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
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:29298 |
| Date | 10 April 2017 |
| Creators | Aurich, Franziska |
| Contributors | Dahmann, Christian, Knust, Elisabeth, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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