Delta-Notch signaling is crucial for development of nearly every tissue in metazoans. Signals received by the Notch receptor influence transcription of select target genes that ultimately restrict the developmental fate of the signal receiving cell with respect to its neighbors. The Notch pathway also functions in contexts of abnormal proliferation and differentiation, e.g. cancer and inflammation. Therefore, understanding the regulation of signaling through the Notch receptor protein at the cellular and molecular level is of great significance. In this dissertation, I investigated three ways in which Notch signaling is regulated, namely (1) proteolysis of the Delta ligand; (2) endocytosis of the Delta ligand; and (3) proteolysis of the Notch receptor.. The Delta protein has three functions. First, Delta is a ligand for Notch when bound to it from an adjacent cell. Second, Delta is an inhibitor of Notch when coexpressed with it in the same cell. Third, Delta is hypothesized to be a receptor and, upon binding to Notch, signals to nucleus. Delta undergoes proteolysis by ADAM proteases and there are two contradictory models for the role of Delta cleavage: (1) cleavage disables Delta function; and (2) cleavage activates Delta function. Overall, the results presented in this dissertation strengthen the first model and weaken the second one. Consistent with the first model, we showed that preventing Delta cleavage strengthens its ligand function. As well, when co-expressed in the same with Notch, Delta cleavage is upregulated therefore disabling Delta function as inhibitor of Notch. In contrast to the second model, we showed that Delta proteolysis does not follow a previously established pattern of cleavages typical of cell surface proteins that are activated by proteolysis. Delta also undergoes endocytosis. Two general models have emerged that are again contradictory: (1) endocytosis downregulates cell surface expression of Delta and therefore diminishes its ability to bind Notch; (2) endocytosis of Delta invokes activation of Notch signaling. Overall, our results strengthen the first model and weaken the second one. In support of the first model, we first demonstrated that Notch activation shows a linear relationship to the amount of Delta ligand present on the cell surface and that subsequent inhibition of cell surface expression of Delta leads to its loss of function. In contrast to the second model, we showed that endocytosis of Delta is not required to activate Notch. We also resolved that earlier evidence in support for this model stemmed from misinterpretations of the properties of a Delta mutant protein. Proteolysis of Notch activates the signaling cascade. Binding of Delta to Notch was previously regarded as a requisite regulatory step to invoke receptor proteolysis. We identified the ability of Kuzbanian and TACE, ADAM proteases that cleave Notch in response to Delta stimulation, to activate Notch in a ligand-independent manner. Altogether, our results demonstrate that proteolysis and endocytosis of Delta are independent mechanisms that act to downregulate Delta function and are therefore an important means of attenuating the Notch signal. Alternatively, we find a novel means of enhancing Notch signals in specific contexts, namely through ligand-independent Notch activation by the ADAMs Kuzbanian and TACE. With respect to the latter observation, Kuzbanian and TACE expression is known to be elevated in several human diseases, and thus predicts that engagement of Notch signaling is a contributing factor in these pathologies.

Development requires the acquisition of different cell fates. A major conserved pathway required for cell fate determination is the Notch signalling pathway. Neuralized is a key regulator of the Notch pathway and is essential for embryonic development in Drosophila melanogaster. I have been studying the role of Neuralized during Drosophila development, focusing on the regulation of this protein. Neuralized is an E3 ubiquitin ligase that targets Notch ligands for ubiquitination and endocytosis in the signal sending cell. This endocytic event is required for signal transduction, and cells lacking Neuralized fail to signal through Notch. I have identified a conserved interaction between Neuralized and phosphoinositides that is essential for the ability of Neuralized to promote ligand endocytosis and Notch signalling. Interactions between Neuralized and phosphoinositides are not required for ligand ubiquitination, identifying a role for Neuralized in downstream aspects of ligand trafficking. I have also determined that Neuralized is dynamically regulated through a combination of tissue specific expression, subcellular trafficking, protein interactions and posttranslational modification. Neuralized contains two related protein domains of unknown function called Neuralized homology repeats (NHR). To gain insight into the function of the NHR domain, I characterized another NHR containing protein, CG3894. CG3894 is required for development and preliminary data indicate that NHR domains dimerize, suggesting a possible interaction between Neuralized and CG3894. The study of Neuralized in Drosophila has contributed to our understanding of this essential protein both at a developmental and cellular level, and has provided a means through which to ask questions about regulation of Notch signalling in a relatively simple context. Given the importance of Notch signalling to development, and contributions that aberrations in signalling make to cancer and diseases of the nervous system, expanding our understanding of the regulation of Notch signalling is essential to understanding how this important pathway functions.

