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 work presented in the thesis was carried with the aim to understand how Widerborst (Wdb) regulate planar cell polarity in Drosophila wing. In search of proteins interacting with Wdb I carried a Yeast Two Hybrid screen and identified a protein, bitesize, with tandem C2 domains in its C terminus interacting with Wdb. Wdb also interacts with btsz genetically and removal of one copy each of Wdb and btsz enhances the truncated hair phenotype observed in Wdb EMS mutants and btsz P element insertion mutants. There are at least three predicted isoforms of bitesize and loss of the btsz-II isoform is lethal. Clonal analysis of a btsz mutant, btszJ5-2, which removes the btsz II isoform resulted in truncated wing hair outgrowth. On the other hand over expression of a myc-btsz-II construct resulted in hair duplication phenotype. However, over expression of the GFP-CT is sufficient to give wing hair duplication phenotype and this hair duplication phenotype is stronger than that caused by myc-btsz-II over expression. The Myc tagged btsz-II protein shows apical localization. Though most of the protein is confined to cytoplasm, btsz-II also marks the plasma membrane. The GFP-CT construct marks the plasma membrane strongly and is enriched in the apical region. The over expression of CT domain is sufficient to give hair duplication phenotype and the strong difference observed in the localization pattern of full length btsz-II protein and GFP-CT together suggest that regulation of membrane localization of btsz through its CT region is important to regulate hair morphogenesis. As the loss of function (truncated wing hair) and gain of function (hair duplication) both affect the process of hair morphogenesis, it can be said that btsz is a positive regulator of hair morphogenesis. Since no defect in cortical polarization of Fmi was observed in cells lacking btsz-II, btsz is required for establishment of cortical domains. However with the present study it remains unknown how exactly the C2 domains might regulate hair morphogenesis and whether Wdb targets btsz for dephophorylation to PP2A catalytic subunit.

During the development of multicellular organisms, organs grow to well-defined shapes and sizes. The proper size and patterning of tissues are ensured by signaling molecules as e.g. morphogens. Secreted from a restricted source, morphogens spread into the adjacent target tissue where they form a graded concentration profile. Upon binding of the morphogens to receptors on the cell surfaces, the morphogenetic signal is transduced inside the cell via the phosphorylation of transcription factors, which subsequently regulate the expression of different target genes. Thus, cell fates are determined by the local concentration of such morphogens. In this work, we investigate three key aspects of morphogenetic signaling processes in growing tissues. First, we study the mechanics of tissue growth via cell division and cell death. We examine the rearrangements of cells on large scales and times by developing a continuum theory which describes the growing tissue as an active complex fluid. In our description we include anisotropic stresses generated by oriented cell division, and we show that average cellular trajectories exhibit anisotropic scaling behaviors. Our description is used to study experimentally measured shape changes of the developing wing disk of the fruit fly Drosophila melanogaster. Second, we focus on the spreading of morphogens in growing tissues. We show that the flow field of cell movements due to oriented cell division and cell death causes a drift term in the morphogen transport equation, which captures the stretching and dilution of the concentration profile. Comparing our theoretical results to recent experiments in the Drosophila wing disk, we find that the transport of the morphogen Dpp is mainly intracellular. We moreover show that the decay length of the Dpp gradient increases during development as a result of changing degradation rate and diffusion coefficient, whereas the drift of molecules due to growth is negligible. Thus growth does not affect the decay length of the gradient, but the decay length of the gradient might affect the tissue growth rate as discussed in this work. Finally, we develop a microscopic theoretical description of the intracellular transduction machinery of morphogenetic signals within an individual cell. Our description captures the kinetics of the trafficking of proteins between different cellular compartments in response to receptor-bound signaling molecules. Analyzing experimental data at the Drosophila neuromuscular junction, we show that the morphogenetic signaling is modulated by synaptic signaling via neuronal action potentials.

