Pour comprendre les mécanismes de l'immunité innée, nous utilisons Caenorhabditis elegans comme un organism model host et coniospora Drechmeria comme un pathogène. D. coniospora adhère à la cuticule de C. elegans pour infecter son épiderme. Le ver répond par une régulation de gènes de défense multiples, y compris des gènes codant pour des peptides antimicrobiens (AMP) comme nlp-29. En utilisant des vers transgéniques portant des constructions rapporteurs fluorescents comme nlp-29p :: gfp, nous pouvons suivre l'expression des gènes in vivo AMP et de chercher des gènes nécessaires à l'induction de gènes AMP à travers les écrans génétiques. Le but de mon projet était de caractériser des mutants qui ont été identifiés dans un nouvel écran génétique saturée, où 57 nouveaux allèles Nipi qui manquent nlp-29 d'induction après l'infection ont été isolés. Adaptation d'une nouvelle cartographie combinée SNP et toute stratégie de séquençage du génome, nous avons pu isoler 15 allèles de gènes nouveaux Nipi déjà connus et 12 allèles de 6 «nouveaux» gènes. Notre travail a confirmé le rôle principal de la MAPK PKCδ/p38 dans la régulation de l'expression nlp-29 AMP après l'infection, ainsi que le facteur de transcription STA-2/STAT et le transporteur SNF-12/SLC6. Nous faire progresser nos connaissances en identifiant NIPI-4 comme un régulateur positif de l'expression des gènes nlp peptide antimicrobien après l'infection. NIPI-4 est un membre de la famille des kinases nématode spécifique et est prévu pour être un pseudokinase. Nous avons montré qu'il agit dans l'épiderme partie aval de la MAPK p38. / To understand the mechanisms of innate immunity, we use Caenorhabditis elegans as a model host and Drechmeria coniospora as a fungal pathogen. D. coniospora adheres to the cuticle of C. elegans to infect its epidermis. The worm responds by an up-regulation of multiple defence genes, including genes encoding anti-microbial peptides (AMP) like nlp-29. Using transgenic worms carrying fluorescent reporter constructs like nlp-29p::gfp, we can follow AMP gene expression in vivo and look for genes required for the induction of AMP genes through genetic screens. The aim of my project was to characterize mutants that have been identified in a new saturated genetic screen, where 57 new Nipi alleles that lack nlp-29 induction after infection were isolated. Adapting a new combined SNP mapping and whole genome sequencing strategy we were able to isolate 15 new alleles of previously known Nipi genes and 12 alleles of 6 “new” genes. Our work confirmed the primary role of the PKCδ/p38 MAPK in the regulation of nlp-29 AMP expression after infection, as well as the STA-2/STAT transcription factor and the SNF-12/SLC6 transporter. We further advance our knowledge by identifying NIPI-4 as a positive regulator of nlp antimicrobial peptide genes expression after infection. NIPI-4 is a member of a nematode-specific kinase family and is predicted to be a pseudokinase. We showed that it acts in the epidermis partially downstream of the p38 MAPK. It also controls the constitutive expression of antimicrobial peptide genes of the cnc family that are targets of TGFß regulation. Together these suggested that NIPI-4 acts with STA-2 and SNF-12 to regulate AMP gene expression in the epidermis.

The E3 ubiquitin ligase RNF43 (RING finger protein 43) is an important negative modulator of the WNT signalling pathway that acts at the plasma membrane by targeting Frizzled and its co-receptor LRP for degradation. In the small intestine, this prevents uncontrolled expansion of the stem cell compartment and so it is essential to the maintenance of normal tissue homeostasis. However, despite its crucial role in fine-tuning the WNT pathway and its role as a tumour suppressor, it is unclear whether RNF43 has further binding partners and what their functional relevance is to the modulation of WNT signalling. Here, I describe the development of a new screening strategy which combines CRISPR/Cas9 technology with 3D-intestinal organoid culture for the identification of novel molecular interactors of RNF43. Overall, this study and the technology developed provide a tool to enable the detailed description of the mechanism of action of RNF43, which is important not only in order to increase our understanding of WNT pathway regulation but also to gain potential new insights into RNF43 paralogs, by analogy. The investigation of paralogs is crucial as RNF43 belongs to a newly identified family of E3 ubiquitin ligases, named the PA-RING family, whose members are still poorly characterised. The majority of PA-RING family members have not been linked to any signalling pathway, most of their targets are still unknown and in many cases their in vivo function has not been addressed. In this context, my work has specifically focused on the investigation of the potential involvement of additional PA-RING family members in WNT pathway modulation and also on target identification for selected members. The results summarised in this dissertation show that no other PA-RING family member plays a prominent role in WNT pathway modulation aside from Rnf43 and its homologue Znrf3, however, different classes of adhesion molecules are likely to be regulated by certain of these E3 ligases. In conclusion, my work has contributed to unravelling previously unexplored aspects of this protein family, with particular regard to RNF43 and its mechanism of action. Thanks to this original approach, it was possible to identify potential new players involved either in membrane clearance of Frizzled or in RNF43 maturation. In particular, my thesis focuses on the characterisation of the role of DAAM in RNF43-mediated Frizzled internalisation.

