TY - JOUR AB - T cells develop in the thymus in a highly specialized cellular and extracellular microenvironment. The basement membrane molecule, laminin-5 (LN-5), is predominantly found in the medulla of the human thymic lobules. Using high-resolution light microscopy, we show here that LN-5 is localized in a bi-membranous conduit-like structure, together with other typical basement membrane components including collagen type IV, nidogen and perlecan. Other interstitial matrix components, such as fibrillin-1 or -2, tenascin-C or fibrillar collagen types, were also associated with these structures. Three-dimensional (3D) confocal microscopy suggested a tubular structure, whereas immunoelectron and transmission electron microscopy showed that the core of these tubes contained fibrillar collagens enwrapped by the LN-5-containing membrane. These medullary conduits are surrounded by thymic epithelial cells, which in vitro were found to bind LN-5, but also fibrillin and tenascin-C. Dendritic cells were also detected in close vicinity to the conduits. Both of these stromal cell types express major histocompatibility complex (MHC) class II molecules capable of antigen presentation. The conduits are connected to blood vessels but, with an average diameter of 2 mum, they are too small to transport cells. However, evidence is provided that smaller molecules such as a 10 kDa dextran, but not large molecules (>500 kDa), can be transported in the conduits. These results clearly demonstrate that a conduit system, which is also known from secondary lymphatic organs such as lymph nodes and spleen, is present in the medulla of the human thymus, and that it might serve to transport small blood-borne molecules or chemokines to defined locations within the medulla. AU - Drumea-Mirancea, Mihaela AU - Wessels, Johannes T AU - Müller, Claudia A AU - Essl, Mike AU - Eble, Johannes A AU - Tolosa, Eva AU - Koch, Manuel AU - Reinhardt, Dieter P AU - Michael Sixt AU - Sorokin, Lydia AU - Stierhof, York-Dieter AU - Schwarz, Heinz AU - Klein, Gerd ID - 3934 IS - Pt 7 JF - Journal of Cell Science TI - Characterization of a conduit system containing laminin-5 in the human thymus: a potential transport system for small molecules VL - 119 ER - TY - JOUR AB - Integrins regulate cell behavior through the assembly of multiprotein complexes at the site of cell adhesion. Parvins are components of such a multiprotein complex. They consist of three members (alpha-, beta-, and gamma-parvin), form a functional complex with integrin-linked kinase (ILK) and PINCH, and link integrins to the actin cytoskeleton. Whereas alpha- and beta-parvins are widely expressed, gamma-parvin has been reported to be expressed in hematopoietic organs. In the present study, we report the expression pattern of the parvins in hematopoietic cells and the phenotypic analysis of gamma-parvin-deficient mice. Whereas alpha-parvin is not expressed in hematopoietic cells, beta-parvin is only found in myeloid cells and gamma-parvin is present in both cells of the myeloid and lymphoid lineages, where it binds ILK. Surprisingly, loss of gamma-parvin expression had no effect on blood cell differentiation, proliferation, and survival and no consequence for the T-cell-dependent antibody response and lymphocyte and dendritic cell migration. These data indicate that despite the high expression of gamma-parvin in hematopoietic cells it must play a more subtle role for blood cell homeostasis. AU - Chu, Haiyan AU - Thievessen, Ingo AU - Michael Sixt AU - Lämmermann, Tim AU - Waisman, Ari AU - Braun, Attila AU - Noegel, Angelika A AU - Fässler, Reinhard ID - 3935 IS - 5 JF - Molecular and Cellular Biology TI - γ-Parvin is dispensable for hematopoiesis, leukocyte trafficking, and T-cell-dependent antibody response VL - 26 ER - TY - JOUR AB - At least eight of the twelve known members of the beta1 integrin family are expressed on hematopoietic cells. Among these, the VCAM-1 receptor alpha4beta1 has received most attention as a main factor mediating firm adhesion to the endothelium during blood cell extravasation. Therapeutic trials are ongoing into the use of antibodies and small molecule inhibitors to target this interaction and hence obtain