Epithelial sheets line the surfaces of the body, forming a barrier between the external environment and internal tissues. During development, regulation of epithelial architechture can drive morphogenesis and build the three-dimensional structures of the body. Epithelial form and function derive from the polarized morphology of epithelial cells, which have apical surfaces that face the external environment, lateral surfaces containing cell-cell junctions and basal surfaces that connect to the underlying tissue. A network of polarity proteins establishes the apico-basolateral axis, while a system of polarized membrane traffic ensures delivery of specialized cargo to distinct membrane surfaces. How these systems of polarity and trafficking are integrated is still poorly understood. The focus of my study was to investigate how the apical polarity proteins Cdc42, Par6, Bazooka and aPKC (the “Par complex”) regulate polarity and adherens junction (AJ) integrity during Drosophila development. Upon perturbation of Cdc42/Par activity during embryogenesis, apical membrane proteins accumulate in sorting endosomes. This trafficking defect occurs throughout the ectoderm, but in the ventral neuroectoderm (VNE) is accompanied by a concomitant depletion of the apical proteins from the plasma membrane (PM) and a loss of AJ integrity. I have demonstrated that the VNE phenotype is a consequence of the relatively high morphogenetic activity of this tissue. Furthermore, I have shown that the AJ defects are likely a downstream consequence of the depletion of important apical polarity factors, such as Crumbs, from the PM. To further characterize the mechanism of apical trafficking, I searched for interactors of Cdc42/Par in the membrane trafficking machinery. I describe interactions between several trafficking genes and Cdc42/Par and provide evidence that Vps26, a component of the retromer complex that retrieves proteins from endosomal membrane and delivers them to the Golgi for re-secretion, is phosphorylated by aPKC and acts as an aPKC effector in the recycling of apical membrane proteins. I propose that Cdc42/Par regulate the retromer to promote the PM localization of apical proteins, which is important to maintain AJ integrity in morphogenetically active tissues.

Epithelial sheets line the surfaces of the body, forming a barrier between the external environment and internal tissues. During development, regulation of epithelial architechture can drive morphogenesis and build the three-dimensional structures of the body. Epithelial form and function derive from the polarized morphology of epithelial cells, which have apical surfaces that face the external environment, lateral surfaces containing cell-cell junctions and basal surfaces that connect to the underlying tissue. A network of polarity proteins establishes the apico-basolateral axis, while a system of polarized membrane traffic ensures delivery of specialized cargo to distinct membrane surfaces. How these systems of polarity and trafficking are integrated is still poorly understood. The focus of my study was to investigate how the apical polarity proteins Cdc42, Par6, Bazooka and aPKC (the “Par complex”) regulate polarity and adherens junction (AJ) integrity during Drosophila development. Upon perturbation of Cdc42/Par activity during embryogenesis, apical membrane proteins accumulate in sorting endosomes. This trafficking defect occurs throughout the ectoderm, but in the ventral neuroectoderm (VNE) is accompanied by a concomitant depletion of the apical proteins from the plasma membrane (PM) and a loss of AJ integrity. I have demonstrated that the VNE phenotype is a consequence of the relatively high morphogenetic activity of this tissue. Furthermore, I have shown that the AJ defects are likely a downstream consequence of the depletion of important apical polarity factors, such as Crumbs, from the PM. To further characterize the mechanism of apical trafficking, I searched for interactors of Cdc42/Par in the membrane trafficking machinery. I describe interactions between several trafficking genes and Cdc42/Par and provide evidence that Vps26, a component of the retromer complex that retrieves proteins from endosomal membrane and delivers them to the Golgi for re-secretion, is phosphorylated by aPKC and acts as an aPKC effector in the recycling of apical membrane proteins. I propose that Cdc42/Par regulate the retromer to promote the PM localization of apical proteins, which is important to maintain AJ integrity in morphogenetically active tissues.

