Abdominal sepsis is a common, life-threatening condition in the critically ill patients. The c-Jun N-terminal kinase (JNK) is known as a stress-activated protein kinase, in order to study the role of JNK on peritonitis-induced lung injury, the changes of plasma dihydrorhodamine 123 (DHR 123) oxidation level; the myeloperoxidase (MPO) and extravasations of Evans blue dye (EBD) of lung in wild-type (WT) mice with P. aeruginosa-induced peritonitis were determined first. Second, the specific JNK inhibitor, SP600125 or lefunomide, was given to WT mice immediately after P. aeruginosa injection and DHR oxidation, MPO activity, and EBD extravasations were examined. Third, JNK1-/- mice and JNK1+/- mice were subjected to peritonitis and assayed for DHR 123 oxidation, MPO activity, EBD extravasations, and reactive oxygen species (ROS). Fourth, chimeric mice (WT ¡÷ WT, JNK1-/- ¡÷ WT, WT¡÷JNK1-/-) were generated and used to determine the role of hematopoietic cells in peritonitis-induced lung damage. The results show that peritonitis induced DHR 123 oxidation; MPO activity and EBD extravasations in lungs and administration of specific JNK inhibitor decreased the peritonitis-induced DHR oxidation and lung damage. Also, both JNK1-/- and JNK1+/- mice showed a decreased DHR oxidation and lung damage after peritonitis. Finally, the decrease of DHR 123 oxidation, ROS, and lung damage in JNK1-/- ¡÷ WT chimeric mice suggests that that peritonitis-induced expression of iNOS and subsequent peroxynitrite production and lung damage depends on the JNK1 signaling of the hematopoietic cells.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20237号 / 医博第4196号 / 新制||医||1019(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 松原 和夫, 教授 渡邉 大, 教授 伊佐 正 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

Francisella tularensis is a highly virulent Gram-negative bacterium causing the zoonotic disease tularemia. Arthropod-borne transmission plays an important role in transferring the disease to humans. F. tularensis induces very low amounts of pro-inflammatory cytokines during infection, due to inhibition of immune signaling pathways and an unusual structure of its lipopolysaccharide (LPS). To date, there is no vaccine available that is approved for public use, although an attenuated live vaccine strain (LVS) is commonly used as a model of the more infectious Francisella strains. To produce an effective vaccine it is important to understand the lifecycle of F. tularensis, including the interaction with the arthropod hosts. Drosophila melanogaster is a widely used model organism, which is increasingly being used in host-pathogen interaction studies as the immune pathways in flies are evolutionary conserved to the immune pathways in humans. An important part of the immune defense of D. melanogaster as well as of arthropods in general is the production of antimicrobial peptides. These peptides primarily target the bacterial membrane, inhibiting bacterial proliferation or directly killing the bacteria. The aim of this thesis was to establish D. melanogaster as a model for F. tularensis infection and as a model for arthropod vectors of F. tularensis. Also, to use D. melanogaster to further study the interaction between F. tularensis and arthropod vectors, with specific regard to the host immune signaling and arthropod antimicrobial peptides. F. tularensis LVS infects and kills D. melanogaster in a dose-dependent manner. During an infection, bacteria are found inside fly hemocytes, phagocytic blood cells, similar as in human infections. In mammals genes of the intracellular growth locus (igl) are important for virulence. In this work it is shown that the igl genes are also important for virulence in flies. These results demonstrate that D. melanogaster can be used as a model to study F. tularensis-host interactions. LVS induces a prolonged activation of several immune signaling pathways in the fly, but seem to interfere with the JNK signaling pathway, similarly as in mammals. Overexpression of the JNK pathway in flies has a protective effect on fly survival. Relish mutant flies, essentially lacking a production of antimicrobial peptides, succumb quickly to a F. tularensis infection, however, F. tularensis is relatively resistant to individual D. melanogaster antimicrobial peptides. Overexpressing antimicrobial peptide genes in wildtype flies has a protective effect on F. tularensis infection, suggesting that a combination of several antimicrobial peptides is necessary to control F. tularensis. The production of numerous antimicrobial peptides might be why D. melanogaster survives relatively long after infection. An intact structure of the lipid A and of the Kdo core of Francisella LPS is necessary for resistance to antimicrobial peptides and full virulence in flies. These results are similar to previous studies in mammals. In contrast to studies in mammals, genes affecting the O-antigen of F. tularensis LPS are not necessary for virulence in flies. In conclusion, this thesis work shows that D. melanogaster can be used as a model for studying F. tularensis-host interactions. LVS activates several immune pathways during infection, but interfere with the JNK pathway. Overexpressing the JNK pathway results in increased survival of flies infected with LVS. Despite rather high resistance to individual antimicrobial peptides, exposure to a combination of several D. melanogaster antimicrobial peptides reduces the virulence of F. tularensis.

