Almost all animal cells maintain a thin layer of actin filaments and associated proteins underneath the cell membrane. The actomyosin cortex is subject to internal stress patterns which result from the spatiotemporally regulated activity of non-muscle myosin II motors in the actin network. We study how these active stresses drive changes in cell shape and flows within the cortical layer, and how these cytoskeletal deformations and flows govern processes such as cell migration, cell division and organelle transport. Following a continuum mechanics approach, we develop theoretical descriptions for three different cellular processes, to obtain - in collaboration with experimental groups - a detailed and quantitative understanding of the underlying cytoskeletal mechanics. We investigate the forces and cortex flows involved in adhesion-independent cell migration in confinement. Many types of cell migration rely on the extension of protrusions at the leading edge, where the cells attach to the substrate with specific focal adhesions, and pull themselves forward, exerting stresses in the kPa range. In confined environments however, cells exhibit migration modes which are independent of specific adhesions. Combining hydrodynamic theory, microfluidics and quantitative imaging of motile, non-adherent carcinosarcoma cells, we analyze the mechanical behavior of cells during adhesion-independent migration. We find that the accumulation of active myosin motors in the rear part of these cells results in a retrograde cortical flow as well as the contraction of the cell body in the rear and expansion in the front, and we describe how both processes contribute to the translocation of the cells, depending on the geometric and mechanical parameters of the system. Importantly, we find that the involved propulsive forces are several orders of magnitude lower than during adhesive motility while the achieved migration velocities are similar. Moreover, the distribution of forces on the substrate during non-adhesive migration is fundamentally different, giving rise to a positive force dipole. In contrast to adhesive migration modes, non-adhesive cells move by exerting pushing forces at the rear, acting to expand rather than contract their substrate as they move. These differences may strongly affect hydrodynamic and/or deformational interactions between collectively migrating cells. In addition to the work outlined above, we study contractile ring formation in the actin cytoskeleton before and during cell division. While in disordered actin networks, myosin motor activity gives rise to isotropic stresses, the alignment of actin filaments in the cortex during cell division introduces a preferred direction for motor-filament interactions, resulting in anisotropies in the cortical stress. Actin filaments align in myosin-dependent shear flows, resulting in possible feedback between motor activity, cortical flows and actin organization. We investigate how the mechanical interplay of these different cortical properties gives rise to the formation of a cleavage furrow during cell division, describing the level of actin filament alignment at different points on the cortex with a nematic order parameter, in analogy to liquid crystal physics. We show that cortical anisotropies arising from shear-flow induced alignment patterns are sufficient to drive the ingression of cellular furrows, even in the absence of localized biochemical myosin up-regulation. This mechanism explains the characteristic appearance of pseudocleavage furrows in polarizing cells. Finally, we study the characteristic nuclear movements in pseudostratified epithelia during development. These tissues consist of highly proliferative, tightly packed and elongated cells, with nuclei actively travelling to the apical side of the epithelium before each cell division. We explore how cytoskeletal properties act together with the mechanics of the surrounding tissue to control the shape of single cells embedded in the epithelium, and investigate potential mechanisms underlying the observed nuclear movements. These findings form a theoretical basis for a more detailed characterization of processes in pseudostratified epithelia. Taken together, we present a continuum mechanics description of the actomyosin cell cortex, and successfully apply it to several different cell biological processes. Combining our theory with experimental work from collaborating groups, we provide new insights into different aspects of cell mechanics.

Die initialen immunologischen Prozesse, die zur Entwicklung autoimmuner Lebererkrankungen führen, sind weitgehend unbekannt. Deshalb wurden in dieser Arbeit die Antigenpräsentation, die Migration sowie der Phänotyp in vivo aktivierter CD8 T-Zellen in der Leber anhand eines Mausmodells der autoimmunen Hepatitis untersucht. Es konnte gezeigt werden, dass hepatische dendritische Zellen an der Entstehung von CD8 Effektor-T-Zellen und an der Inflammation der Leber beteiligt sind. Kupffer-Zellen dagegen nehmen im autoimmunen Kontext in der Leber eine tolerogene Funktion ein. Die in vivo in der Leber aktivierten CD8 T-Zellen zeigten spezifische Oberflächenmarker und ein ungewöhnliches Migrationsverhalten. So wurde zum einen mit Neuropilin-1 ein weitgehend unbekannter Oberflächenmarker identifiziert, zum anderen spricht die Expression von bekannten Markern, die den Aktivierungsstatus der CD8 T-Zellen definieren, für einen hybriden Phänotyp. Sie besitzen sowohl Charakteristika von naiven CD8 T-Zellen als auch von Effektorzellen, eine Eigenschaft, die auch bei zentralen Gedächtniszellen gefunden wird. In der Leber aktivierte CD8 T-Zellen können nicht nur proinflammatorische Zytokine ausschütten und somit eine Inflammation in der Leber auslösen, sondern sind außerdem in der Lage durch Lymphknoten zu zirkulieren. Dagegen ist ihnen der Zugang zum Darm verwehrt, womit eine direkte regulatorische Funktion im Darm ausgeschlossen werden kann. Obwohl auf in der Leber aktivierten CD8 T-Zellen spezifische Adhäsionsmoleküle identifiziert wurden, existiert keine exklusive gewebespezifische Migration in die Leber, wie sie etwa für im Darm aktivierte CD8 T-Zellen nachgewiesen wurde. Im darmassoziierten lymphatischen Gewebe aktivierte CD8 T-Zellen akkumulieren in der Leber und tragen möglicherweise zur Schädigung der Leber im Rahmen chronisch entzündlicher Darmerkrankungen bei. Diese Arbeit trägt somit zum besseren Verständnis der Entstehung autoimmuner Prozesse in der Leber bei. / Initial immunological processes leading to autoimmune liver diseases are largely