Understanding the establishment of polarity and the cell fate specification of epithelial cells is important for developmental biology, regenerative medicine and the study of cancer. In this thesis, models of pre-implantation epithelial development are used to investigate the relationship between these two processes. The trophoblast is an extraembryonic epithelial tissue which contributes to the placenta. Addition of BMP4 to mouse and human embryonic stem (mES) cells grown in culture has been suggested to induce differentiation of cells to the trophoblast lineage. The use of this differentiation method was investigated as a possible model of trophoblast polarisation and cell fate specification. Unfortunately, with the protocol and reagents available this model did not appear to physiologically recapitulate trophoblast development and was not reliable. The primitive endoderm is an epithelium which arises from the inner cell mass during mammalian pre-implantation development. It faces the blastocoel cavity and later gives rise to the extraembryonic parietal and visceral endoderm. When mES cells are grown in suspension they form aggregates of differentiating cells known as embryoid bodies. The outermost cell layer of an embryoid body is an epithelial cell type comparable to the primitive endoderm. Embryoid bodies were used here to study the polarisation and cell fate specification of the primitive endoderm. The outer cells of these embryoid bodies were found to gradually acquire the hallmarks of polarised epithelial cells and express markers of primitive endoderm cell fate. The acquisition of epithelial polarity occurred prior to the maximal expression of cell fate markers. Fgfr/Erk signalling is known to be required for specification of the primitive endoderm, but its role in polarisation of this tissue is less well understood. To investigate the function of this pathway in the primitive endoderm, embryoid bodies were cultured in the presence of a small molecule inhibitor of Mek. This inhibitor caused a loss of expression of markers of primitive endoderm cell fate and maintenance of the pluripotency marker Nanog. In addition, a mislocalisation of apico-basolateral markers and disruption of the epithelial barrier which normally blocks free diffusion across the epithelial cell layer occurred. Two inhibitors of the Fgf receptor elicited similar phenotypes, suggesting that Fgf receptor signalling promotes Erkmediated polarisation. This data shows that the formation of a polarised primitive endoderm layer in embryoid bodies requires the Fgfr/Erk signalling pathway.

ASPP2 has been identified as a haploinsufficient tumour suppressor in mice, and an activator of the apoptotic function of the p53 family. Yeast two-hybrid experiments have also shown that ASPP2 interacts with a large number of proteins involved in other major signalling pathways. The mechanism(s) of action of ASPP2 are therefore complex, and likely to involve more than just the stimulation of the apoptotic programme. Since a study previously conducted in our laboratory revealed that the deletion of ASPP2 in vivo leads to severe hydrocephalus in the J129/C57BL6 background (Vives et al., 2006), it can be hypothesised that ASPP2 safeguards the normal development of the mammalian central nervous system (CNS), in addition to its role as a tumour suppressor. Deletion of ASPP2 leads to the development of hydrocephalus, most probably by affecting tight junctions (TJs) in the choroid plexus, thereby impairing its blood-cerebrospinal fluid (CSF) barrier function. TJ defects are likely to be the underlying cause of the loss of cell polarity observed in the neuroepithelium of several areas of the CNS. As cell polarity plays a key role in multiple aspects of CNS development, ASPP2 appears to be required for the proper lamination of the cerebral cortex and retina.

The relationship between MUC1 and EGFR has been characterized by our lab to be highly tumorigenic. A peptide therapeutic was developed in our lab to block the cytoplasmic interaction of MUC1 and EGFR by competing with the EGFR-binding domain of MUC1. The peptide, PMIP, reduced invasion and proliferation in vitro and reduced tumor growth and metastasis in vivo. These studies demonstrated the potency of MUC1/EGFR interactions in tumor progression, and we sought to explore this concept further. We wanted to clarify a mechanism by which MUC1 and EGFR together drive breast cancer metastasis, and we identified c-Met as a mediator of MUC1 and EGFR-driven cell motility. In two separate assays, we demonstrated that c-Met activity was necessary for MUC1 and EGFR to promote migration and invasion. In addition, we wanted to identify the role of EGFR membrane localization in membrane identity and tumor initiation. We established several EGFR localization mutants to compare to wild-type basolateral EGFR and we performed proof-of-concept experiments to show that these mutants will be useful in future studies. Finally, we studied the effect of MUC1 and EGF loss on tissue architecture and function in the lactating mammary gland. EGF is the primary ligand for EGFR during lactation, and MUC1 is highly expressed during this period of mammary development. In addition, it has been shown that EGFR and MUC1 interact at the apical cell surface of lactating mammary ducts, yet there is no link between lactation and tumor formation. We hypothesized that MUC1 and EGFR interaction may have a role in maintaining tissue architecture and lactation function in the mouse mammary gland. We found instead that the loss of MUC1 and EGF had no noticeable effect on lactation and did not result in tissue defects. These studies further clarified the relationship between MUC1 and EGFR in several different contexts, showing a role for their interaction in metastatic progression, and showing that their ablation has no effect in the lactating mammary gland. Future studies will elucidate the role of MUC1 and EGFR interaction in tumor initiation, and we have taken several steps in our studies toward that goal.