Pectic polysaccharides, a class of plant cell wall polymers, form one of the most complex networks known in nature. Despite their complex structure and their importance in plant biology, little is known about the molecular mechanism of their biosynthesis, modification, and turnover, particularly their structure-function relationship. One way to gain insight into pectin metabolism is the identification of mutants with an altered pectin structure. Those were obtained by a recently developed pectinase-based genetic screen. Arabidopsis thaliana seedlings grown in liquid medium containing pectinase solutions exhibited particular phenotypes: they were dwarfed and slightly chlorotic. However, when genetically different A. thaliana seed populations (random T-DNA insertional populations as well as EMS-mutagenized populations and natural variations) were subjected to this treatment, individuals were identified that exhibit a different visible phenotype compared to wild type or other ecotypes and may thus contain a different pectin structure (pec-mutants). After confirming that the altered phenotype occurs only when the pectinase is present, the EMS mutants were subjected to a detailed cell wall analysis with particular emphasis on pectins. This suite of mutants identified in this study is a valuable resource for further analysis on how the pectin network is regulated, synthesized and modified. Flanking sequences of some of the T-DNA lines have pointed toward several interesting genes, one of which is PEC100. This gene encodes a putative sugar transporter gene, which, based on our data, is implicated in rhamnogalacturonan-I synthesis. The subcellular localization of PEC100 was studied by GFP fusion and this protein was found to be localized to the Golgi apparatus, the organelle where pectin biosynthesis occurs. Arabidopsis ecotype C24 was identified as a susceptible one when grown with pectinases in liquid culture and had a different oligogalacturonide mass profile when compared to ecotype Col-0. Pectic oligosaccharides have been postulated to be signal molecules involved in plant pathogen defense mechanisms. Indeed, C24 showed elevated accumulation of reactive oxygen species upon pectinase elicitation and had altered response to the pathogen Alternaria brassicicola in comparison to Col-0. Using a recombinant inbred line population three major QTLs were identified to be responsible for the susceptibility of C24 to pectinases. In a reverse genetic approach members of the qua2 (putative pectin methyltransferase) family were tested for potential target genes that affect pectin methyl-esterification. The list of these genes was determined by in silico study of the pattern of expression and co-expression of all 34 members of this family resulting in 6 candidate genes. For only for one of the 6 analyzed genes a difference in the oligogalacturonide mass profile was observed in the corresponding knock-out lines, confirming the hypothesis that the methyl-esterification pattern of pectin is fine tuned by members of this gene family. This study of pectic polysaccharides through forward and reverse genetic screens gave new insight into how pectin structure is regulated and modified, and how these modifications could influence pectin mediated signalling and pathogenicity. / Pektin Polysaccharide, eine Klasse pflanzlicher Zellwand Polymere, formen eine der komplexesten natürlichen Strukturen. Trotz seiner immensen Bedeutung in der Biologie der Pflanzen sind die Kenntisse über die molekularen Mechanismen der Pektin Biosynthese, dessen Modifikation und Abbau überraschend gering. Eine Möglichkeit neue Einblicke in den pflanzlichen Pektin Metabolismus zu erhalten, ist die Identifizierung von Mutanten mit veränderter Pektinstruktur. Solche Mutanten konnten durch ein neuatiges Selektionsverfahren gefunden werden. Zieht man Keimlinge der Ackerschmalwand (Arabidopsis thaliana) in Flüssigmedium mit Pektinase an, so lässt sich ein typischer Phänotyp beobachten: Die Pflanzen sind kleinwüchsig und leicht chlorotisch. Diesem Verfahren wurden Populationen verschiedener Genotypen (Insertions Linien, EMS Mutanten, natürlich vorkommende Varianten) ausgesetzt. Auf diese Weise wurden Individuen identifiziert, die gegenüber der Pektinase Behandlung eine verminderte oder erhöhte Resistenz aufweisen, was auf eine veränderte Pektinstruktur hindeutet. Die EMS Mutanten wurden einer detaillierten Zellwand Analyse unterzogen. die so in dieser Arbeit identifizierte Kollektion von Mutanten stellt eine wertvolle Ressource für weitere Forschungsansätze zur Regulation, Biosynthese und Modifikation des Pektins dar. Die Lokalisation der Insertionen in den T-DNA Linien führte zur Identifikation interessanter Gene, zu denen der putative Zuckertransporter PEC100 gehört. Dieses Gen steht vermutlich in Verbindung mit der Synthese von Rhamnogalakturonan-I, einem Bestandteil des Pektins. In dieser Arbeit konnte PEC100 im Golgi Apparat, dem Ort der Pektin Biosynthese, lokalisiert werden. Die natürlich vorkommende Variante C24 ist besonders empfindlich gegenüber der Pektinase. Diese Empfindlichkeit konnte anhand rekombinanter Inzucht Linien auf drei bedeutende quantitative Merkmalsloci (QTL) eingegrenzt werden. C24 zeigte zudem ein gegenüber der Referenz verändertes Massenprofil der Oligogalakturonide. Diese werden derzeit als Signalmoleküle in der pflanzlichen Pathogenabwehr diskutiert, was mit der in dieser Arbeit geseigten Resistenz von C24 gegenüber Schwarzfleckigkeit verursachende Pilz (Alternaria brassicicola) korreliert. In einem revers-genetischen Ansatz wurden zudem Mitglieder der Pektin Methyltransferase Familie als potentielle Enzyme getestet, die die Pektin Methylesterifikation beeinflussen könnten. Diese Mutation in einer dieser Methyltransferasen führte zu Veränderungen des Oligogalakturonid Massenprofils. Dies bestätigt die Hypothese, dass Mitglieder dieser Genfamilie an der Regulation der Methylesterifikation von Pektin beteiligt sind. Die vorliegende Studie, in der ein genetishen Selektionverfahren und Methoden der reversen Genetik kombiniert wurden, hat neue Einblicke in die Regulation und Modifikation von Pektin geliefert.

