Proefschrift Universiteit van Amsterdam. / Met lit. opg. - Met samenvatting in het Nederlands.

Univ., Diss.--Halle, 1994. / Literaturverz. Bl. 81 - 102.

Hohenheim, University, Diss., 2007.

Hohenheim, Univ., Diss., 2007.

University, Diss., 2001--Giessen.

Die Mikroglia entspricht in ihrer Gesamtpopulation und Funktion den ortsansässigen Makrophagen des zentralen Nervensystems. Sie nehmen sowohl in der Neuroinflammation und -degeneration als auch bei der Entwicklung und Erhaltung der neuronalen Funktion eine wichtige Rolle ein. Entwicklungsgeschichtlich stammen die Mikroglia, im Gegensatz zu anderen Makrophagen, wohl mehrheitlich von primitiven Dottersackmakrophage ab, welche in der frühen embryonalen Entwicklung in das zentrale Nervensystem immigrieren und dort zur endgültigen Mikrogliapopulation differenzieren. Dieses Konzept basiert vor allem auf den Arbeiten von Ginhoux et al. und Schulze et al. In diesen Arbeiten, die auf sogenannten Fate-Mapping-Experimenten basieren, kann die Abstammung der Mikroglia von Dottersackmakrophagen zwar demonstriert, der Einfluss anderer Vorläuferzellen jedoch nicht vollständig ausgeschlossen werden. Um den Einfluss einer nicht aus dem Dottersack stammenden Zelllinie auf die adulte Mikroglia zu untersuchen, nutzten wir daher Mäuse mit einem homozygoten knock-out der RNS Endoribonuklease Dicer, welche an die Expression von Vav1 gebunden ist. Der Vav1-Lokus wird exklusiv in hämatopoetischen Zellen aktiviert und dies zu einem Zeitpunkt, an dem die Rekrutierung der Mikroglia aus den Dottersackmakrophagen bereits vollständig abgeschlossen ist. Verglichen wurden daher in der vorgelegten Arbeit Vav1-Cre+:dicer knock-out Mäuse mit den entsprechenden Wildtyp Geschwistertieren zu prä- und postnatalen Zeitpunkten hinsichtlich der Anzahl an Iba-1 positiven Zellen. In makroskopischer Betrachtung konnten keine wesentlichen Unterschiede zwischen den Tieren beobachtet werden. Die Mäuse sind von vergleichbarem Gewicht und Größe. Im Erscheinungsbild zu postnatalen Zeitpunkten imponieren die knock-out Mäuse jedoch blass. Entsprechend hierzu stellt sich das Knochenmark mit deutlicher fibröser Involution dar, was in Blutuntersuchung als eine Panzytopenie der kock-out Mäuse diagnostiziert werden kann. Ein Umstand, der durch den Dicer knock-out in hämatopoetischen Zellen zu erwarten war. In lichtmikroskopischen Schnitten stellte sich die Gewebearchitektur des zentralen Nervensystems vergleichbar dar. In immunhistochemischen Färbung der Mikroglia mit Iba-1 als spezifisches Zielgen waren Form und Verteilung zwischen den Mäusen zwar vergleichbar, jedoch war die Anzahl an Mikroglia in den knock-out Tieren zum Zeitpunkt P1 um rund 40% vermindert. Es konnte ausgeschlossen werden, dass dies durch unterschiedliche Proliferation oder Apoptose der Mikroglia erklärt werden kann. Eine Zeitreihe mit den Zeitpunkten E16.8, E18.5, P0 und P1 konnte aufzeigen, dass sich bis einschließlich P0 kein Unterschied in der absoluten Mikrogliaanzahl zwischen Wildtyp und knock-out Mäusen festzustellen war. Untersucht wurden pro Zeitpunkt und Maus je vier Areale (Hippocampus, Mesencephalon, genu corporis callosi und ventrale hippocampale Kommissur). Ferner konnte gezeigt werden, dass die Mikroglia in den knock-out Mäusen die Mikroglia typischen Marker F4/80 und PU.1 exprimieren. Schlussendlich demonstriert die vorgelegte Arbeit, dass Störungen der definitiven Hämatopoese einen Einfluss auf die Mikrogliapopulation haben, was gegen die Annahme einer exklusiven Rekrutierung aller Mikroglia von Dottersackmakrophagen spricht.

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

Le sujet de ma thèse était de décrire sur le plan moléculaire le processus d'embryogenèse somatique chez la vigne. Pour cela, les étapes-clés d'entrée et de sortie du cycle d'embryogenèse secondaire ont été caractérisées par l'analyse de l'expression de quelques gènes impliqués dans le développement ou la défense, en particulier les gènes PR10.Grâce à l'exploitation de la séquence complète du génome de Vitis vinifèra disponible sur le site du Genoscope, j'ai pu caractériser exhaustivement la famille multigènique des PR10. Celle-ci est composée de 17 séquences disposées en tandem et formant un cluster compact sur le chromosome 5, dont 3 pseudogènes et au moins 13 séquences transcrites. L'expression de 10 de ces gènes a d'abord été analysée par RT-PCR semi-quantitative dans différents organes de la plante et dans des tissus traités au 2,4-D. Elle suggère une diversification fonctionnelle marquée. De plus, le niveau d'expression de plusieurs gènes PR10 est élevé dans les cals embryogènes, suggérant qu'ils pourraient jouer un rôle lors de l'embryogenèse somatique. L'étude de l'expression des gènes PR10 par RT-PCR quantitative en temps réel dans différents tissus ayant montré une capacité embryogénique variable lorsqu'ils sont soumis à un traitement par le 2,4-D met en évidence que le niveau d'expression varie entre les gènes et selon les tissus. L'expression de certains gènes est fortement induite par le 2,4-D dans les tissus à capacité embryogénique et seulement faiblement dans les tissus ne donnant jamais d'embryons somatiques, ce qui suggère fortement que ceux-ci pourraient être des marqueurs de la capacité embryogénique chez la vigne.

Universiẗat, Diss.--Halle, 1994.