Background

The developing epithelium of the Drosophila wing imaginal disc (WID) is a well-established model system to study the intracellular trafficking of Wnt proteins. Imaginal discs are epithelial sac-like structures of Drosophila larvae that give rise to adult body parts during pupal transformation. Development of the fly wing from WID depends on the correct formation of several wing axes. To this end, several morphogen gradients are established. The dorsal-ventral wing axis is defined by Wg (Drosophila Wnt-1) secretion from a narrow cell stripe of Wg-expressing cells along the dorsal-ventral boundary. Upon secretion, Wg travels across the WID epithelium over short and long distances to activate target gene transcription in a dose-dependent manner. To ensure Wg gradient formation and proper wing development, Wg secretion and spread from the polarized WID epithelial monolayer are tightly regulated.

Upon translation, Wg is lipid-modified in the endoplasmic reticulum (ER) and transported via the Golgi toward the apical plasma membrane with its cargo receptor Evi/Wls (Bänziger et al., 2006; Bartscherer et al., 2006). While Wg can be transferred to neighboring cells by cell-cell contacts (Alexandre et al., 2014), long-range Wg secretion and signal transduction depend on Wg entry into the endosomal system (Strigini and Cohen, 2000; Pfeiffer et al., 2002). To this end, Wg re-enters the cell after apical presentation through a dynamin-independent endocytic route that delivers Wg into endosomal compartments (Hemalatha et al., 2016). Basal routes for secondary Wg secretion have also been described (Yamazaki et al., 2016) and Wg colocalizes with Rab4-recycling endosomes on the apical WID cell side (Gao et al., 2017; Linnemannstöns et al., 2020; Witte et al., 2020). Apical, secondary Wg release from Rab4-recycling endosomes could therefore fuel the Wg gradient independently of apicobasal transport (Witte et al., 2020). Consequently, interference with the apical release of Wg significantly hinders Wg gradient formation (Chaudhary and Boutros, 2019; Linnemannstöns et al., 2020).

The visualization of Wg trafficking to the cell surface and in post-endocytic endosomal compartments is an important tool to understand how the long- and short-range Wg signal is generated. Here, we present three main methods that have been established to unravel which of the proposed secretory routes feed the Wg signal: (1) the analysis of steady-state extracellular Wg levels [initially established by Strigini and Cohen (2000)]; (2) the visualization of intracellular Wg transport after endocytic uptake [modified from Hemalatha et al. (2016)]; and (3) the visualization of Wg recycling (Witte et al., 2020) (Figure 1). All three methods are based on immunofluorescence staining of dissected Drosophila WID, but different approaches are used to visualize Wg in the respective WID compartment (Figure 2). Briefly, while (1) and (3) make use of non-permeabilizing staining protocols, albeit at different temperatures, to specifically visualize surface bound extracellular Wg, (2) uses the endocytic uptake of antibody-labeled Wg to visualize its endosomal transport. Using UAS/Gal4-driven gene expression in combination with an engrailed promotor to drive genetic manipulations specifically in posterior WID allows the direct quantitation of anterior and posterior phenotypes within the same WID.

Multiple intracellular trafficking routes have been proposed to contribute to Wg signal transfer. It is therefore especially interesting to see how extracellular and intracellular Wg distributions react to interference with different aspects of the Wg trafficking machinery. As genetic manipulations are comparatively easy in Drosophila, the WID provides an appropriate means to probe this system. We hope that the methods presented herein contribute to gaining further insight into the evolutionarily conserved secretory Wg/Wnt trafficking pathway in order to fully understand how Wnt signals can be transferred over short and long distances. Moreover, these protocols are not limited to Wg and can be used to investigate the trafficking of other morphogens, such as Hedgehog (Hh) or Decapentaplegic (Dpp).

Figure 2. Overview of the different staining approaches. After WID dissection, extracellular Wg staining is performed on ice without permeabilization. The Wg uptake assay is performed at 22°C and includes acid washing to remove extracellularly bound signal, as well as permeabilization. Wg recycling is monitored at 22°C in the absence of permeabilization. After staining, WID samples are again dissected and mounted for downstream imaging and quantitation.