Search

Gradients of morphogens, secreted signaling molecules, are crucial for providing cells with positional information during animal development. While the processes of forma- tion and interpretation of these gradients have been extensively studied, the impact of morphogenetic events on patterning through morphogen gradients remains largely unex- plored. This thesis aims to understand the interplay and feedback mechanisms between tissue shape and morphogen gradients formation. To address this, we developed an analysis pipeline using MATLAB to accurately measure morphogen gradients in curved epithelia. By computationally deforming confocal images of curved tissues, we quantified the levels of a protein of interest at a specified distance from a reference point along the apico-basal axis. Applying our pipeline to the Hedgehog morphogen gradient in the Drosophila eye and wing imaginal discs, which serve as model systems for folded and flat epithelial tissues, respectively, we made an intriguing discovery. Despite the distinct morphologies of these tissues, the decay rate of the Hedgehog gradient remained com- parable. This led us to investigate the robustness of Hedgehog gradient formation by manipulating the morphology of the wing and eye discs. We induced ectopic fold forma- tion at the boundary between the source and receiver tissue of Hedgehog in the wing disc. We found that the decay rate of the Hedgehog gradient remained unchanged even in the wing disc with perturbed morphology, supporting the notion that the Hedgehog gradient is robust towards variability in tissue shapes. Additionally, we locally flattened the eye disc by introducing a mutation that inhibited depolymerization of F-actin. This resulted in the inability of cells to form the morphogenetic furrow and in an expansion of the Hedgehog range compared to the wild-type. However, according to our quantifica- tion, the expansion in the Hedgehog range is to be attributed to a shift in its source rather than a c

Epithelial tissues are one of the most abundant tissues in our body. They make up essential organs like the gut, heart and eyes. These organs take up their complex 3D shapes during normal development of the embryo. Our understanding of such large-scale 3D shape changes is limited mainly due to the technical difficulties of imaging and quantifying such developmental events. In this thesis, I study two events in which epithelial monolayers change their 3D shape. In both the projects, I use data from light-sheet microscopic images of developmental events. These data are provided by my collaborators. In this thesis, I further analyzed them using quantitative approaches and interpreted them using computational models. In the first project, I study a case of a developing tissue inside a rigid confinement. A perfect model system for this is the Drosophila embryo which consists of an epithelial monolayer (blastoderm) inside a rigid shell (vitelline membrane). During gastrulation, the blastoderm is under compressional stresses due to tissue proliferation and compression from the germband extension. During this time, an invagination separating the future head and the trunk region appears. This is known as the cephalic furrow (CF). As the CF disappears after some time, its relevance in the normal development of the embryo is unclear. To understand its role, my collaborators image the blastoderm in mutant embryos which lack CF. These mutant embryos have either of the genes even-skipped (eve) or buttonhead (btd) knocked down. In the absence of CF, temporary ectopic folds appear in the blastoderm in locations which vary between embryos. Unlike the CF, ectopic folds appear suddenly and hence look like buckling events. I hypothesize that ectopic folds appear because of the compressive stresses generated in the blastoderm during the germband extension or by the compression of tissues that are adjacent to mitotic domains. Moreover, in normal embryos, CF, which is a controlled invagin

