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

Summary

We describe a protocol for measuring contacts between cells in adjacent epithelial layers in live Drosophila wing imaginal discs using a GFP reconstitution-based approach.

Abstract

Embryonic tissue growth and patterning are largely controlled by signals exchanged locally between cell populations within the tissues themselves. Cytonemes are a type of signaling filopodia first identified in Drosophila that connect and mediate exchange between signal-producing and signal-receiving cells. In the developing Drosophila wing imaginal disc, cytonemes are involved in signal exchange between distinct populations of cells within the disc proper (DP) epithelium, which will form the adult wing, as well as between DP cells and cells in adjacent disc-associated tissues. Cytonemes synapse with target cells to form intimate membrane contacts.

Here, we present a protocol for quantifying cytoneme-mediated contact between DP cells and cells of the adjacent peripodial membrane (PerM) epithelium, which is separated from the DP cells by the disc lumen, using a GFP reconstitution approach in live wing discs. Using the GAL4-UAS and LexA-LexAop systems, complementary fragments of split-GFP (spGFP1-10, spGFP11), each fused to the transmembrane domain of CD4, are expressed on either side of the disc lumen. Imaging of reconstituted GFP fluorescence in live wing disc preparations by confocal microscopy is then used to generate image stacks from which reconstituted GFP fluorescence can be localized and quantified. Using this system it is possible to co-express protein-coding or RNA interference transgenes in either cytoneme-producing or target cells to gauge their effect on DP-PerM cell contacts. This system, easily adaptable to other tissues, thus enables the identification of factors important for cytoneme formation or function.

Introduction

The development of embryonic tissues is controlled by cells located in 'organizing centers' that signal to distant cells within a tissue, controlling their decisions to proliferate (i.e., grow and divide) or adopt particular fates¹. This cell non-autonomous signaling is mediated by ligands produced by organizing center cells that form concentration gradients through the tissues and elicit concentration-dependent responses. In many cases, these ligands are either delivered or picked up through long actin-based signaling filopodia called cytonemes that connect signal-sending and -receiving cells in tissues^2,3. First discovered in the Drosophila wing imaginal disc⁴, cytonemes have also been identified in mammals and other vertebrates^5,6,7,8,9. A better understanding of the role of cytonemes in cell non-autonomous signaling, while at an early stage, is crucial to deciphering how cells communicate to organize into tissues and how these communication lines are modified in various pathological conditions, including developmental malformations and cancer.

Cytonemes can extend from source cells to deliver ligands to target cells or from target cells to receive ligands close to their sources^2,3. Cytonemes make intimate contacts with their targets, where they are thought to form synapse-like structures, where ligand transfer can occur^3,10,11. This contact can occur between the tips of source and target cytonemes or between cytonemes and cell bodies³. Although not extensively characterized, cell adhesion through adhesion molecules or through receptor-ligand interactions is, in some cases, needed for the proper activation of downstream signaling events^12,13,14, making this an important aspect of cytoneme biology.

Several studies have applied the "GFP reconstitution across synaptic partners" (GRASP) technique to the analysis of cytoneme contacts. This method was developed for identifying and mapping synaptic partners in complex nervous systems¹⁵. It is based on the expression of the two complementary fragments of split-GFP (spGFP1-10 and spGFP11), each fused to the extracellular region of a transmembrane domain (e.g. of CD4), in different populations of cells. If the plasma membranes of cells in those two populations come into direct contact, it brings the complementary domains of spGFP into proximity, leading to the reconstitution of GFP fluorescence. This approach has been used in Drosophila to identify the existence of cytoneme contacts between cells within the wing disc and between the wing disc and other closely apposed tissues^{12,16,17,18,19,20,21}.

This paper describes the application of GRASP to the characterization of contacts between two morphologically distinct epithelial layers of the Drosophila wing imaginal disc, the disc proper (DP) and the peripodial membrane (PerM). These epithelial layers form a sac surrounding a central lumen, with the pseudostratified columnar DP cells located on one side and the squamous PerM cells on the other, both with their apical membranes facing inwards towards the lumen (Figure 1A). There is some evidence for translumenal signaling between the two layers^22,23,24,25, and we recently documented signaling from the DP to control the proliferation of PerM cells that is mediated by apical cytonemes in the DP²¹. This protocol involves using the GAL4/UAS and LexA/LexAop transgene expression systems to express complementary fragments of spGFP fused to CD4 on the membranes of DP and PerM cells. It uses reconstituted GFP fluorescence to readout membrane contact between the two cell populations.

Protocol

The nubbin-GAL4 driver is used to express CD4-spGFP1-10 specifically in the wing pouch region of the DP (Figure 1A). The PerM-LexA driver²¹ is used to express CD4-spGFP11 specifically in the PerM (Figure 1A). These two expression systems are independent of one another, allowing simultaneous and specific expression of different transgenes in DP and PerM (Figure 1B,C).