unknown. Therefore this thesis analyzed the antigen presentation, the migration as well as the phenotype of in vivo activated CD8 T cells in the liver by employing a mouse model for autoimmune hepatitis. It was shown that hepatic dendritic cells are effective antigen-presenting cells, which contribute to the induction of functional effector CD8 T cells in the liver and hepatitis. In contrast, Kupffer cells have a tolerogenic role during autoimmune processes in the liver. CD8 T cells that were in vivo activated in the liver display specific surface markers and unusual migration patterns. On the one hand an unusual surface molecule Neuropilin-1 was identified, on the other hand expression of well-known markers defining the activation-status of CD8 T cells suggests a hybrid phenotype. They reflect aspects of naive and effector T cells, characteristics also found on central memory T cells. Liver-primed CD8 T cells do not only produce pro-inflammatory cytokines leading to hepatitis, but they also retain their ability to circulate through lymph nodes. However, they have no access to the gut, which suggests that a direct regulatory function in the gut can be excluded. Although specific adhesion molecules on CD8 T cells activated in the liver were identified, no exclusive tissue-specific migration into the liver exists, as was shown for CD8 T cells primed in the gut. CD8 T cells activated in the gut-associated lymphoid tissue accumulate in the liver, in principle enabling them to induce liver pathology in the context of inflammatory bowel disease. Thus, the here described findings contribute to the understanding of initial immunological processes in autoimmune liver diseases.

During zebrafish gastrulation, highly coordinated cellular rearrangements lead to the formation of the three germ layers, ectoderm, mesoderm and endoderm. Recent studies have identified silberblick (slb/wnt11) as a key molecule that regulates gastrulation movement through a conserved pathway, which shares significant similarity with a signalling pathway that establishes epithelial planar cell polarity (PCP) in Drosophila (Heisenberg et al., 2000; Veeman et al., 2003), suggesting a role for cell polarity in regulating gastrulation movements. However, the cellular and molecular mechanisms by which slb/wnt11 functions during zebrafish gastrulation are still not fully understood. In the first part of the thesis, the three-dimensional movement and morphology of individual cells in living embryos during the course of gastrulation were recorded and analysed using high resolution confocal microscopy. It was shown that in slb/wnt11 mutant embryos, hypoblast cells within the forming germ ring display slower, less directed migratory movements at the onset of gastrulation, which are accompanied by defects in the orientation of cellular processes along the individual movement directions of these cells. The net movement direction of the cells is not changed, suggesting that slb/wnt11-mediated orientation of cellular processes serves to facilitate and stabilize cell movements during gastrulation. By using an in vitro reaggregation assay on mesendodermal cells, combined with an analysis of the endogenous expression levels and distribution of E-cadherin in zebrafish embryos at the onset of gastrulation, E-cadherin mediated adhesion was found to be a downstream mechanism regulating slb/wnt11 function during gastrulation. Interestingly, the effects of slb/wnt11 on cell adhesion appear to be dependent on Rab5-mediated endocytosis, suggesting endocytic turnover of cell-cell contacts as one possible mechanism through which slb/wnt11 mediates its effects on gastrulation movements. - Die Druckexemplare enthalten jeweils eine CD-ROM als Anlagenteil: QuickTimeMovies (ca. 23 MB)- Übersicht über Inhalte siehe Dissertation S. 92 - 93"

Prominin-1 (alias CD133) has received considerable interest because of its expression by several stem and progenitor cells originating from various sources, including the neural and hematopoietic systems. As a cell surface marker, prominin-1 is now used for somatic stem cell isolation. Its expression in cancer stem cells has broadened its clinical value, as it might be useful to outline new prospects for more effective cancer therapies by targeting tumor-initiating cells. Cell biological studies of this molecule have demonstrated that it is specifically concentrated in various membrane structures that protrude from the planar areas of the plasmalemma. Prominin-1 binds to the plasma membrane cholesterol and is associated with a particular membrane microdomain in a cholesterol-dependent manner. Although its physiological function is not yet determined, it is becoming clear that this cell surface protein, as a unique marker of both plasma membrane protrusions and membrane microdomains, might reveal new aspects of the cell biology of rare stem and cancer stem cells. The aim of this review is to outline the recent discoveries regarding the dynamic reorganization of the plasma membrane of rare CD133+ hematopoietic progenitor cells during cell migration and division. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

This thesis describes a highly interdisciplinary approach to discern the differing impact of scaffold dimensionality and physical space restrictions on the behavior of single cells. Rolled-up nanotechnology is employed to fabricate three-dimensional (3D) SiO/SiO2 microtube geometries of varied diameter, that after a biofunctionalization step are shown to support the growth of U2OS and six different types of stem cells. Cell confinement quantifiable through the given microtube diameter is tolerated by U2OS cells through a remarkable elongation of the cell body and nucleus down to a certain threshold, while the integrity of the DNA is maintained. This confinement for NSPCs also leads to the approaching of the in vivo morphology, underlining the space-restrictive property of live tissue. The dimensionality of the cell culture scaffold however is identified as the major determiner of NSPC migration characteristics and leads to a morphologically distinct mesenchymal to amoeboid migration mode transition. The 3D microtube migration is characterized by exclusively filopodia protrusion formation, a higher dependence on actin polymerization and adopts aspects of in vivo-reported saltatory movement. The reported findings contribute to the determination of biomaterial scaffold design principles and advance our current understanding of how physical properties of the extracellular environment affect cell migration characteristics.