anti-inflammatory effects. However, extravasation is only one possible process that is mediated by beta1 integrins and there is evidence that they also mediate leukocyte retention and positioning in the tissue, lymphocyte activation and possibly migration within the interstitium. Genetic mouse models where integrins are selectively deleted on blood cells have been used to investigate these functions and further studies will be invaluable to critically evaluate therapeutic trials. AU - Michael Sixt AU - Bauer, Martina AU - Lämmermann, Tim AU - Fässler, Reinhard ID - 3936 IS - 5 JF - Current Opinion in Cell Biology TI - β1 integrins: zip codes and signaling relay for blood cells VL - 18 ER - TY - JOUR AB - Wnt11 is a key signal, determining cell polarization and migration during vertebrate gastrulation. It is known that Wnt11 functionally interacts with several signaling components, the homologues of which control planar cell polarity in Drosophila melanogaster. Although in D. melanogaster these components are thought to polarize cells by asymmetrically localizing at the plasma membrane, it is not yet clear whether their subcellular localization plays a similarly important role in vertebrates. We show that in zebrafish embryonic cells, Wnt11 locally functions at the plasma membrane by accumulating its receptor, Frizzled 7, on adjacent sites of cell contacts. Wnt11-induced Frizzled 7 accumulations recruit the intracellular Wnt signaling mediator Dishevelled, as well as Wnt11 itself, and locally increase cell contact persistence. This increase in cell contact persistence is mediated by the local interaction of Wnt11, Frizzled 7, and the atypical cadherin Flamingo at the plasma membrane, and it does not require the activity of further downstream effectors of Wnt11 signaling, such as RhoA and Rok2. We propose that Wnt11, by interacting with Frizzled 7 and Flamingo, modulates local cell contact persistence to coordinate cell movements during gastrulation. AU - Witzel, Sabine AU - Zimyanin, Vitaly AU - Carreira Barbosa, Filipa AU - Tada, Masazumi AU - Heisenberg, Carl-Philipp J ID - 4140 IS - 5 JF - Journal of Cell Biology TI - Wnt11 controls cell contact persistence by local accumulation of Frizzled 7 at the plasma membrane VL - 175 ER - TY - JOUR AB - The detection of microRNAs (miRNAs) at single-cell resolution is important for studying the role of these posttranscriptional regulators. Here, we use a dual-fluorescent green fluorescent protein (GFP)-reporter/monomeric red fluorescent protein (mRFP)-sensor (DFRS) plasmid, injected into zebrafish blastomeres or electroporated into defined tissues of mouse embryos in utero or ex utero, to monitor the dynamics of specific miRNAs in individual live cells. This approach reveals, for example, that in the developing mouse central nervous system,, miR-124a is expressed not only in postmitotic neurons but also in neuronal progenitor cells. Collectively, our results demonstrate that acute administration of DFRS plasmids.offers an alternative to previous in situ hybridization and transgenic approaches and allows the monitoring of miRNA appearance and disappearance in defined cell lineages during vertebrate development. AU - Tonelli, Davide AU - Calegari, Frederico AU - Fei, Ji AU - Nomura, Tadashi AU - Osumi, Noriko AU - Heisenberg, Carl-Philipp J AU - Huttner, Wieland ID - 4145 IS - 6 JF - Biotechniques TI - Single-cell detection of microRNAs in developing vertebrate embryos after acute administration of a dual-fluorescence reporter/sensor plasmid VL - 41 ER - TY - JOUR AB - During vertebrate gastrulation, a well-orchestrated series of morphogenetic changes leads to the formation of the three germ layers: the ectoderm, mesoderm and endoderm. The analysis of gene expression patterns during gastrulation has been central to the identification of genes involved in germ layer formation. However, many proteins are regulated on a translational or post-translational level and are thus undetectable by gene expression analysis. Therefore, we developed a 2D-gel-based comparative proteomic approach to target proteins involved in germ layer morphogenesis during zebrafish gastrulation. Proteomes of ectodermal and mesendodermal progenitor cells