Endocytic trafficking of Wingless and its receptors, Arrow and DFrizzled-2, in the Drosophila wing

Abstract

During animal development, Wnt/Wingless (Wg) signaling is required for the patterning of multiple tissues. While insufficient signal transduction is detrimental to normal development, ectopic activation of the pathway can be just as devastating. Thus, numerous controls exist to precisely regulate Wg signaling levels. Endocytic trafficking of pathway components has recently been proposed as one such control mechanism. Here, we characterize the vesicular trafficking of Wg and its receptors, Arrow and DFrizzled-2 (DFz2), and investigate whether trafficking is important to regulate Wg signaling during dorsoventral patterning of the larval wing. We demonstrate a role for Arrow and DFz2 in Wg internalization. Subsequently, Wg, Arrow and DFz2 are trafficked through the endocytic pathway to the lysosome, where they are degraded in a hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)-dependent manner. Surprisingly, we find that Wg signaling is not attenuated by lysosomal targeting in the wing disc. Rather, we suggest that signaling is dampened intracellularly at an earlier trafficking step. This is in contrast to patterning of the embryonic epidermis, where lysosomal targeting is required to restrict the range of Wg signaling. Thus, signal modulation by endocytic routing will depend on the tissue to be patterned and the goals during that patterning event.

Endocytosis of Wingless via a dynamin-independent pathway is necessary for signaling in Drosophila wing discs

“…Endosomes with both Wg and DFz2 (described in players (Dsh, Axin) to the plasma membrane and initiate canonical Wnt signaling (59). However, endocytosis of Wnt3A in L cells (60) and Wg in Drosophila wing discs (13,14,61) has been shown to be necessary for canonical Wnt signaling as the downstream players are recruited to endosomes (13,62). In our experiments, Wg interacts productively with DFz2 in the acidified endosome (or when the cell surface is exposed to acidic pH).…”

“…Endocytosis in the signal-receiving cells may either be important in shaping the distribution of secreted Wg across the wing disc (11) or in a cell-autonomous fashion affect signaling by promoting the interaction of Wg and DFz2 within an endosome (12,13). Endocytosis also mediates Arrow-directed degradation necessary for the observed Wg distribution and signaling (14). However, rescue of patterning is observed even upon replacement of the endogenous Wg with a transmembrane-tethered Wg, thus raising questions on the importance of a secreted Wg gradient (15).…”

“…Regardless, inhibition of endocytosis in the recipient cells, by using the dominant-negative (DN) or the temperature-sensitive form of shibire, demonstrates the importance of dynamin-dependent endocytosis in Wg-mediated signaling (11,13). Interestingly, when examined carefully, Wg is observed in endosomes even in null clones of its signaling receptors Frizzled (Fz) and Arrow (14,(16)(17), suggesting that other receptors or pathways may be important for its internalization.…”

Frizzled and LRP5/6 Receptors for Wnt/ -Catenin Signaling

“…Thus, some cautions are warranted, particularly when perturbations of general endocytic pathways are performed and thus broad cellular events beyond Wnt signaling are affected (Gagliardi et al 2008). Indeed, manipulating endocytic molecules in Drosophila has also yielded contradictory results regarding Wg signaling (Piddini et al 2005;Rives et al 2006;Seto and Bellen 2006). Finally, some of the cell biological studies of LRP6 and Wnt signaling have relied on overexpression of LRP6 and other components.…”

“…(2) Because GSK3 levels do not overtly decrease during Wnt signaling, it requires a mechanism for GSK3 subsequently to escape MVBs, which normally shuttles the cargo to lysosomes for degradation. (3) It suggests that genes involved in MVB formation are required for Wnt signaling, for which supporting evidence exists in Xenopus but not in Drosophila (Piddini et al 2005;Rives et al 2006;Seto and Bellen 2006;Taelman et al 2010). (4) It predicts that most GSK3 substrates (in fact, 20% of cellular proteins) in addition to b-catenin are under Wnt regulation (Taelman et al 2010), thus departing from the prevailing view that roles of GSK3 in Wnt and other signaling (such as insulin) pathways are insulated from one another.…”