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2011 Joint Autumn Meeting of the BSCDB/BSM
“Advanced Microscopy Meets Cell and Developmental Biology”
October 22, 2011
Universiteit Antwerpen, Campus Groenenborger (Building T)
Local Organizers: Jean-Pierre Timmermans, Dirk Adriaensen & Peter Ponsaerts
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2011 Joint Autumn Meeting of the BSCDB/BSM
“Advanced Microscopy Meets Cell and Developmental Biology” !
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INVITED SPEAKERS
Invited Speakers
BSCDB & BSM – October 22, 2011 in Antwerp Invited Speakers "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
PROLIFERATE OR DIFFERENTIATE. INSIGHTS INTO THE ROLE OF BMP SIGNALING IN ENS DEVELOPMENT USING ZEBRAFISH.
Iain T. Shepherd.
Department of Biology, Emory University, Atlanta GA, USA. A fundamental question in developmental biology is how does a functional nervous system form during embryogenesis? We investigate the cellular, and molecular mechanisms involved in this process by studying the development of the vertebrate enteric nervous system (ENS). The ENS is derived from the neural crest. ENS neural crest precursors interact with themselves and their surrounding tissues to determine the migratory pathway the precursor cells will follow to and along the intestine, the number of enteric neurons/glia that the precursors will generate, and the final type of ENS neuron or glia that they will become. We have developed the zebrafish as a model system to study ENS development. We have identified and characterized several novel zebrafish ENS mutants many of which also exhibit abnormal endoderm development. Our studies suggest that endoderm derived factors directly and indirectly regulate ENS precursors migration, proliferation and differentiation. One of the most striking mutants we have identified is the lessen (lsn) mutant. Lsn mutants have a significant reduction in the number of enteric neurons as a result of a null mutation in the zebrafish MED24 gene, a component of the Mediator co-transcriptional activation complex. To identify which signaling pathways may be directly responsible for the lsn mutant‟s ENS phenotype we undertook a microarray study to identify changes in the gene expression profile in lsn mutant intestines as compared to wild type embryos. This data has revealed that there are specific changes in the expression of components of the BMP signaling pathway. This pathway has been previously implicated in ENS development in other species however the precise molecular mechanism by which this pathway elicits its effects on ENS development has not been determined. I will present data from my lab that shows BMP signaling regulates the expression of the inhibitory of differentiation (ID) genes in the intestine and ENS precursors. I will propose a new model for how BMP can apparently have different effects on ENS precursors depending on the developmental stage of the embryo and the concentration of BMP.
Invited Speakers
BSCDB & BSM – October 22, 2011 in Antwerp Invited Speakers "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
Short C.V. of Iain T. Shepherd I. Education 1990 B.Sc. with Honors, in Biochemistry from Imperial College of Science
Technology and Medicine University of London UK and Associate of the Royal College of Science. London UK
1994 D.Phil. in Physiological Sciences. University of Oxford UK “The Early Development of Tectal Efferent Projections in the Chick”
II. Scientific career 1990-1994 Pre-doctoral Fellow in the laboratory of Dr. Jeremy Taylor, Department
of Human Anatomy, University of Oxford, UK. Early development of the axon scaffold in the avian CNS.
1994- 1998 Post-doctoral fellow in the laboratory of Dr. Jonathan Raper, Department of Neuroscience, University of Pennsylvania, Philadelphia. Functional analysis of Sempahorin (collapsin) family axon guidance molecules during development.
1998 - 2002 Senior Research Fellow in the laboratory of Dr. David Raible, Department of Biological Structure, University of Washington, Seattle. Cellular molecular and genetic analysis of zebrafish ENS development.
2002- 2008 Assistant Professor in the Department of Biology, Emory University Atlanta.
2008-present Associate Professor with tenure in the Department of Biology, Emory University Atlanta.
III. Grants and awards 1990-1993 Medical Research Council UK Graduate Studentship 1998 Wellcome Trust UK Career Development Award 2002 Emory University Research Council Award 2002 Emory University Center for Teach and Curriculum Course Development
Grant 2003 March of Dimes Basil O'Connor New Investigator Award 2004 NIH RO1 from the NIDDK 1RO1 DK067285 " Development of Zebrafish
Enteric Nervous System 2009 Emory University Research Council Award
Invited Speakers
BSCDB & BSM – October 22, 2011 in Antwerp Invited Speakers "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
MAKING CRYO-ELECTRON TOMOGRAPHY MORE WIDELY APPLICABLE IN CELL BIOLOGY
Alexander Rigort, Elizabeth Villa, Felix Bäuerlein, David Günther, Jürgen M. Plitzko and Wolfgang Baumeister.