Decapentaplegic (Dpp), a member of the TGF-[Beta] superfamily, acts as a morphogen to direct cell differentiation, determine cell fate and promote cell survival and proliferation in Drosophila wing development. To investigate the role of Dpp in Drosophila wing development, three aspects of the patterning role of Dpp have been analyzed. First, I investigated the cellular responses to Dpp signaling by a loss of function strategy. The consequences of lacking Dpp signal transduction on cell morphology and tissue integrity were analyzed. Second, I investigated whether Dpp signaling is down-stream of Hh signaling to maintain the normal cell segregation at the A/P boundary by clonal analysis. Third, I investigated whether cross talk among the Hh, Dpp and Wg signaling pathways exists and what its relevance for wing patterning is. To investigate the role of Dpp in Drosophila wing development, the general strategies are to look at the phenotypes of loss-of-function and gain-of-function. Mutant clones lacking Dpp signal transduction by knock down Dpp receptor Thick veins (Tkv) do not survive in wing blade due to JNK dependent apoptosis. To get larger mutant clones for analysis, JNK pathway was inhibited by knock down bsk (encodes JNK) in mutant clones lacking Dpp signaling using FLP-FRT system. Clones double mutant for tkv and bsk did not undergo apoptosis, but recovered at very low frequencies compared to sibling clones. Here, I showed that the low recovery of tkv bsk double mutant clones are due to the extrusion of mutant cells. The extrusion of tkv bsk double mutant cells correlated with changes in the actin cytoskeleton and a dramatic loss of the apical microtubule web normally present in these cells. These results suggest that Dpp signaling is required for cell morphogenesis in Drosophila wing development. We propose that Dpp acts as a survival factor in the wing disc epithelium by orchestrating proper cytoskeletal organization and maintaining normal cell-cell contact. Drosophila wing is subdivided into anterior (A) and posterior (P) compartments. This developing into adjacent compartments is crucial for the patterning of Drosophila wing. Previous study has shown that Hedgehog (Hh) signaling is required in A cells to maintain the A/P boundary and is sufficient to specify A type cell sorting. A previous study has in addition implicated the signaling molecule Decapentaplegic (Dpp) in maintaining the A/P boundary. However, this study did not address whether and in which cells, A and/or P, Dpp signal transduction was required to maintain this boundary. Here, I have analyzed the role of components of the Dpp signal transduction pathway and the relation of Dpp and Hh signaling in maintaining the A/P boundary by clonal analysis. I showed that Dpp signaling mediated by the Dpp target gene, T-box protein Optomotor-blind (Omb), is required in A cells, but not in P cells, to maintain the normal position of the A/P boundary. During patterning formation, it is essential for cells to receive precise positional information to pattern the tissue. It has been proposed for a long time that different signaling pathways such as Hedgehog (Hh), Dpp and Wingless (Wg) signaling pathways provide positional information for tissue patterning in an integrated manner. Recently, evidence of interactions between Hh and Dpp as well as Wg and Hh signaling pathways has been reported in Drosophila wing. Here, I have identified additional interactions among Hh, Dpp and Notch/Wg signaling. We propose that the selector gene engrailed, Hh and Dpp signaling interact with each other to regulate target genes expression and thus to pattern the wing along the A/P axis. Further more, I showed that Dpp signaling is also participating in the patterning along the D/V axis by interaction with the selector gene apterous and Notch/Wg signaling.

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.

The neuromeric concept of brain formation has become a well-established model to explain how order is created in the developing vertebrate central nervous system. The most important feature of neuromeres is their compartmentalization on the cellular level: Each neuromere comprises a lineage-restricted population of cells that does not intermingle with cells from neighboring compartments. The units of the vertebrate hindbrain, the rhombomeres, serve as the best-studied examples of neuromeres. Here, the lineage restriction mechanism has been found to function on the basis of differentially expressed adhesion molecules. To date, hard evidence for the existence of other lineage restricted regions in more anterior parts of the brain is still scarce. The focus of this study is the midbrain-hindbrain boundary (mhb) region, where the juxtaposition of the mesencephalon and metencephalon gives rise to a signaling center, termed the midbrain-hindbrain or isthmic organizer. Evidence for lineage restriction boundaries in the mhb region is still controversial, with some very recent studies supporting the existence of a lineage boundary between the mesencephalon and metencephalon and others rejecting this. Here, I present data strongly supporting the existence of a compartment boundary between the posterior midbrain and anterior hindbrain territory. I base this proposition on cell-tracing experiments with single cell resolution. By connecting the traces to a molecular midbrain marker, I establish a link between cell fate and behavior. In the second part, I present a novel tissue explant method for the zebrafish that has the potential to serve numerous developmental studies, especially imaging of so far inaccessible regions of the embryo.