During morphogenesis, epithelial tissues undergo dramatic changes in shape, transitioning from flat sheets to three-dimensional folded structures. This remarkable transformation relies on dynamic changes in mechanical tension at both their apical and basal surfaces. While it is well-established that the generation of mechanical tension at the apical side is driven by the actomyosin network, research on this process has often overlooked the generation of mechanical tension at the basal surface. Moreover, the mechanical response to stress, encompassing both elastic (spring-like) and viscous (fluid-like) properties, is important for epithelial transformations, yet this mechanical response is poorly understood for the basal cell surface. In this thesis, we investigated how basal tension is influenced by the basement membrane - an extracellular matrix layer which has been widely regarded as a passive scaffold for cells. We probed the material mechanical response of the basement membrane and directly measured and analyzed basal tension in the wing imaginal disc epithelium of Drosophila. To study the mechanical response, I used long-term confocal imaging and fluorescence recovery after photobleaching (FRAP) to analyze the turnover and mobility of Collagen IV, a component of the basement membrane. The low Collagen IV mobility and turnover (≈ 40 hours) suggest a solid-like behavior of the basement membrane at the time scale of hours. Moreover, Atomic Force Microscopy (AFM) force-indentation curves reveal low hysteresis and an elastic solid-like response. To measure basal mechanical tension, I probed the basement membrane with an AFM. Interpreting the results of AFM shallow indentations on the basal side of explanted wing discs as indenting into a fixed, elastic, stretched thin film, I investigated in control conditions and after molecular perturbations basal mechanical tension. Mechanical tension was ≈ 0.4 mN/m. The removal of collagen IV by collagenase significantly reduced

The basement membrane is a specialized extracellular matrix (ECM) that is crucial for the development of epithelial tissues and organs. In Drosophila, the mechanical properties of the basement membrane play an important role in the proper elongation of the developing egg chamber; however, the molecular mechanisms contributing to basement membrane mechanical properties are not fully understood. Here, we systematically analyze the contributions of individual ECM components towards the molecular composition and mechanical properties of the basement membrane underlying the follicle epithelium of Drosophila egg chambers. We find that the Laminin and Collagen IV networks largely persist in the absence of the other components. Moreover, we show that Perlecan and Collagen IV, but not Laminin or Nidogen, contribute greatly towards egg chamber elongation. Similarly, Perlecan and Collagen, but not Laminin or Nidogen, contribute towards the resistance of egg chambers against osmotic stress. Finally, using atomic force microscopy we show that basement membrane stiffness mainly depends on Collagen IV. Our analysis reveals how single ECM components contribute to the mechanical properties of the basement membrane controlling tissue and organ shape.

Pancreatic cancer is the seventh leading cause of cancer related mortalities worldwide and incidences are increasing. The prognosis remains poor as the 5-year survival rate is below 10%. This can be partly explained by the silent progression of disease as most patients present with advanced disease at time of diagnosis. In turn, surgical resection, the only potential curative measure, is not possible in nearly 80% of cases due to the occurrence of distant metastasis and/or infiltration of major vessels in close proximity to the pancreas. In patients with localized but advanced disease, resectability can be achieved in some cases by initiation of a neoCTx. However, as neoCTx is commonly conducted by administering multi-drug treatments, severe side effects occur frequently, which require an adaption of drug doses administered. In this study, we revealed the negative impact of these drug dose changes during neoCTx on the patients´ treatment outcome. R0 resections were significantly less frequently observed, and the N-status significantly impacted by the tumor regression grade, which in turn trended towards minor response in the cohort of patients that did not sustain full dose course prior surgery. In turn, treatment of LA PDAC could be improved by increasing the proportion of patients that undergo neoCTx without any changes of the treatment schedule. Patient-derived PDAC organoid could serve as an avatar of patients´ tumor disease on which optimal treatment protocols could be tested. In this study, a large living PDAC PDO biobank successfully has been established from surgical resection specimens as well as EUS guided FNA samples. Subsequently, a new protocol for molecular subtyping of PDAC on organoids was established by assessing the expression level of KRT81 and CFTR, as a replacement for HNF1a, using IF staining. Strikingly, we observed identical PDAC subtypes in PDOs and their respective tissue of origin in nearly all cases. This observation allowed the assumptio