Hematopoiesis is an intricately regulated homeostatic process that maintains all of the differentiated blood cell lineages. N-ethyl-N-nitrosurea (ENU) is a powerful mutagen that induces point mutations randomly in the genome. ENU was used in a dominant forward genetic screen to identify novel mutations in regulators of hematopoiesis and to create new mouse models of hematopoietic disease. The objectives of this thesis were to characterize two mutants that originated from the dominant screen (7192 and 7238) and to develop a pharmacologically sensitized screen that would detect a unique set of mutations undetectable in the dominant screen. The 7192 mutant from the ENU dominant screen presented with elevated microcytic red blood cells (RBC) and increased polychromasia. The causative mutation was identified as a nonsense mutation in Ank1 (Q895X) that coded for a truncated ANK1 protein. Ank17192 is a novel mouse model of hereditary spherocytosis (HS), a human disease that results from increased RBC fragility. We have demonstrated that Ank17192/+ mice model a mild HS and Ank17192/7192 mice model severe HS. The 7238 mutant from the dominant ENU screen was macrothrombocytic and carried a missense mutation in Myh9 (Q1443L). The Myh97238/7238 mice are viable and have a more severe phenotype of macrothrombocytopenia. Myh97238 is the first mouse model for Myh9 related disorders that accurately models the genetic origins and the systemic manifestations of the disorder. A pharmacologically sensitized screen using chemotherapeutic drugs was designed to induce stress hematopoiesis to detect mutations that alter cell cycle of hematopoietic progenitors or stress hematopoiesis. Analysis of both peripheral blood and progenitor recovery kinetics, determined that 5-fluorouracil (5FU) and phenylhydrazine were good candidates for a pharmacologically sensitized screen. 5FU was successfully incorporated into an ENU dominant screen, and 13 platelet recovery outliers were detected. From these outliers, three mutant lines were successfully established.

