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.

Comprendre comment les facteurs génétiques et environnementaux interagissent au cours du développement est une question fondamentale en biologie. Je me suis intéressée à cette question en utilisant le réseau de développement de la vulve du nématode C. elegans comme système modèle. L’objectif de mon projet était une étude quantitative de la modulation par l’environnement des voies de signalisation impliquées dans ce processus telles que, Ras, Delta-Notch et Wnt. J’ai tout d’abord analysé comment un facteur environnemental spécifique (la carence nutritionnelle) modifie les activités et les interactions entre les voies de signalisation sous-jacentes au développement vulvaire chez C. elegans. J’ai ainsi mis en évidence que l’augmentation de l’induction vulvaire par la carence passe par une augmentation de l’activité de la voie Ras et est indépendante de la voie Wnt. Cet effet de l’environnement est assuré par la détection de la diminution de l’apport en nutriments, probablement par l’action de la voie TOR, et affecte l’induction vulvaire en parallèle ou en amont du récepteur à l’EGF. J’ai ensuite examiné la sensibilité environnementale du système de développement de la vulve de Caenorhabditis dans une perspective évolutive et ce, grâce à l'analyse comparative de différents isolats. J’ai pu observer que l’exposition à des températures extrêmes induit des variants et des défauts de manière fortement dépendante de la souche et de l’espèce. L’occurrence de certains défauts développementaux induits par la température révèlent en outre que certaines cellules précurseurs de la vulve et les voies de signalisation associées présentent une sensibilité environnementale différente. / How genetic and environmental factors interact during development is a key question in current biology, yet little is known about how molecular and cellular processes integrate environmental information. In my PhD research I aimed to address this problem using the network of C. elegans vulval signalling pathways as a model system. The principal objective of my project was to quantitatively examine how involved major signalling pathways, EGF-Ras-MAPK, Wnt and Delta-Notch, are modulated by specific environmental signals. First, I analysed how a specific environmental factor (starvation) alters activities and interplay of signalling pathways underlying C. elegans vulval cell fate patterning. I found that starvation consistently increased vulval induction through upregulation of the EGF-Ras-MAPK pathway activity independent of the Wnt pathway. This environmental effect is mediated by internal sensing of nutrient deprivation, likely acting through the TOR pathway, and affects vulval induction at the level or upstream of the EGF receptor. Second, I examined the environmental sensitivity of the Caenorhabditis vulval developmental system from an evolutionary perspective through comparative analysis of different C. elegans and C. briggsae isolates. I found that extreme temperature induced diverse developmental variants and defects, which were strongly genotype- and species-dependent. The occurrence of certain developmental defects induced by temperature extremes further revealed that vulval precursor cells and associated fates differ in temperature sensitivity, and this cell-specific sensitivity shows evolutionary variation.

Au cours de ce projet de thèse, j’ai étudié le lien fonctionnel entre la morphogénèse épithéliale et la signalisation Delta-Notch, dans le cadre de la formation du tube neural chez le poisson-zèbre. La signalisation Delta-Notch est primordiale pour le développement embryonnaire et le maintien de l’homéostasie des tissus adultes. De façon inattendue, j’ai observé suite à la perte-de-fonction de Mib une perte de la polarité apico-basale dans le neuro-épithélium de la moelle épinière embryonnaire. L’analyse plus poussée de ce phénotype m’a ensuite permis de montrer que l’activité de l’intégralité de la signalisation Notch est requise pour l’établissement de la polarité apico-basale dans le tube neural de poisson-zèbre. En effet, l’inhibition des ligands de Notch et des activateurs transcriptionnels situés en aval, Rbpja et Rbpjb, résulte en l’interruption de la polarité apico-basale. De plus, l’activation ectopique de Notch entraîne un sauvetage complet de la polarité apico-basale dans les embryons déplétés pour Mib. Finalement, le mutant Mib échoue à activer la transcription de protéines de polarité apicale Crumbs1 et Crumbs2a au cours de la formation du tube neural, ce qui suggèrerait que la signalisation Notch agit en amont des complexes de polarité. De façon surprenante, nous avons également montré que le composant de la signalisation Notch, Mib, affecte les mouvements de convergence-extension et la division cellulaire orientée, appelée C-divisions, durant la neurulation et la gastrulation à travers la signalisation PCP. Cet effet de Mib sur la PCP est indépendant de son rôle sur la signalisation Notch. Généralement, cette étude révèle un double-rôle de Mib. / In this Ph.D. project, I study the functional link between epithelial polarity and Delta-Notch signaling in the context of zebrafish neural tube morphogenesis. Notch signaling, one of the major signaling pathways and of prime importance in neurogenesis, has been widely studied for its function in cell fate specifications. Surprisingly, I found that the Notch signaling component Mindbomb (Mib) loss-of-function led to a loss of apico-basal polarity in the neuroepithelium of the embryonic spinal cord. I further explored that the activity of the entire Notch signaling pathway is actually required for the earliest steps of establishment of apico-basal polarity in the zebrafish neural tube. Indeed, inhibition of Notch ligands and downstream transcriptional activators Rbpja and Rbpjb resulted in a disruption of apico-basal polarity. Moreover, ectopic activation of Notch ensued to a complete rescue of apico-basal polarity in Mib loss of function embryos. Furthermore, Mib mutant embryos fail to upregulate the transcription of the apical polarity proteins Crumbs1 and Crumbs2a in the course of neural tube formation, suggesting that Notch signalling might act upstream of polarity complexes. Moreover, I found that Mib affects convergent-extension movements and oriented cell divisions during neurulation and gastrulation through an effect on planar cell polarity. Remarkably, this effect of Mib on PCP is independent of its role in Notch signaling. These results indicate a novel role of Mib in the regulation of PCP signaling. Altogether, this study revealed a dual role of Mib in the epithelial morphogenesis of the zebrafish neural tube.