Mammalian cells can initiate intracellular signalling pathways that activate pro-survival changes to maintain their integrity following their exposure to a range of extracellular stresses. One group of changes preserves cellular integrity through the regulation of cytoskeletal organization. Despite the recognised importance of maintaining microtubule (MT) networks, the specific mechanisms regulating cytoskeleton organisation in response to stress remain relatively poorly explored. Among the numerous proteins that regulate MT organisation, stathmin (STMN) is a key MT destabilising protein that regulates MT disassembly through its ability to bind tubulin dimers. The actions of STMN can be regulated by a number of growth factor-activated and cell cycle regulatory protein kinases. In preliminary work, our studies suggest the potential regulation of STMN by c-Jun N-terminal Kinase (JNK) in cells exposed to stress. Specifically, we observed changes in STMN phosphorylation which were coordinated with JNK activation. / This project has explored the contribution of stress-activated c-Jun N-terminal Kinase (JNK) to STMN phosphorylation observed during osmotic stress. More detailed in vitro biochemical analysis has revealed that JNK directly phosphorylates STMN. In addition, we have compared STMN phosphorylation by different MAPK family member. In particular, our results illustrated that JNK predominantly phosphorylate STMN on serine residue 38 (S38) whereas ERK most likely targeted STMN S25. By examining specifically the phosphorylation of the four regulatory serine residues in vitro, we proposed a model of hierarchical phosphorylation among STMN serine residues. Specifically, our results demonstrated that phosphorylation of S38 was a pre-requisite for S25 phosphorylation by JNK in vitro. Furthermore, our results also demonstrated the impacts of JNK binding domain (JBD) and tubulin on STMN phosphorylation in vitro. Overall, this project identified STMN as a novel JNK substrate. The results have broadened our understanding on the JNK-mediated STMN phosphorylation as the first step to provide deeper insights into the different functions of JNK in the mammalian stress response.

The development of a stereotyped pattern of neural connectivity depends upon the behavior of growth cones, motile structures at the tips of axons that propel axon growth and steer the axon to its targets. When growth cones reach their appropriate target cells, they halt and ultimately remodel into stationary presynaptic boutons. The influence of extracellular cues in directing growth cones to their targets is well studied, but cell-intrinsic factors are also increasingly appreciated for their role in driving much of growth cone behavior. Dual leucine zipper kinases (DLKs) promote growth cone motility and must be kept in check to ensure normal development. PHR (Pam/Highwire/RPM-1) ubiquitin ligases therefore target DLK for proteosomal degradation unless axon injury occurs. Overall DLK levels decrease during development, but how DLK levels are regulated within a developing growth cone has not been examined. We analyzed the expression of the fly DLK Wallenda (Wnd) in R7 photoreceptor growth cones as they halt at their targets and as they remodel into presynaptic boutons. We found that Wnd protein levels are repressed by the PHR protein Highwire (Hiw) during R7 growth cone halting, as has been observed in other systems. However, during remodeling, Wnd levels are further repressed by a temporally-expressed transcription factor, Tramtrack69 (Ttk69). Previously unobserved negative feedback from JNK also contributes to Wnd repression. We conclude that maturing neurons progressively deploy additional mechanisms to keep DLK off and thereby protect their connectivity. We use live imaging to directly probe the effects of Wnd and Ttk69 on remodeling R7 growth cones and conclude that Ttk69 coordinates multiple regulators of this process. Preliminary results indicate that excess Wnd signaling requires the transcription factor Fos to disrupt growth cone remodeling in R7s. This opens up new strategies to identify how Wnd exerts its motility-promoting effects on growth cone cytoskeletons. Additional findings point to a later requirement for Wnd in normal R7 synapse development, suggesting that Wnd expression is not fully silenced in R7s. Further investigation into these findings would greatly advance our understanding of how the neuronal cytoskeleton is regulated as neurons undergo profound morphological and functional changes while developing. This dissertation includes both unpublished and published co-authored material. This dissertation also includes supplemental movie files, which can be found online and are described in Appendix B.