Während der Gewebeentwicklung beeinflusst die Anpassung der mechanischen Spannung bei Zell-zu-Zell-Kontakten das Gewebewachstum, die Musterbildung und die Morphogenese. Die Erzeugung und Kontrolle der mechanischen Spannung hängt von Komponenten des Zytoske- letts wie dem Aktomyosin und den Mikrotubuli-Netzwerken ab. Die Bildung von Komparti- mentgrenzen ist ein wichtiger Entwicklungsprozess, der auf der Anpassung mechanischer Spannungen beruht. Kompartimentgrenzen sind Abstammungsbeschränkungen, die Zellen mit unterschiedlichen Funktionen und Identitäten innerhalb von Geweben trennen. Zellverbindun- gen entlang der Kompartimentgrenzen sind häufig durch eine Anreicherung von filamentösen (F-) Aktin sowie nicht-muskulären Myosin II (Myosin II) Motorprotein und erhöhter mechani- scher Spannung gekennzeichnet. Die Mechanismen, durch die F-Aktin und Myosin II an die- sen Verbindungsstellen angereichert werden, sind jedoch kaum verstanden. Hier zeigen wir, dass an der sich bildenden anteroposterioren Kompartimentgrenze der Puppenepidermis von Drosophila melanogaster F-Aktin und Myosin II vorübergehend angereichert werden. Die An- reicherung von F-Aktin scheint nicht von mechanischer Spannung abzuhängen. Die Fluores- zenzerholung nach Photobleichversuchen (Fluorescence recovery after photobleaching, FRAP) weist eher darauf hin, dass Myosin II vorzugsweise an Zellübergängen entlang der Komparti- mentgrenze stabilisiert wird. Darüber hinaus zeigen wir unter Verwendung einer photokonver- tierbaren Form von Myosin II, dass Myosin II vorzugsweise aus einem zytosolischen Pool an Zellverbindungen entlang der Kompartimentgrenze rekrutiert wird. Um die Rolle des Mikro- tubuli-Netzwerks bei der Bildung von Kompartimentgrenzen zu testen, haben wir außerdem dessen Organisation in der Puppenepidermis charakterisiert. Wir zeigen, dass sich Mikrotubuli und das Mikrotubuli-Minus-Ende-bindende Protein Patronin in einem Streifen anteriorer Zellen entlang der Kompartimentgrenze ansammeln. In

Die Dissertationsarbeit untersucht genetische Varianten in Zellkulturmodellen des metastatischen und kastrationsresistenten Prostatakarzinoms. Außerdem werden Mechanismen der Chemoresistenz, insbesondere der Resistenz gegen Cisplatin und Docetaxel in diesen Zelllinien untersucht., This Dissertation evaluates genetic variants found in cell culture models of metastatic castration resistant prostate cancer. Furthermore, mechanisms of resistance against the chemotherapeutic drugs cisplatin and docetaxel are investigated in these cell lines.

Sexual reproduction requires meiosis, a specialized cell division program that halves the chromosome number of germ cells in order to generate gametes. Chromosome number reduction is achieved by two successive rounds of cell divisions after a single round of DNA replication. In meiosis I, homologous chromosomes (homologs) recombine to produce at least one reciprocal DNA exchange, called crossover (CO), in each chromosome. COs ensure proper segregation, as the resulting tetrad chromosomes are bisected during the first division to form dyads. In meiosis II, dyads split allowing segregation of single chromatids, resembling mitosis. During prophase I, the earliest stage of meiosis I, each pair of sister chromatids is arranged in series of loops tethered longitudinally by a structure called the chromosome axis, which serves as a scaffold for the machinery that promotes the formation of programmed DNA double-strand breaks (DSBs). Recombination is initiated when the DSBs are resected to produce single-stranded DNA (ssDNA) overhangs that seek out their homologs, promoting pairing. Then the synaptonemal complex (SC) forms, physically linking homolog axes through transverse filaments. In the context of the SC, meiotic recombination repairs the DSBs and turns a small fraction of them into COs. DSB formation will continue on unsynapsed axes and it is only terminated by the complete synapsis of all homolog pairs in pachytene stage. Meiocytes that fail to complete recombination and/or synapsis are eliminated (spermatocytes by mid-pachytene; oocytes from late prophase I, but at or before follicle formation), as these defects can cause chromosomal abnormalities that will be passed down to the offspring, causing severe diseases or death. Our group and others suggest that there are two simultaneous pachytene checkpoints, one that is activated by persistent DSBs and the other by asynapsis. This issue has proven very difficult to approach, since defective DSB formation/repair will inev