Mammals have two orthologs, Mll and Trx2, for the Drososphila protein Trithorax (TRX), which is the founding member of the trithorax group (TrxG) of epigenetic regulators. TrxG proteins are characterized by an evolutionary conserved SET domain. A major function of all SET domain- containing proteins is to modulate gene activity, but the underlying mechanisms are poorly understood. Apparently TRX, Mll and Trx2 are histone H3 lysine 4 specific methyltransferases. So far all evidence points to roles in expression of specific target genes. However, target genes and function of the epigenetic regulator Trx2 were still unknown. Homozygous trx2 mutant embryos arrest in development because of severe and widespread defects {Glaser, 2005 #296}. Thus mouse embryonic stem (ES) cells carrying a null mutation of trx2 were used as an alternative model system to address the implication of Trx2 in differentiation. This study showed that Trx2 is redundant for ES cell self-renewal. Homozygous trx2 knockout ES cells did not exhibit cell cycle defects. However, loss of Trx2 resulted in reduced proliferation and increased apoptosis rates in trx2-/- ES cells. Due to the fact that differentiation requires an appropriate rate of population growth, trx2-/- cells were affected adversely upon in vitro differentiation. Neurogeneic differentiation of trx2 mutant cells generated fewer mature neurons than wild type cells. Moreover a temporal delay in the developmental progression to differentiation became apparent. Cardiac differentiation of trx2-/- cells confirmed the developmental defect and temporal delay. Notably differentiation of trx2-/- cells was merely delayed or impaired but it was not absent, implying that Trx2 is not required for gene expression programs specific for neurons or cardiac myocytes. We propose that differentiation of trx2-/- ES cells is impaired because apoptosis is disturbing differentiation. Apart from analyzing the phenotype of trx2 mutant cells, this work was focused on the identification of Trx2 target genes. Oligonucleotide expression arrays were used to identify genes whose expression levels were affected by the absence of Trx2. In general, loss of Trx2 function resulted in more genes with decreased than increased expression levels. This is consistent with the hypothesis that Trx2 functions as a transcriptional activator. Comparison of gene expression profiles for constitutive and conditional trx2 mutant cells enabled a distinction between direct and indirect target genes for Trx2. As a result Magoh2 was identified as the key candidate target gene for Trx2. Interaction between Trx2 and Magoh2 suggested a potential regulatory role for Trx2 in alternative splicing. Furthermore this work provided evidence that Trx2 could be involved in the maintenance of CpG island promoter gene expression, thus providing a potent regulatory mechanism for ubiquitously expressed genes.

Cell polarity is required for asymmetric division, a mechanism to generate cell diversity by distributing fate determinants unequally to daughter cells. The establishment of polarity requires the evolutionarily conserved partitioning-defective (PAR) proteins as well as the actin cytoskeleton. In Caenorhabditis elegans one-cell embryos, the PAR proteins are segregated into an anterior (PAR-3, PAR-6) and a posterior (PAR-1, PAR-2) corticaldomain. The formation of PAR polarity correlates with anterior-posterior differences in the contractile activity of the cortex, known as "contractile polarity". It is thought that regulation of contractile polarity controls the establishment of PAR polarity, but detailed evidence to support this idea is lacking. To investigate how modulation of the actomyosin cytoskeleton affects polarity establishment, the acto-myosin cytoskeleton was perturbed by RNA-mediated interference (RNAi) of two Rho GTPases, CDC-42 and RHO-1. To examine how Rho GTPases are implemented in actin remodeling, it is important to analyze how their activity is controlled and how different activities affect polarity formation. The role of two putative Rho GTPase regulators, the Rho GTPase exchange factor (GEF) ECT-2 and the Rho GTPase activating protein (GAP) K09H11.3 were analyzed with respect to polarity formation. The formation of polarity was analyzed by using GFP-labeled proteins, and several different tracking methods were used to investigate the establishment of contractile and PAR polarity in more detail.This study demonstrates that both RHO-1 and CDC-42 are involved in polarity establishment in C. elegans embryos. But importantly, both act by different mechanisms. RHO-1 organizes the acto-myosin cytoskeleton into a contractile network, and therefore is essential for the formation of contractile polarity. The organization of the acto-myosin cytoskeleton is critical to ensure proper PAR protein distribution. Furthermore, a balance of RHO-1 activity by the GEF ECT-2 and the GAP K09H11.3 appears to be important for cortical contractility, for PAR protein domain size and for mutual exclusion of the PAR proteins. Although CDC-42 was shown to be a universal regulator of the actin cytoskeleton, CDC-42 acts downstream of contractile polarity. CDC-42 is required for linking PAR-6 to the cortex. In the absence of RHO-1 and ECT-2, PAR-6 and CDC-42 are not localized to the anterior cortex. This suggests that RHO-1, by organizing the actomyosin cytoskeleton into a contractile network, regulates the segregation of CDC-42 to the anterior cortex, and concomitantly PAR-6 localization. This study shows that the distribution of PAR is related to cortical activity and supports the model that the actin cytoskeleton plays an important role in polarity establishment.