Selon une étude précédente, une limitation à l'efficacité anticancéreuse du 5-Fluorouracile (5-FU) repose sur la sécrétion d'IL-1β par des cellules myéloïdes immunosuppressives (MDSC). La libération d'IL-1β mature provient de l'activation de NLRP3 induite par le 5- FU et de l’augmentation de l’activité de la caspase-1 dans les MDSC, qui favorise la reprise de la croissance tumorale chez des souris traitées avec 5-FU. L'acide docosahexaénoïque (DHA) appartient à la famille des acides gras oméga-3 et possède des propriétés anticancéreuses et anti-inflammatoires qui pourraient améliorer la chimiothérapie à base de 5-FU. Dans ces travaux, nous démontrons que le DHA inhibe la sécrétion d'IL 1β induite par le 5 FU dans une lignée cellulaire de MDSC (MSC-2). Chez des souris porteuses de tumeurs traitées par 5 FU, nous avons montré qu'un régime alimentaire enrichi en DHA réduit la concentration d'IL 1β circulante et la récidive tumorale après une injection de 5 FU. Le traitement par 5 FU conduit à l'activation de JNK dans les MDSC et l'inhibiteur de JNK SP600125 diminue la sécrétion d’IL-1β. De plus, le DHA est capable de contrecarrer l'activation de JNK induite par 5-FU dans les MDSC, entraînant la chute de la libération de l’IL 1β. De plus, nous avons montré que la supplémentation en DHA dans les MDSC exposées au 5 FU diminuait l’activité de la caspase-1 ainsi que la modification des interactions entre NLRP3 et la caspase-1, ASC ou β-arrestine-2. De manière inattendue, la régulation de l'activité de la caspase-1 par le DHA était indépendante de JNK, ce qui suggère que le DHA pourrait contrôler la sécrétion de l’IL 1β par le biais de l'inflammasome NLRP3 et de la voie JNK. Enfin, nous avons trouvé une corrélation négative entre la teneur en DHA dans le plasma et l'induction du niveau d'IL 1β ou de la caspase-1 dans le sang de patients traités par chimiothérapie à base de 5-FU.L’ensemble de ces données fournissent de nouvelles informations sur la régulation de la sécrétion de l’IL-1β par le DHA et son bénéfice potentiel dans la chimiothérapie à base de 5-FU. / A limitation to 5-Fluorouracil (5-FU) anti-cancer efficacy relies on the secretion of IL-1β by myeloid-derived suppressor cells (MDSC) according to a previous pre-clinical report. The release of mature IL-1β originates from 5 FU mediated NLRP3 activation with increased caspase-1 activity in MDSC and sustains tumor growth recovery in 5 FU treated mice. Docosahexaenoic acid (DHA) belongs to omega-3 fatty acid family and harbors both anti cancer and anti inflammatory properties which might could improve 5 FU chemotherapy. Here, we demonstrate that DHA inhibits 5 FU induced IL 1β secretion produced by a MDSC cell line (MSC-2). In tumor-bearing mice treated with 5 FU, we showed that a DHA enriched diet reduces circulating IL 1β concentration and tumor recurrence after 5 FU injection. 5 FU treatment led to JNK activation in MDSC and JNK inhibitor SP600125 decreased IL 1β secretion. Moreover, DHA was able to counteract 5 FU mediated JNK activation in MDSC leading to the drop of IL 1β release. In addition, we showed that DHA supplementation in 5 FU exposed MDSC decreases caspase-1 activity along with a modification of the interactions between NLRP3 and caspase-1, ASC or β arrestin-2. Unexpectedly, the regulation of caspase-1 activity by DHA was independent of JNK which suggests that DHA could control IL 1β secretion through both NLRP3 inflammasome and JNK pathway. Interestingly, we found a negative correlation between DHA content in plasma and the induction of circulating IL 1β level or caspase-1 activity in patients treated with 5 FU based chemotherapy.Together, these data provide new insights on the regulation of IL 1β secretion by DHA and its potential benefit in 5-FU based chemotherapy.

