No / P-cadherin serves as a major topobiological cue in mammalian epithelium. In human hair follicles (HFs), it is prominently expressed in the inner hair matrix that harbors the HF pigmentary unit. However, the role of P-cadherin in normal human pigmentation remains unknown. As patients with mutations in the gene that encodes P-cadherin show hypotrichosis and fair hair, we explored the hypothesis that P-cadherin may control HF pigmentation. When P-cadherin was silenced in melanogenically active organ-cultured human scalp HFs, this significantly reduced HF melanogenesis and tyrosinase activity as well as gene and/or protein expression of gp100, stem cell factor, c-Kit, and microphthalmia-associated transcription factor (MITF), both in situ and in isolated human HF melanocytes. Instead, epidermal pigmentation was unaffected by P-cadherin knockdown in organ-cultured human skin. In hair matrix keratinocytes, P-cadherin silencing reduced plasma membrane β-catenin, whereas glycogen synthase kinase 3 beta (GSK3β) and phospho-β-catenin expression were significantly upregulated. This suggests that P-cadherin-GSK3β/Wnt signaling is required for maintaining the expression of MITF to sustain intrafollicular melanogenesis. Thus, P-cadherin-mediated signaling is a melanocyte subtype-specific topobiological regulator of normal human pigmentation, possibly via GSK3β-mediated canonical Wnt signaling.

No / The aim of this study was to develop a synthetic hydrogel to act as a corneal substitute capable of selectively supporting the adhesion and proliferation of limbal epithelial cells (LECs) while inhibiting growth of limbal fibroblasts. Deficiency of LECs causes conjunctival epithelial cells to move over the cornea, producing a thick scar pannus. Unilateral defects can be treated using LEC cultured from the unaffected eye, transplanting them to the affected cornea after scar tissue is removed. The underlying wound bed is often damaged, however, hence the need to develop a corneal inlay to aid in corneal re-epithelialization. Transparent epoxy-functional polymethacrylate networks were synthesized using a combination of glycerol monomethacrylate, ethylene glycol dimethacrylate, lauryl methacrylate and glycidyl methacrylate that produced two different bulk hydrogel compositions with different equilibrium water contents (EWCs): Base 1 and Base 2, EWC=55% and 35%, respectively. Two sets of amine-functional hydrogels were produced following reaction of the epoxide groups with excesses of either ammonia, 1,2-diamino ethane, 1,3-diamino propane, 1,4-diamino butane or 1,6-diamino hexane. Neither series of hydrogels supported the proliferation of limbal fibroblasts irrespective of amine functionalization but they both supported the adhesion and proliferation of limbal epithelial cells, particularly when functionalized with 1,4-diamino butane. With Base 1 hydrogels (less so with Base 2) a vigorous epithelial outgrowth was seen from small limbal explants and a confluent epithelial layer was achieved in vitro within 6days. The data support the development of hydrogels specific for epithelial formation.

Yes / A major challenge in the production of 3D tissue engineered skin is the recreation of the basement membrane region to promote secure attachment and yet segregation of keratinocytes from the dermal substitute impregnated with fibroblasts. We have previously shown that simple electrospun scaffolds provide fibres on which the cells attach, proliferate, and self-sort into epithelium and dermis. In a development of this in this study tri-layered scaffolds were then electrospun from poly L-lactic acid and poly hydroxybutyrate-co-hydroxyvalerate. In these a central layer of the scaffolds comprising nano-porous/nano-fibrous poly hydroxybutyrate-co-hydroxyvalerate fibres was interwoven into the bulk micro-porous poly L-lactic acid microfibers to mimic the basement membrane. Keratinocytes and fibroblasts seeded onto these scaffolds and cultured for 2 weeks showed that neither cell type was able to cross the central nano-porous barrier (shown by SEM, and fluorescence monitoring with CellTracker™) while the micro-fibrous poly L-lactic acid provided a scaffold on which keratinocytes could create an epithelium and fibroblasts could create a dermal substitute depositing collagen. Although cells did not penetrate this barrier the interaction of cells was still evident-essential for epithelial development.

