No description available.

No description available.

Many organs and structures formed during the embryonic morphogenesis of animals derive from epithelia. Epithelia are made up of apicobasally polarized cells which adhere to and communicate with each other, allowing for epithelial integrity and plasticity. During embryonic morphogenesis, epithelia change their shape and migrate in a coordinated manner. How these epithelial processes are regulated is still not fully understood. In a forward genetic screen using the embryo of the fruit fly Drosophila melanogaster, candidate genes influencing the morphogenesis of epithelial structures were identified. Three genes, CG17364, CG17362 and CG9040 were identified as possible regulators of lumen stability in the salivary glands, tubular organs deriving from the embryonic epithelium. Furthermore, the gene Cdep was found to play a crucial role in epithelial sheet migration during dorsal closure of the embryo. Embryos carrying genomic insertions that could affect the expression of CG17364, CG17362 and CG9040 show a luminal penotype of the embryonic salivary glands characterized by alternating bloated and seemingly closed sections. Therefore, one of these genes or a combination of them likely plays a role in stabilizing the salivary gland lumen. However, neither CG17364 nor CG17362 or CG9040 contain any known protein domains, hence their molecular roles remain unknown. Cdep (Chondrocyte-derived ezrin-like protein) is a member of the FERM-FA subclass of proteins. Proteins of the FERM family have been shown to interact with the plasma membrane and membrane-bound proteins as well as cytoskeleton components. Accordingly, they have been implicated in stabilizing the cell cortex, and some of them are involved in signal transduction mechanisms. In addition to a FERM domain, Cdep also contains a RhoGEF domain, although is still not clear whether it actually exerts GEF activity. Genomic insertions in the Cdep locus cause defects in embryonic dorsal closure and atypical migratory behaviour in epithelial tubes. In order to study the molecular role of Cdep, the CRISPR/Cas9 system was employed to establish loss-of-function mutants of Cdep. The mutants show aberrations in germ band retraction, dorsal closure and head involution. Moreover, I found that two mutants carrying a premature STOP codon in the Cdep ORF, CdepE16X and CdepG17X, rescue the defects observed in embryonic cuticles mutant for two other FERM-FA members yurt (yrt) and coracle (cora). A deletion of the full Cdep ORF did not rescue those defects. I hypothesize that CdepE16X and CdepG17X encode Cdep variants with increased activity, which compensates for the loss of yrt or cora function, respectively. In conclusion, this leads to a model in which Cdep acts in parallel to Yrt and Cora during Drosophila embryonic morphogenesis. Many of the defects described in this study are reminiscent of phenotypes found in embryos mutant for components and downstream effectors of the Jun-N-terminal Kinase (JNK) pathway. Hence, my work supports an earlier hypothesis according to which a mouse homologue of Cdep, Farp2, acts as an upstream activator of the JNK pathway during epithelial cell migration in vitro (Miyamoto et al., 2003) The data provided here shows that Cdep plays a role in the morphogenesis of a great number of epithelia-derived organs and structures in vivo. My study therefore elucidates a missing link between cell migration cues and JNK pathway activation.:1 Introduction 1 1.1 Epithelial cell polarity 1 1.1.1 Cellularization and formation of the primary epithelium 1 1.1.1.1 Establishment of epithelial polarity and adhesion 2 1.1.2 The epithelial polarity network 3 1.1.3 Cell-cell adhesion 5 1.1.3.1 Adherens junctions 5 1.1.3.2 Septate junctions 6 1.2 Epithelial movements in Drosophila embryonic morphogenesis 6 1.2.1 Epithelial tube formation