were compared and 35 significantly regulated proteins were identified by mass spectrometry, including several proteins with predicted functions in cytoskeletal organization. A comparison of our proteomic results with data obtained in an accompanying microarray-based gene expression analysis revealed no significant overlap, confirming the complementary nature of proteomics and transcriptomics. The regulation of ezrin2, which was identified based on a reduction in spot intensity in mesendodermal cells, was independently validated. Furthermore, we show that ezrin2 is activated by phosphorylation in mesendodermal cells and is required for proper germ layer morphogenesis. We demonstrate the feasibility of proteomics in zebrafish, concluding that proteomics is a valuable tool for analysis of early development. AU - Link, Vinzenz AU - Carvalho, Lara AU - Castanon, Irinka AU - Stockinger, Petra AU - Shevchenko, Andrej AU - Heisenberg, Carl-Philipp J ID - 4176 IS - 10 JF - Journal of Cell Science TI - Identification of regulators of germ layer morphogenesis using proteomics in zebrafish VL - 119 ER - TY - JOUR AB - Background: Zebrafish (D. rerio) has become a powerful and widely used model system for the analysis of vertebrate embryogenesis and organ development. While genetic methods are readily available in zebrafish, protocols for two dimensional (2D) gel electrophoresis and proteomics have yet to be developed. Results: As a prerequisite to carry out proteomic experiments with early zebrafish embryos, we developed a method to efficiently remove the yolk from large batches of embryos. This method enabled high resolution 2D gel electrophoresis and improved Western blotting considerably. Here, we provide detailed protocols for proteomics in zebrafish from sample preparation to mass spectrometry (MS), including a comparison of databases for MS identification of zebrafish proteins. Conclusion: The provided protocols for proteomic analysis of early embryos enable research to be taken in novel directions in embryogenesis. AU - Link, Vinzenz AU - Shevchenko, Andrej AU - Heisenberg, Carl-Philipp J ID - 4173 JF - BMC Developmental Biology TI - Proteomics of early zebrafish embryos VL - 6 ER - TY - JOUR AB - Detailed reconstruction of the spatiotemporal history of embryonic cells is key to understanding tissue formation processes but is often complicated by the large number of cells involved, particularly so in vertebrates. Through a combination of high-resolution time-lapse lineage tracing and antibody staining, we have analyzed the movement of mesencephalic and metencephalic cell populations in the early zebrafish embryo. To facilitate the analysis of our cell tracking data, we have created TracePilot, a software tool that allows interactive manipulation and visualization of tracking data. We demonstrate its utility by showing novel visualizations of cell movement in the developing zebrafish brain. TracePilot (http://www.mpi-cbg.de/tracepilot) is Java-based, available free of charge, and has a program structure that allows the incorporation of additional analysis tools. AU - Langenberg, Tobias AU - Dracz, Tadeusz AU - Oates, Andrew AU - Heisenberg, Carl-Philipp J AU - Brand, Michael ID - 4178 IS - 4 JF - Developmental Dynamics TI - Analysis and visualization of cell movement in the developing zebrafish brain VL - 235 ER - TY - JOUR AB - Epithelial morphogenesis depends on coordinated changes in cell shape, a process that is still poorly understood. During zebrafish epiboly and Drosophila dorsal closure, cell-shape changes at the epithelial margin are of critical importance. Here evidence is provided for a conserved mechanism of local actin and myosin 2 recruitment during theses events. It was found that during epiboly of the zebrafish embryo, the movement of the outer epithelium (enveloping layer) over the yolk cell surface involves the constriction of marginal cells. This process depends on the recruitment of actin and myosin 2 within the yolk cytoplasm along the margin of the enveloping layer. Actin and myosin 2 recruitment within the yolk cytoplasm requires the Ste20-like kinase Msn1, an orthologue of Drosophila Misshapen. Similarly, in Drosophila, actin and myosin 2 