PiT1/SLC20A1 a été identifiée pour la première fois comme récepteur rétroviral, puis de nombreuses autres études réalisées in vitro ont permis de révéler ces différentes fonctions. PiT1 est notamment un transporteur de phosphate-sodium dépendant. Par le biais de cette activité de transporteur de phosphate inorganique (Pi), PiT1 est impliqué dans plusieurs processus cellulaires comme la minéralisation osseuse, la calcification vasculaire (dans certaines pathologies), et la réabsorption rénale et intestinale de Pi. Dans notre laboratoire, afin de mieux caractériser les fonctions physiologiques de PiT1, un Knock Out (KO) total pour cette protéine a été généré. Les souris présentent un phénotype létal embryonnaire et une atteinte hépatique. Au vu de ces résultats, d’autres études, réalisées dans le laboratoire, ont mis en évidence l’implication de PiT1 dans la prolifération et l’apoptose cellulaire. Ces fonctions nouvellement décrites sont spécifiques à PiT1 et indépendantes de sa fonction de transporteur de Pi.Les objectifs majeurs de mon travail de thèse ont consisté à mieux comprendre le rôle de PiT1 dans les mécanismes apoptotiques déclenchés par le TNF-α. Pour cela, j’ai étudié la cascade d’activation du TNF-α dans les cellules Hela exprimant de manière stable un shPiT1 ou un shScramble. En effet, mes résultats suggèrent que l’association de TRAF2 ; un élément clé de l’activation de la voie des MAPKs ; à PiT1 par le bisais de sa large boucle intracellulaire, permettrait la dissociation des kinases en amont de la voie des MAPKs, GCK (MAP4K) et MLK3 (MAP3K), induisant leur désactivation et ainsi la régulation négative de JNK. La désactivation de JNK induit à son tour à l’inhibition des caspases et donc la signalisation apoptotique. Par ailleurs, j’ai montré que la suractivation de JNK dans les cellules invalidées pour PiT1 corrèle avec la dissocition antérieur du complexe TRAF2-cellular Inhibitor of Apoptosis Proteins (cIAPs). Ainsi, le complexe pro-apoptotique formé de la caspase-8, la protéine FAS-Associated via Death Domaine (FADD) et du Receptor Interactinf Protein 1 (RIP1) était plus actif.Par ailleurs, la boucle PiT1 et la boucle PiT2 ; son homologue ; ont été échangées, permettant ainsi l’obtention des protéines chimères P2-BclP1 et P1-BclP2. Celles-ci représenteraient des outils intéressants pour mieux comprendre les mécanismes cellulaires engagés dans cette voie apoptotique. Enfin, le rôle de PiT1 dans l’apoptose induite par le TNF-α étudié dans des modèles cellulaires a été confirmé dans deux modèles murins faisant ainsi et pour la première fois un lien entre PiT1 et une pathologie inflammatoire, l’hépatite fulminante. Ces résultats montrent que l’absence de PiT1 sensibilise le foie à l’apoptose; à l’appui d’autres résultats ; PiT1 pourrait être considéré comme une cible thérapeutique dans des pathologies inflammatoires et cancéreuses. / PiT1/SLC20A1 was identified for the first time as retroviral receptor then phosphate inorganic-dependent sodium transporter activity. Through this more a function of phosphate inorganic (Pi) transporter, PiT1 is involved in multiple cellular processes such as bone mineralization, vascular calcification, renal and intestinal reabsorption of Pi. In our laboratory, a total mice Knock Out (KO) for this gene encoding for PiT1 was generated to characterize its physiological functions. The embryonic mice PiT1 KO have a lethal phenotype through liver damage. We have previously found that additional transport-independent functions. PiT1 is involeved in proliferation and in the regulation of tumour necrosis factor (TNF)-induced apoptosis. Modulated cells was mediated by an increased activation of c-Jun N-terminal Kinase (JNK).The aim of my study was to define the role of PiT1 in apoptotic mechanisms of TNF-α signaling. For that, I have studied the regulation cascade of TNF-α pathway in Hela cells expressing shPiT1 or shScramble. My results suggest that intra-cytoplasmic loop domain of PiT1 was interact with TRAF2 ; a key element in the MAPK pathway activation. Furthermore, we also have shown that TNF-induced association of two JNK upstream kinases (Germinal Centre Kinase (GCK or MAP4K) and Mixed Lineage Kinase 3 (MLK3 or MAP3K) to PiT1 suggesting that PiT1 inducing their deactivation and thus down-regulation of JNK. Furthermore, we have shown that JNK increased signalling in PiT1-depleted cells correlates with the earlier dissociation of TRAF2 – cellular Inhibitor of Apoptosis Proteins (cIAPs) complexes. Thus, the apoptotic complex formed by caspase-8, Fas-Associated protein via Death Domain (FADD) and Receptor Interacting Protein 1 (RIP1) was more effectively activated. Moreover, PiT1 and PiT2 loops, were exchanged, thus allowing obtaining chimeric proteins BclP1-P2 and P1-BclP2. These proteins represent valuable tools to explore the mechanisms involved in the apoptotic pathway. Finally, we confirmed the relevance of these observations in vitro and showed that PiT1 gene conditional deletion in the liver of adult mice increases their sensitivity to fulminant hepatitis TNF-induced. These results are the first report of the involvement of PiT1 in a fatal pathology.