during Drosophila embryogenesis 7 1.2.2 Coordinated migration of epithelial sheets during Dros. embryogenesis 7 1.2.2.1 FERM domain proteins in epithelial migration 9 1.2.2.2 Cdep 10 1.3 Mutagenesis with the CRISPR/Cas9 system 12 2 Aim of My PhD Thesis Work 15 3 Preliminary Work 17 4 Results 19 4.1 A screen for novel regulators in Drosophila embryonic morphogenesis 19 4.1.1 Deficiencies on the left arm of chromosome 3 cause defects in SG lumen morphology 19 4.1.2 A locus in the overlap of two deficiencies on the right arm of chromosome 3 causes defects in Drosophila embryonic dorsal closure 20 4.2 Two uncharacterized genes regulate SG lumen diameter in Drosophila embryos 22 4.2.1 Mutations in two uncharacterized genes on chromosome 3 cause intermittent tube closure in the Drosophila SG 22 4.2.2 CG17362/ CG9040/ CG17364 play a role in the maintenance of SG lumen width after lumen expansion 24 4.2.3 CG17362 is exclusively expressed in the embryonic SG 25 4.2.4 CG17362 and CG9040 do not contain known protein domains and are only conserved in Drosophilidae 28 4.3 Loss of Cdep causes different defects in Drosophila embryonic morphogenesis 30 4.3.1 Insertions in the ORF of Cdep cause defects in DC and HI 30 4.3.2 Embryos transheterozygous for PBac{5HPw+}CdepB122 and Mi{MIC} CdepMI00496 show defects in the LE during DC 34 4.3.3 Insertions in the ORF of Cdep cause defects in tracheal and Malpighian tubule morphogenesis 34 4.3.4 The CRISPR/Cas9 system was used to generate loss-of-function mutants of Cdep 35 4.3.5 LOF mutants of Cdep show a variety of phenotypes 37 4.3.6 LOF mutants of Cdep cause denticle belt defects and ventral holes in Drosophila larval cuticles 38 4.3.7 Phenotypes in Cdep−/− mutant embryos are likely not caused by maternal defects 44 4.3.8 LOF mutants of Cdep cause segments to fuse 44 4.3.9 Defects in Cdep−/− mutants are not due to actin mislocalization 51 4.3.10 Cdep genetically interacts with yurt 51 4.3.11 Cdep genetically interacts with cora 55 5 Discussion 57 5.1 The role of CG17362 and CG9040 in SG lumen stability 57 5.1.1 Impairments in the expression of CG17362 and CG9040 could be the cause for the intermittent tube closure in the embryonic SGs 57 5.1.2 CG17362 and CG9040 could be necessary for SG lumen dilation and stability of lumen diameter 57 5.2 The role of Cdep in Drosophila embryonic morphogenesis 59 5.2.1 Cdep is a regulator of the JNK pathway 60 5.2.1.1 Mutations in TGF-_ pathway components cause LE bunching and gaps in the tracheal dorsal trunk 60 5.2.1.2 The JNK pathway, like Cdep, is instrumental in GBR, DC and HI 61 5.2.1.3 Mouse Farp2 acts upstream of the JNK pathway 61 5.2.2 Does Cdep act as a GEF? 62 5.2.3 Cdep might regulate epithelial migration in parallel to Yrt and Cora 63 5.3 Conclusion and Outlook 64 6 Materials and Methods 67 6.1 Cell strains, plasmids and DNA constructs 67 6.2 Culture media 68 6.2.1 Lysogeny Broth (Bertani, 1951) 68 6.2.2 Super Optimal Catabolite repression (SOC) medium (Hanahan, 1983) 68 6.3 Molecular biology methods 69 6.3.1 Amplifying DNA by standard PCR technology 69 6.3.2 Molecular cloning 70 6.3.2.1 Cloning with restriction endonucleases (Old and Primrose, 1980) 70 6.3.2.2 Gibson Assembly (Gibson et al., 2009) 70 6.3.3 Detecting DNA via agarose gel electrophoresis 71 6.3.4 Purifying DNA from E.coli 71 6.3.5 Extracting DNA from Drosophila adults 71 6.3.5.1 Extracting mRNA from Drosophila embryos and reverse transcription into cDNA 72 6.3.6 Making mRNA probes for in situ hybridization 72 6.3.7 Measuring DNA concentrations using the NanoDrop 72 6.3.8 DNA sequencing 72 6.3.9 Transformation 73 6.3.10 Mutagenesis of Cdep with the CRISPR-Cas9 system 73 6.3.10.1 CRISPR/Cas9 tools for mutagenesis in D.melanogaster 73 6.3.11 Strategies for CRISPR/Cas9 mutagensis 74 6.3.11.1 vectors and oligomers used for mutagenesis of Cdep via CRISPR-Cas9 system 75 pCFD3-dU6:3-CdepEx2gRNA 