Almost all animal cells maintain a thin layer of actin filaments and associated proteins underneath the cell membrane. The actomyosin cortex is subject to internal stress patterns which result from the spatiotemporally regulated activity of non-muscle myosin II motors in the actin network. We study how these active stresses drive changes in cell shape and flows within the cortical layer, and how these cytoskeletal deformations and flows govern processes such as cell migration, cell division and organelle transport. Following a continuum mechanics approach, we develop theoretical descriptions for three different cellular processes, to obtain - in collaboration with experimental groups - a detailed and quantitative understanding of the underlying cytoskeletal mechanics. We investigate the forces and cortex flows involved in adhesion-independent cell migration in confinement. Many types of cell migration rely on the extension of protrusions at the leading edge, where the cells attach to the substrate with specific focal adhesions, and pull themselves forward, exerting stresses in the kPa range. In confined environments however, cells exhibit migration modes which are independent of specific adhesions. Combining hydrodynamic theory, microfluidics and quantitative imaging of motile, non-adherent carcinosarcoma cells, we analyze the mechanical behavior of cells during adhesion-independent migration. We find that the accumulation of active myosin motors in the rear part of these cells results in a retrograde cortical flow as well as the contraction of the cell body in the rear and expansion in the front, and we describe how both processes contribute to the translocation of the cells, depending on the geometric and mechanical parameters of the system. Importantly, we find that the involved propulsive forces are several orders of magnitude lower than during adhesive motility while the achieved migration velocities are similar. Moreover, the distribution of forces on the substrate during non-adhesive migration is fundamentally different, giving rise to a positive force dipole. In contrast to adhesive migration modes, non-adhesive cells move by exerting pushing forces at the rear, acting to expand rather than contract their substrate as they move. These differences may strongly affect hydrodynamic and/or deformational interactions between collectively migrating cells. In addition to the work outlined above, we study contractile ring formation in the actin cytoskeleton before and during cell division. While in disordered actin networks, myosin motor activity gives rise to isotropic stresses, the alignment of actin filaments in the cortex during cell division introduces a preferred direction for motor-filament interactions, resulting in anisotropies in the cortical stress. Actin filaments align in myosin-dependent shear flows, resulting in possible feedback between motor activity, cortical flows and actin organization. We investigate how the mechanical interplay of these different cortical properties gives rise to the formation of a cleavage furrow during cell division, describing the level of actin filament alignment at different points on the cortex with a nematic order parameter, in analogy to liquid crystal physics. We show that cortical anisotropies arising from shear-flow induced alignment patterns are sufficient to drive the ingression of cellular furrows, even in the absence of localized biochemical myosin up-regulation. This mechanism explains the characteristic appearance of pseudocleavage furrows in polarizing cells. Finally, we study the characteristic nuclear movements in pseudostratified epithelia during development. These tissues consist of highly proliferative, tightly packed and elongated cells, with nuclei actively travelling to the apical side of the epithelium before each cell division. We explore how cytoskeletal properties act together with the mechanics of the surrounding tissue to control the shape of single cells embedded in the epithelium, and investigate potential mechanisms underlying the observed nuclear movements. These findings form a theoretical basis for a more detailed characterization of processes in pseudostratified epithelia. Taken together, we present a continuum mechanics description of the actomyosin cell cortex, and successfully apply it to several different cell biological processes. Combining our theory with experimental work from collaborating groups, we provide new insights into different aspects of cell mechanics.

