to a fresh vial of food.To collect the eggs, flip the crossed flies into the vial and allow them to lay for three to four hours before removing them. Keep the egg collection vials at 25 degrees Celsius for approximately 144 hours until the larvae are at the third instar stage. Then select the appropriate larvae for dissection.First, choose larvae that are non-TB which will be normal length rather than short and fat. To select the DPP-GFP expressing larvae, view them under a fluorescence stereo microscope. All non-TB larvae will have some fluorescence, but the GFP is restricted to the wing discs and is less bright on the DPP-GFP larvae.Prepare modified HL3 media according to manuscript directions which will keep the wing discs alive for imaging. Adjust the pH to 7.1 with HEPES then filter it through a 0.22 micrometer filter. Place two pieces of colorless double-sided tape width wise across a microscope slide leaving a space of about five millimeters between them and making sure that the ends of the tape lie flushed with the edges of the slide.Use a flat edge to press the tape firmly to the slide. Add 25 microliters of the modified HL3 media between the two pieces of tape. When the wing disc is mounted in the media, the pieces of tape will prevent it from being crushed by the coverslip.To dissect the larva, place it in the modified HL3 media and gently tear it in half using two pairs of forceps. Discard the posterior half, then grasp the middle of the anterior half with one pair of forceps and use the other pair to push the mouth hooks back into the larva until it is completely inverted. Look for the sharp bends in the two darker colored primary branches of the trachea that run down both sides of the larva.The wing discs lie directly underneath. Gently remove the discs and put them in the HL3 media on the prepared slide making sure that no pieces of trachea are still attached to the discs. Arrange the wing discs so that the peripodial side of the wing pouch is facing upward with the disc lying flat.Cover the wing with a coverslip and seal with nail polish. Once the polish is dry, immediately proceed with imaging. Image the mounted wing disc using a confocal laser scanning microscope with a 40X oil objective and a 488 nanometer laser.Collect images at a speed of two hertz for two minutes. For best results, set the laser power just strong enough to get a clear image of the DPP-releasing cells to avoid over bleaching. Collect the images as a time series and export them as AVI files to obtain a video of the DPP release.When the protocol is successful, DPP-GFP appears as a stripe down the center of the wing disc with nuclei visible as non-fluorescent circles. DPP-GFP release is visible as fluorescent puncta that appear and disappear both in and far away from the cell bodies. The distinct puncta are likely in cytonemes or at cytonemes synapses.The wing discs are mounted so that the imaging is done from the peripodial side. If the wing disc is imaged from the reverse side, the DPP-GFP fluorescence will appear out of focus and resolution will be poor. When GFP is not fused to DPP, no puncta outside of the cell bodies are observed.This control demonstrates that DPP-GFP behaves differently than GFP alone. Furthermore, no fluorescent puncta appear when DPP-GFP is not expressed. During this procedure, it is important that the wing disc is mounted peripodial side up.If the wing disc is improperly oriented, DPP-GFP will appear out of focus. This method could be used to study the release of other developmental signaling ligands like wingless or hedgehog. We can explore all the different environmental and genetic factors that may affect DPP release.In addition, we can adapt this method to study BMP release from other cell types.

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

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JoVE Journal

Developmental Biology

5.5K ビュー.  University of Colorado School of Medicine. DPPは、細胞の宿主に、細胞内でトランスクリプションに影響を与えるシグナルを送ることによって、細胞の運命に影響を与える。DPPが産生されるが放出されない場合、受容体にアクセスできないため、その役割を果たすことができません。この技術により、研究者はDPP放出ダイナミクスを変化させる遺伝的および環境的要因を調べることができ、DPPリリースを可視化す?...