The vertebrate tail is unique for each species and fulfils a broad spectrum of functions. In the axolotl (Ambystoma mexicanum), a tailed amphibian, the tail constitutes one-third of the full body length and is necessary for swimming. Despite its size, most of the tail's tissues are derived from the posterior neural plate of the neurula. Although giving rise to neuronal structures of the central nervous system along most of its length, the most posterior part of the neural plate develops preponderantly into presomitic mesoderm (PSM) which forms muscle, bone and cartilage of the tail and posterior trunk. During development, the posterior neural plate reverses its orientation during an anterior turn movement (Taniguchi et al., 2017). Cells of the most posterior plate region become now localised in an anterior position while previously more anterior neural plate cells land at a more posterior site. Simultaneously, the axial neural tube and notochord extend themselves posteriorly. The PSM, developing bilaterally to the central axis, is integrated into posterior tail expansion while forming new somites at its anterior end. It is still elusive which morphological changes the PSM undergoes to facilitate tail formation and posterior elongation of the embryo. Furthermore, it remains enigmatic in what way PSM cells change their shape, orientation, migration behaviour and distribution to meet the requirements needed for adjusting PSM and somite morphology. With homotopic tissue transplantations of posterior neural plate cells from a gfp-expressing donor to a white (d/d) recipient, enabled specific labelling of all mesodermal cells of the tail. Otherwise, mesodermal cells of the trunk and tail can not be distinguished, neither genetically nor morphologically. With this cell labelling approach, the entire tail mesoderm could be imaged in toto. Thus, measurements of the morphological changes of the PSM and cell tracking in 3D was possible during development. With this technique, p, Der Schwanz der Wirbeltiere ist bei jeder Art einzigartig und erfüllt ein breites Spektrum an Funktionen. Beim Salamander Axolotl (Ambystoma mexicanum), macht der Schwanz ein Drittel der gesamten Körperlänge aus und ist zum Schwimmen notwendig. Trotz seiner Größe stammen die meisten Gewebe des Schwanzes von der posterioren Neuralplatte der Neurula ab. Obwohl der größte Teil der Neuralplatte neuronale Strukturen des Zentralnervensystems hervorbringt, entwickelt sich der posteriore Teil der Neuralplatte überwiegend zu präsomitischem Mesoderm (PSM), das Muskeln, Knochen und Knorpel des Schwanzes und des hinteren Rumpfes bildet. Während der Entwicklung kehrt die posteriore Neuralplatte ihre Orientierung in einer anterioren Drehbewegung um (Taniguchi et al., 2017). Zellen der hintersten Plattenregion werden in eine anteriore Position verschoben, während zuvor anteriorere Neuralplattenzellen an einer posterioren Stelle landen. Gleichzeitig verlängert sich das axiale Neuralrohr und das Notochord nach posterior. Das PSM, das sich bilateral zur Zentralachse entwickelt, ist im Prozess der Schwanzverlängerung involviert, während es gleichzeitig an seinem vorderen Ende neue Somiten bildet. Es ist immer noch unklar, welche morphologischen Veränderungen das PSM durchläuft, um die Schwanzbildung und die posteriore Ausdehnung des Embryos zu ermöglichen. Darüber hinaus ist unbekannt, auf welche Weise PSM-Zellen ihre Form, Orientierung, ihr Migrationsverhalten und ihre Verteilung ändern, die für eine Veränderung der PSM- und Somitenmorphologie erforderlich sind. Mit homotopen Gewebetransplantationen von posterioren Neuralplattenzellen von einem gfp-exprimierenden Spender auf einen weißen (d/d) Empfänger, konnte eine spezifische Markierung aller mesodermalen Zellen des Schwanzes erreicht werden. Andernfalls können mesodermale Zellen des Rumpfes und des Schwanzes weder genetisch noch morphologisch unterschieden werden. Mit diesem Zellmarkierungsansatz konnte das gesamte Schwanzmesoderm