Oscillations are present at many different levels of biological organization. The cell cycle that directs the division of individual cells, the regular depolarization of neurons in the sinu-atrial node which underlies the regular beating of the heart, the circadian rhythms that govern the daily activity cycles of virtually all organisms, and the clocks that make entire populations of fireflies flash on and off in unison feature as prominent examples of biological clocks. During development, biological clocks regulate the patterning of growing tissues, as is the case in vertebrate somitogenesis, and potentially also in vertebrate limb outgrowth and axial segmentation of invertebrate embryos. During vertebrate segmentation, the embryonic axis is subdivided along its anterior-posterior axis into epithelial spheres of cells called somites. This rhythmic process is thought to be driven by a multicellular oscillatory gene network, the so-called segmentation clock. Oscillations of hairy and enhancer of split gene products have been proposed to constitute the core clockwork in individual cells, and these oscillators are coupled to each other by Delta-Notch intercellular signaling. The interaction of the segmentation clock with a posteriorly-moving arrest wavefront then translates the temporal information encoded by the clock into a spatial pattern of segments. In the framework of this Clock and Wavefront model, segment length is determined by both clock period and arrest wavefront velocity. How the period of the segmentation clock is regulated is presently unknown, and understanding the mechanism of period setting might yield insight into the nature and function of the segmentation clock. In this study, two different but complementary approaches were pursued to investigate how period is regulated in the zebrafish segmentation clock. First, it has been reported that zebrafish mind bomb (mib) mutant embryos form somites more slowly than their wt siblings, suggesting that Mib might be implicated in period setting. Mib is an E3 ubiquitin ligase required for ubiquitination and endocytosis of the Notch ligand Delta, and Notch signaling is impaired in mutants with defective Mib. It has been suggested that the mechanistic basis for the requirement of Delta endocytosis in Notch signaling is a need for Delta to enter a particular endocytic compartment, potentially a recycling endosome, in a ubiquitin-dependent manner, where its signaling ability might be established or amplified by an as yet unknown posttranslational modification. In the present study, Delta trafficking through the endocytic pathway was analyzed in the PSM of wt and mib embryos through colocalization studies with endocytic markers. The rationale of this approach was that if Delta gained access to a particular endocytic compartment through Mib-dependent endocytosis, the presence of Delta in this compartment would be expected to be reduced in mutants with defective Mib, thereby revealing the compartment’s identity. However, no qualitative changes in colocalization with different endocytic markers could be detected in mib mutants, and the methods available did not allow for quantification of colocalization in wt or mutant backgrounds. However, Delta colocalized with 13 markers of recycling endosomes, consistent with the hypothesis that these are functionally important in Notch signaling. More refined techniques will be necessary for a quantitative analysis of normal as compared to impaired Delta trafficking. A genetic approach to period regulation proved to be successful for the Drosophila circadian clock, where the identification of period mutants advanced the understanding of the clock’s genetic circuitry. This motivated a screen for period mutants of the segmentation clock, which was carried out by measuring somitogenesis period, segment length and arrest wavefront velocity in a pool of candidate mutants. A subset of Delta-Notch mutants, and embryos treated with a small-molecule inhibitor that impairs Notch signaling, displayed correlated increases in somitogenesis period and segment length, while there was no detectable change in arrest wavefront velocity. Combined, these findings suggested that segmentation clock period is increased in experimental conditions with impaired Delta-Notch signaling. Using a theoretical description of the segmentation clock as an array of coupled phase oscillators, the delay in the coupling and the autonomous frequency of individual cells were estimated from the direction and magnitude of the period changes. The mutants presented here are the first candidates for segmentation clock period mutants in any vertebrate. The nature of the molecular lesions in these mutants, all of which affect genes implicated in intercellular Delta-Notch signaling, suggests that communication between oscillating PSM cells is a key factor responsible for setting the period of the segmentation clock.

Comprendre comment les facteurs génétiques et environnementaux interagissent au cours du développement est une question fondamentale en biologie. Je me suis intéressée à cette question en utilisant le réseau de développement de la vulve du nématode C. elegans comme système modèle. L'objectif de mon projet était une étude quantitative de la modulation par l'environnement des voies de signalisation impliquées dans ce processus telles que, Ras, Delta-Notch et Wnt. J'ai tout d'abord analysé comment un facteur environnemental spécifique (la carence nutritionnelle) modifie les activités et les interactions entre les voies de signalisation sous-jacentes au développement vulvaire chez C. elegans. J'ai ainsi mis en évidence que l'augmentation de l'induction vulvaire par la carence passe par une augmentation de l'activité de la voie Ras et est indépendante de la voie Wnt. Cet effet de l'environnement est assuré par la détection de la diminution de l'apport en nutriments, probablement par l'action de la voie TOR, et affecte l'induction vulvaire en parallèle ou en amont du récepteur à l'EGF. J'ai ensuite examiné la sensibilité environnementale du système de développement de la vulve de Caenorhabditis dans une perspective évolutive et ce, grâce à l'analyse comparative de différents isolats. J'ai pu observer que l'exposition à des températures extrêmes induit des variants et des défauts de manière fortement dépendante de la souche et de l'espèce. L'occurrence de certains défauts développementaux induits par la température révèlent en outre que certaines cellules précurseurs de la vulve et les voies de signalisation associées présentent une sensibilité environnementale différente.