Max-Planck-Institut für Biochemie, Abt. Molekulare Strukturbiologie, D-82152 Martinsried, Germany.
Cryo-electron tomography (CET) allows to visualize the interior of cells and to investigate large protein complexes close to their native state and within the functional context of the cytoplasm. The application of this technique to complex cellular systems (i.e. large cells or tissues) is commonly referred to as “cellular tomography”. However, only sufficiently small structures (<500 nm) can be examined in toto by CET with a resolution that allows detection and identification of macromolecular structures using molecular pattern recognition methods. The bulk volume of most eukaryotic cells exceeds the thickness accessible by today‟s intermediate voltage transmission electron microscopes. It is therefore essential to be able to routinely prepare good quality thin specimens under cryo conditions, in order to apply CET efficiently in cell biology. For this purpose, tools and techniques providing controlled access to structural features buried deep inside cellular volumes have to be developed. Recently, focused ion beam (FIB) milling is receiving increasing attention as a technique for thinning frozen-hydrated biological specimens. In contrast to mechanical cryo-sectioning, thinning of the frozen-hydrated specimen occurs through the process of sputtering with focused ions, typically gallium. Pilot studies have shown that cryo-FIB milling can indeed be applied to frozen-hydrated material, rendering samples, which contained bacterial cells transparent enough for transmission electron microscopy [1, 2]. Here, we demonstrate an advanced FIB-based approach for the in situ preparation of thin TEM lamellas from frozen-hydrated cellular samples which opens up large windows into the crowded interior of cells. In order to make CET more widely applicable in cell biology not only specimen preparation is of importance, but also subsequent data analysis. For a deeper analysis of structural features in the complex settings of cellular landscapes, it is necessary to characterize them by parameters based on statistics. Consequently large data sets must be analyzed (similar to what is done e.g. in live cell imaging) – a task calling for automation. The three-dimensional organization of actin networks in the context of unperturbed cellular environments serves as an example for this approach. For a quantitative characterization of these networks, the tomograms must be segmented in a reproducible manner. We demonstrate this by introducing a novel automated procedure for the segmentation of actin filaments [3], which combines template matching with a new tracing algorithm. References [1] Marko et al., Nat. Methods. (2007), 4: 215-7. [2] Rigort et al., J Struct. Biol. (2010), 172:169-79. [3] Rigort et al., J Struct. Biol. (2011), doi:10.1016/j.jsb.2011.08.012 This research was supported by the European Commission in the 7th Framework Program (grant agreement HEALTH-F4-2008-201648/PROSPECTS)..
Invited Speakers
BSCDB & BSM – October 22, 2011 in Antwerp Invited Speakers "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
Short C.V. of Alexander Rigort I. Education 10/1996 – 04/2002 Diploma studies in Biology University of Bonn, Germany 04/2002 Diploma in Biology Major: Cell Biology, Minor: Genetics and
Physics 10/2003 – 10/2004 Studies in Computer Science University of Applied
Sciences Trier, Germany Department of Design and Informatics Degree: Certified Courses in Database Systems and Programming
04/2002 – 03/2005 Doctoral studies in Cell Biology Institute for Cell Biology, University of Bonn. Supervised by Prof. Dr. V. Herzog Dissertation Topic: “Migration tracks of human epidermal keratinocytes as biological nanostructures: structure, analysis and potential biological relevance”
03/2005 Dr. rer. nat. Faculty of Mathematics and Natural Sciences, University of Bonn, Germany Degree with honors („summa cum laude“)
II. Scientific career 2000 – 2005 Teaching assistant in practical courses of General Biology,
Animal Physiology and Cell Biology for undergraduate students, University of Bonn, Germany
2002 – 08/2005 Research fellow and teaching assistant at the Institute for Cell Biology, University of Bonn, Germany
09/2005 – 2008 Postdoctoral research at the Max-Planck-Institute of Biochemistry, Dept. of Molecular Structural Biology, Martinsried, Germany
2006 – 2008 Postdoctoral research at the Ben-Gurion University, Dept. of Life Sciences and the National Institute for Biotechnology in the Negev (NIBN), Beer-Sheva, Israel (extended stays)
Since 2008 Team leader of the Focused Ion Beam (FIB) microscopy group, Dept. of Molecular Structural Biology, Max-Planck-Institute of Biochemistry, Martinsried, Germany
Invited Speakers
BSCDB & BSM – October 22, 2011 in Antwerp Invited Speakers "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
ADVANCED MICROSCOPIC VISUALIZATION OF MITOCHONDRIAL FUNCTION: LIVE-CELL IMAGING OF THE CELL BIOLOGICAL CONSEQUENCES OF ISOLATED COMPLEX I DEFICIENCY.