The basic genetic scheme involves crossing flies to generate larvae of the genotype nub-GAL4/UAS-CD4-spGFP1-10;PerM-LexA/LexAop-CD4-spGFP11. As a negative control, we leave out the LexAop-CD4-spGFP11 transgene. Other transgenes (e.g., protein-coding, double-stranded RNA) can be expressed as desired in the DP layer (under the control of UAS sequences) or in the PerM (under the control of the LexA operator).

This is a live imaging protocol that cannot be interrupted. Material preparation is estimated at ~10 min. Dissections should not be performed for more than 20 min at a time before imaging. Imaging takes ~30 min and should not last more than ~1 h. For multiple conditions or a high number of samples, the procedure must be carried out in multiple rounds to ensure the best results.

1. Material preparation

1. Ahead of dissection, clean tweezers, a 9-well glass depression spot plate (or other container suitable for collecting and cleaning larvae), and a reusable Sylgard (silicone)-coated Petri dish used for dissection with 70% ethanol.
2. Prepare microscope slides by performing a 70% ethanol wash and then sticking an imaging spacer to the dried slide. Cut individual wells out of the 8-well strip with scissors and cut in half (Figure 2A). Remove the protective backing from one side of each half and stick spacers to the slide, spaced slightly apart to create a sheltered space in the middle where the discs will be placed (Figure 2B).
   NOTE: In our experience, this simplifies sample mounting as the precise volume of medium transferred to the slide is less critical. Any small excess volume will simply flow out through the gap upon coverslipping. As we image for only a relatively short time (generally ~1 h), we did not experience any problems with medium drying.
3. Thaw live-imaging medium (unsupplemented Schneider's Drosophila Medium stored in frozen 10 mL aliquots) and keep it readily accessible in an ice bucket.
4. In a 2 mL microcentrifuge tube, mix 1.5 mL of medium with 20 µL of 200 µg/mL Hoechst 33342 (final concentration 2.7 µg/mL). Keep the tube on ice.
   NOTE: This specific form of Hoechst dye is used because it is highly membrane-permeable. CAUTION: Gloves should be used when handling Hoechst 33342. Tools should be washed after use.

2. Wing disc dissection and slide preparation

1. Following genetic crossing and rearing at 25°C, pick wandering third-instar larvae from the tube and wash them in cold live-imaging medium in a 9-well glass depression spot plate.
   NOTE: Cytonemes are very fragile. In the following steps, care should be taken to manipulate tissues around the discs, trying to avoid direct contact between the discs and dissection tools. We prepare three or four larvae at a time, as damaging a few discs is unavoidable, and this may only become noticeable during the imaging steps.
2. Under the dissecting microscope, transfer the larvae into a drop of live-imaging medium on a silicone-coated Petri dish and start dissecting using two dissecting forceps. To do so, first hold the head and anterior body part steady by pinching the larvae with one pair of forceps at about one-third the body length (from the anterior). Using the second pair of forceps, pinch the body just posterior to the first forceps. Then, pull the posterior part of the larvae away to isolate the anterior "half".
3. Invert the anterior half by securing it with one pair of tweezers on either side of the cut end and pushing the head through with the other pair (like turning a sock inside out), to expose the internal structures, including imaginal discs, trachea, salivary glands, fat body, and gut.
4. Carefully remove the salivary glands, fat body, and gut, being careful not to disturb the lateral trunks of the trachea, which should overlie and protect the wing discs.
5. Use forceps to carefully transfer the cleaned anterior halves with attached wing discs into a drop of clean live-imaging medium with Hoechst, placed between the spacers on a prepared slide (Figure 2C). Isolate the wing discs from the rest of the tissues by gently blunt-dissecting them away from the fine branches of the trachea that connect to the disc, using either one blade of the dissecting forceps or a fine tungsten wire attached to a dissecting needle holder. Discard the rest of the carcass.
6. Orient the discs using one blade of the dissecting forceps or tungsten wire dissecting needle. Due to their flattened tear-drop-like shape, wing discs will fall with either the DP or PerM facing up. For this protocol, orient all discs with the PerM side up.
7. Adjust the medium volume to fill the well entirely, to slightly above the level of the imaging spacer, by either adding or removing the medium as needed. Remove the protective backing from the top side of each imaging spacer half and then carefully lower a coverslip over the sample. Gently press on the coverslip where it contacts the imaging spacers (e.g., with the rounded end of dissecting forceps) to ensure that the coverslip adheres to the spacer.
   NOTE: The medium level should be slightly above the spacer height to avoid trapping air bubbles, but not so high as to generate significant flow when the coverslip is lowered.

