Orientador : Maria Luiza Silveira Mello / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-14T03:12:34Z (GMT). No. of bitstreams: 1 Avancini_RitaMariaPereira_D.pdf: 3157954 bytes, checksum: d5d7b827a0faad06d88206cde7070cb9 (MD5) Previous issue date: 1988 / Resumo: Os núcleos das células tróficas proximais de ovário de Chrysomya putoria, nas diferentes fases do desenvolvimento ovariano, foram examinados em preparações submetidas à reação de Feulgen. Tinha-se como objetivo conhecer as alterações cromatínicas/cromossômicas durante o desenvolvimento das células relacionadas ao processo de formação dos ovos. A alteração mais marcante encontrada foi a organização do material cromatínico sob a forma de cromossomos politênicos típicos. Nessa fase são vistas 6 unidades cromossômicas, nas quais, em determinado momento do seu processo de compactação, pode ser visualizado o padrão de bandas e interbandas característico de politênicos de dípteros. Após essa fase, os cromossomos tornam-se mais e mais encurtados, até que, quando todos apresentam praticamente o mesmo comprimento, os filamentos começam a se separar, originando numerosas unidades, de tamanhos diferentes entre elas que, espalham-se por todo o espaço nuclear. A partir daí, até o final do desenvolvimento, o núcleo tem aparência interfásica, apresentando um ponto heterocromático fortemente corado. A porção eucromática do cromossomo X sofre descompactação precoce, em relação aos autossomos e à sua própria porção heterocromática. Se a fêmea adulta estiver sob uma dieta de açúcar e água, os cromossomos politênicos podem permanecer como tal por um período muito mais longo do que quando sob dieta padrão. Através de microespectrofotometria mostrou-se que os núcleos tornam-se altamente endopoliplóides durante a ovogênese, atingindo o valor máximo de 2048 C, resultado de 10 ciclos de duplicação do DNA. Valores menores, a cada fase, foram obtidos nos núcleos distais, mostrando que há assincronia nos ciclos endorreplicativos das diferentes células tróficas do folículo. O volume ocupado pela cromatina corada aumenta cerca de 12 a 15 vezes durante o desenvolvimento. Os valores Feulgen-DNA bem como a área ocupada pelo corpo heterocromático não acompanham, proporcionalmente, o crescimento do restante da cromatina. As células epitelias do folículo mostraram tornarem-se também endopoliplóides, passando por até 4 ciclos de replicação, após o início da vitelogenese / Abstract: The nuclei of proximal nurse cells of the ovary of Chrysomya putoria in the different stages of ovarian development were examined in preparations submitted to the Feulgen reaction. The objective was to follow the chromatin/chromosomal alterations occurring during the development of those cells involved in the process of egg formation. The most marked alteration encountered was the organizatian of the chromatin in the form of typical polytene chromosomes. In this stage 6 chromosomes could be seen, in which, at a specific point in their process of compactation, the pattern of bands and interbands characteristic of dipteran polytene chromosomes, could be seen. After this stage the chromosomes became increasingly shorter until, when all were practically the same length, their constituent filaments began to separate and scatter throughout the nuclear space. From this point on, up to the end of development, the nucleus exhibited an interphasic appearance, presenting one strongly staining heterochromatic body. The euchromatic region of the X chromosome underwent premature decompactation as compared to the autosomes and to its own heterochromatic segment. If the adult female were maintained on a sugar and water diet, the polytene chromosomes would remain as such for a much longer period than when maintained on a standard diet. Microspectrophotometry showed that the nuclei became highly endopolyploidy during oogenesis, attaining a maximum value of 2048 C, resulting from 10 cycles of DNA duplication. At each stage, lower values were found in the distal nuclei, showing the existence of asynchrony in the endoreplicative cycles of the different nurse cells of the follicle. The volume occupied by the stained chromatin increased 12 or 15 times during development. Neither this Feulgen-DNA values nor the area occupied by the heterochromatic body proportionally followed the growth of the rest of the chromatin. The epithelial cells of the follicle also showed endopolyploidization, passing through up to 4 cycles of replication after the start of vitellogenesis / Doutorado / Doutor em Ciências