Peter H.G.M. Willems and Werner J.H. Koopman.
Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
Inborn errors of metabolism have a collective incidence of 1 in 1,500 live births and in 1 of 10,000 cases the defect is of mitochondrial origin. A recent literature survey linked mutations in 221 genes in the nuclear DNA (nDNA) and 13 genes in the mitochondrial DNA (mtDNA) to a primary mitochondrial disorder. In the majority of patients with a mitochondrial disorder the disease is caused by a defect in the oxidative phosphorylation (OXPHOS) system, which is the main source of cellular ATP. Dysfunction of this system gives rise to a variety of clinical manifestations, particularly in organs and tissues with high-energy demand such as brain (encephalopathies), heart (cardiomyopathies), skeletal muscle (myopathies) and liver (hepatopathies). The OXPHOS system is built of ~85 proteins encoded by both the mtDNA (13 proteins) and the nDNA. These proteins are organized in 5 multisubunit enzyme complexes, which are embedded in the mitochondrial inner membrane. Complex I (CI) is the largest OXPHOS complex. It consists of 45 subunits, 7 encoded by the mtDNA and the remainder by the nDNA. Its main function is to abstract electrons from NADH, transport them along its internal redox pathway, using the energy released during this transport to translocate protons from the matrix to the intermembrane space and, finally, donate them to coenzyme Q. One-third of the patients with an inherited OXPHOS disease is diagnosed with isolated CI deficiency. Thus far, mutations have been identified in all 7 mtDNA- and 12 of the nDNA-encoded structural subunits as well as in a rising number of nDNA-encoded CI assembly factors. Mutations in nuclear genes encoding structural CI subunits have a dramatic effect on neurodevelopment and overall patient survival. The majority of patients present with Leigh disease, an early-onset, fatal neurodegenerative disorder that is typically characterized by symmetrical lesions of necrosis and capillary proliferation in variable regions of the central nervous system. Clinical signs and symptoms include muscular hypotonia, dystonia, developmental delay, abnormal eye movements, seizures, respiratory irregularities, failure to thrive and lactic acidemia. As far as our patient cohort is concerned, two children died in the neonatal period, whereas another six did not reach the age of one. Current treatment regimens of these children are purely empirical and most lack success. Therefore, with the final aim of developing new treatment strategies, we use live-cell luminometry and imaging microscopy to unveil the cell biological consequences of CI deficiency and to evaluate the effect of drug interventions. Because there is hardly any muscle biopsy material available for research purposes, we use primary skin fibroblasts, which are more readily accessible. All patient fibroblast lines used in our studies are from children, in whom an isolated complex I deficiency has been confirmed in both muscle tissue and cultured skin fibroblasts. An overview of our recent work will be presented, showing that mitochondrial membrane potential, Ca2+ handling and ATP production are to a variable extent impaired among a large cohort of CI-deficient patient fibroblast lines. From the results obtained, the picture emerges that a reduction in cellular CI activity leads to a depolarization of the mitochondrial membrane potential, resulting in a decreased supply of mitochondrial ATP to the Ca2+-ATPases of the intracellular stores and thus to a reduced Ca2+ content of these stores. In addition, the possible involvement of increased reactive oxygen production will be discussed. References -Smeitink J, van den Heuvel L, DiMauro S. The genetics and pathology of oxidative phosphorylation. Nat Rev Genet. 2001;2:342-52.