In den letzten Jahren gab es Fortschritte im Verständnis der Gefäßbildung bei Ereignissen wie Keimen, Lumenbildung und Gefäßstabilisierung. Nach der Bildung eines primitiven Plexus ist die Gefäßoptimierung und hierarchische Umwandlung der morphologischen Gefäße in einen reifen Plexus durch vaskuläres "Pruning" wenig verstanden. Unterschiedliche Blutflussprofile in nebeneinander angeordneten Gefäßen können Asymmetrien in der Scherspannung verursachen, was die Zellmigration in Bereichen mit höherem Fluss fördert und die Destabilisierung von Segmenten mit geringem Fluss induziert. Diese Studie basiert auf der Hypothese, dass funktionelle Gefäßnetzwerke und Umbildung durch flussgesteuerte Endothelzellmigration ausgelöst werden. Wie die zelluläre Erfassung physikalischer Kräfte integriert ist, um Informationen zu übertragen und das Verhalten von Zellen zu modifizieren, ist noch unbekannt. Die Studie untersucht die Regulation und Koordination durch RhoGTPase-Signale während der kollektiven endothelialen Migration aufgrund von Flüssigkeitskräften. RhoGTPasen ermöglichen die räumlich-zeitliche Koordination, langfristige Anpassung an den Fluss und morphologische Umgestaltung. Beeinträchtigungen von RhoGTPasen zeigen Defekte bei Zellmigration und kollektiver Koordination in Umgebungen mit freiem Rand und strömungsgetriebener Migration. Die Studie erläutert den Einfluss der RhoGTPase-Regulation der Verbindungsdynamik in Verbindung mit der Aktinorganisation, die für die mechanische Kopplung und endotheliale Reaktionsfähigkeit erforderlich ist. Insgesamt betont die Studie die Relevanz der räumlich-zeitlichen RhoGTPase-Kontrolle und der Aufrechterhaltung der mechanischen Kopplung zwischen Strömung und Migration für die kollektive Koordination als Reaktion auf hämodynamische Kräfte. / In recent years, there have been significant advances in understanding how new vessels form during events like sprouting, lumen formation, and vessel stabilization. Yet, after the formation of a basic network, the crucial step of rearranging vessels into a mature structure, known as vascular pruning, needs further investigation. It's suggested that different blood flow profiles in nearby vessels create imbalances in shear stress, leading to cell migration toward higher flow regions, destabilizing low-flow segments, and causing the collapse of redundant segments. This study, in line with existing literature, proposes that functional vascular networks and remodeling result from the flow-driven migration of endothelial cells. However, how cells precisely sense physical forces to regulate their behavior and coordinate migration in response to flow remains unknown. I explore the regulation and coordination of Rho GTPases during collective endothelial migration under fluid forces. Rho GTPases' coordination allows long-term adaptation to flow and morphological remodeling. Impairments in Rho GTPases reveal defects in cell migration and collective coordination during free-edge and flow-driven migration. Finally, I explain how Rho GTPases' regulation influences junctional dynamics and actin organization, crucial for mechanical coupling and endothelial responsiveness to flow. Overall, this study emphasizes the importance of controlling Rho GTPases over time and maintaining mechanical coupling between flow and migration for collective coordination in response to fluid forces.

Tissue elongation is an important requirement for proper tissue morphogenesis during animal development. The Drosophila egg chamber is an excellent model to study the molecular processes underlying tissue elongation. An egg chamber is composed of germline cells that are enveloped by a somatic follicle epithelium. While the egg chamber matures, the drastic increase of the egg chamber’s volume is accompanied by a shape change from round to oval. Egg chamber elongation coincides with a circumferential alignment of F-actin filaments, microtubules, and fibrils of the extracellular matrix (ECM). Additionally, egg chambers rotate around their future long axis. It has been proposed that this rotation aligns F-actin filaments and ECM fibrils. The circumferentially aligned F-actin and ECM fibrils form a molecular corset that promotes egg chamber elongation. The atypical cadherin Fat2 is required for egg chamber rotation, the circumferential alignment of F-actin, microtubules, and ECM fibrils and for egg chamber elongation. However, the molecular mechanisms by which Fat2 influences egg chamber elongation remain unknown. In my thesis I performed a structure-function analysis of Fat2. I generated a Fat2 version that lacks the intracellular region and a second version, which lacks both intracellular region and the transmembrane domain and tested their ability to compensate for