75 CdepSTOP-ssODN 76 pCFD4-CdepExc_2sgRNAs 77 pDsRed-5’HA-3’HA 78 6.3.11.2 Screening for successful mutagenesis events in flies modified with the CRISPR/Cas9 system 79 Insertion of an in-frame stop codon into the Cdep ORF 79 Replacing the entire Cdep locus with dsRed 80 6.3.12 Extracting protein from Drosophila embryos 83 6.3.13 Detecting and separating proteins by mass via SDS-PAGE 83 6.3.14 Detection of specific polypeptides by Western blot analysis (Towbin et al., 1979) 84 6.3.14.1 Transferring polypeptides to nitrocellulose membrane 84 6.3.14.2 Detecting specific polypeptides via immonublotting 85 6.4 Online tools for prediction of protein domains, interactions, sequence alignments, etc. 86 6.5 Cell culture growth and harvest 86 6.5.1 Growing E.coli cells for vector amplification 86 6.5.2 Cell harvest 86 6.6 Work with Drosophila melanogaster 87 6.6.1 Fly stocks used: deficiencies, alleles, duplications and balancers 88 6.6.2 Recombining alleles located on the same chromosome 93 6.6.3 Collecting Drosophila melanogaster embryos 93 6.6.4 Histochemistry 94 6.6.4.1 Embryo dechorionation 94 6.6.4.2 Embryo fixation for light microscopy of whole specimens 94 Heat fixation of Drosophila embryos 94 Formaldehyde fixation of Drosophila embryos 94 6.6.4.3 Embryo fixation for light microscopy of semi-thin sections (Tepass and Hartenstein, 1994) 95 6.6.4.4 Antibody staining of embryos for Immunofluorescence 95 6.6.4.5 Phalloidin and DAPI staining of embryos 96 6.6.4.6 Embryo mounting for analysis via fluorescence microscopy 96 6.6.4.7 Embryo mounting for live imaging 97 6.6.4.8 Embedding of embryos for semi-thin sectioning 97 6.6.4.9 Cuticle preparation from hatched and unhatched Drosophila larvae 97 6.6.4.10 Preparation and staining of ovarian follicles from Drosophila females 98 6.6.4.11 mRNA in situ hybridization of whole-mount Drosophila embryos 99

Morphogens are secreted signalling molecules that are expressed in restricted groups of cells within the developing tissue. From there, they are secreted and travel throughout the target field and form concentration gradients. These concentration profiles endow receiving cells with positional information. A number of experiments in Drosophila demonstrated that the morphogen Decapentaplegic (Dpp) forms activity gradients by inducing the expression of several target genes above distinct concentration thresholds at different distances from the source. This way, Dpp contributes to developmental fates in the target field such as the Drosophila wing disc. Although the tissue distribution as well as the actual shape and size of the Dpp morphogen concentration gradient has been visualized, the cell biological mechanisms through which the morphogen forms and maintains a gradient are still a subject of debate. Two hypotheses as to the dominant mechanism of movement have been proposed that can account for Dpp spreading throughout the Drosophila wing imaginal target tissue: extracellular diffusion and planar transcytosis, i. e. endocytosis and resecretion of the ligand that is thereby transported through the cells. Here, I present data indicating that implications of a theoreticalanalysis of Dpp spreading, where Dpp transport through the target tissue is solely based on extracellular diffusion taking into account receptor binding and subsequent internalization, are inconsistent with experimental results. By performing Fluorescence Recovery After Photobleaching (FRAP) experiments, I demonstrate a key role of Dynamin-mediated endocytosis for Dpp gradient formation. In addition, I show that most of GFP-Dpp traffics through endocytic compartments at the receiving epithelial cells, probably recycled through apical recycling endosomes (ARE). Finally, a Dpp recycling assay based on subcellular photouncage of ligand is presented to address specifically the Dpp recycling event at the receiving cells.