During animal development precise coordination and regulation of cell proliferation, differentiation and cell death is required for proper tissue organization. This is achieved through specific cell communication by intercellular signals. Cell death, for example, is a mechanism utilized by multicellular organisms for several developmental processes such as elimination of damaged cells or morphological shaping. Apoptosis can be induced by intrinsic signals generated within the cells or from extrinsic signals received from the surrounding environment. This work centered on the analysis of the mechanisms and signals that trigger apoptosis during Drosophila oogenesis. Drosophila ovaries are composed of approximately 16-20 ovarioles, each of which contains a series of egg chambers that are proceeding through the 14 stages of oogenesis moving from the germarium toward the oviduct. For the mature egg to be formed cell death has to occur at specific stages both in the germline (nurse cells) and in the somatic cells (follicle cells). However, whether this apoptosis is caused by intrinsic or extrinsic signals is not known. In addition to this developmentally controlled cell death, apoptosis can also be induced by environmental cues. Under starvation, for example, there is an increase of apoptosis at particular stages: in the germarium and at mid-oogenesis. Mid-oogenesis (stage8/9) is when vitellogenesis starts. At these stages the state of the egg chambers are checked in order to eliminate, through apoptosis of the germline, defective egg chambers. In starved flies, through activation of this “check-point”, oogenesis is blocked before vitellogeneis starts. It is not clear what are the signals that prevent apoptosis at mid-oogenesis in well-fed flies. In this study I have analyzed the function of a newly identified signaling molecule CG7447 during Drosophila oogenesis. My results indicate that CG7447 is required to prevent apoptosis at mid-oogenesis in well-fed flies. CG7447 RNA is only detectable in cells of the germarium, but not at later stages of oogenesis. When an HA tagged version of CG7447 (CG7447-HA) was expressed in the follicle cells of the germarium, this protein was found to be enriched in the oocyte during stages 2-8. These data suggest that CG7447 might be a secreted protein produced in the germarium, which is then secreted into the oocyte. To test the function of CG7447 during oogenesis, I generated a mutant allele. The mutation in CG7447 reduced female fertility. Mutant ovaries showed a block of egg chamber development at mid-oogenesis and this block correlated with apoptosis of the follicle cells. These mutant phenotypes could be reversed by expression of CG7447-HA, showing that these defects are due to the mutation in CG7447. Surprisingly, expression of CG7447-HA in the follicle cells only from stage 9 onward could restore fertility and normal oogenesis in a CG7447 mutant, indicating that CG7447 is required for follicle cell survival at later stages. Proper nutritional conditions are required to prevent apoptosis in the germline. Our data suggest that CG7447 is instead required to prevent apoptosis in the follicle cells. Thus, our analysis appears to have identified a novel signaling pathway that prevents survival of follicle cells in well-fed flies. Finally, our bioinformatic analysis showed that CG7447 is homologous to vertebrate EGF-like domain 7 (Egfl7) and EGF-like domain 8 (Egfl8) proteins. Importantly, expression of mouse Egfl7 or Egfl8 were able to confer normal oogenesis and fertility to CG7447 mutant flies. We therefore conclude that CG7447 is an evolutionary conserved protein and that CG7447 and Egfl7/8 share a common molecular function. CG7447 is a newly identified signaling molecule required during Drosophila oogenesis to promote the survival of follicle cells and to allow entry into vitellogenesis. Identification of the signaling cascade triggered by CG7447 will be important to more precisely understand its function during oogenesis. It may also help to reveal the molecular role of Egfl7/8 during vertebrate development.