Bones are essential vertebrate structures that arise during embryonic development from mesenchymal condensations. In the skull vault, bones arise from condensations above the eye, which later expand to cover the brain. Contrarily to most bones in the axial skeleton, which develop through an intermediate cartilage template, the skull vault develops from the direct differentiation of mesenchymal progenitors into bone cells, or osteoblasts. The molecular details of this process have however remained elusive, since systems allowing the dynamical observation of cells while they undergobone differentiation have been lacking. Recent data in the Tabler lab, acquired by imaging skull caps ex vivo, has uncovered that differentiation takes place during expansion stages, and that a progressive wave of differentiation underlies the presence of intermediate states between progenitors and osteoblasts. In this thesis, we focused on the cranial mesenchyme to study the differentiation trajectory of skull bone progenitors at single cell resolution. By harnessing single cell RNA-Sequencing, we elucidated the heterogeneity of cells in the cranial mesenchyme, describing in molecular terms meningeal, dermal, osteoblastic and progenitor populations. By modelling dynamics from single cell RNA-Seq data, we inferred that a population expressing intermediate levels of cartilage specific genes, such as Col2a1, underlies differentiation towards dermal, meningeal and bone fate. Using single cell resolution in situ RNA hybridisation, we mapped the anatomical location of progenitors in a layer between the meninges and the bone. To better understand the relationship between the phenotype of these progenitors and differentiated cartilage, we examined the presence of cartilage-like ECM in the tissue. Finally, we asked whether similar progenitors may be present in other intramembranous bones outside the skull by re-applying these tools on the clavicle. Taken together, our data lead us to hypothesise a

The shape of a tissue or entire organ is important for its biological function. Tissue and organ shapes arise from molecular activities that control and execute cellular processes, such as oriented cell divisions, cell shape changes or cell rearrangements. However, how molecular activities control cellular processes during the shaping of organs is not well understood. This thesis spotlights two aspects of organ shaping based on Drosophila egg chambers as model tissue. One focus lies on three-dimensional imaging of cellular mechanics during development and the other aspect dissects the molecular function of the fat2 gene, that is crucial for tissue elongation in Drosophila egg chambers.:TABLE OF CONTENTS 1 SUMMARY ................................................................................................................... I 2 ZUSAMMENFASSUNG ...............................................................................................III 3 TABLE OF CONTENTS ................................................................................................V 4 LISTS ..........................................................................................................................10 5 INTRODUCTION ........................................................................................................16 6 AIMS OF THE THESIS ...............................................................................................40 7 MATERIALS AND METHODS .....................................................................................41 8 RESULTS ....................................................................................................................58 9 DISCUSSION .............................................................................................................92 10 ACKNOWLEDGEMENTS .......................................................................................103 11 REFERENCES ...........................................................