localization and cell constriction at the margin of the epidermis mediate dorsal closure and are controlled by Misshapen. Thus, this study has characterized a conserved mechanism underlying coordinated cell-shape changes during epithelial morphogenesis. AU - Köppen, Mathias AU - Fernández, Beatriz AU - Carvalho, Lara AU - Jacinto, António AU - Heisenberg, Carl-Philipp J ID - 4184 IS - 14 JF - Development TI - Coordinated cell-shape changes control epithelial movement in zebrafish and Drosophila VL - 133 ER - TY - JOUR AB - The molecular and cellular mechanisms governing cell motility and directed migration in response to the chemokine SDF-1 are largely unknown. Here, we demonstrate that zebrafish primordial germ cells whose migration is guided by SDF-1 generate bleb-like protrusions that are powered by cytoplasmic flow. Protrusions are formed at sites of higher levels of free calcium where activation of myosin contraction occurs. Separation of the acto-myosin cortex from the plasma membrane at these sites is followed by a flow of cytoplasm into the forming bleb. We propose that polarized activation of the receptor CXCR4 leads to a rise in free calcium that in turn activates myosin contraction in the part of the cell responding to higher levels of the ligand SDF-1. The biased formation of new protrusions in a particular region of the cell in response to SDF-1 defines the leading edge and the direction of cell migration. AU - Blaser, Heiko AU - Reichman Fried, Michal AU - Castanon, Irinka AU - Dumstrei, Karin AU - Marlow, Florence AU - Kawakami, Koichi AU - Solnica Krezel, Lilianna AU - Heisenberg, Carl-Philipp J AU - Raz, Erez ID - 4218 IS - 5 JF - Developmental Cell TI - Migration of zebrafish primordial germ cells: A role for myosin contraction and cytoplasmic flow VL - 11 ER - TY - JOUR AB - The growth function of populations is central in biomathematics. The main dogma is the existence of density-dependence mechanisms, which can be modelled with distinct functional forms that depend on the size of the Population. One important class of regulatory functions is the theta-logistic, which generalizes the logistic equation. Using this model as a motivation, this paper introduces a simple dynamical reformulation that generalizes many growth functions. The reformulation consists of two equations, one for population size, and one for the growth rate. Furthermore, the model shows that although population is density-dependent, the dynamics of the growth rate does not depend either on population size, nor on the carrying capacity. Actually, the growth equation is uncoupled from the population size equation, and the model has only two parameters, a Malthusian parameter rho and a competition coefficient theta. Distinct sign combinations of these parameters reproduce not only the family of theta-logistics, but also the van Bertalanffy, Gompertz and Potential Growth equations, among other possibilities. It is also shown that, except for two critical points, there is a general size-scaling relation that includes those appearing in the most important allometric theories, including the recently proposed Metabolic Theory of Ecology. With this model, several issues of general interest are discussed such as the growth of animal population, extinctions, cell growth and allometry, and the effect of environment over a population. (c) 2005 Elsevier Ltd. All rights reserved. AU - de Vladar, Harold ID - 4237 IS - 2 JF - Journal of Theoretical Biology TI - Density-dependence as a size-independent regulatory mechanism VL - 238 ER - TY - JOUR AU - Harold Vladar AU - González,J. A ID - 4235 JF - Journal of Theoretical Biology TI - Dynamic response of cancer under the influence of immunological activity and therapy ER - TY - JOUR AB - In finite populations, genetic drift generates interference between selected loci, causing advantageous alleles to be found more often on different chromosomes than on the same chromosome, which reduces the rate of adaptation. This “Hill–Robertson effect” generates indirect selection to increase recombination rates. We present a new method to quantify the strength of this selection. Our model represents a new beneficial allele (A) entering a population as a single