Invited Speakers
BSCDB & BSM – October 22, 2011 in Antwerp Invited Speakers "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
-Willems P, Valsecchi F, Distelmaier F, Verkaart S, Visch H, Smeitink J, Koopman W. Mitochondrial Ca2+ homeostasis in human NADH:ubiquinone oxidoreductase deficiency. Cell Calcium 2008;44:123-33. -Distelmaier F, Koopman W, van den Heuvel L, Rodenburg R, Mayatepek E, Willems P, Smeitink J. Mitochondrial complex I deficiency: from organelle dysfunction to clinical disease. Brain. 2009; 132:833-42. -Koopman W, Nijtmans L, Dieteren C, Roestenberg P, Valsecchi F, Smeitink J, Willems P. Mammalian mitochondrial complex I: biogenesis, regulation, and reactive oxygen species generation. Antioxid Redox Signal. 2010;12:1431-70. -Koopman W, Willems, P and Smeitink J. Mitochondrial Medicine. N Engl J Med, revised version submitted. Short C.V. of Peter H.G.M. Willems I. Education 1977 Higher Vocational Education, Zoology, Physiology, Pharmacology, OLAN,
Nijmegen, The Netherlands 1980 B. Sc Biology, Radboud University, Nijmegen, The Netherlands 1984 M. Sc Biology, Radboud University, Nijmegen, The Netherlands 1988 PhD, Mechanisms of Signal Transduction in the Exocrine Pancreas,
Biochemistry, Radboud University, Nijmegen, The Netherlands II. Scientific career 1974-1977 Laboratory assistant, Organon International bv, Oss, The Netherlands 1997-2001 Assistant professor, Biochemistry, Radboud University Nijmegen Medical
Centre, The Netherlands 2001-present Associate professor, Vital Cell Imaging, Biochemistry, Nijmegen Centre
for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
III Grants and awards 1992-1997 Research fellow of the Royal Netherlands Academy of Arts and Sciences
(KNAW), Biochemistry, Radboud University Nijmegen Medical Centre, The Netherlands
IV. Brief research interests Cell biological understanding of mitochondrial disorders and development of rational therapeutics. Focus lies on life cell analysis of cell biological consequences of inherited oxidative phosphorylation defects. Dynamic mathematical models are developed to simulate the effect of metabolic and/or pharmaceutic interventions.
SHORT COMMUNICATIONS
Selected short oral communications
BSCDB & BSM – October 22, 2011 in Antwerp Short communications "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
MORNING SESSION Poster abstract 10:
EPITHELIAL-ENDOTHELIAL CROSS-TALK DURING THYROID FOLLICLE FORMATION.
Anne-Christine Hick, Céline Forez, Pierre J. Courtoy and Christophe E. Pierreux.
Université Catholique de Louvain, de Duve Institute, CELL Unit, Brussels. Poster abstract 7:
QUANTITATIVE ANALYSIS OF GLIAL CELL BEHAVIOUR FOLLOWING MESENCHYMAL STROMAL CELL IMPLANTATION IN THE CENTRAL NERVOUS SYSTEM OF IMMUNOCOMPETENT MICE.
Nathalie De Vocht (1,2), Dan Lin (3), Jelle Praet (1,2), Kristien Reekmans (1), Jasmijn Daans (1), Niel Hens (3), Patrick Pauwels (4), Zwi Berneman (1), Annemie Van der Linden (2), Peter Ponsaerts (1).
1: Laboratory of Experimental Hematology, Vaccine and Infectious Disease Institute (Vaxinfectio), University of Antwerp, Antwerp;
2: BioImaging Laboratory, University of Antwerp, Antwerp; 3: Center for Statistics, I-Biostat, University of Hasselt, Hasselt; 4: Laboratory of Pathology, University of Antwerp, Antwerp. Poster abstract 27:
FUNCTIONAL AND MORPHOLOGICAL VISUALIZATION OF IN VITRO NEURONAL NETWORK MATURATION.
Peter Verstraelen (1), Isabel Pintelon (1), Rony Nuydens (2), Sofie Thys (1), Inge Keuleers (1), Frans Cornelissen (2), Christoph Tombeur (2), Theo Meert (2) and Jean-Pierre Timmermans (1).
1: Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, B-2020 Antwerp;
2: Johnson & Johnson Pharmaceutical Research and Development, Neuroscience Department, B-2340 Beerse.
Selected short oral communications
BSCDB & BSM – October 22, 2011 in Antwerp Short communications "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
AFTERNOON SESSION Poster abstract 8:
REPETITIVE DISRUPTIONS OF THE NUCLEAR ENVELOPE INVOKE TEMPORARY LOSS OF CELLULAR COMPARTMENTALIZATION IN LAMINOPATHIES.