Complexity in multi-cellular life arises from the ability of tissues to communicate with each other; tissue cross-talk during development coordinates growth and ultimately gives rise to organs with appropriate form and function. Drosophila larvae use systemic signaling mediated by endocrine factors and hormones to successfully grow and transition through developmental stages. Larvae sense nutrition primarily through the secretion of Drosophila insulinlike peptides (DILPs) 2 and 5 produced in the larval brain. Secreted DILPs bind to receptors in target tissues and activate the insulin/insulin-like signaling (IIS) pathway, which in imaginal tissues like the wing disc regulate adult wing size and cell number. The IIS pathway also directly regulates timing of developmental transitions through the production of the molting hormone ecdysone. Upon nutritional deprivation, growth is repressed but larvae manage to transition to pupal stages. This robust physiological adaptation allows Drosophila to successfully develop when grown on inadequate nutrient sources. However, the role of IIS in development under starvation is not clearly understood. In the first part of the thesis, I show that DILPs 2 and 5 are taken up in the corpora cardiaca (CC). No protein apart from the Drosophila insulin receptor (dInR) has been previously implicated in cellular uptake of DILPs in any tissue. I demonstrate that DILPs bind to non-canonical insulin-binding protein IMPL2 and are endocytosed in a clathrin-dependent manner. The CC produce adipo-kinetic hormone (AKH), a functional analog of glucagon. I show that up-taken DILPs are copackaged with AKH in secretory vesicles. CC is known to secrete AKH upon larval starvation. I find that upon starvation, CC cells also secrete stored DILPs to the prothoracic gland(PG) in a paracrine fashion, through neuronal projections running along the ‘lumen’ of the latter. DILP secretion from CC to PG reveals a novel type of hormonal signaling and provides a mecha

Many animals have the extraordinary ability to replace lost body parts, even so we humans do not. One critical but poorly understood aspect of this phenomenon is how wounds tailor the regeneration response to the particular target structure that needs to be regrown. In my thesis work I have attempted to address this problem in the champions of regeneration, the planarian flatworms. If one of these animals is cut into tiny pieces, each of the pieces will regenerate a head at the anterior end and tail at the posterior end. For over a century investigators have searched for the intrinsic polarity cue underlying this regeneration polarity, but until now its mechanistic basis is not known. The explicit goal of my thesis work was to identify this cue. The general approach that I have taken toward identification of the intrinsic polarity is to systematically compare two different planarian species with subtle variations in the establishment of regeneration polarity, Schmidtea mediterranea and Girardia tigrina. First, I demonstrate through systematic comparison of different amputation paradigms that regeneration polarity is dependent not only on species, but also on piece length, body size and anteroposterior axis position. Second, given that these findings are consistent with a gradient- based intrinsic polarity cue as prevalent hypothesis in the field, I tested whether the recently identified tail-to-head gradient of canonical Wnt (cWnt) signalling could be mechanistic basis of regeneration polarity. As precondition for doing so, I developed new approaches to measure and manipulate cWnt signalling in planaria. The data acquired with these tools suggest that the cWnt gradient may contribute to the observed position-dependence of regeneration polarity but is overall not the (only) intrinsic polarity cue. Third, I present my initial efforts to test whether the longitudinal muscle fibres (LMFs) in which notum is exclusively activated are an intrinsic polarity cue. My results

Correct nuclear position is crucial for cellular function. The cytoskeletal mechanisms of nuclear positioning have been studied intensely in cultured cells. However, it is less clear if and how tissue morphology can influence nuclear positioning in developing tissues. To address this question, this thesis compares nuclear migration in straight and curved neuroepithelia of the developing zebrafish. Neuroepithelial nuclei occupy different apicobasal positions in interphase but migrate to the apical surface before mitosis, a process essential for epithelial development. While apical migration in the straight hindbrain and the curved retina depends on actomyosin, it is unclear how the necessary forces are generated and if tissue morphology influences the force generation mechanisms. Remarkably, this study demonstrates that in neuroepithelia of different shape nuclei move with distinct kinetics and undergo distinct deformations. Such differences are explained by the action of disparate forces that propel hindbrain and retinal nuclei. In agreement with this conclusion, hindbrain and retinal cells display distinct actomyosin distribution and regulation during nuclear migration. Apical movement is shown to depend on Rho-ROCK activity in the hindbrain and formin activity in the retina. Therefore, hindbrain and retinal cells employ distinct actin-dependent mechanisms of nuclear positioning. Comparison of nuclear movements in another pair of straight and curved neuroepithelia shows that in tissues with similar morphology nuclei have conserved modes of apical migration. The different mechanisms of apical migration used in tissues of different shape argue that tissue morphology can indeed influence the mechanism of nuclear positioning. The findings in this thesis suggest that different mechanisms arise due to differences in actin arrangements during development of tissues with distinct curvature. Furthermore, they emphasize the importance of developmental context, tissue and cel