The establishment and maintenance of cell polarity during development is an active process that requires specific protein sorting and targeting to apical and basolateral regions of the cell. Our lab has identified an apical early endosomal marker, endotubin, in developing rat intestine, which we have used to label specialized apical endosomal tubules, and to probe for components of the apical sorting machinery. Studies with endotubin have implicated the small GTPase Rab14 as part of the sorting machinery for apical targeting. The current work pursues further study of the interaction between Rab14 and endotubin, as well as the role for Rab14 in the establishment of cell asymmetry. Interestingly, even nonpolarized cells may utilize polarized trafficking components for proper sorting and dynamics of endotubin.

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.

L'établissement de la polarité apico-basale dans les tissus épithéliaux est étroitement lié à la division cellulaire, mais les mécanismes moléculaires sous-jacents n'ont pas encore été établis. A l'aide d'un modèle de culture en 3 dimensions de cellules rénales (MDCK), j'ai montré que lors du développement d'un cyst, la GTPase Rab35 joue un rôle majeur dans l'établissement de la polarité et le positionnement du lumen pendant la première division cellulaire. Au niveau moléculaire, Rab35 permet de coupler l'initiation de la polarité apico-basale avec la cytocinèse via l'attachement au sillon de clivage de vésicules intracellulaires contenant des déterminants clé de l'établissement de la polarité. Ces vésicules transportent notamment les protéines aPKC, Cdc42, Crumbs3 ainsi que le facteur d'ouverture de la lumière Podocalyxin. De plus, l'attachement de ces vésicules au sillon de clivage dépend de l'interaction directe entre Rab35 et la queue cytoplasmique de Podocalyxin. Par conséquence, l'inactivation de Rab35 entraine une inversion complète de la polarité apico-basale des kystes 3D. J'ai mis en évidence un nouveau mécanisme de ciblage des vésicules intracellulaire au site de clivage dépendant de la protéine Rab35 impliqué à la fois dans l'initiation de la polarité apico-basale et dans l'ouverture de la lumière au centre du cyst. / Establishment and maintenance of apico-basal polarity in epithelial organs must be tightly coupled with cell division, but the underlying molecular mechanisms are largely unknown. Using 3D cultures of renal MDCK cells (cysts), I found that the Rab35 GTPase plays a crucial role in polarity initiation and apical lumen positioning during the first cell division of cyst development. At the molecular level, Rab35 physically couples cytokinesis with the initiation of apico-basal polarity by tethering intracellular vesicles containing key apical determinants at the cleavage site. These vesicles transport aPKC, Cdc42, Crumbs3 and the lumen promoting factor Podocalyxin, and are tethered through a direct interaction between Rab35 and the cytoplasmic tail of Podocalyxin. Consequently, Rab35 inactivation leads to complete inversion of apico-basal polarity in 3D cysts. This novel and unconventional mode of Rab-dependent vesicle targeting provides a simple mechanism for triggering both initiation of apico-basal polarity and lumen opening at the centre of cysts.