5-Oxo-ETE ist ein chemotaktischer Faktor für Granulozyten, der von der NADP+-abhängigen Dehydrogenase 5h-dh aus dem 5-Lipoxygenaseprodukt 5-HETE gebildet wird. Ziel dieser dreiteiligen Studie war es, die der 5-oxo-ETE-Produktion zugrunde liegenden Regulationsmechanismen aufzuklären. I. Einfluß von myeloider Zelldifferenzierung auf die Expression von 5h-dh in HL-60 und U-937 Zellen. Undifferenzierte HL-60 und U-937 Zellen produzieren vergleichbare Mengen von 5-oxo-ETE wie Monozyten oder Granulozyten. Differenzierung von U-937 Zellen mit PMA verdreifacht die Enzymaktivtät von 5h-dh, während die Behandlung von HL-60 Zellen mit dh-VitD3 diese verdoppelte. Der Einfluß von PMA auf 5h-dh wurde darüber hinaus in Mikrosomen von U-937 Zellen untersucht. Die Behandlung PMA verdreifachte Vmax, liess aber KM unbeeinflußt. II. Regulation der 5-oxo-ETE-Produktion durch oxidativen Stress und Glukose. Da der GSH-Redoxzyklus die Produktion von NADP+ zur Folge hat, stimulierten die Hydroperoxide H2O2 und tBOOH die Synthese von 5-oxo-ETE in U-937 Zellen. Aufgrund seiner Verarbeitung durch den Pentosephosphat Zyklus, der NAD+ in NADPH umwandelt, inhibierte Glucose diesen Effekt von H2O2. Die Synthese von 5-oxo-ETE wurde durch H2O2 auch in humanem Monozyten, Lymphocyten und Thrombozyten, aber nicht in Neutrophilen angeregt. Im Gegensatz zu Monozyten zeigten sich Thrombozyten und Lymphozyten allerdings glukose-resistent. T-BOOH ehöhte auch die Produktion von 5-oxo-ETE nach Zugabe von Ionophore und Arachidonsäure zu mononukleären Blutzellen. III. 5h-dh-Expression in human Strukturzellen. Zunächst rasterten wir mehrere sekundäre Epithelzelllinien und fanden 5h-dh in allen Zellen. Drei Indizien lassen vermuten, daß die epithele 5h-dh der myeloiden entspricht: (i) die enzymatische Aktivtät liegt vor allem in der mikrosomalen Fraktion vor, (ii) bei dem Kofaktor handelt es sich um NADP+ und nicht um NAD+, und (iii) 5S-HETE ist das bevorzugte Substrat. Weitere Studien zeigten, daß auch primäre humane Aorta-Endothelzellen 5h-dh expremieren. Vergleichbar zu Entzündungszellen wird die Produktion von 5-oxo-ETE auch in Endothel- und in Epithelzellen durch oxidativen Stress angeregt. / 5-Oxo-ETE is a highly potent granulocyte chemoattractant that is formed by the NADP+-dependent dehydrogenase 5h-dh by oxidation of the 5-lipoxygenase product 5-HETE. The objective of this study was to investigate underlying regulatory mechanisms of 5-oxo-ETE production in human cells. This matter was addressed from three directions. I. Expression of 5h-dh in HL-60 and U-937 cells and its activity changes during myeloid cell differentiation. Undifferentiated U-937 and HL-60 cells produce similar amounts of 5-oxo-ETE compared to monocytes or neutrophils. Differentiation of U-937 cells with PMA resulted in a 3-fold increase in 5-oxo-ETE production. Similarly, incubation of HL-60 cells with dh-VitD3 induced a 2-fold increase in 5-oxo-ETE production. The impact of PMA on 5h-dh was also investigated in the microsomal fraction of U-937 cells and compared to neutrophil microsomes. PMA treatment leads to a increase of Vmax but does not affect KM. II. Regulation of 5-oxo-ETE by oxidative stress and glucose levels. We found that H2O2 and t-butyl hydroperoxide strongly stimulate 5-oxo-ETE formation by U-937 cells through the GSH redox cycle by providing NADP+. Glucose inhibited the response to H2O2 through its metabolism by the pentose phosphate pathway, which converts NADP+ back to NADPH. 5-Oxo-ETE synthesis was also strongly stimulated by hydroperoxides in blood monocytes, lymphocytes, and platelets, but not neutrophils. Unlike monocytic cells, lymphocytes and platelets were resistant to the inhibitory effects of glucose. 5-Oxo-ETE synthesis following incubation of peripheral blood mononuclear cells with arachidonic acid and calcium ionophore was also strongly enhanced by t-BOOH. III. Expression of 5h-dh in human structural cells. We screened several secondary epithelial cell lines and detected 5h-dh in all cell lines. Epithelial 5h-dh and the inflammatory cell 5h-dh are identical: (i) the enzymatic activity is localized in microsomes, (ii) the cofactor is NADP+, and (iii) 5S-HETE is the preferred substrate. We also found that primary human aortic endothelial cells express 5h-dh. 5-oxo-ETE production by both endothelial and epithelial cells is regulated by oxidative stress in a manner similar to inflammatory cells.