Epithelial folding is an important morphogenetic process that is essential in transforming simple sheets of cells into complex three-dimensional tissues and organs during animal development (Davidson, 2012). Epithelial folding has been shown to rely on constriction forces generated by the apical actomyosin network (Martin et al., 2009; Roh-Johnson et al., 2012; Sawyer et al., 2010). However, the contributions of mechanical forces acting along lateral and basal cell surfaces to epithelial folding remain poorly understood. Here we combine live imaging with force measurements of epithelial mechanics to analyze the formation of two epithelial folds in the Drosophila larval wing imaginal disc. We show that these two neighboring folds form via two distinct mechanisms. These two folds are driven either by decrease of basal tension or increase of lateral tension, none of them depends on apical constriction. In the first fold, a local decrease in extracellular matrix (ECM) density in prefold cells results in a reduction of mechanical tension on the basal cell surface, leading to basal expansion and fold formation. Consistent with that, a local reduction of ECM by overexpression of Matrix metalloproteinase II is sufficient to induce ectopic folding. In the second fold a different mechanism is at place. Here basal tension is not different with neighboring cells, but pulsed dynamic F-actin accumulations along the lateral interface of prefold cells lead to increased lateral tension, which drives cell shortening along the apical-basal axis and fold formation. In this thesis I described two distinct mechanisms driving epithelial folding, both basal decrease and lateral increase in tension can generate similar morphological changes and promote epithelial folding in the Drosophila wing discs., Die Faltung von Epithelien ist ein wichtiger morphogenetischer Prozess, der die Entstehung komplexer, dreidimensionaler Gewebe und Organe aus einfachen Zellschichten ermöglicht (Davidson, 2012). Es ist bekannt, dass Kräfte erzeugt durch das apikale Aktomyosin-Netzwerk wichtig sind für die erfolgreiche Faltung von Epithelien (Martin et al., 2009; Roh-Johnson et al., 2012; Sawyer et al., 2010). Die Rolle von mechanischen Kräften, die entlang der lateralen und basalen Seite wirken, ist jedoch kaum verstanden. Wir verbinden Lebendmikroskopie mit der Messung von mechanischen Eigenschaften, um die Entstehung von 2 Epithelfalten in den Imaginalscheiben von Drosophila zu verstehen. Wir können dadurch zeigen, dass die beiden Falten durch unterschiedliche Mechanismen entstehen. Sie entstehen entweder durch eine Verringerung der Spannung auf der basalen Seite oder durch eine Erhöhung der Spannung auf der lateralen Seite, aber keine von beiden entsteht durch zusammenziehende Kräfte auf der apikalen Seite. Die erste Falte entsteht durch eine lokale Verringerung der extrazellulären Matrix in den Vorläuferzellen, was zu einer Reduktion der Spannung auf der basalen Seite und zur Ausbildung der Falte führt. Die zweite Falte wird durch einen anderen Mechanismus ausgebildet. Hier ist nicht die Spannung auf der basalen Seite reduziert sondern dynamische Anreicherungen von F-Aktin auf der lateralen Seite resultieren in einer erhöhten lateralen Spannung, die zu einer Verkürzung der Zellen und damit zur Ausbildung einer Falte führt. In meiner Arbeit zeige ich 2 neue Mechanismen zur Entstehung von Epithelfalten auf, durch Absenken der Spannung auf der basalen oder Erhöhen auf der lateralen Seite.