Die Zebrafischmutation heart and mind (had), welche die alpha1B1-Untereinheit der Na+,K+ ATPase betrifft, verzögert die Streckung des Herzschlauches und führt zu weiteren Entwicklungsanomalien, die an andere Zellpolaritätsmutanten wie nagie oko (nok) und heart and soul (has) erinnern. In dieser Arbeit habe ich die Funktion und Regulation von Had/Na+,K+ ATPase während der Herzmorphogenese des Zebrafisches und seine möglichen Interaktionen mit Has/Prkci und Nok/Mpp5 untersucht. In konnte nachweisen, dass genetische Interaktionen zwischen had und nok in der Aufrechterhaltung von Zonula-Occludens-1-(ZO-1)-positiven Adhäsionsbändern Adhäsionsbändern in myokardialen Zellen während der Herzentwicklung nachweisen. Meine Ergebnisse deuten darauf hin, dass die Interaktion zwischen Nok/Mpp5 und Had/Na+,K+ ATPase zur Aufrechterhaltung der myokardialen ZO-1-Adhäsionsbändern die Funktion der Na-Pumpe erfordert und dass die korrekte Ionengradienten zur Aufrechterhaltung der myokardialen Integrität beiträgt. Meine Ergebnisse zeigen eine Phosphorylierung des N-terminalen Endes von Had/ Na+,K+ ATPase durch PKCs. PKCs wurden bereits mit der Regulation der Na-Pumpen-Funktion durch Phosphorylierung von N-terminalen Resten in Verbindung gebracht. Meine Ergebnisse legen die Möglichkeit nahe, dass dieser Mechanismus im Zebrafisch konserviert ist. Die Analyse der subzelluläre Lokalisation einer Phosphorylierungs-defizienten Form von Had/Na+,K+ ATPase legt nahe, dass während Herzschlauch-elongation die Had/Na+,K+ ATPase-Aktivität an der Zellmembran durch die Phosphorylierung an einer amino-terminalen Amino-säure reguliert wird. Frühere Studien legen nahe, dass die Herzmorphogenese durch direkte Phosphorylierung von Has/Prkci-Zielen gesteuert wird. Die Identifikation von Has/Prkci-Phosphorylierungs-Zielen könnte dazu beitragen, Herzmorphogenese besser zu verstehen. Aus diesem Grund wurde ein chemisch-genetischer Ansatz entwickelt, um direkte Phosphorylierungs-Ziele von Has/Prkci zu identifizieren. / The zebrafish heart and mind (had) mutation which disrupts the alpha1B1 subunit of Na+,K+ ATPase causes heart tube elongation defects and other developmental abnormalities that are reminiscent of several epithelial cell polarity mutants including nagie oko (nok) and heart and soul (has). In this work, I investigated the function and regulatory mechanisms of Had/Na+,K+ ATPase during zebrafish cardiac morphogenesis, as well as its´ possible interactions with Has/Prkci and Nok/Mpp5. In this study, I demonstrate genetic interactions between had and nok in maintaining Zonula occludens-1 (ZO-1) positive junction belts within myocardial cells during heart development. My results strongly suggest that the interaction between Nok/Mpp5 and Had/Na+,K+ ATPase in the maintenance of myocardial ZO-1 junction belts requires the Na pump function and that the correct ionic balance contributes to the maintenance of myocardial integrity. My results show phosphorylation of the N-terminal intracellular tail of Had/Na+,K+ ATPase by PKCs. PKCs have previously been implicated in the regulation of the Na pump function via phosphorylation of N-terminal residues. Therefore, my results raise the possibility that this mechanism is conserved in the zebrafish embryo. The analysis of the subcellular distribution of a phosphorylation-deficient form of Had/Na+,K+ ATPase suggests that, during heart tube elongation, Had/Na+,K+ ATPase activity is regulated at the membrane via phosphorylation at an amino-terminal site. Previous studies suggest that heart morphogenesis is driven via direct phosphorylation of Has/Prkci targets. Therefore, identification of Has/Prkci phosphorylation targets would contribute to better understand cardiac morphogenesis. For this purpose, a chemical genetic approach was designed to identify Has/Prcki direct phosphorylation targets.