Winnok De Vos (1), Frederik Houben (2), Erik Manders (1,3), Valerie Verstraeten (2), Maurice Van Steensel (2), Carlo Marcelis (4), Arthur Van Den Wijngaard (2), Ashraf Malhas (5), David Vaux (5), Frans Ramaekers (2) and Jos Broers (2).
1: Dept. Molecular Biotechnology, Ghent University, Belgium; 2: Maastricht University Medical Center, The Netherlands; 3: Center for Advanced Microscopy, University of Amsterdam, The Netherlands; 4: Department of Clinical Genetics, Radboud University Nijmegen Medical Center, The
Netherlands; 5: Sir William Dunn School of Pathology, Oxford University, UK. Poster abstract 19:
TOWARDS AN OPTIMISED LUNG SLICE MODEL FOR CONFOCAL LIVE CELL IMAGING OF THE PULMONARY NEUROEPITHELIAL BODY MICROENVIRONMENT USING GAD-GFP MICE.
Kathy Schnorbusch, Inge Brouns, Robrecht Lembrechts, Isabel Pintelon, Jean-Pierre Timmermans and Dirk Adriaensen.
Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Groenenborgerlaan 171, BE-2020 Antwerp.
Poster abstract 15:
DYNAMICS AND ORGANIZATION OF ALPHA3-CONTAINING GLYCINE RECEPTORS USING SINGLE MOLECULE DETECTION.
Kristof Notelaers (1,2), Susana Rocha (2), Nick Smisdom (1), Daniel Janssen (1), Jochen C. Meier (3), Jean-Michel Rigo (1), Johan Hofkens (2) and Marcel Ameloot (1).
1: Biomedical Research Institute, Hasselt University and School of Life Sciences, transnational University Limburg, Agoralaan building C, 3590 Diepenbeek;
2: Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, B-3001 Heverlee;
3: RNA Editing and Hyperexcitability Disorders Helmholtz Group, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Strasse 10, 13092 Berlin, Germany.
POSTER ABSTRACTS
Poster 1
BSCDB & BSM – October 22, 2011 in Antwerp Alpaerts Katrien "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
1: Alpaerts Katrien presenting author ; [email protected]
VISUALIZATION OF MURINE INTESTINAL DENDRITIC CELLS DURING INTESTINAL SCHISTOSOMIASIS: A MORPHOLOGICAL GLANCE AT THE SUBSETS IN THE LAMINA PROPRIA.
Katrien Alpaerts (1), Roeland Buckinx (1), Nathalie Cools (3), Leela R. Avula (1), Viggo F. Van Tendeloo (3,4), Luc Van Nassauw (1,2) and Jean-Pierre Timmermans (1).
1: Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, B-2020 Antwerp.
2: Laboratory of Human Anatomy and Embryology, Faculty of Medicine, University of Antwerp, B- 2020 Antwerp.
3: Laboratory of Experimental Hematology, Vaccine & Infectious Disease Institute (Vaxinfectio), Faculty of Medicine, University of Antwerp, B-2020 Antwerp.
4: Center for Cellular Therapy and Regenerative Medicine, Antwerp University Hospital, B-2020 Antwerp.
The gastrointestinal (GI) tract is heavily exposed to a large number of food antigens, bacteria, viruses and toxins. This infers the need to maintain a delicate balance between tolerance to self-antigens and active immune responses towards pathogenic agents. Intestinal dendritic cells (DCs) are key regulators of intestinal immune homeostasis as they have a major influence on T-cell responses. In the past, intestinal DCs were simply defined as one population of CD11c+MHCII+ cells. At present, based on the expression of other cell surface markers (eg. CD103 and CX3CR1), diverse DC subsets were recognized in the lamina propria (LP) and organized lymphoid tissues. So, two main subsets of intestinal LP-DCs, i.e. a migratory subset expressing CD11c/CD103 and a resident subset expressing CD11c/CX3CR1, each with distinct functions, were described. The aim of the present study was to reveal the distribution of both LP-DC subsets in the healthy and infected murine ileum to unravel the role of these DCs in mucosal immune responses and neuro-immune interactions. We used Schistosoma mansoni-infected mice because intestinal schistosomiasis is associated with impaired epithelial barrier function. Using immunohistochemistry, we visualized and characterized intestinal LP-DCs during inflammation with antibodies against CD11c, CD103 and CX3CR1. CD11c+ cells were observed in the LP of normal and infected ileum. CD103+ cells were only detected in the LP of infected ileum, while CX3CR1+ cells were found in the LP of control and inflamed ileum. Double immunolabeling with antibodies against CD11c and the neuronal marker β-tubulin, revealed in the LP neuronal fibers lying in very close proximity to CD11c+ cells in the LP, suggesting neuron – DC communication. In order to accurately quantify DC subtypes, we created and optimized a dissociation and isolation protocol based on enzymatical and mechanical disaggregation of ileal tissue. Flow cytometric analyses showed an average viability of 75% with 10-15% CD11c+ cells in the isolate. In the future, this method will be refined using multi-parametric immunolabeling to identify LP-DC subtypes and to evaluate the effect of inflammation on DC phenotypes. At present, we conclude that during intestinal schistosomiasis, migratory CD103+ DCs are attracted to the LP and that a close interaction between neurons and DCs is plausible. This study was supported by the FWO-grant G.0179.08N.