Morphogens are signaling molecules that play a key role in animal development. They spread from a restricted source into an adjacent target tissue forming a concentration gradient. The fate of cells in the target tissue is determined by the local concentration of such morphogens. Morphogen transport through the tissue has been studied in experiments which lead to the suggestion of several transport mechanisms. While diffusion in the extracellular space contributes to transport, recent experiments on the morphogen Decapentaplegic (Dpp) in the fruit fly Drosophila provide evidence for the importance of a cellular transport mechanism that was termed "planar transcytosis". In this mechanism, morphogens are transported through cells by repeated rounds of internalization and externalization. Starting from a microscopic theoretical description of these processes, we derive systems of nonlinear transport equations which describe the interplay of transcytosis and passive diffusion. We compare the results of numerical calculations based on this theoretical description of morphogen transport to recent experimental data on the morphogen Dpp in the Drosophila wing disk. Agreement with the experimental data is only achieved if the parameters entering the theoretical description are chosen such that transcytosis contributes strongly to transport. Analyzing the derived transport equations, we find that transcytosis leads to an increased robustness of the created gradients with respect to morphogen over-expression. Indications for this kind of robustness have been found in experiments. Furthermore, we theoretically investigate morphogen gradient formation in disordered systems. Here, an important question is how the position of concentration thresholds can be defined with high precision in the noisy environment present in typical developing tissues. Among other things, we find that the dimensionality of the system in which the gradient is formed plays an important role for the precision. Comparing gradients formed by transcytosis to those formed by extracellular diffusion, we find substantial differences that may result in a higher precision of gradients formed by transcytosis. Finally, we suggest several experiments to test the theoretical predictions of this work.

No description available.

Embryonic development depends on the precise coordination of cell fate specification, patterning and morphogenesis. Although great strides have been made in the molecular understanding of each of these processes, how their interplay governs the formation of complex tissues remains poorly understood. New techniques for experimental manipulation and image quantification enable the study of development in unprecedented detail, resulting in new hypotheses on the interactions between known components. By expressing these hypotheses in terms of rules and equations, computational modeling and simulation allows one to test their consistency against experimental data. However, new computational methods are required to represent and integrate the network of interactions between gene regulation, signaling and biomechanics that extend over the molecular, cellular and tissue scales. In this thesis, I present a framework that facilitates computational modeling of multiscale multicellular systems and apply it to investigate pancreatic development and the formation of vascular networks. This framework is based on the integration of discrete cell-based models with continuous models for intracellular regulation and intercellular signaling. Specifically, gene regulatory networks are represented by differential equations to analyze cell fate regulation; interactions and distributions of signaling molecules are modeled by reaction-diffusion systems to study pattern formation; and cell-cell interactions are represented in cell-based models to investigate morphogenetic processes. A cell-centered approach is adopted that facilitates the integration of processes across the scales and simultaneously constrains model complexity. The computational methods that are required for this modeling framework have been implemented in the software platform Morpheus. This modeling and simulation environment enables the development, execution and analysis of multi-scale models of multicellular systems. These models are represented in a new domain-specific markup language that separates the biological model from the computational methods and facilitates model storage and exchange. Together with a user-friendly graphical interface, Morpheus enables computational modeling of complex developmental processes without programming and thereby widens its accessibility for biologists. To demonstrate the applicability of the framework to problems in developmental biology, two case studies are presented that address different aspects of the interplay between cell fate specification, patterning and morphogenesis. In the first, I focus on the interplay between cell fate stability and intercellular signaling. Specifically, two studies are presented that investigate how mechanisms of cell-cell communication affect cell fate regulation and spatial patterning in the pancreatic epithelium. Using bifurcation analysis and simulations of spatially coupled differential equations, it is shown that intercellular communication results in a multistability of gene expression states that can explain the scattered spatial distribution and low cell type ratio of nascent islet cells. Moreover, model analysis shows that disruption of intercellular communication induces a transition between gene expression states that can explain observations of in vitro transdifferentiation from adult acinar cells into new islet cells. These results emphasize the role of the multicellular context in cell fate regulation during development and may be used to optimize protocols for cellular reprogramming. The second case study focuses on the feedback between patterning and morphogenesis in the context of the formation of vascular networks. Integrating a cell-based model of endothelial chemotaxis with a reaction-diffusion model representing signaling molecules and extracellular matrix, it is shown that vascular network patterns with realistic morphometry can arise when signaling factors are retained by cell-modified matrix molecules. Through the validation of this model using in vitro assays, quantitative estimates are obtained for kinetic parameters that, when used in quantitative model simulations, confirm the formation of vascular networks under measured biophysical conditions. These results demonstrate the key role of the extracellular matrix in providing spatial guidance cues, a fact that may be exploited to enhance vascularization of engineered tissues. Together, the modeling framework, software platform and case studies presented in this thesis demonstrate how cell-centered computational modeling of multi-scale and multicellular systems provide powerful tools to help disentangle the complex interplay between cell fate specification, patterning and morphogenesis during embryonic development.

