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Abstract

The highly conserved Notch signaling pathway governs a plethora of cellular processes and developmental transitions. Unlike other cell surface receptors that rely on elaborate cytoplasmic cascades to adjust pathway activity, Notch receptors transmit messages directly to the nucleus. Despite this deceivingly straightforward pathway architecture, a variety of mechanisms exists to amplify or limit Notch inductive inputs, thereby ensuring precise pathway output and appropriate biological functions. This dissertation investigates three developmentally relevant mechanisms of Notch pathway regulation. One operates intracellularly, affecting both ligand-exposed and unexposed cells (Chapter 2), another relies on dynamic ligand-receptor interactions between neighboring cells (Chapter 3), and the third effectively activates the pathway in the absence of ligand induction (Chapter 4). Using the Drosophila melanogaster wing and S2 cells, I first show how the Abelson kinase and the E3 ubiquitin ligases Su(dx) and Nedd4Lo cooperatively regulate the passage of Notch through late endosomes/multivesicular bodies (LEs/MVBs). Whether as part of the same pathway or as parallel inputs, these proteins divert Notch from the membrane into the lumen of LEs, a subcellular domain that restricts signaling. I discuss how this regulatory network modulates Notch activity in the developing pupal wing epithelium. Second, I uncover how a Delta-Notch feedback mechanism continuously refines noisy Notch activity to establish vein-intervein boundaries in the pupal wing. Through genetic manipulations, I demonstrate that this feedback is robust, uses multi-step sequential intercellular interactions, and can be activated anywhere in the tissue under inappropriate levels of signaling. Finally, I provide evidence that low levels of Notch signaling actively repress vein fate within intervein domains, where cells are distant from a significant ligand source. Genetic mosaic analyses confirm that intervein cells sustain pathway activation in a Delta/Serrate-independent manner. This represents the first report of a developmental role for ligand-independent Notch signaling in non-circulating cells. In conclusion, this work provides critical insights into how intracellular and intercellular inputs are integrated to achieve precise spatiotemporal Notch activation during wing vein patterning. The interplay between local cell-autonomous mechanisms and longer-range cell-cell interactions is essential to establishing vein/intervein tissue domains and boundaries. While local regulatory events can drive fast-scale adjustments to Notch induction within individual cells, intercellular Delta-Notch feedback interactions coordinate signaling across longer distances, refining pathway deployment and tissue patterns over time. Together, these complementary modes of regulation optimize Notch activity, ensuring the precision and robustness of its cellular and developmental functions.

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