Poster 2
BSCDB & BSM – October 22, 2011 in Antwerp Ameloot Marcel "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
2: Ameloot Marcel presenting author ; [email protected]
FLUORESCENCE LIFETIME FILTERED RASTER IMAGE CORRELATION SPECTROSCOPY.
Ben De Clercq and Marcel Ameloot.
Biomedical Research Institute, Hasselt University and School of Life Sciences, transnational University Limburg, Agoralaan building C, 3590 Diepenbeek.
The heterogeneity in composition and interaction within the cellular membrane translates into a wide range of diffusion coefficients of its constituents. Therefore, several complementary techniques such as fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT), each with its own range of temporal en spatial scales, have to be applied to explore the dynamics of membrane components. Raster image correlation spectroscopy (RICS) offers a much wider dynamic range than each of these methods separately and allows for spatial mapping of the dynamic properties. RICS is implemented on a confocal laser-scanning microscope. The wide dynamic range is achieved by exploiting the inherent time information carried by the scanning laser beam in the generation of the confocal images. The diffusion coefficient is retrieved from the spatial autocorrelation function of the image. Measurements are done using a confocal microscope with two-photon illumination and photon counting detection. The micro time (i.e. the time since the excitation pulse) and the macro time (i.e. the time since the start of the acquisition) of every detected photon are registered. The time correlated single photon counting (TCSPC) in micro time mode allows measuring fluorescence lifetimes at the (sub)nanosecond time scale. The simultaneous registration of micro and macro times allows for lifetime filtering in an FCS experiment. This means that an autocorrelation function can be obtained for each lifetime component present in the sample. This filtering can be applied to emitters that have overlapping spectra and requires only one detection channel. In this work we aim to apply the lifetime filtering to the RICS method. Application to dyes with an environment dependent lifetime should allow for a mapping of the diffusion coefficient based on the fluorescent lifetimes. The spatial autocorrelation function is decomposed in separate correlation functions. Each of them is analyzed for a diffusion coefficient. The implicit fluorescence lifetime imaging allows then to associate a local diffusion coefficient to each pixel. Preliminary results obtained with homogeneous samples are presented.
Poster 3
BSCDB & BSM – October 22, 2011 in Antwerp Boeykens Nele "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
3: Boeykens Nele presenting author ; [email protected]
CHARACTERIZATION OF A NASH/RESECTION MODEL TO EVALUATE CELLULAR THERAPY FOR LIVER DISEASE.
Nele Boeykens (1), Peter Ponsaerts (2), Dirk Ysebaert (1) and Kathleen De Greef (1).