copy, while another beneficial allele (B) is sweeping at another locus. A third locus affects the recombination rate between selected loci. Using a branching process model, we calculate the probability distribution of the number of copies of A on the different genetic backgrounds, after it is established but while it is still rare. Then, we use a deterministic model to express the change in frequency of the recombination modifier, due to hitchhiking, as A goes to fixation. We show that this method can give good estimates of selection for recombination. Moreover, it shows that recombination is selected through two different effects: it increases the fixation probability of new alleles, and it accelerates selective sweeps. The relative importance of these two effects depends on the relative times of occurrence of the beneficial alleles. AU - Roze, Denis AU - Nicholas Barton ID - 4248 IS - 3 JF - Genetics TI - The Hill-Robertson effect and the evolution of recombination VL - 173 ER - TY - GEN AB - A recent analysis has shown that divergence between human and chimpanzee varies greatly across the genome. Although this is consistent with ‘hybridisation’ between the diverging human and chimp lineages, such observations can be explained more simply by the null model of allopatric speciation. AU - Nicholas Barton ID - 4250 IS - 16 T2 - Current Biology TI - Evolutionary Biology: How did the human species form? VL - 16 ER - TY - CONF AU - Thomas Wies AU - Kuncak, Viktor AU - Lam,Patrick AU - Podelski,Andreas AU - Rinard,Martin ID - 4359 TI - Field Constraint Analysis ER - TY - CONF AU - Maler, Oded AU - Dejan Nickovic AU - Pnueli,Amir ID - 4373 TI - Real Time Temporal Logic: Past, Present, Future ER - TY - CONF AU - Maler, Oded AU - Dejan Nickovic AU - Pnueli,Amir ID - 4374 TI - From MITL to Timed Automata ER - TY - CONF AB - We propose and evaluate a new algorithm for checking the universality of nondeterministic finite automata. In contrast to the standard algorithm, which uses the subset construction to explicitly determinize the automaton, we keep the determinization step implicit. Our algorithm computes the least fixed point of a monotone function on the lattice of antichains of state sets. We evaluate the performance of our algorithm experimentally using the random automaton model recently proposed by Tabakov and Vardi. We show that on the difficult instances of this probabilistic model, the antichain algorithm outperforms the standard one by several orders of magnitude. We also show how variations of the antichain method can be used for solving the language-inclusion problem for nondeterministic finite automata, and the emptiness problem for alternating finite automata. AU - De Wulf, Martin AU - Doyen, Laurent AU - Thomas Henzinger AU - Raskin, Jean-François ID - 4406 TI - Antichains: A new algorithm for checking universality of finite automata VL - 4144 ER - TY - CONF AU - Alur, Rajeev AU - Pavol Cerny AU - Zdancewic,Steve ID - 4401 TI - Preserving Secrecy Under Refinement ER - TY - CONF AB - The synthesis of reactive systems requires the solution of two-player games on graphs with ω-regular objectives. When the objective is specified by a linear temporal logic formula or nondeterministic Büchi automaton, then previous algorithms for solving the game require the construction of an equivalent deterministic automaton. However, determinization for automata on infinite words is extremely complicated, and current implementations fail to produce deterministic automata even for relatively small inputs. We show how to construct, from a given nondeterministic Büchi automaton, an equivalent nondeterministic parity automaton that is good for solving games with objective . The main insight is that a nondeterministic automaton is good for solving games if it fairly simulates the equivalent deterministic automaton. In this way, we omit the determinization step in game solving and reactive synthesis. The fact that our automata are nondeterministic makes them surprisingly simple, amenable to symbolic implementation, and allows an incremental search for winning strategies. AU - Thomas Henzinger AU - Piterman, Nir ID - 4437 TI - Solving games without determinization VL - 4207 ER -