Le tube digestif est la première barrière contre les agresseurs présents dans la nourriture (virus, bactéries, produits chimiques, pesticides etc...). Il doit donc maintenir au mieux son intégrité structurale et fonctionnelle tout au long de la vie de l'individu. Bien que l'impact délétère d'une intoxication aiguë puisse être surmonté par la capacité de défense et de régénération de la muqueuse digestive, une agression prolongée ou répétée peut compromettre l'équilibre physiologique (l'homéostasie) du tube digestif. Parmi les agresseurs pouvant être ingérés avec la nourriture, on trouve la bactérie Bacillus thuringiensis (Bt) et représente 70% des ventes de bioinsecticides. Bt est une bactérie Gram+ sporulante qui produit, pendant la sporulation, des toxines nommées Cry. Parmi les différentes souches de Bt, certaines ont été sélectionnées pour la spécificité d'action de leurs toxines Cry contre des nuisibles et sont commercialisées sous forme de sporanges. Certaines de ces souches sont utilisées en agriculture biologique en France et l'accroissement de leur utilisation fait qu'elles sont de plus en plus présentes dans la nourriture, source de contamination potentielle pour l'homme et l’environnement. La question qui se pose maintenant est de savoir si un tel accroissement de l’utilisation de Bt peut avoir des impacts sur des espèces non cibles. Mon projet de thèse a consisté en l’étude des conséquences de l'ingestion de Bt (sous forme végétative ou de sporanges) sur la physiologie intestinale de la drosophile (animal non sensible à Bt en termes de toxicité aigüe / The digestive tract is continuously subjected to multiple aggressions through virus, bacteria, toxins and chemicals mixed in the feed. Therefore the gut lining has established a mechanism of replenishment in order to maintain the physiological function of the organ called the gut homeostasis. Although the deleterious impact of acute poisoning can be overcome by the defense capacity and regeneration of the gut mucosa, prolonged or repeated intoxication can impair its homeostasis. Among the aggressors hidden in the feed, there is the bacterium Bacillus thuringiensis (Bt). Bt is worldwide used as bioinsecticide. Indeed the multitude of Bt strains produces a broad range of crystalline toxins, named Cry toxins, which certain have been selected in organic farming owing to their lethal properties against specific pests. Because of incentive programs for sustainable development, the use of Bt bioinsecticides as an alternative to chemical pesticides will further increase in the next decades. Although the specificity of the acute toxicity of Cry toxins has been proved since many years, data are scarce on adverse effects that could result from chronic exposure. The question now is how far non-target organisms will be potentially impacted by the resulting augmentation of the Bt bacterium and its Cry toxins in the environment. To answer this challenge, I used Drosophila (a non-target organism) to study the impacts of Bt bioinsecticides on the gut physiology because 1/ the digestive tract is the main entrance for feed contaminated by Bt bioinsecticides and 2/ Bt and its toxins are known to impair the gut epithelium of sensitive pests

Many organs and structures formed during the embryonic morphogenesis of animals derive from epithelia. Epithelia are made up of apicobasally polarized cells which adhere to and communicate with each other, allowing for epithelial integrity and plasticity. During embryonic morphogenesis, epithelia change their shape and migrate in a coordinated manner. How these epithelial processes are regulated is still not fully understood. In a forward genetic screen using the embryo of the fruit fly Drosophila melanogaster, candidate genes influencing the morphogenesis of epithelial structures were identified. Three genes, CG17364, CG17362 and CG9040 were identified as possible regulators of lumen stability in the salivary glands, tubular organs deriving from the embryonic epithelium. Furthermore, the gene Cdep was found to play a crucial