1: Lab of Experimental Surgery, University of Antwerp; 2: Lab of Experimental Hematology, University of Antwerp, Belgium. Background: Surgical resection is currently the only treatment modality for patients with primary or secondary liver tumours. Although healthy liver tissue is able to fully regenerate after 70 % resection of liver mass, tumours are unfortunately most often found in (pre)cirrhotic, non-alcoholic steatohepatitic (NASH) and/or post-chemotherapy livers, which are less able to regenerate upon resection. In this study, we investigate a combination of two established liver injury models, partial hepatectomy (PHx) and NASH, as a pre-clinical animal model to investigate potential beneficial effects of cell therapy on the (endogenous) recovery of damaged liver tissue following resection. Materials & Methods: In a first experiment, we evaluated the PHx model by performing a 70% partial hepatectomy in rats and scrutinized the endogenous recovery for 6 weeks. In a second experiment, rats were fed by a methionine/choline deficient diet (MCDD) for 5 weeks followed by 70% PHx. In a third experiment, rats were fed by a MCDD for 4 weeks, followed by 70% PHx, feeding by a standard diet for 1 week and a MCDD for 3 weeks. Afterwards endogenous liver recovery was monitored. In all experiments, rats were weighted weekly and cholinesterase levels were evaluated in blood samples. At the end of the experiment, the liver was dissected and prepared for histology. Liver sections were stained with haematoxylin/eosin (H/E) and Oil Red O to evaluate liver architecture and steatosis, respectively. Results: In the first experiment, blood cholinesterase was significantly decreased after PHx and returned to normal values after 6 weeks. In the second experiment, all animals died during PHx, suggesting that 5 weeks MCDD is too long for combining with PHx. In the third experiment, blood cholinesterase was significantly decreased and steatosis was histologically observed in all rats after 4 weeks of MCDD. After PHx at this time point, the blood cholinesterase level remained decreased, suggesting a longer period needed for endogenous recovery. Currently, cholinesterase values for combined NASH/PHx are monitored long-term and liver tissue is examined at different time points. Conclusion: A combination of two liver injury models, PHx and NASH, was evaluated for suitability to monitor efficacy of pre-clinical cell therapy protocols on endogenous liver regeneration. Although further research is needed, preliminary data demonstrate the feasibility of this combined model over single injury models.
Poster 4
BSCDB & BSM – October 22, 2011 in Antwerp Bronckaers Annelies "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
4: Bronckaers Annelies presenting author ; [email protected]
ULTRASTRUCTURAL AND IMMUNOHISTOCHEMICAL CHARACTERISATION OF STEM CELLS FROM APICAL PAPILLA.
A. Bronckaers (1,*), W. Martens (1,*),P. Hilkens (1), T. Struys (1), C. Politis (1,2) and I. Lambrichts (1).
1: Laboratory of Morphology, Biomedical Research Institute (BIOMED), Hasselt University, Diepenbeek;
2: Department of Maxillo-Facial Surgery, Hospital of East Limburg (ZOL), Genk; *: these authors contributed equally to this research. Identification and isolation of high-quality human postnatal stem cells from accessible resources is an important goal in stem cell research. In this study, we describe the mesenchymal stem cell properties of stem cells of apical papillae (SCAP), obtained from extracted third molars. Besides classical immunohistochemical and histological staining methods, transmission electron microscopy (TEM) was used to study the differentiation potential of these stem cells, as TEM provides detailed ultrastructural information and incontestable evidence of differentiation. Undifferentiated SCAP displayed typical mesenchymal stem cell characteristics: adherence to a plastic surface, a spindle-shaped fibroblast-like morphology and the capacity to form colonies in vitro. Furthermore, the cells expressed the mesenchymal stem cell markers CD29, CD44, CD105, CD117, CD146 and Stro-1. TEM analysis showed the ultrastructural characteristics of SCAP: a bipolar morphology with euchromatic nuclei, cell organelles spread throughout the cell cytoplasm and absence of extracellular matrix depositions. After incubation in adipogenic differentiation medium, SCAP were able to differentiate into adipogenic cells: immune-reactivity of fatty acid binding protein-4 was observed in differentiated cells. Both Oil red O staining and TEM analysis demonstrated an accumulation of numerous intracellular lipid droplets. Induction of SCAP in chondrogenic medium, resulted in cells that were immune-reactive for aggrecan. At the ultrastructural level, the cells possessed intracellular vesicles and numerous striated collagen fibres were found in the extracellular matrix. Osteogenic stimulation of SCAP resulted in the occurrence of calcified matrix deposits which could be visualised with Alizarin Red S staining. Furthermore, the de novo expression of alkaline phosphatase was observed. TEM analysis revealed that the SCAP had acquired an osteoblast-like morphology with an eccentric nucleus. Furthermore, striated collagen fibres, mineralized nuclei together with the presence of hydroxyapatite needles were found in the extracellular matrix, which confirmed real mineralization after differentiation. In conclusion, our study is the first study that provides ultrastructural evidence that SCAP represent an easy-accessible mesenchymal-like stem cell source which is capable of differentiating into cells of the mesodermal lineage. Our data establish the foundation for further studies to determine the efficacy of the application of SCAP in cell-based therapies for the reconstruction of fat, cartilage and bone.
