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,

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-sp....

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