role in epithelial sheet migration during dorsal closure of the embryo. Embryos carrying genomic insertions that could affect the expression of CG17364, CG17362 and CG9040 show a luminal penotype of the embryonic salivary glands characterized by alternating bloated and seemingly closed sections. Therefore, one of these genes or a combination of them likely plays a role in stabilizing the salivary gland lumen. However, neither CG17364 nor CG17362 or CG9040 contain any known protein domains, hence their molecular roles remain unknown. Cdep (Chondrocyte-derived ezrin-like protein) is a member of the FERM-FA subclass of proteins. Proteins of the FERM family have been shown to interact with the plasma membrane and membrane-bound proteins as well as cytoskeleton components. Accordingly, they have been implicated in stabilizing the cell cortex, and some of them are involved in signal transduction mechanisms. In addition to a FERM domain, Cdep also contains a RhoGEF domain, although is still not clear whether it actually exerts GEF activity. Genomic insertions in the Cdep locus cause defects in embryonic dorsal closure and atypical migratory behaviour in epithelial tubes. In order to study the molecular role of Cdep, the CRISPR/Cas9 system was employed to establish loss-of-function mutants of Cdep. The mutants show aberrations in germ band retraction, dorsal closure and head involution. Moreover, I found that two mutants carrying a premature STOP codon in the Cdep ORF, CdepE16X and CdepG17X, rescue the defects observed in embryonic cuticles mutant for two other FERM-FA members yurt (yrt) and coracle (cora). A deletion of the full Cdep ORF did not rescue those defects. I hypothesize that CdepE16X and CdepG17X encode Cdep variants with increased activity, which compensates for the loss of yrt or cora function, respectively. In conclusion, this leads to a model in which Cdep acts in parallel to Yrt and Cora during Drosophila embryonic morphogenesis. Many of the defects described in this study are reminiscent of phenotypes found in embryos mutant for components and downstream effectors of the Jun-N-terminal Kinase (JNK) pathway. Hence, my work supports an earlier hypothesis according to which a mouse homologue of Cdep, Farp2, acts as an upstream activator of the JNK pathway during epithelial cell migration in vitro (Miyamoto et al., 2003) The data provided here shows that Cdep plays a role in the morphogenesis of a great number of epithelia-derived organs and structures in vivo. My study therefore elucidates a missing link between cell migration cues and JNK pathway activation.

Many organs and structures formed during the embryonic morphogenesis of animals derive from epithelia. Epithelia are made up of apicobasally polarized cells which adhere to and communicate with each other, allowing for epithelial integrity and plasticity. During embryonic morphogenesis, epithelia change their shape and migrate in a coordinated manner. How these epithelial processes are regulated is still not fully understood. In a forward genetic screen using the embryo of the fruit fly Drosophila melanogaster, candidate genes influencing the morphogenesis of epithelial structures were identified. Three genes, CG17364, CG17362 and CG9040 were identified as possible regulators of lumen stability in the salivary glands, tubular organs deriving from the embryonic epithelium. Furthermore, the gene Cdep was found to play a crucial role in epithelial sheet migration during dorsal closure of the embryo. Embryos carrying genomic insertions that could affect the expression of CG17364, CG17362 and CG9040 show a luminal penotype of the embryonic salivary glands characterized by alternating bloated and seemingly closed sections. Therefore, one of these genes or a combination of them likely plays a role in stabilizing the salivary gland lumen. However, neither CG17364 nor CG17362 or CG9040 contain any known protein domains, hence their molecular roles remain unknown. Cdep (Chondrocyte-derived ezrin-like protein) is a member of the FERM-FA subclass of