Palmer, LR; (2014) The route taken by Wingless in secreting cells. Doctoral thesis , UCL (University College London).

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Abstract

Wingless (Wg), the major Drosophila Wnt, contributes to patterning, growth and cell survival during development. Wg is produced in a stripe at the dorsal-ventral boundary of the wing imaginal disc, a pseudostratified epithelium. Whole mount staining of permeabilised discs reveal that the Wg protein is tightly localized in the apical region of secreting cells. By contrast, extracellular Wg is barely detectable at the apical surface. Instead, extracellular Wg is mostly found at the basolateral surface of secreting, as well as surrounding, cells. These observations suggest that, in secreting cells, apically produced Wg traffics to the basolateral surface for release and gradient formation. This possibility has not been formally investigated. Nevertheless, specific regulators of Wg secretion have been identified. For example, Wntless (Wls)/Evenness interrupted (Evi) binds Wg in the ER and transports it to the plasma membrane. Without Evi, Wg accumulates within the secretory pathway. To test the transcytosis model, I have designed means of tracking Evi and Wg in secreting cells, using classical secretory and endocytic markers as guideposts. I have constructed tagged versions of Wg and Evi, which rescue wg or evi mutants when expressed at an endogenous level. In one set of experiments, I have produced a step of Wg expression and fixed discs at subsequent time points. With this approach I have determined that Wg moves from its apical production site towards the basolateral surface. This was confirmed with experiments utilising a temperature-sensitive dynamin mutant (shibirets) to control endocytosis. With this genetic tool, I have obtained data suggesting that Evi too transits through the apical surface of expressing cells before progressing basally. Unlike Wg, Evi is not released at the basolateral surface. I suggest that instead, it is recycled to replenish the secretory pathway, where it can escort more Wg to the apical surface.

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