Fat2 functions in fat2-/- mutant egg chambers. My results reveal that the intracellular region is required for the microtubule alignment, and for egg chamber rotation. In contrast, the intracellular region is not required for F-actin and ECM alignment, and for egg chamber elongation. Hence, my findings for the first time demonstrate that egg chamber rotation is not required for F-actin and ECM fibril alignment and that egg chamber elongation can occur independently from egg chamber rotation. My work uncouples some of the parallel processes that take place during oogenesis and changes the view on the mechanisms that drive tissue elongation in this important model system. / Das Strecken von Geweben ist ein wichtiger Prozess bei der Gestaltbildung während der Entwicklung von Organismen. Die Eikammer von Drosophila ist ein hervorragendes Modellsystem, um die Gewebestreckung zu untersuchen. Eine Eikammer besteht aus Keimbahnzellen und einem einschichtigen Follikelepithel, das die Keimbahn umschließt. Während die Eikammer heranwächst durchläuft sie eine drastische Gestaltveränderung von rund nach oval. Zeitgleich zur Streckung der Eikammer weist das Follikelepithel parallel angeordnete F-actin−Filamente, Mikrotubuli und Fasern der extrazellulären Matrix (ECM) auf, welche die Eikammer ringsum umlaufen. Zudem rotieren die Eikammern um ihre zukünftige Längsachse. Bisher nahm man an, die Rotation würde für die Ausrichtung der F-actin−Filamente, Microtubuli und ECM-Fasern gebraucht werden. Die Anordnung der F-actin−Filamente und ECM-Fasern bilden dann ein molekulares Korsett, das die Gewebestreckung fördert. Das atypische Cadherin Fat2 wird für die Rotation der Eikammern, die umlaufende Anordnung der F-actin–Filamente, Microtubuli und ECM-Fasern sowie für die Streckung der Eikammern benötigt. Die Mechanismen, mit denen Fat2 die Gewebestreckung beeinflusst, sind allerdings unbekannt. In meinem Projekt führte ich eine Struktur-Funktions-Analyse von Fat2 durch. Ich generierte eine Version von Fat2 mit einer Deletion der kompletten intrazellulären Region und eine zweite, die weder die intrazelluläre Region noch die Transmembran-Domäne besitzt und testete, ob diese Versionen die Funktionen von Fat2 in fat2-/- mutanten Eikammern kompensieren können. Meine Ergebnisse zeigen, dass die intrazelluläre Region für die Anordnung der Mikrotubuli und für die Rotation der Eikammern gebraucht wird. Die intrazelluläre Region wird jedoch weder für die Anordnung von F-actin–Filamenten und den ECM-Fasern noch für die Streckung der Eikammer benötigt. Meine Erkenntnisse zeigen erstmalig, dass die Streckung der Eikammern ohne Rotation stattfinden kann. Meine Arbeit entkoppelt damit mehrere parallel stattfindende Prozesse während der Entwicklung der Eikammer und eröffnet einen neuen Einblick in die Mechanismen der Gewebestreckung in diesem wichtigen Modellsystem.

Tissue elongation is an important requirement for proper tissue morphogenesis during animal development. The Drosophila egg chamber is an excellent model to study the molecular processes underlying tissue elongation. An egg chamber is composed of germline cells that are enveloped by a somatic follicle epithelium. While the egg chamber matures, the drastic increase of the egg chamber’s volume is accompanied by a shape change from round to oval. Egg chamber elongation coincides with a circumferential alignment of F-actin filaments, microtubules, and fibrils of the extracellular matrix (ECM). Additionally, egg chambers rotate around their future long axis. It has been proposed that this rotation aligns F-actin filaments and ECM fibrils. The circumferentially aligned F-actin and ECM fibrils form a molecular corset that promotes egg chamber elongation. The atypical cadherin Fat2 is required for egg chamber rotation, the circumferential alignment of F-actin, microtubules, and ECM fibrils and for egg chamber elongation. However, the molecular mechanisms by which Fat2 influences egg chamber elongation remain unknown. In my thesis I performed a structure-function analysis of Fat2. I generated a Fat2 version that lacks the intracellular region and a second version, which lacks both intracellular region and the transmembrane domain and tested their ability to compensate for Fat2 functions in fat2-/- mutant egg chambers. My results reveal that the intracellular region is required for the microtubule alignment, and for egg chamber rotation. In contrast, the intracellular region is not required for F-actin and ECM alignment, and for egg chamber elongation. Hence, my findings for the first time demonstrate that egg chamber rotation is not required for F-actin and ECM fibril alignment and that egg chamber elongation can occur independently from egg chamber rotation. My work uncouples