Imaging Dpp Release from a Drosophila Wing Disc

Summary

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.

Abstract

The transforming Growth Factor-beta (TGF-β) superfamily is essential for early embryonic patterning and development of adult structures in multicellular organisms. The TGF-β superfamily includes TGF-β, 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-β 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.

Introduction

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 wing^1,2,3,4. 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 disc^5,6,7,8. Dpp binds to a complex of type 1 and type 2 serine/threonine kinase receptors^9,10. 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 differentiation^4,11.

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 defects^12,13. Several ion channels impact development of the Drosophila wing and associated structures^14,15. 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

Protocol

1. Collecting Eggs to Generate Larvae for Dissection

1. 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.
2. To collect eggs, flip the crossed flies into a fresh vial of food and allow them to lay for 3–4 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.
3. Keep the egg collection vials at 25 °C for approximately 144 h until larvae are at the third instar (wandering) stage.
4. 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.
5. 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.

2. Preparing Solution for Larval Imaging and Dissection

1. 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.
2. Adjust pH to 7.1 using HEPES and filter with a 0.22 μm filter. Use this media for both the wing disc dissections and as the mounting media for live imaging.

3. Preparing Slides for Wing Disc Mounting

1. 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.
2. 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.
3. Place 25 μL 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).

4. Dissecting and Mounting Wing Discs for Imaging

1. Dissect larvae in the prepared modified HL3 media²⁶.
2. Gently tear larvae in half using two pairs of forceps and discard the posterior half of the larva.
3. 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.
4. 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.
5. Gently remove the wing discs and transfer them to the 25 μL 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.
6. 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).
7. 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.

5. Imaging Dpp Release

1. 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.
2. 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.

Results

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...

Discussion

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’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 cytonemes^15,24,25. These data suggest that developmental cell-cell ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Baker's yeast	Red Star
CaCl2 dyhydrate	Fisher Scientific	C79-500
Coverslips	VWR	484-457
Double-sided tape	Scotch
Drosophila Agar Type II	Apex	66-104
Drosophila melanogaster: Dpp-GAL4/TM6 Tb Hu			This stock will soon be made available at Bloomington Drosophila Stock Center
Drosophila melanogaster: Sp/CyO-GFP; UAS-Dpp-GFP/TM6 Tb Hu			This stock will soon be made available at Bloomington Drosophila Stock Center
Dumont Tweezers #5	World Precision Instruments	500233	Forceps for dissecting
HEPES	Sigma Aldrich	H3375
KCl	Fisher Scientific	AC193780010
Light Corn Syrup	Karo
Malt Extract	Breiss
MgCl2	Fisher Scientific	AC223210010
Microscope slides	Sigma Aldrich	S8400
NaCl	Fisher Scientific	S271-500
NaHCO3	RPI	S22060-1000.0
Nail polish	Electron Micsroscopy Sciences	72180
Propionic Acid	VWR	U330-09
Soy Flour	ADM Specialty Ingredients	062-100
Sucrose	Fisher Scientific	S5-3
Sucrose	Fisher	S512
Tegosept	Genesee Scientific	20-259
Trehalose dyhydrate	Chem-Impex International, Inc.	00766
Yellow Corn Meal	Quaker
Zeiss LSM 780 confocal microscope	Zeiss		Microscope for live imaging
Zeiss SteREO Discovery.V8 microscope	Zeiss		Microscope for dissections