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.
