Pfeiffer, Sven; (2001) Wingless transport in the embryonic epidermis of Drosophila. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The wingless gene encodes a secreted glycoprotein of the Wnt family. Wingless is required for many cell fate decisions during Drosophila development and has been proposed to function as a classic morphogen. Morphogens are defined as localised factors that pattern fields of cells in a concentration dependent manner (Wolpert, 1981). The mechanisms through which morphogens in general, and Wingless in particular, build their activity gradients have been unclear, with evidence for both restricted diffusion and intracellular transport. This thesis describes investigations of Wingless movement across the Drosophila embryonic epidermis. In chapter 2 I demonstrate an alternative mode of transport for Wingless. In embryos, wingless is transcribed in narrow stripes of cells abutting the source of Hedgehog. I show that these cells or their progeny leave the domain of wingless expression towards the anterior. As they do so, they no longer receive the Hedgehog signal and stop transcribing wingless. They can, however, retain inherited Wingless protein in secretory vesicles and carry it over a distance of up to 4 cell diameters from the site of transcription. Experiments with a membrane-tethered form of Wingless show that this mechanism is sufficient to account for the normal range of Wingless. However, if the contribution of protein inheritance is removed, Wingless can still reach distant target cells by diffusion/transport. I suggest that both transport mechanisms operate in the wild type. In chapter 3 I develop a method for the detailed study of epidermal cell movement in living embryos, inspired by the findings of chapter 2 that demonstrate the importance of cell lineage for intrasegmental patterning. I apply these methods to the study of cell movement during germband retraction, mainly because this is the time when Wingless specifies epidermal cell fates. Although it is assumed that there is no mitotic activity during this stage, I find a surprising amount of cell divisions. A patterned behaviour appears to be present in the orientation of mitoses. In chapter 4 I use a biologically active GFP-tagged Wingless (GFP-Wingless) to study Wingless trafficking. I find that GFP-Wingless (as well as Wingless) remains tightly associated with secreting cells and this may explain the short range seen in embryos. At the anterior of each stripe of wingless expression, where Wingless specifies bald cuticle, extracellular Wingless is readily endocytosed and can be recycled back to the cell surface (the first direct observation of ligand recycling in live embryos). I suggest that, within this region of the ectoderm, endocytosis and recycling serve to sustain high level signalling.

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