Hematopoiesis is an intricately regulated homeostatic process that maintains all of the differentiated blood cell lineages. N-ethyl-N-nitrosurea (ENU) is a powerful mutagen that induces point mutations randomly in the genome. ENU was used in a dominant forward genetic screen to identify novel mutations in regulators of hematopoiesis and to create new mouse models of hematopoietic disease. The objectives of this thesis were to characterize two mutants that originated from the dominant screen (7192 and 7238) and to develop a pharmacologically sensitized screen that would detect a unique set of mutations undetectable in the dominant screen. The 7192 mutant from the ENU dominant screen presented with elevated microcytic red blood cells (RBC) and increased polychromasia. The causative mutation was identified as a nonsense mutation in Ank1 (Q895X) that coded for a truncated ANK1 protein. Ank17192 is a novel mouse model of hereditary spherocytosis (HS), a human disease that results from increased RBC fragility. We have demonstrated that Ank17192/+ mice model a mild HS and Ank17192/7192 mice model severe HS. The 7238 mutant from the dominant ENU screen was macrothrombocytic and carried a missense mutation in Myh9 (Q1443L). The Myh97238/7238 mice are viable and have a more severe phenotype of macrothrombocytopenia. Myh97238 is the first mouse model for Myh9 related disorders that accurately models the genetic origins and the systemic manifestations of the disorder. A pharmacologically sensitized screen using chemotherapeutic drugs was designed to induce stress hematopoiesis to detect mutations that alter cell cycle of hematopoietic progenitors or stress hematopoiesis. Analysis of both peripheral blood and progenitor recovery kinetics, determined that 5-fluorouracil (5FU) and phenylhydrazine were good candidates for a pharmacologically sensitized screen. 5FU was successfully incorporated into an ENU dominant screen, and 13 platelet recovery outliers were detected. From these outliers, three mutant lines were successfully established.

Chez Drosophila melanogaster, l’orientation horaire (dextrale) des organes est déterminée par un gène unique codant la Myosine non conventionnelle de type ID (MyoID). Un crible génétique modificateur en contexte sensibilisé pour myoID nous a permis d’identifier klp64D comme un gène interagissant génétiquement avec myoID. Celui-ci code l’une des sous-unités motrices du complexe moteur hétérotrimérique Kinésine-2 (Kin-2) constitué d’une autre sous-unité motrice Klp68D et d’une sous-unité adaptatrice Kap3. Nous montrons que klp68D interagit génétiquement avec myoID lors de la mise en place de l’axe D/G. Ceci suggère donc un rôle de l’ensemble du complexe Kin-2 dans l’asymétrie D/G. Chez les vertébrés, Kin-2 participe à l’assemblage des cils impliqués dans la détermination D/G lors de la gastrulation. Or, chez la drosophile, les cils ne sont pas requis dans la détermination D/G. MyoID et Kin-2 sont requis de manière synchrone dans la voie dextrale lors de la détermination D/G. En outre, Kin-2 joue un rôle important dans la rotation horaire du génitalia et l’enroulement dextral de l’intestin postérieur adulte (hindgut). Kin-2 est requise dans l’organisateur D/G de l’hindgut adulte pour l’orientation biaisée des cellules qui n’expriment pas MyoID. Par ailleurs, nos résultats suggèrent que l’activité de Kin-2 n’est pas requise dans le sous-ensemble de cellules qui exprime MyoID. Enfin, le rôle joué par Kin-2 dans l’asymétrie D/G semble indépendant de la polarité apico-basale et des jonctions adhérentes. Kin-2 pourrait donc jouer un rôle non ciliaire dans la phase de propagation de l’information directionnelle induite par MyoID. / In nature most of the bilateralia are left/right (L/R) asymmetric. In Drosophila, asymmetry is apparent in the directional looping of gut and terminalia. Dextral orientation of organs is controlled by the activity of a single gene myosin ID (myoID) whose mutation induces a fully inverted L/R axis. To date little is known of how the initial L/R cue induced by MyoID is propagated and maintained through the rest of the architecture of the L/R organizer. Here we present the identification of klp64D and klp68D as new myoID interacting genes. These genes encodes the two motor sub-units of the Drosophila Kinesin-2 motor complex. Interestingly, this microtubule-based motor plays a ciliary function in vertebrate L/R morphogenesis. However, we show that in Drosophila cilia are not involved in L/R asymmetry. We demonstrate that Kinesin-2 acts during L/R determination in the dextral pathway. Furthermore Kinesin-2 is required for proper L/R patterning both of male genitalia and of adult hindgut. L/R activity of Kinesin-2 is restricted to cells that do not express MyoID suggesting a role for this motor in propagation of the L/R cue. Our findings show for the first time a non ciliary role for Kinesin-2 in L/R axis determination. Thus, these results shed light on an evolutionary conservation between Drosophila and vertebrate L/R determination.

