Two new distinct mechanisms drive epithelial folding in Drosophila wing imaginal discs

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 ‘dance’ of life: visualizing metamorphosis during pupation in the blow fly Calliphora vicina by X-ray video imaging and micro-computed tomography

© 2017 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. The attached file is the published version of the article

Genetic analysis of Drosophila adult muscle type specification

Muscles of all higher animals comprise different muscle types adapted to perform distinct functions in the body. These express different sets of genes controlled by distinct combinations of transcriptional programs and extracellular signals, and thus differ in their myofibrillar organization and contractile properties. Despite major progress in our understanding of myogenesis, the genetic pathways controlling the formation and function of different muscle types are still largely uncharacterized. Flying insects possess specialized flight muscles enabling wing oscillations with frequencies of up to 1000 Hz together with high power outputs of 80 W per kg muscle. To achieve these parameters, flight muscles contain stretch-activated myofibrils with a unique fibrillar organization, whereas all other, more slowly contracting muscles, such as leg muscles, display a tubular morphology. To delineate the genetic regulation of muscle development and function, and, in particular, muscle type specification, we performed a genome-wide RNA interference (RNAi) screen in Drosophila, in which we systematically inactivate genes exclusively in muscle tissue. We uncovered more than 2000 genes with putative roles in muscles, many of which we were able to assign to specific functions in muscle, myofibril or sarcomere organization by phenotypic characterization. Muscle-specific knockdown of 315 genes resulted in viable, but completely flightless animals, indicating a specific function of those genes in fibrillar flight muscles. Detailed morphological analysis of these 315 genes revealed a striking phenotype upon knockdown of the zinc finger transcription factor spalt major (salm): the fibrillar flight muscles are switched to tubular muscles, whereas tubular leg muscles are wild type, demonstrating that salm is a key determinant of fibrillar muscle fate. We could show that the transcription factor vestigial (vg) acts upstream of salm to induce its expression specifically in fibrillar flight muscles. Importantly, salm is not only required but also sufficient to induce the fibrillar muscle fate upon ectopic expression in other muscle types. Microarray analysis, comparing mRNA expression from adult wild-type flight and leg muscles to salm knockdown flight muscles, indicates that salm instructs most features of fibrillar muscles by regulating both gene expression as well as alternative splicing. Remarkably, we could show that spalt’s function in programming stretch-activated fibrillar muscles is conserved in insect species separated by 280 million years of evolution. Interestingly, in mouse two of the four spalt-like (sall) genes are expressed in heart, a stretch-activated muscle, sharing some features with insect fibrillar flight muscles. Since heart abnormalities observed in patients suffering from the Towns-Brocks syndrome are caused by a mutation in SALL1, it is possible that Spalt’s function to determine a fibrillar, stretch-modulated muscle type is conserved to vertebrates

Studying fibroblast growth factor (FGF) mediated cell migration in "Drosophila" larval air sacs