Orientador : Angelo Pires do Prado / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-14T03:12:21Z (GMT). No. of bitstreams: 1 Avancini_RitaMariaPereira_M.pdf: 3969485 bytes, checksum: a69e9c937e04cea1b5e8f1041b1f12eb (MD5) Previous issue date: 1984 / Resumo: Neste estudo foram investigados alguns aspectos básicos da biologia reprodutiva de C. putoria mosca varejeira, da família Calliphoridae, de introdução recente no Brasil. As moscas foram mantidas em gaiolas de criação sob dieta padrão composta por fígado bovino cru (3 horas/dia) e água à vontade. Constatou-se que são necessários, no mínimo 6 dias para que as fêmeas tenham seus folículos ovarianos totalmente desenvolvidos o que ocorre de maneira sincrôni-ca. Este desenvolvimento é possível quando a fêmea recebe dieta protéica adequada, pois do contrário não se inicia a deposição de vitelo nos folículos. Portanto, sob dieta constituída por açúcar e água, C. putoria manifestou-se como a nautógena. Porém, mesmo após um período prolongado sob dieta aproteica, C. putoria manteve seu potencial reprodutivo, pois desenvolveu normalmente os folículos quando passou ter acesso ao fígado.Esta espécie foi capaz de desenvolver os folículos da segunda camada mesmo quando os primeiros não tinham ainda sido ovipostos, multiplicando dessa forma o mero de ovos a ser deixados numa postura. Os fatos acima mencionados podem ter uma ampla repercussão nas populações naturais, pois se aí também ocorrerem, sabemos que: em ausência de matéria orgânica protéica, as fêmeas não desenvolvem ovos, mas que quando o alimento volta a existir, o processo é retomado e, conseqüentemente, novos indivíduos surgirão; além disto, em ausência de condições ideais para oviposição, o estoque dos folícu-los maduros irá sendo multiplicado. Os ovários são do tipo meroístico politrófico contando em média com 76 folículos por ovário. Existem 15 células tróficas por folículo e o núcleo destas pode apresentar uma ploidia final de até 13 vezes a inicial. estimativa feita a partir das áreas das projeções dos núcleos obtidas de montagem total com coloração de Feulgen. No início do processo de multiplicação desse material nuclear são visíveis cromossomos politênicos típicos. Esses núcleos a-presentam um corpúsculo de DNA bastante evidente o qual não encontramos descrito para outras espécies da família. C putoria apresenta desenvolvimento ovariano bastante semelhante ao que ocorre para a espécie na Tan-zânia (África oriental) e com ela produz descendentes férteis.Este fato nos fez pensar na hipótese de sua origem a partir da África oriental e não da ocidental como anteri-ormente suposto. Julgamos que com o presente estudo serão oferecidos subsídios para bem fundamentar futuros programas de controle dessa espécie-praga / Abstract: This study was carried out to investigate some basic aspects concerning the reproductive biology of a blowfly, Chrydomya putoria (Ca11iphoridae) which has recently been introduced in Brazil.Adu1t flies were kept in cages and were fed on fresh cattle liver during 3 hours a day and water "adlibitum".Results showed that females presented a synchronic development of ovarian follicles, a process which took 6 days at least to be completed.No yolk deposition in the follicles was started out unless a proper protein diet has been supplied. Therefore, C. putoria was an anautogenous species when fed only with sugar and water. However C.putoria maintained its reproductive potential presenting normal follicle growth when liver was available even after along time under an aproteic diet. The concerned species was able to develop the second batch of eggs when the first one had not been laid yet. Thus, the arnount of eggs of a certain oviposition could be multiplied. Great changes in populations structure can be expected if the facts above also occur in the field since females do not complete oogenesis without proteic matter, but when it is present again in the field, eggs can be produced and, consequently, new flies will be risen. In addition, when ideal conditions for egg laying do not occur, there will be mature follicle batches multiplication. In this species, ovaries are classified as meroistic polytrophic, each one presenting 76 follicles in average. There are 15 nurse cells in a follicle and their nucleous may present a final ploidy 13 times greater than the initial one, which was estimated in view of the nucleous areas projections obtained in Feulgen-stained whole mounts. Typical polytenic chromosomes can be seen at the begining of this nuclear material multiplication. Those nuclei present a very remarkable DNA body which we did not find described for other alliphoridae species. C. putoria ovarian developrnent pattern is very similar to that of Tanzania (Eastern Africa) species. When these two species are crossed, fertile progeny is produced. This leads to the hypothesis of its origin from the Eastern Africa instead of the Western one as previously described. / Mestrado / Mestre em Biologia