3. Imaging

1. Image samples at room temperature using a confocal microscope (argon ion laser with a 488 nm wavelength for GFP, UV laser with 350 nm wavelength for Hoechst) equipped with a 40x/1.30 NA oil immersion lens and microscopy software. Acquire image stacks with 1 µm step size that span from just above the highest level of the PerM (where Hoechst fluorescence diminishes) to the basal side of the DP, which generally works out to more than 100 images per stack. Acquire repeated image stacks to generate timelapse movies.
   NOTE: We image a slide for no more than ~1-2 h.
2. For the microscope software settings, acquire images using 8-bit coding, line averaging of 2, detector gain generally between 650 and 750, and separated channel tracks to reduce signal bleedthrough. Use identical laser and detector settings for all conditions, predetermined by performing tests on each condition beforehand.
   NOTE: At 40x magnification, our images have a resolution of 5.0386 pixels per µm (pixel size: 0.1985 x 0.1985 µm²). In our setup, the optimal confocal microscope settings included the highest laser intensity possible (without damaging the tissue) to capture as much signal as possible, while at the same time yielding little to no saturation; and the lowest possible noise as assessed on negative controls, achieved by reducing detector offset and acquisition speed (if needed). We generally use less than 5% laser power and maximum scan head speed ('9' in the Zen software settings). A single image stack typically takes ~1 min 45 s to acquire, and images are taken every 2-3 min for timelapse analyses.

4. Image analysis

1. Project stacks using the maximum intensity projection function of the ImageJ software package (click on image | Stacks | Z project, select Maximum intensity in the menu).
2. In the MIP images, determine the wing pouch area based on the pattern of wing disc folding (using the Hoechst channel) using Polygon Selection. Use the same selection on the GFP channel with the Clear Outside Function (click on Edit | Clear outside) to set every outside pixel value to 0.
3. In the MIP images, use the Hoechst channel to clean the GFP images, specifically removing any artefactual spots that appear in both channels by setting the concerned pixel value to 0 (click on Polygon Selection, then Edit | clear).
4. In the cleaned MIP images, use the Histogram function (click on Analyze | Histogram | List) to obtain and copy the list of all pixel values into a table in a spreadsheet.
5. Set a threshold value based on the analysis of the background signal in negative controls (and then verified in other samples; see the discussion).
6. Calculate the percentage of GFP-positive surface using the following formula:
   %GFP-positive surface = × 100
7. Normalize the data at the end by dividing each value by the mean of the reference condition (which is set to 1).
   NOTE: Valid pixel = pixel with a value ≥ 1, effectively ignoring any deleted pixel whose value was set to 0. Pixel values of 0 do not occur naturally.
8. For determining where in the discs the reconstituted GFP signal localizes, use XZ-reslices of image stacks to look at the apicobasal axis of the disc epithelium. Perform the reslices in Fiji without interpolation, and increase the image height in the Z-axis using the Scale function with bilinear interpolation to improve visibility.

Results

To test the usefulness of the GRASP procedure for measuring contacts between DP and PerM cells, we examined wing discs of four different genotypes: wild-type negative-control discs (genotype: w¹¹¹⁸) which will only display background levels of autofluorescence in the GFP channel; discs expressing the CD4-spGFP1-10 in the DP layer, but lacking the CD4-spGFP11 transgene, which will show the level of fluorescence produced by GFP1-10 alone (which we expected to be negligible, as GFP1-10 should not fluores...

Discussion

Cytonemes play an important role in the distribution of ligands, controlling the growth and organization of developing tissues. Signal exchange takes place where cytoneme tips make intimate membrane contacts with their targets. In this protocol, we describe a simple method for analyzing cytoneme-mediated contacts between epithelial layers in the wing disc using the GRASP technique.

The technique presented here requires, at a minimum, four components-a GAL4 driver, a LexA driver, and the two tr...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Discovery V12 dissection microscope	Zeiss		dissection microscope
Dumont #55 forceps, Biology tips	Fine Science Tools	11255-20	dissecting forceps
EP-Slik (slik20358)	BDSC	Panneton et al. 2015	fly strain for expressing Slik
FIJI		Schindelin J. et al. (2012)	image analysis software
Hoechst 33342	ThermoFisher Scientific	H3570	live imaging nuclear stain
LexAop-CD4-spGFP11	BDSC	93018	fly strain
LSM 700 confocal microscope	Zeiss		confocal microscope
nub-GAL4	Bloomington Drosophila Stock Centre (BDSC)	86108	fly strain
PerM-LexA		Rambaud, Joseph et al., 2025	fly strain
PYREX 9-depression glass spot platesell	Corning Life Sciences	7220-85	for collecting and washing larvae
Schneider's Drosophila Medium	ThermoFisher Scientific	21720024	live-imaging medium
SecureSeal imaging spacers, 8-well, 0.12 mm thick	Grace Bio-Labs	654008	spacer
SYLGARD 184 silicone elastomer kit	Sylgard	3097358-1004	for making dissection plates
UAS-CD4-spGFP1-10	BDSC	93017	fly strain
Zen Black	Zeiss		acquisition software