proteins. Proteins of the FERM family have been shown to interact with the plasma membrane and membrane-bound proteins as well as cytoskeleton components. Accordingly, they have been implicated in stabilizing the cell cortex, and some of them are involved in signal transduction mechanisms. In addition to a FERM domain, Cdep also contains a RhoGEF domain, although is still not clear whether it actually exerts GEF activity. Genomic insertions in the Cdep locus cause defects in embryonic dorsal closure and atypical migratory behaviour in epithelial tubes. In order to study the molecular role of Cdep, the CRISPR/Cas9 system was employed to establish loss-of-function mutants of Cdep. The mutants show aberrations in germ band retraction, dorsal closure and head involution. Moreover, I found that two mutants carrying a premature STOP codon in the Cdep ORF, CdepE16X and CdepG17X, rescue the defects observed in embryonic cuticles mutant for two other FERM-FA members yurt (yrt) and coracle (cora). A deletion of the full Cdep ORF did not rescue those defects. I hypothesize that CdepE16X and CdepG17X encode Cdep variants with increased activity, which compensates for the loss of yrt or cora function, respectively. In conclusion, this leads to a model in which Cdep acts in parallel to Yrt and Cora during Drosophila embryonic morphogenesis. Many of the defects described in this study are reminiscent of phenotypes found in embryos mutant for components and downstream effectors of the Jun-N-terminal Kinase (JNK) pathway. Hence, my work supports an earlier hypothesis according to which a mouse homologue of Cdep, Farp2, acts as an upstream activator of the JNK pathway during epithelial cell migration in vitro (Miyamoto et al., 2003) The data provided here shows that Cdep plays a role in the morphogenesis of a great number of epithelia-derived organs and structures in vivo. My study therefore elucidates a missing link between cell migration cues and JNK pathway activation.:1 Introduction 1 1.1 Epithelial cell polarity 1 1.1.1 Cellularization and formation of the primary epithelium 1 1.1.1.1 Establishment of epithelial polarity and adhesion 2 1.1.2 The epithelial polarity network 3 1.1.3 Cell-cell adhesion 5 1.1.3.1 Adherens junctions 5 1.1.3.2 Septate junctions 6 1.2 Epithelial movements in Drosophila embryonic morphogenesis 6 1.2.1 Epithelial tube formation during Drosophila embryogenesis 7 1.2.2 Coordinated migration of epithelial sheets during Dros. embryogenesis 7 1.2.2.1 FERM domain proteins in epithelial migration 9 1.2.2.2 Cdep 10 1.3 Mutagenesis with the CRISPR/Cas9 system 12 2 Aim of My PhD Thesis Work 15 3 Preliminary Work 17 4 Results 19 4.1 A screen for novel regulators in Drosophila embryonic morphogenesis 19 4.1.1 Deficiencies on the left arm of chromosome 3 cause defects in SG lumen morphology 19 4.1.2 A locus in the overlap of two deficiencies on the right arm of chromosome 3 causes defects in Drosophila embryonic dorsal closure 20 4.2 Two uncharacterized genes regulate SG lumen diameter in Drosophila embryos 22 4.2.1 Mutations in two uncharacterized genes on chromosome 3 cause intermittent tube closure in the Drosophila SG 22 4.2.2 CG17362/ CG9040/ CG17364 play a role in the maintenance of SG lumen width after lumen expansion 24 4.2.3 CG17362 is exclusively expressed in the embryonic SG 25 4.2.4 CG17362 and CG9040 do not contain known protein domains and are only conserved in Drosophilidae 28 4.3 Loss of Cdep causes different defects in Drosophila embryonic morphogenesis 30 4.3.1 Insertions in the ORF of Cdep cause defects in DC and HI 30 4.3.2 Embryos transheterozygous for PBac{5HPw+}CdepB122 and Mi{MIC} CdepMI00496 show defects in the LE during DC 34 4.3.3 Insertions in the ORF of Cdep cause defects in tracheal and Malpighian tubule morphogenesis 34 4.3.4 The CRISPR/Cas9 system was used to generate loss-of-function mutants of Cdep 35 4.3.5 LOF mutants of Cdep show a variety of phenotypes 37 4.3.6 LOF mutants of Cdep cause denticle belt defects and ventral holes in Drosophila larval cuticles 38 4.3.7 