Die alveoläre Echinokokkose ist eine, vorrangig in der nördlichen Hemisphäre verbreitete, parasitäre Erkrankung. Verursacht wird sie beim Menschen durch das Larvenstadium des Fuchsbandwurms. Homeoboxgene sind hochkonservierte Gene, die die Morphogenese von Lebewesen steuern. Die Anzahl und die Bedeutung von Homeoboxgenen in der Entwicklung von E.multilocularis waren bislang unbekannt. Im Rahmen dieser Arbeit konnten mit Hilfe von Sequenzanalysen im Genom des Fuchsbandwurms erstmals Homeoboxgene identifiziert und deren Expressionsmuster mittels PCR in verschiedenen Larvenstadien charakterisiert werden. Von insgesamt 23 gefundenen Homeoboxgenen wurden 15 Gene auf ihre larvenstadienspezifische Expression untersucht. Neun der untersuchten Gene zeigten in dem gewählten Versuchsaufbau eine Expression in den untersuchten Larvenstadien, fünf davon zeigten eine verstärkte Expression in den späten Larvenstadien. Für acht dieser neun Gene ließen sich darüber hinaus Hinweise auf eine Prozessierung ihrer mRNA über den Mechanismus des Trans-Spleißens finden. Vorangehende Versuche der Arbeitsgruppe von Prof. Brehm hatten einen Zusammenhang zwischen einer Stimulation früher Entwicklungsstadien der Parasitenlarven mit dem Zytokin BMP-2 und dessen rascherer Entwicklung in spätere Entwicklungsstadien nahegelegt. Die Auswirkung einer Behandlung früher Entwicklungsstadien mit dem Zytokin BMP-2 auf die jeweilige Genexpression wurde daher für die ausgewählten 15 Gene überprüft. Fünf Gene zeigten unter dessen Einfluss eine verstärkte Expression. Zwei darunter waren solche, die eine stärkere Expression in späten Larvenstadien aufwiesen. Diese zwei Gene stellen nun Kandidaten dar, die an der Entwicklung von E.multilocularis maßgeblich beteiligt sein könnten. Durch die Untersuchungen dieser Arbeit ergaben sich wichtige Hinweise auf die Entwicklung und die Regulationsmechanismen der Genexpression von E. multilocularis. Sie bilden eine Grundlage, die Rolle der Homeoboxgene für den Fuchsbandwurm näher zu beschreiben und die hormonelle Kreuzregulation zwischen Parasit und Wirt weiter zu studieren. / Alveolar Echinococcosis is a rather rare parasitic disease, mainly occuring in the northern hemisphere. It is caused by the larval stage of the flatworm Echinococcus multilocularis. Homeoboxgenes are highly conserved genes, that play a crucial role in the development of organism. Until now neither the number in nor the importance of these genes for E.multilocularis was known. Within this study we could identify the homeoboxgenes in the genome of E.multilocularis and analyse the expression pattern in different larval stages. All together we found a number of 23 homeoboxgenes, the expression pattern in different larval stages was analysed for 15 genes. Within the chosen experimental setup nine of the genes were expressed, five showed higher expression in later larval stages. Furthermore there was a strong hint for the processing of mRNA through trans-splicing for eight genes. Previous experiments of the working group Brehm had shown a relation between a stimulation of young metacestodes with the cytokine BMP-2 and a faster development towards later larval stages. Therefore we anaylsed the effect of the cytokine BMP-2 on the geneexpression pattern of fifteen genes. Under its influence five genes showed higher expression rates. Two belonged to the group of genes that had shown higher expression in later larval stages. These two genes could potentially be candidates with a high significance in the development of E.multilocularis. Due to this work we could elucidate important hints for the development of E.multilocularis and the regulation of its gene expression. Based on these findings it will be possible to study the role of homeoboxgenes and the host-parasite cross-regulation in E.multilocularis in more detail.

Techn. Universiẗat, Diss., 2003--Darmstadt. / Dateien im PDF-Format.