Crohns sjukdom är en kronisk inflammatorisk tarmsjukdom av okänd orsak. Det tidigaste tecknet på Crohns sjukdom är mikroskopiska sår i det s.k. follikelassocierade epitelet (FAE) som täcker ansamlingar av immunceller i tarmen. FAE är specialiserat för att fånga innehåll från tarmen och transportera det till underliggande immunvävnad. Denna funktion är viktig för att inducera skyddande immunsvar, men den utgör också en ingångsväg för sjukdomsalstrande bakterier. Crohns sjukdom är associerat med ett kraftigt ökat immunsvar mot bakterier, och sjukdomsförloppet kan ändras av stress. Det övergripande syftet med avhandlingen var att studera effekterna av stress på FAE samt att undersöka rollen av FAE vid utvecklingen av tarminflammation, särskilt vid Crohns sjukdom. Inledningsvis studerades effekterna av psykologisk stress på FAE. Stressade råttor uppvisade ökad genomsläpplighet av bakterier efter stress, och passagen var högre i FAE än i vanligt epitel. Efterföljande experiment visade att stressförändringarna i slemhinnan regleras via kortikotropinfrisättande hormon och mastceller. Vidare visade det sig att vasoaktiv intestinal peptid kunde efterlikna stressens effekter på genomsläppligheten, och att detta kunde förhindras genom att blockera mastcellerna. Studier av tunntarmsslemhinna från patienter med icke-inflammatorisk tarmsjukdom och friska kontroller visade en högre passage av bakterier i FAE än i vanligt epitel. Hos patienter med Crohns sjukdom var bakteriepassagen genom FAE betydligt ökad jämfört med kontroller. Resultaten från detta avhandlingsarbete visar att stress kan förändra upptaget av bakterier från tarmen via FAE, med mekanismer som innefattar kortikotropinfrisättande hormon och mastceller. Detta har gett nya kunskaper kring regleringen av slemhinnebarriären. Vidare presenterar denna avhandling nya insikter i sjukdomsuppkomsten vid Crohns sjukdom genom att påvisa en tidigare okänd defekt i barriärfunktionen i FAE. / The earliest observable signs of Crohn’s disease are microscopic erosions in the follicle-associated epithelium (FAE) covering the Peyer’s patches. The FAE, which contains M cells, is specialised in sampling of luminal content and delivery to underlying immune cells. This sampling is crucial for induction of protective immune responses, but it also provides a route of entry for microorganisms into the mucosa. Crohn’s disease is associated with an increased immune response to bacteria, and the disease course can be altered by stress. The overall aim of this thesis was to study the effects of stress on the FAE and elucidate the role of FAE in the development of intestinal inflammation, specifically Crohn’s disease. Initially, rats were submitted to acute and chronic water avoidance stress to study the effects of psychological stress on the FAE. Stressed rats showed enhanced antigen and bacterial passage, and the passage was higher in FAE than in regular villus epithelium (VE). Further, stress gave rise to ultrastructural changes. Subsequent experiments revealed the stress-induced increase in permeability to be regulated by corticotropin-releasing hormone and mast cells. Furthermore, vasoactive intestinal peptide (VIP) mimicked the stress effects on permeability, and the VIP effects were inhibited by a mast cell stabiliser. Human studies of ileal mucosa from patients with non-inflammatory disease and healthy controls showed a higher antigen and bacterial passage in FAE than in VE. In patients with Crohn’s disease, the bacterial passage across the FAE was significantly increased compared to non-inflammatory and inflammatory controls (ulcerative colitis). Furthermore, there was an enhanced uptake of bacteria into dendritic cells, and augmented TNF-α release in Crohn’s disease mucosa. Taken together this thesis shows that stress can modulate the uptake of luminal antigens and bacteria via the FAE, through mechanisms involving CRH and mast cells. It further shows that human ileal FAE is functionally distinct from VE, and that Crohn’s disease patients exhibit enhanced FAE permeability compared to inflammatory and non-inflammatory controls. This thesis presents novel insights into regulation of the FAE barrier, as well as into the pathophysiology of Crohn’s disease by demonstrating a previously unrecognised defect of the FAE barrier function in ileal Crohn’s disease.

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.