some of the parallel processes that take place during oogenesis and changes the view on the mechanisms that drive tissue elongation in this important model system.:1 ABSTRACT I 2 ZUSAMMENFASSUNG II 3 TABLE OF CONTENTS III 4 LISTS 7 4.1 List of Abbreviations 7 4.2 List of figures 9 5 INTRODUCTION 11 5.1 Tissue morphogenesis during development 11 5.1.1 Tissue organization by differential cell affinity 11 5.1.2 Cell adhesion is mediated by cadherins12 5.1.3 The cytoskeleton drives cell shape changes 13 5.1.4 Planar polarity is required for tissue-level directionality 17 5.2 Models of tissue elongation 19 5.2.1 Germ-band extension in Drosophila melanogaster 19 5.2.2 Primitive streak formation in the chick embryo 21 5.2.3 Neural tube formation in Xenopus 22 5.3 Drosophila egg chamber as a model system to study tissue morphogenesis 24 5.3.1 Oogenesis in Drosophila 24 5.3.2 Egg chamber as a model for tissue elongation 27 5.3.3 Planar polarized organization of the F-actin cytoskeleton in the follicle epithelium 29 5.3.4 Egg chamber elongation requires a link between extracellular matrix and F-actin cytoskeleton 32 5.3.5 Egg chamber rotation is proposed to be a requisite for egg chamber elongation 34 5.3.6 The atypical cadherin Fat2 provides a key role during egg chamber elongation 35 6 AIMS OF THE THESIS 38 7 MATERIALS AND METHODS 39 7.1 Fly husbandry 39 7.2 Used fly stocks 39 7.3 Phenotypic markers 40 7.4 Ovary dissection for fixation 40 7.5 Antibody stainings 41 7.6 Used antibodies 42 7.7 Drug treatment 42 7.8 Microscopy of fixed samples 43 7.9 Live imaging 43 7.9.1 Imaging of the basal F-actin oscillations 43 7.9.2 Imaging of egg chamber rotation 44 7.10 Generation of the transgenic fosmid constructs 44 7.10.1 General materials required for molecular genetics in E.coli 46 7.10.2 Step 1: Amplification of the tagging cassette 47 7.10.3 Step 2: Transformation of the helper plasmid pRedFlp4 49 7.10.4 Step 3: Red-operon driven insertion of the tagging cassette 50 7.10.5 Step 4: Removal of the KanR gene 50 7.10.6 Step 5: DNA isolation and verification of the correct transgenic construct 50 7.10.7 Step 6: Integration of the transgene into the fly genome 52 7.11 Image analysis and quantifications 53 7.11.1 Statistics 53 7.11.2 Aspect ratio measurements 54 7.11.3 Quantification of GFP localization55 7.11.4 Quantification of tissue-wide Collagen IV alignment 55 7.11.5 Quantification of tissue-wide angles of F-actin and microtubules 57 7.11.6 Analysis of periodicity of F-actin oscillations 58 7.11.7 Quantification of the rotation velocity of egg chambers 60 8 RESULTS 61 8.1 Expression of full-length fat2-GFP gene fully rescues all aspects of the fat258D mutant phenotype 61 8.1.1 Expression of the fat2-GFP gene rescues the fat258D mutant egg shape and sterility 61 8.1.2 Using an ‘Alignment parameter’ SAP to quantify the directionality of cytoskeletal structures and extracellular matrix fibrils 63 8.1.3 Expression of the fat2-GFP gene rescues microtubule alignment of fat258D mutant egg chambers65 8.1.4 Expression of the fat2-GFP gene rescues F-actin and Collagen IV alignment of fat258D mutant egg chambers 67 8.2 Generation of different fat2 mutant transgenes by homologous recombineering 70 8.3 The intracellular region of Fat2 is dispensable for some specific aspects of the Fat2 functions 73 8.3.1 The egg chamber elongation is independent of the intracellular region of Fat2 73 8.3.2. Localization of Fat2 protein depends on the intracellular region of Fat2 76 8.3.3 The alignment of microtubules is dependent on the intracellular region of the protein 78 8.3.4 The intracellular region of Fat2 is required for proper early F-actin and Collagen IV fibril alignment 81 8.3.5 The intracellular region of Fat2 is required for late F-actin and Collagen IV fibril alignment 85 8.3.6 F-actin filaments and ECM fibrils co-align in fat258D mutant stage 8 egg chambers 88 8.3.7 F-actin filaments and ECM fibrils do not co-align in fat258D mutant stage 10 egg chambers 90 8.3.8 The stability of basal F-actin fibers and Collagen IV fibrils mutually depend on each other at stage 8 92 8.3.9 The contractile pulses of F-actin in stage 9 egg chambers are independent of the intracellular region of Fat293 8.3.10 The intracellular region of Fat2 is required for proper egg chamber rotation in the early developmental stages96 8.3.11 The intracellular region of Fat2 is required for proper egg chamber rotation in later developmental stages 99 9 DISCUSSION 103 9.1 Egg chamber elongation can be uncoupled from egg chamber rotation 104 9.2 Egg chamber elongation correlates with a functional molecular corset 107 9.3 Fat2 promotes egg chamber elongation by its extracellular region 109 9.4 Alternative mechanisms potentially drive egg chamber