Invertebrates and vertebrates use FGF signaling in many developmental processes. Mesoderm formation, limb outgrowth but also the development of the vascular system and the lung rely on FGF ligands. We have chosen to study the Drosophila FGF signaling pathway that has been shown to be required for mesodermal- as well as tracheal cell migration. We aimed at a better understanding of FGF signaling to elucidate how the extracellular information, provided by the FGF/Bnl ligand is interpreted in tracheal cells. Using Downstream of FGFR (Dof), an adaptor protein of the FGF signaling pathway, as an entry point, we have previously identified interacting proteins and focused on one prime candidate as a potential linker of FGFR to the cytoskeleton. This candidate protein Receptor of protein kinase C (Rack1) is conserved throughout evolution. rack1 is expressed in the early embryonic tracheal system and has been proposed to play important roles in cell migration as well as in the regulation of the actin cytoskeleton. We have identified and characterized rack1 mutants; these mutants are zygotic lethal but neither show a detectable embryonic- nor any other larval phenotype, due to a very high maternal contribution. Removing the maternal store by generating germline clones results in eggs that fail to develop. This developmental arrest is due to an incomplete transfer of maternal product into the oocyte (nurse cell dumping). In order to characterize the function of rack1 in the context of FGF signaling, we started to characterize the development of third instar larval air sacs. It has been reported that this structure develops via cell migration as well as cell division in response to FGF/Bnl signaling. First we confirm the occurrence of cell division and found that in early air sacs, division is ubiquitous and becomes restricted later to the central part of the air sac. We also documented cell behavior during cell migration using live imaging. To initiate a genetic analysis of rack1 and other candidate target genes in tracheal cell migration, strains and methods were established, allowing the generation of mosaic air sacs consisting of marked wild-type or mutant cells in an otherwise heterozygous background based on the MARCM system. This system was also applied to characterize cellular shape and dynamics of individual or small groups of air sac tracheoblasts in different parts of the air sac. We found that air sac tip cells extend long and dynamic actin based protrusions and further demonstrated that cells not directly located at the tip do form similar protrusions. Finally, we took advantage of the our knowledge of air sac architecture and development to study the cell-autonomous requirement of candidate genes in genetic mosaics. We showed that marked wild-type clones have a preference to be positioned at the tip. Mutants lacking btl or dof, two genes required for embryonic tracheal cell migration, never populate regions at the migratory front. We inferred that air sac tracheoblast cells lacking btl or dof are deficient in migration and take this as a readout for measuring cell migration. Having established criteria for measuring cell migration in air sacs, we tested rack1 mutants for their involvement in air sac tracheoblast migration and find that this gene is not required for this process. We also analyzed other candidate genes as well as components of the FGF signaling pathway and found evidence that Ras plays a dual role during third instar air sac formation. It appears to integrate signaling input from the EGFR pathway to trigger cell division as well as input from the FGF pathway to activate a cell migratory response. In contrast to border cells, mutants affecting the transcription factor Slow border cells (Slbo), the VEGFR (PVR) or DE-Cadherin (Shg) do not impede air sac tracheoblast migration. Components shown to regulate the actin cytoskeleton in response to PVR signaling such as Myoblast city (Mbc) the Drosophila Dock180 homologue or the small Rho family GTPases Rac1, Rac2 and Mig-2-like (Mtl) as well as the effector Chickadee, the Drosophila homologue of Profilin, are essential for air sac tracheoblast migration. Thus, recruitment of these actin cytoskeleton regulators and effectors is mediated via different ligands/receptors in trachea and border cells. Our studies demonstrate that the development of the air sac during late larval stages is a good system to study guided cell migration and allows the genetic dissection of the FGF signaling pathway. The tools we developed allow to assay any candidate gene for which a mutant is available and also laid the foundation for the isolation and characterization of genes in a genome wide EMS screen

Nuclear positioning and functional regulation of endogenous genes and transgenes in the fruit fly Drosophila melanogaster and in mammalian cells