Development requires the acquisition of different cell fates. A major conserved pathway required for cell fate determination is the Notch signalling pathway. Neuralized is a key regulator of the Notch pathway and is essential for embryonic development in Drosophila melanogaster. I have been studying the role of Neuralized during Drosophila development, focusing on the regulation of this protein. Neuralized is an E3 ubiquitin ligase that targets Notch ligands for ubiquitination and endocytosis in the signal sending cell. This endocytic event is required for signal transduction, and cells lacking Neuralized fail to signal through Notch. I have identified a conserved interaction between Neuralized and phosphoinositides that is essential for the ability of Neuralized to promote ligand endocytosis and Notch signalling. Interactions between Neuralized and phosphoinositides are not required for ligand ubiquitination, identifying a role for Neuralized in downstream aspects of ligand trafficking. I have also determined that Neuralized is dynamically regulated through a combination of tissue specific expression, subcellular trafficking, protein interactions and posttranslational modification. Neuralized contains two related protein domains of unknown function called Neuralized homology repeats (NHR). To gain insight into the function of the NHR domain, I characterized another NHR containing protein, CG3894. CG3894 is required for development and preliminary data indicate that NHR domains dimerize, suggesting a possible interaction between Neuralized and CG3894. The study of Neuralized in Drosophila has contributed to our understanding of this essential protein both at a developmental and cellular level, and has provided a means through which to ask questions about regulation of Notch signalling in a relatively simple context. Given the importance of Notch signalling to development, and contributions that aberrations in signalling make to cancer and diseases of the nervous system, expanding our understanding of the regulation of Notch signalling is essential to understanding how this important pathway functions.

Les cellules épithéliales fournissent différentes fonctions biologiques: elles servent de barrière entre l'extérieur et l'intérieur d'un organisme et forment un continuum mécanique à travers les jonctions adhérentes qui les connectent. Au cours du développement, elles subissent des modifications extrêmes pour former l'embryon: elles changent de forme, modifient leur position relative ou perdent leur intégrité épithéliale. La plus part de ces changement se basent sur la modulation de l'actomyosine corticale et jonctionale, et sur la modulation des protéines qui définissent et maintiennent la polarité apico basale. Neuralized (Neur) est une E3 ubiquitine ligase qui est conservée des nématodes jusqu'aux mammifères. Elle a été découverte pour son rôle dans la régulation de la signalisation Delta/Nocth. Dans ce travail on décrit deux autres functions Notch-indépendantes de Neur dans le remodelage des épithéliums. En premier temps, on montre que Neur régule négativement la protéine apicale Crumbs à travers une isoforme de Stardust, ce qui permet le remodelage de l'intestin postérieur de la Drosophile et favorise la migration trans-epithéliale des cellules germinales primordiales. Puis, on présente que, pendant la gastrulation, Neur module la contractilité de l'actomyosine dans le mésoderme, et indirectement dans l'ectoderme, pour contrôler la formation du sillon ventral. / Epithelial cells serve many biological functions: they act as a barrier to separate the interior from the exterior, and form a mechanical continuum through the junctions that interconnect them. During development, they undergo dramatic changes to shape the embryo: they change their shape, modify their relative position or lose their epithelial integrity. Most of these changes rely on the modulation of cortical and junctional actomyosin, and the regulation of the proteins that define and maintain the epithelial apical/basal polarity. Neuralized (Neur) is an E3 ubiquitin ligase conserved from nematodes to mammals. It was first discovered for its role in the regulation of Delta/Notch signalling. Here we describe two Notch independent roles of Neur in epithelial remodelling. First, we show that Neur negatively regulates the apical protein Crumbs though a specific isoform of Stardust. This allows the remodelling of the drosophila posterior midgut and favours the trans-epithelial migration of the primordial germ cells. Finally, we present that Neur modulates actomyosin contractility in the mesoderm, and indirectly in the ectoderm, to control ventral furrow formation during gastrulation.