elongation 111 9.5 New model of egg chamber elongation 114 9.6 Future perspectives 116 9.7 Impact on tissue morphogenesis in general 119 10 ACKNOWLEDGEMENTS 120 11 REFERENCES 121 12 APPENDIX 134 12.1 Script for “FFTAlignment.m" 134 12.2 Script for “Test1” 143 12.3 Script for “AverageCellAlignment.m" 143 / Das Strecken von Geweben ist ein wichtiger Prozess bei der Gestaltbildung während der Entwicklung von Organismen. Die Eikammer von Drosophila ist ein hervorragendes Modellsystem, um die Gewebestreckung zu untersuchen. Eine Eikammer besteht aus Keimbahnzellen und einem einschichtigen Follikelepithel, das die Keimbahn umschließt. Während die Eikammer heranwächst durchläuft sie eine drastische Gestaltveränderung von rund nach oval. Zeitgleich zur Streckung der Eikammer weist das Follikelepithel parallel angeordnete F-actin−Filamente, Mikrotubuli und Fasern der extrazellulären Matrix (ECM) auf, welche die Eikammer ringsum umlaufen. Zudem rotieren die Eikammern um ihre zukünftige Längsachse. Bisher nahm man an, die Rotation würde für die Ausrichtung der F-actin−Filamente, Microtubuli und ECM-Fasern gebraucht werden. Die Anordnung der F-actin−Filamente und ECM-Fasern bilden dann ein molekulares Korsett, das die Gewebestreckung fördert. Das atypische Cadherin Fat2 wird für die Rotation der Eikammern, die umlaufende Anordnung der F-actin–Filamente, Microtubuli und ECM-Fasern sowie für die Streckung der Eikammern benötigt. Die Mechanismen, mit denen Fat2 die Gewebestreckung beeinflusst, sind allerdings unbekannt. In meinem Projekt führte ich eine Struktur-Funktions-Analyse von Fat2 durch. Ich generierte eine Version von Fat2 mit einer Deletion der kompletten intrazellulären Region und eine zweite, die weder die intrazelluläre Region noch die Transmembran-Domäne besitzt und testete, ob diese Versionen die Funktionen von Fat2 in fat2-/- mutanten Eikammern kompensieren können. Meine Ergebnisse zeigen, dass die intrazelluläre Region für die Anordnung der Mikrotubuli und für die Rotation der Eikammern gebraucht wird. Die intrazelluläre Region wird jedoch weder für die Anordnung von F-actin–Filamenten und den ECM-Fasern noch für die Streckung der Eikammer benötigt. Meine Erkenntnisse zeigen erstmalig, dass die Streckung der Eikammern ohne Rotation stattfinden kann. Meine Arbeit entkoppelt damit mehrere parallel stattfindende Prozesse während der Entwicklung der Eikammer und eröffnet einen neuen Einblick in die Mechanismen der Gewebestreckung in diesem wichtigen Modellsystem.:1 ABSTRACT I 2 ZUSAMMENFASSUNG II 3 TABLE OF CONTENTS III 4 LISTS 7 4.1 List of Abbreviations 7 4.2 List of figures 9 5 INTRODUCTION 11 5.1 Tissue morphogenesis during development 11 5.1.1 Tissue organization by differential cell affinity 11 5.1.2 Cell adhesion is mediated by cadherins12 5.1.3 The cytoskeleton drives cell shape changes 13 5.1.4 Planar polarity is required for tissue-level directionality 17 5.2 Models of tissue elongation 19 5.2.1 Germ-band extension in Drosophila melanogaster 19 5.2.2 Primitive streak formation in the chick embryo 21 5.2.3 Neural tube formation in Xenopus 22 5.3 Drosophila egg chamber as a model system to study tissue morphogenesis 24 5.3.1 Oogenesis in Drosophila 24 5.3.2 Egg chamber as a model for tissue elongation 27 5.3.3 Planar polarized organization of the F-actin cytoskeleton in the follicle epithelium 29 5.3.4 Egg chamber elongation requires a link between extracellular matrix and F-actin cytoskeleton 32 5.3.5 Egg chamber rotation is proposed to be a requisite for egg chamber elongation 34 5.3.6 The atypical cadherin Fat2 provides a key role during egg chamber elongation 35 6 AIMS OF THE THESIS 38 7 MATERIALS AND METHODS 39 7.1 Fly husbandry 39 7.2 Used fly stocks 39 7.3 Phenotypic markers 40 7.4 Ovary dissection for fixation 40 7.5 Antibody stainings 41 7.6 Used antibodies 42 7.7 Drug treatment 42 7.8 Microscopy of fixed samples 43 7.9 Live imaging 43 7.9.1 Imaging of the basal F-actin oscillations 43 7.9.2 Imaging of egg chamber rotation 44 7.10 Generation of the transgenic fosmid constructs 44 7.10.1 General materials required for molecular genetics in E.coli 46 7.10.2 Step 1: Amplification of the tagging cassette 47 7.10.3 Step 2: Transformation of the helper plasmid pRedFlp4 49 7.10.4 Step 3: Red-operon driven insertion of the tagging cassette 50 7.10.5 Step 4: Removal of the KanR gene 50 7.10.6 Step 5: DNA isolation and verification of the correct transgenic construct 50 7.10.7 Step 6: Integration of the transgene into the fly genome 52 7.11 Image analysis and quantifications 53 7.11.1 Statistics 53 7.11.2 Aspect ratio measurements 54 7.11.3 Quantification of GFP localization55 7.11.4 Quantification of tissue-wide Collagen IV alignment 55 7.11.5 Quantification of tissue-wide angles of F-actin