The cell is the central functional unit of life. Cell behaviors, such as cell division, movements, differentiation, cell death as well as cell shape and size changes, determine how tissues change shape and grow during regeneration and development. However, a generally applicable framework to measure and describe the behavior of the multitude of cells in a developing tissue is still lacking. Furthermore, the specific contribution of individual cell behaviors, and how exactly these cell behaviors collectively lead to the morphogenesis and growth of tissues are not clear for many developmental and regenerative processes. A promising strategy to fill these gaps is the continuing effort of making developmental biology a quantitative science. Recent advances in methods, especially in imaging, enable measurements of cell behaviors and tissue shapes in unprecedented detail and accuracy. Consequently, formalizing hypotheses in terms of mathematical models to obtain testable quantitative predictions is emerging as a powerful tool. Tests of the hypotheses involve the comparison of model predictions to experimentally observed data. The available data is often noisy and based on only few samples. Hence, this comparison of data and model predictions often requires very careful use of statistical inference methods. If one chooses this quantitative approach, the challenges are the choice of observables, i.e. what to measure, and the design of appropriate data-driven models to answer relevant questions. In this thesis, I applied this data-driven modeling approach to vertebrate morphogenesis, growth and regeneration. In particular, I study spinal cord and muscle regeneration in axolotl, muscle development in zebrafish, and neuron development and maintenance in the adult human brain. To do so, I analyzed images to quantify cell behaviors and tissue shapes. Especially for cell behaviors in post-embryonic tissues, measurements of some cell behavior parameters, such as the proliferation rate, could not be made directly. Hence, I developed mathematical models that are specifically designed to infer these parameters from indirect experimental data. To understand how cell behaviors shape tissues, I developed mechanistic models that causally connect the cell and tissue scales. Specifically, I first investigated the behaviors of neural stem cells that underlie the regenerative outgrowth of the spinal cord after tail amputation in the axolotl. To do so, I quantified all relevant cell behaviors. A detailed analysis of the proliferation pattern in space and time revealed that the cell cycle is accelerated between 3-4 days after amputation in a high-proliferation zone, initially spanning from 800 µm anterior to the amputation plane. The activation of quiescent stem cells and cell movements into the high-proliferation zone also contribute to spinal cord growth but I did not find contributions by cellular rearrangements or cell shape changes. I developed a mathematical model of spinal cord outgrowth involving all contributing cell behaviors which revealed that the acceleration of the cell cycle is the major driver of spinal cord outgrowth. To compare the behavior of neural stem cells with cell behaviors in the regenerating muscle tissue that surrounds the spinal cord, I also quantified proliferation of mesenchymal progenitor cells and found similar proliferation parameters. I showed that the zone of mesenchymal progenitors that gives rise to the regenerating muscle segments is at least 350 µm long, which is consistent with the length of the high-proliferation zone in the spinal cord. Second, I investigated shape changes in developing zebrafish muscle segments by quantifying time-lapse movies of developing zebrafish embryos. These data challenged or ruled out a number of previously proposed mechanisms. Motivated by reported cellular behaviors happening simultaneously in the anterior segments, I had previously proposed the existence of a simple tension-and-resistance mechanism that shapes the muscle segments. Here, I could verify the predictions of this mechanism for the final segment shape pattern. My results support the notion that a simple physical mechanism suffices to self-organize the observed spatiotemporal pattern in the muscle segments. Third, I corroborated and refined previous estimates of neuronal cell turnover rates in the adult human hippocampus. Previous work approached this question by combining quantitative data and mathematical modeling of the incorporation of the carbon isotope C-14. I reanalyzed published data using the published deterministic neuron turnover model but I extended the model by a better justified measurement error model. Most importantly, I found that human adult neurogenesis might occur at an even higher rate than currently believed. The tools I used throughout were (1) the careful quantification of the involved processes, mainly by image analysis, and (2) the derivation and application of mathematical models designed to integrate the data through (3) statistical inference. Mathematical models were used for different purposes such as estimating unknown parameters from indirect experiments, summarizing datasets with a few meaningful parameters, formalizing mechanistic hypotheses, as well as for model-guided experimental planning. I venture an outlook on how additional open questions regarding cell turnover measurements could be answered using my approach. Finally, I conclude that the mechanistic understanding of development and regeneration can be advanced by comparing quantitative data to the predictions of specifically designed mathematical models by means of statistical inference methods.