Biological tubes possessing a curvilinear form and a hollow interior exist in most multicellular eukaryotes. In Eumetazoa, the tubes usually comprise an eminently complex network and enable the transport and exchange of fluids and gases between tissues and organs, but also between organisms and their environment. Thus, tubular structures are both morphologically and physiologically integral parts of the animals. Based on a genetic screen for novel factors involved in heart tube differentiation and morphogenesis in Drosophila, the identified mutants were subdivided into several classes: cardiac hyperplasia (kuz and mam, both involved in the Notch-dependent cardiomyocyte specification, Publication 1); impaired cytokinesis (pav and tum, both components of the centralspindlin complex); a single ptc mutant showing a “truncated” heart (Publication 2); and a single loss-of-function mutant displaying reduced lumen diameter in epithelial tubes and perturbed secretion of ECM-components. The latter allele was mapped to the gene locus gartenzwerg (garz) that encodes a large ArfGEF. Due to its novelty, garz was selected as a central part of the thesis (Publication 3). Although garz seems to be expressed ubiquitously, its transcripts are abundant in active secreting cells of tubular structures. Moreover, mutations of garz abolish Golgi-integrity, cause massive retention of secretory cargo in the ER and arrest the apical transport of lipids and ECM molecules. As a consequence, lumen of the salivary glands and trachea fail to expand and show a decreased diameter. The observed phenotypes in tracheal network and salivary glands phenocopy those of COPI/COPII-subunits as well as actin-dependent secretion mutants, suggesting the underlying mechanism might be common. Thus, it is supposed that proper tubulogenesis needs Garz for initiation of the Arf1-COPI machinery. Furthermore, Golgi-based post-translational modifications, targeted sorting of vesicles, outward transport of proteins, or directed membrane delivery all depend on the secretory pathway, and such processes are essential in establishing polarized cells which build the tubular structures. In conclusion, this mechanism seems to be neither restricted to tubulogenesis nor specific to Drosophila. Due to the presence of garz homologues in every eukaryotic genomes, the Arf1/COPI based secretory pathway may play a universal role in metazoan development.

No description available.

No description available.

Spinal neurons are important in several aspects motor control. For example, the neurons essential for locomotor movements reside in the ventral spinal cord. In this thesis, different motor control functions are being related to neuronal populations defined by their common expression of a gene. First, a targeted disruption of the gene for vesicular glutamate transporter 2 (Vglut2/ Slc17a6) is described. The mutant animals die at birth because of their inability to breathe. The neuronal network in the brainstem, responsible for inspiration, was shown to become non-functional by the targeted deletion of Vglut2. To our surprise, it was still possible to induce rhythmic activity with normal left/right alternation in spinal cords isolated from VGLUT2-null embryos. Inconsistent reports of Vglut1 expression in the spinal cord made us re-evaluate the Vglut1 and Vglut2 expressions. While Vglut2 expression was widespread in the spinal cord, Vglut1 expression was restricted to a few cells dorsal to the central canal.  Taken together, the data suggest that, glutamatergic signaling is mandatory to drive the bilateral breathing, but not needed for coordination of basal alternating spinal locomotor rhythm. Next, a screen for genes with restricted ventral expression was made. Some of the genes found could be connected to the characteristics of specific neuronal cell populations. For example, fast motor neurons were shown to express the genes Calca and Chodl. Further, we found the Chrna2 expression selectively in putative Renshaw cells. It seems likely that the gene product, the alpha2 subunit of the nicotinergic receptor, could be linked to the unique connection of motor neurons to Renshaw cells. We used the Chrna2 promoter to drive expression of Cre recombinase in a transgenic mouse. The Cre activity was present in most neurons labeled with Renshaw cell markers, which should make it a useful tool for functional studies of this population. The studies presented here show how the genes expressed in subsets of neurons can be used to target populations of neurons for functional studies of neuronal systems.