A GFP Complementation-based Dual-expression System for Assessing Cell-Cell Contact Mediated by Cytonemes in Live Drosophila Wing Imaginal Discs

So our research focuses on understanding how distantly located cells in tissues communicate with one another. We are using the Drosophila wing imaginal disc as a model system to try to understand how cytidine are formed, how they function, and what role they play in locally controlling tissue growth. Studies suggest that growth factors are often traveling through specialized cellular projections like actin-based signaling filopodia, called cytonemes in order to be delivered to the target cells.

Cytonemes are very thin and fragile structures that are easily disrupted by fixation protocol. To observe them, you need to use dedicate live imaging approaches with expressed fluorescently tagged protein. This greatly limits the tool and approaches we can use to investigate them.

We identified a protein kinase called Slik that is involved in cytoneme biogenesis. Expression of Slik in one epidural layer in the wing imaginal disc triggers the formation of cytoneme that crossed the disc lumen and stimulates the proliferation of cells in the neighboring epithelial layer. In future, we would like to identify the protein acting downstream of Slik to promote cytoneme formation, to identify the ligand that is delivered via this cytoneme to promote perforation, and to better understand the physiological importance of this mechanism in controlling tissue growth.

To begin, cut individual wells from the eight-well strip using scissors and then cut each well in half. Remove the protective backing from one side of each half and stick the spacers onto the microscope slide, spacing them slightly apart to create a sheltered central space for disc placement. Pick wandering third instar larvae from the rearing tube following genetic crossing and incubation at 25 degrees Celsius.

Under a dissecting microscope, transfer the larvae into a drop of live imaging medium on a silicone-coated Petri dish. Using two dissecting forceps, start dissection by pinching the larvae at about 1/3 body length from the anterior with one pair of forceps to hold it steady. With the second pair, pinch the body just posterior to the first point and pull to separate the posterior half, isolating the anterior section.

Invert the anterior half by gripping both sides of the cut end with tweezers and pushing the head through using a second pair, turning it inside out. This exposes internal structures such as imaginal discs, trachea, salivary glands, fat body, and gut. Remove the salivary glands, fat body, and gut using forceps, taking care not to disturb the lateral tracheal trunks overlying the wing discs.

Using forceps, transfer the cleaned anterior halves with attached wing discs into a drop of clean live imaging medium with hooks placed between the slide spacers. To isolate the wing discs, gently dissect them from the fine tracheal branches with one blade of the dissecting forceps or a fine tungsten wire mounted on a dissecting needle holder. Discard the remaining carcass.

Orient the wing discs using a dissecting forceps blade or tungsten needle so that the peripodial membrane side faces up. Adjust the medium volume so it slightly overfills the well above the spacer level. Remove the top protective backing from the imaging spacer, and gently lower a cover slip over the sample.

Press the cover slip at the spacer contact points using the rounded end of forceps to ensure proper adhesion. To project the image stacks in ImageJ, select Image, then Stacks, followed by Z Project, and choose Maximum Intensity from the menu. In the maximum intensity projection images, identify the wing pouch area based on the wing disc folding pattern using the hooks channel and the polygon selection tool.

Apply the same selection to the GFP channel, and select Edit followed by Clear Outside to eliminate noise outside the selected region. Use the hooks channel in the maximum intensity projection images to identify the artifactual spots in the GFP images. Then, select the area using the polygon selection tool.

Click Edit and choose Clear. In the cleaned maximum intensity projection images, select Analyze, followed by Histogram, and choose List to obtain all pixel values. Copy this list into a spreadsheet table.

To set a threshold value, analyze the background signal in the negative control samples and confirm this value using other sample images. Calculate the percentage of GFP-positive surface by dividing the number of pixels above the threshold by the total number of valid pixels and multiplying the result by 100. Finally, normalize each calculated value after dividing it by the mean of the reference condition, which is set to one.

In wild-type discs lacking both split GFP components, only minimal granular GFP autofluorescence was observed near the center of the wing pouch, and this baseline signal was used to define a fluorescence threshold. Discs expressing only CD4 split GFP1-10 in the disc proper layer showed fluorescence levels indistinguishable from wild-type, confirming the non-fluorescent nature of GFP1-10 alone. Co-expression of CD4 split GFP1-10 in the disc proper and CD4spGFP11 in the peripodial membrane led to a noticeable increase in discrete, bright GFP spots localized to the disc lumen, with a significantly larger fluorescence positive area above threshold than controls.

Slik expression in the disc proper caused a strong increase in peripodial membrane nuclei density and a dramatic rise in GFP signal intensity across the wing pouch region, suggesting that cytonemes induced by Slik establish enhanced contact with peripodial membrane cells.