Post-transcriptional regulatory mechanisms are widely used to influence cell fate decisions in germ cells, early embryos, and neurons. Many conserved cytoplasmic RNA regulatory proteins associate with each other and assemble on target mRNAs, forming ribonucleoprotein (RNP) complexes, to control the mRNAs translational output. How these RNA regulatory networks are orchestrated during development to regulate cell fate decisions remains elusive. We addressed this problem by focusing on Caenorhabditis elegans germline development, an exemplar of post-transcriptional control mechanisms. Here, we report the discovery of GLS-1, a new factor required for many aspects of germline development, including the oocyte cell fate in hermaphrodites and germline survival. We find that GLS-1 is a cytoplasmic protein that localizes in germ cells dynamically to germplasm (P) granules. Furthermore, its functions depend on its ability to form a protein complex with the RNA-binding Bicaudal-C ortholog GLD-3, a translational activator and P granule component important for similar germ cell fate decisions. Based on genetic epistasis experiments and in vitro competition experiments, we suggest that GLS-1 releases FBF/Pumilio from GLD-3 repression. This facilitates the sperm-to-oocyte switch, as liberated FBF represses the translation of mRNAs encoding spermatogenesis-promoting factors. Our proposed molecular mechanism is based on the GLS-1 protein acting as a molecular mimic of FBF/Pumilio. Furthermore, we suggest that a maternal GLS-1/GLD-3 complex in early embryos promotes the expression of mRNAs encoding germline survival factors. Our work identifies GLS-1 as a fundamental regulator of germline development. GLS-1 directs germ cell fate decisions by modulating the availability and activity of a single translational network component, GLD-3. Hence, the elucidation of the mechanisms underlying GLS-1 functions provides a new example of how conserved machinery can be developmentally manipulated to influence cell fate decisions and tissue development.

No description available.

Post-transcriptional regulatory mechanisms are widely used to influence cell fate decisions in germ cells, early embryos, and neurons. Many conserved cytoplasmic RNA regulatory proteins associate with each other and assemble on target mRNAs, forming ribonucleoprotein (RNP) complexes, to control the mRNAs translational output. How these RNA regulatory networks are orchestrated during development to regulate cell fate decisions remains elusive. We addressed this problem by focusing on Caenorhabditis elegans germline development, an exemplar of post-transcriptional control mechanisms. Here, we report the discovery of GLS-1, a new factor required for many aspects of germline development, including the oocyte cell fate in hermaphrodites and germline survival. We find that GLS-1 is a cytoplasmic protein that localizes in germ cells dynamically to germplasm (P) granules. Furthermore, its functions depend on its ability to form a protein complex with the RNA-binding Bicaudal-C ortholog GLD-3, a translational activator and P granule component important for similar germ cell fate decisions. Based on genetic epistasis experiments and in vitro competition experiments, we suggest that GLS-1 releases FBF/Pumilio from GLD-3 repression. This facilitates the sperm-to-oocyte switch, as liberated FBF represses the translation of mRNAs encoding spermatogenesis-promoting factors. Our proposed molecular mechanism is based on the GLS-1 protein acting as a molecular mimic of FBF/Pumilio. Furthermore, we suggest that a maternal GLS-1/GLD-3 complex in early embryos promotes the expression of mRNAs encoding germline survival factors. Our work identifies GLS-1 as a fundamental regulator of germline development. GLS-1 directs germ cell fate decisions by modulating the availability and activity of a single translational network component, GLD-3. Hence, the elucidation of the mechanisms underlying GLS-1 functions provides a new example of how conserved machinery can be developmentally manipulated to influence cell fate decisions and tissue development.

No description available.

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