The development of an organism and its adult homeostasis rely on regulatory mechanisms that control the underlying gene expression programs. In certain biological contexts, such as germ cell development, gene expression regulation is largely executed at the post-­‐transcriptional level. This relies on RNA-­‐binding proteins (RBPs), whose activity and expression are also heavily controlled. While the RNA-­‐binding potential of RBPs is currently of intense scrutiny, surprisingly little is known to date about the molecular mechanisms that control RNA-­‐binding proteins abundance in the context of germ cell development. This work identifies the molecular mechanisms that shape expression patterns of two evolutionarily conserved RNA-­‐binding proteins, CPB-­‐3 and GLD-­‐ 1, which belong to CPEB and STAR protein family, respectively. By focusing on their regulation in the C. elegans germ line, this work reveals an involvement of the proteasome in reducing levels of CPB-­‐3/CPEB and GLD-­‐1/STAR at the pachytene-­‐to-­‐diplotene transition during meiotic prophase I. Furthermore, it documents that CPB-­‐3 and GLD-­‐1 are targeted to proteasomal degradation by a conserved SCF ubiquitin ligase complex that utilises SEL-­‐10/Fbxw7 as a substrate recognition subunit. Importantly, destabilisation of both RBPs is likely triggered by their phosphorylation, which is regulated by the mitogen-­‐activated protein kinase, MPK-­‐1, and restricted to the meiotic timepoint of pachytene exit. Lastly, this work investigates the potential consequences of target mRNA regulation upon delayed RBP degradation. Altogether, the collected data characterise a molecular pathway of CPEB and STAR protein turnover, and suggest that MPK-­‐1 signaling may couple RBP-­‐mediated regulation of gene expression to progression through meiosis during oogenesis.

Stem cell niches are specialized signalling microenvironments that allow maintenance of the stem cells. According to the traditional model of the stem cell niche, the niche signalling input is integrated by a cell towards a binary decision between stemness and differentiation. I have studied the regulation of somatic cyst stem cell (CyCS) proliferation in the testicular stem cell niche of Drosophila melanogaster by performing the DamID screen for targets of the transcriptional regulator Zfh1, a shared target of Jak/STAT and Hedgehog niche signalling. I have found that Zfh1 binds to the regulatory regions of kibra and salvador, tumour suppressors of the Hippo/Yorkie pathway, and downregulates them, restricting Yorkie activity to the Zfh1 positive CySCs. Clonal inactivation of the Hippo pathway is sufficient for CySC proliferation, but does not affect their differentiation ability. I therefore proposed a different stem cell niche model, whereby the niche signalling directly “micromanage” stem cell behavior, not involving the cell fate decision making.

The past decade has witnessed a breakthrough in the field of structural DNA nanotechnology, which utilizes DNA molecules as a construction material rather than as simple carriers of the genetic information. With the superior programmability of DNA, sub-nanometer precision in the self-assembly of various complex two- and three-dimensional nanostructures is achievable. It also allows a site-specific placement of different objects and functional groups onto the formed structures. This has enabled the assembly of highly sophisticated nanostructures for various applications. While the field of structural DNA nanotechnology has been astonishingly advancing, many nanoelectronics-relevant structures are made of inorganic materials, and DNA-based nanostructures have shown rather low conductivity. This has limited the use of DNA structures in nanoelectronics and reflected the need for a similar programmable route for the inorganic nanofabrication. A conceivable solution would use DNA nanostructures in a way that will precisely transfer the structural information of the DNA shapes into fabricated metallic nanostructures. One way to do that is to use the DNA nanostructures as templates for external material deposition onto the DNA surface. While this strategy has been effective in proving the concept of DNA-shape transfer, metallic nanostructures fabricated this way have shown some drawbacks, such as showing rough surface morphologies and lacking the required homogeneity for the fabricated metallic structures. An alternative strategy would be to design DNA mold structures that can dictate the shape of metal that is “cast” inside such a DNA container. The main topic of my thesis concerns the second strategy. To discuss this in detail, the structure and some of the important properties of DNA are introduced in section 1.1. In section 1.2 the main milestones in the development of the DNA-nontechnology field are discussed and section 1.3 focuses on previous fabrication approaches o