Les voies aériennes humaines sont bordées d'un épithélium pseudostratifié composé principalement de cellules basales et de cellules pyramidales parmi lesquelles figurent les cellules sécrétrices de mucus et les cellules multiciliées. Toutes ces cellules contribuent à la clairance mucociliaire des voies respiratoires. Cet épithélium se régénère lentement dans des conditions homéostatiques, mais il est capable de se régénérer rapidement après agression grâce à des processus de prolifération, de migration, de polarisation et de différenciation. Chez les patients atteints de maladies respiratoires chroniques telles que la broncho-pneumopathie chronique obstructive, l'asthme ou la mucoviscidose, la réparation tissulaire est souvent défectueuse, caractérisée par une perte de cellules multiciliées et une hyperplasie des cellules sécrétrices, ayant pour conséquence une clairance mucociliaire affectée. La séquence des événements cellulaires conduisant à un tissu fonctionnel ou remodelé est encore mal décrite. Notre principal objectif a été d’identifier les types cellulaires successifs mis en jeu lors de la régénération tissulaire et les événements moléculaires responsables d'une régénération saine ou pathologique. Nous avons analysé la composition cellulaire de l’épithélium des voies respiratoires à plusieurs stades de différenciation en utilisant un modèle de culture 3D in vitro qui reproduit la composition cellulaire in vivo. En appliquant une méthode de transcriptomique sur cellule unique couplée à des méthodes bioinformatiques, nous avons établi les hiérarchies cellulaires permettant de reconstruire les différentes trajectoires cellulaires mises en jeu lors de la régénération de l’épithélium des voies respiratoires humaines. Après avoir confirmé les lignages cellulaires qui ont été précédemment décrits, nous avons découvert une nouvelle trajectoire reliant les cellules sécrétrices de mucus aux cellules multiciliées. Nous avons également caractérisé de nouvelles populations cellulaires et de nouveaux acteurs moléculaires impliqués dans le processus de régénération de l'épithélium des voies respiratoires humaines. Enfin, grâce à ces approches, nous avons mis en évidence des réponses spécifiques de chaque type cellulaire survenant dans des situations pathologiques d’hyperplasie sécrétoire. Ainsi, nos données, en apportant d'importantes contributions à la compréhension de la dynamique de différenciation de l’épithélium des voies respiratoires humaines, ouvrent de nouvelles voies vers l’identification de cibles thérapeutiques. / Human airways are lined by a pseudostratified epithelium mainly composed of basal and columnar cells, among these cells we can find multiciliated, secretory cells and goblet cells. All these cells work together in the mucociliary clearance of the airways. This epithelium regenerates slowly under homeostatic conditions but is able to recover quickly after aggressions through proliferation, migration, polarization and differentiation processes. However, in patients with chronic pulmonary diseases such as chronic obstructive pulmonary disease, asthma or cystic fibrosis, epithelial repair is defective, tissue remodeling occurs, leading to loss of multiciliated cells and goblet cell hyperplasia, impairing correct mucociliary clearance. The sequence of cellular events leading to a functional or remodelled tissue are still poorly described. Hence, we aim at identifying the successive cell types appearing during tissue regeneration and the molecular events that are responsible for healthy or pathological regeneration. We have analysed airway epithelial cell composition at several stages of differentiation using an in vitro 3D culture model which reproduces in vivo epithelial cell composition. Applying single cell transcriptomics and computational methods, we have identified cell lineage hierarchies and thus constructed a comprehensive cell trajectory roadmap in human airways. We have confirmed the cell lineages that have been previously described and have discovered a novel trajectory linking goblet cells to multiciliated cells. We have also discovered novel cell populations and molecular interactors involved in the process of healthy human airway epithelium regeneration. Using these approaches, we have finally shed light on cell-type specific responses involved in pathological goblet cell hyperplasia. Our data, by bringing significant contributions to the understanding of differentiation’s dynamics in the context of healthy and pathological human airway epithelium, may lead to the identification of novel therapeutic targets.

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

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In patients with age-related macular degeneration, a monolayer of cells in the eyes called retinal pigment epithelium differ from healthy ones in morphology. It is therefore important to quantify the morphological changes, which will help us better understand the physiology, disease progression and classification. Classification of the RPE morphometry has been accomplished with whole tissue data. In this work, we focused on the spatial aspect of RPE morphometric analysis. We used the second-order spatial analysis to reveal the distinct patterns of cell clustering between normal and diseased eyes for both simulated and experimental human RPE data. We classified the mouse genotype and age by the k-Nearest Neighbors algorithm. Radially aligned regions showed different classification power for several cell shape variables. Our proposed methods provide a useful addition to classification and prognosis of eye disease noninvasively.