The goal of the work was to address the role of higher order nuclear architecture in the functional regulation of endogenous genes and transgenes in different species. In the first part of the work, 3D distance measurements were performed to analyze in WT flies and in different transgenic lines of Drosophila melanogaster (Cavalli and Paro, 1998; Zink and Paro, 1995) the nuclear localization of endogenous genes and transgenic constructs containing the Polycomb Response Element (PRE) Fab-7 relative to the nuclear periphery and heterochromatin. Transgenic constructs containing the Fab-7 element and three endogenous genes, Abd-B, sd, and Ubx, were first analyzed at their inactive state. The results showed that they were preferentially associated with the nuclear periphery and did not display specific associations with heterochromatin. The localization of the transgenic Fab-7 element was further analyzed at different states of activity. Activation of the transgenic Fab-7 element resulted in frequent (up to ~50%) association with the boundary of the heterochromatic domain. The percentages of such associations were tissue- and fl y line-dependent. Further investigations of the boundary of heterochromatin showed that this region has a complex organization, where euchromatic sites enriched in the active form of RNA pol II and trimH3K4, sites enriched in dimH3K4 and Pc-binding sites, as well as pericentromeric satellite DNA are exposed and juxtaposed towards each other. The concentration and specific architecture of such sites at the boundary of heterochromatin might help to maintain the equilibrium between activation and repression at this boundary. This specific environment might be favourable for maintaining PREs in the active state. I also investigated in three transgenic lines whether endogenous and transgenic copies of the Fab-7 element interact physically, using 3D distance measurements. In five tissues analyzed, no pairing between endogenous and transgenic copies of the Fab-7 element was observed. Also enhancement of Pc-mediated silencing did not induce pairing. Additionally, the general organization of Pc-binding sites was addressed in six larval tissues. We used different methods of microscopy and image analyses to count the numbers of Pc foci in nuclei from these tissues. Our data did not indicate clustering of Pc-binding sites and formation of so called „Pc bodies“. However, corresponding analyses are not without problems at the current stage of methodology and results must be interpreted carefully. Together with the results of previously published studies, which investigated pairing between PREs (Bantignies et al., 2003; Vazquez et al., 2006), my data demonstrated that such pairing is a highly tissue-specific phenomenon and is likely not involved in the regulation of PREs in various tissues. Activity-related positioning of transgenes was also addressed in transgenic porcine cell lines (Hofmann et al., 2003). Results of 2D erosion analyses showed that the LV-PGK transgene was associated with the nuclear periphery in its inactive state, while it occupied more interior positions in its active state. This corresponds to my results obtained with transgenic Drosophila lines. My data also suggested that the active LV-PGK construct might be associated with heterochromatin in one case. However, further experiments would be necessary to confirm such associations. The results obtained with Drosophila and porcine cells suggested conserved mechanisms for tethering inactive loci to the nuclear periphery. These were further addressed using the human CFTR locus as a model, which is closely associated with the nuclear periphery in its inactive state (Zink et al., 2004). The question was addressed whether Tpr, a protein associated with the nuclear basket, plays a role in the perinuclear localization of the inactive CFTR locus. CFTR showed a high degree of association with the nuclear periphery in control cells in accordance with previous data (Zink et al., 2004). After knock-down of Tpr via RNAi CFTR displayed a more interior positioning. This suggests that Tpr is involved in the organization of inactive gene loci at the nuclear periphery. Moreover, since Drosophila Tpr has a high level of homology to the mammalian Tpr (Zimowska et al., 1997) and as it has been shown that the yeast Tpr homologs Mlp1 and Mlp2 are involved in tethering of inactive loci to the nuclear periphery in yeast cells (Galy et al., 2000), it is possible that Tpr is a part of a conserved mechanism anchoring inactive loci to the nuclear periphery in eukaryotic cells

MorphoGraphX:A platform for quantifying morphogenesis in 4D

Morphogenesis emerges from complex multiscale interactions between genetic and mechanical processes. To understand these processes, the evolution of cell shape, proliferation and gene expression must be quantified. This quantification is usually performed either in full 3D, which is computationally expensive and technically challenging, or on 2D planar projections, which introduces geometrical artifacts on highly curved organs. Here we present MorphoGraphX (www.MorphoGraphX.org), a software that bridges this gap by working directly with curved surface images extracted from 3D data. In addition to traditional 3D image analysis, we have developed algorithms to operate on curved surfaces, such as cell segmentation, lineage tracking and fluorescence signal quantification. The software’s modular design makes it easy to include existing libraries, or to implement new algorithms. Cell geometries extracted with MorphoGraphX can be exported and used as templates for simulation models, providing a powerful platform to investigate the interactions between shape, genes and growth.DOI: http://dx.doi.org/10.7554/eLife.05864.001Author keywordsResearch organis