and microtubules 57 7.11.6 Analysis of periodicity of F-actin oscillations 58 7.11.7 Quantification of the rotation velocity of egg chambers 60 8 RESULTS 61 8.1 Expression of full-length fat2-GFP gene fully rescues all aspects of the fat258D mutant phenotype 61 8.1.1 Expression of the fat2-GFP gene rescues the fat258D mutant egg shape and sterility 61 8.1.2 Using an ‘Alignment parameter’ SAP to quantify the directionality of cytoskeletal structures and extracellular matrix fibrils 63 8.1.3 Expression of the fat2-GFP gene rescues microtubule alignment of fat258D mutant egg chambers65 8.1.4 Expression of the fat2-GFP gene rescues F-actin and Collagen IV alignment of fat258D mutant egg chambers 67 8.2 Generation of different fat2 mutant transgenes by homologous recombineering 70 8.3 The intracellular region of Fat2 is dispensable for some specific aspects of the Fat2 functions 73 8.3.1 The egg chamber elongation is independent of the intracellular region of Fat2 73 8.3.2. Localization of Fat2 protein depends on the intracellular region of Fat2 76 8.3.3 The alignment of microtubules is dependent on the intracellular region of the protein 78 8.3.4 The intracellular region of Fat2 is required for proper early F-actin and Collagen IV fibril alignment 81 8.3.5 The intracellular region of Fat2 is required for late F-actin and Collagen IV fibril alignment 85 8.3.6 F-actin filaments and ECM fibrils co-align in fat258D mutant stage 8 egg chambers 88 8.3.7 F-actin filaments and ECM fibrils do not co-align in fat258D mutant stage 10 egg chambers 90 8.3.8 The stability of basal F-actin fibers and Collagen IV fibrils mutually depend on each other at stage 8 92 8.3.9 The contractile pulses of F-actin in stage 9 egg chambers are independent of the intracellular region of Fat293 8.3.10 The intracellular region of Fat2 is required for proper egg chamber rotation in the early developmental stages96 8.3.11 The intracellular region of Fat2 is required for proper egg chamber rotation in later developmental stages 99 9 DISCUSSION 103 9.1 Egg chamber elongation can be uncoupled from egg chamber rotation 104 9.2 Egg chamber elongation correlates with a functional molecular corset 107 9.3 Fat2 promotes egg chamber elongation by its extracellular region 109 9.4 Alternative mechanisms potentially drive egg chamber elongation 111 9.5 New model of egg chamber elongation 114 9.6 Future perspectives 116 9.7 Impact on tissue morphogenesis in general 119 10 ACKNOWLEDGEMENTS 120 11 REFERENCES 121 12 APPENDIX 134 12.1 Script for “FFTAlignment.m" 134 12.2 Script for “Test1” 143 12.3 Script for “AverageCellAlignment.m" 143

During zebrafish gastrulation, highly coordinated cellular rearrangements lead to the formation of the three germ layers, ectoderm, mesoderm and endoderm. Recent studies have identified silberblick (slb/wnt11) as a key molecule that regulates gastrulation movement through a conserved pathway, which shares significant similarity with a signalling pathway that establishes epithelial planar cell polarity (PCP) in Drosophila (Heisenberg et al., 2000; Veeman et al., 2003), suggesting a role for cell polarity in regulating gastrulation movements. However, the cellular and molecular mechanisms by which slb/wnt11 functions during zebrafish gastrulation are still not fully understood. In the first part of the thesis, the three-dimensional movement and morphology of individual cells in living embryos during the course of gastrulation were recorded and analysed using high resolution confocal microscopy. It was shown that in slb/wnt11 mutant embryos, hypoblast cells within the forming germ ring display slower, less directed migratory movements at the onset of gastrulation, which are accompanied by defects in the orientation of cellular processes along the individual movement directions of these cells. The net movement direction of the cells is not changed, suggesting that slb/wnt11-mediated orientation of cellular processes serves to facilitate and stabilize cell movements during gastrulation. By using an in vitro reaggregation assay on mesendodermal cells, combined with an analysis of the endogenous expression levels and distribution of E-cadherin in zebrafish embryos at the onset of gastrulation, E-cadherin mediated adhesion was found to be a downstream mechanism regulating slb/wnt11 function during gastrulation. Interestingly, the effects of slb/wnt11 on cell adhesion appear to be dependent on Rab5-mediated endocytosis, suggesting endocytic turnover of cell-cell contacts as one possible mechanism through which slb/wnt11 mediates its effects on gastrulation movements. - Die Druckexemplare enthalten jeweils eine CD-ROM als Anlagenteil: QuickTimeMovies (ca. 23 MB)- Übersicht über Inhalte siehe Dissertation S. 92 - 93"