Phenotypes in Cdep−/− mutant embryos are likely not caused by maternal defects 44 4.3.8 LOF mutants of Cdep cause segments to fuse 44 4.3.9 Defects in Cdep−/− mutants are not due to actin mislocalization 51 4.3.10 Cdep genetically interacts with yurt 51 4.3.11 Cdep genetically interacts with cora 55 5 Discussion 57 5.1 The role of CG17362 and CG9040 in SG lumen stability 57 5.1.1 Impairments in the expression of CG17362 and CG9040 could be the cause for the intermittent tube closure in the embryonic SGs 57 5.1.2 CG17362 and CG9040 could be necessary for SG lumen dilation and stability of lumen diameter 57 5.2 The role of Cdep in Drosophila embryonic morphogenesis 59 5.2.1 Cdep is a regulator of the JNK pathway 60 5.2.1.1 Mutations in TGF-_ pathway components cause LE bunching and gaps in the tracheal dorsal trunk 60 5.2.1.2 The JNK pathway, like Cdep, is instrumental in GBR, DC and HI 61 5.2.1.3 Mouse Farp2 acts upstream of the JNK pathway 61 5.2.2 Does Cdep act as a GEF? 62 5.2.3 Cdep might regulate epithelial migration in parallel to Yrt and Cora 63 5.3 Conclusion and Outlook 64 6 Materials and Methods 67 6.1 Cell strains, plasmids and DNA constructs 67 6.2 Culture media 68 6.2.1 Lysogeny Broth (Bertani, 1951) 68 6.2.2 Super Optimal Catabolite repression (SOC) medium (Hanahan, 1983) 68 6.3 Molecular biology methods 69 6.3.1 Amplifying DNA by standard PCR technology 69 6.3.2 Molecular cloning 70 6.3.2.1 Cloning with restriction endonucleases (Old and Primrose, 1980) 70 6.3.2.2 Gibson Assembly (Gibson et al., 2009) 70 6.3.3 Detecting DNA via agarose gel electrophoresis 71 6.3.4 Purifying DNA from E.coli 71 6.3.5 Extracting DNA from Drosophila adults 71 6.3.5.1 Extracting mRNA from Drosophila embryos and reverse transcription into cDNA 72 6.3.6 Making mRNA probes for in situ hybridization 72 6.3.7 Measuring DNA concentrations using the NanoDrop 72 6.3.8 DNA sequencing 72 6.3.9 Transformation 73 6.3.10 Mutagenesis of Cdep with the CRISPR-Cas9 system 73 6.3.10.1 CRISPR/Cas9 tools for mutagenesis in D.melanogaster 73 6.3.11 Strategies for CRISPR/Cas9 mutagensis 74 6.3.11.1 vectors and oligomers used for mutagenesis of Cdep via CRISPR-Cas9 system 75 pCFD3-dU6:3-CdepEx2gRNA 75 CdepSTOP-ssODN 76 pCFD4-CdepExc_2sgRNAs 77 pDsRed-5’HA-3’HA 78 6.3.11.2 Screening for successful mutagenesis events in flies modified with the CRISPR/Cas9 system 79 Insertion of an in-frame stop codon into the Cdep ORF 79 Replacing the entire Cdep locus with dsRed 80 6.3.12 Extracting protein from Drosophila embryos 83 6.3.13 Detecting and separating proteins by mass via SDS-PAGE 83 6.3.14 Detection of specific polypeptides by Western blot analysis (Towbin et al., 1979) 84 6.3.14.1 Transferring polypeptides to nitrocellulose membrane 84 6.3.14.2 Detecting specific polypeptides via immonublotting 85 6.4 Online tools for prediction of protein domains, interactions, sequence alignments, etc. 86 6.5 Cell culture growth and harvest 86 6.5.1 Growing E.coli cells for vector amplification 86 6.5.2 Cell harvest 86 6.6 Work with Drosophila melanogaster 87 6.6.1 Fly stocks used: deficiencies, alleles, duplications and balancers 88 6.6.2 Recombining alleles located on the same chromosome 93 6.6.3 Collecting Drosophila melanogaster embryos 93 6.6.4 Histochemistry 94 6.6.4.1 Embryo dechorionation 94 6.6.4.2 Embryo fixation for light microscopy of whole specimens 94 Heat fixation of Drosophila embryos 94 Formaldehyde fixation of Drosophila embryos 94 6.6.4.3 Embryo fixation for light microscopy of semi-thin sections (Tepass and Hartenstein, 1994) 95 6.6.4.4 Antibody staining of embryos for Immunofluorescence 95 6.6.4.5 Phalloidin and DAPI staining of embryos 96 6.6.4.6 Embryo mounting for analysis via fluorescence microscopy 96 6.6.4.7 Embryo mounting for live imaging 97 6.6.4.8 Embedding of embryos for semi-thin sectioning 97 6.6.4.9 Cuticle preparation from hatched and unhatched Drosophila larvae 97 6.6.4.10 Preparation and staining of ovarian follicles from Drosophila females 98 6.6.4.11 mRNA in situ hybridization of whole-mount Drosophila embryos 99