Poster 5
BSCDB & BSM – October 22, 2011 in Antwerp Buckinx Roeland "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
5: Buckinx Roeland presenting author ; [email protected]
INTRACELLULAR IMMUNOREACTIVITY OF TRPV1 IN THE GASTROINTESTINAL TRACT.
Roeland Buckinx, Luc Van Nassauw, Leela Avula, Katrien Alpaerts, Leen Uyttebroek and Jean-Pierre Timmermans.
Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, B-2020 Antwerp.
The transient receptor potential vanilloid 1 (TRPV1) or the capsaicin receptor is a Ca2+-permeant non-selective cation channel responding to noxious stimuli such as low pH (<5.9) and hyperthermia (>43°C). It is well characterized in a subset of sensory neurons in dorsal root and trigeminal ganglia and it plays a crucial role in pain mechanisms. In line with this sensory role, TRPV1 has been described in extrinsic afferent nerve fibers throughout the gastrointestinal tract (GIT). To date, intrinsic expression of TRPV1 has been the subject of debate. Although mRNA expression of TRPV1 in GIT has been described and some studies in several mammalian species report on the TRPV1 expression in enteric neurons, it is generally assumed that in the GIT, the major cellular sources of TRPV1 are the nerve fibers of extrinsic primary afferent neurons. This is rather surprising given the increasing evidence for sensory capacity of enteric neurons. In the present study, we aimed to unravel the distribution and expression of TRPV1 in the non-inflamed and inflamed murine GIT using antibodies directed against different TRPV1 immunogens. The best results were obtained with two antibodies raised in different species and directed against different immunogens in the C-terminus of rat TRPV1, but a distinct expression profile of TRPV1 immunoreactivity throughout the gastrointestinal tract was observed. Using the guinea-pig antibody, immunoreactivity was detected intracellularly in almost all somata of enteric neurons, while using the rabbit antibody, immunoreactivity was present on nerve fibres throughout the GIT and on a few somata of enteric neurons. Both antibodies have been raised against the C-terminus of the channel, which harbors two phosphorylation sites (T704 and S800) and binding domains for phosphatidylinositol 4,5-bisphosphate (PIP2) and calmodulin. We suggest that allosteric changes due to phosphorylation of TRPV1 or to binding of calmodulin on TRPV1, reveal the difference in cellular distribution of immunoreactivity. Furthermore, our data clearly indicate that almost all enteric neurons express TRPV1, albeit intracellularly. An ectopic but functional form of TRPV1 in the endoplasmic reticulum has been described in spinal neurons. Intracellular TRPV1 is suggested to be mainly involved in neuronal health.
Poster 6
BSCDB & BSM – October 22, 2011 in Antwerp De Rycke Riet "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
6: De Rycke Riet presenting author ; [email protected]
THE DMBR/PSB TRANSMISSION ELECTRON MICROSCOPY (TEM) CORE FACILITY VIB-UGENT.
Riet De Rycke (1,2), Dana Demedts (1,2), Tom Beeckman (2), Chris Guérin (1), Frans Van Roy (1) and Dirk Inzé (2).
1: VIB-Dept. for Molecular Biomedical Research, VIB and Ghent University, B-9052 Ghent;
2: VIB-Dept. of Plant Systems Biology, VIB and Ghent University, B-9052 Ghent. The TEM facility located on the campus „Ledeganck‟ of the UGent is providing expertise in the use and application of transmission electron microscopy to serve biological or biomedical research. The facility was established in 2003 and uses the equipment of the Department of Biology. EQUIPMENT AVAILABLE: Jeol JEM1010, Leica ultramicrotome UC6, Leica high-pressure freezer (EM-Pact), Leica AFS (automatic freeze substitution), Ditabis Imaging Plate scanner. The aim of the facility is to support research projects of all scientific groups of VIB1, Department for Molecular Biomedical Research (DMBR), http://www.dmbr.ugent.be, including projects in cell biology, inflammation, glycobiology, virology, cancer biology, cell death, developmental biology, immunology…and VIB2, Department of Plant Systems Biology (PSB), http://www.psb.ugent.be, including projects dealing with cell cycle, cell death, gene regulation, plant microbes, root development, secondary metabolites… New approaches such as high-pressure freezing of various types of cells and tissues have been developed and standard TEM methods for morphological analysis and immuno-gold electron microscopy (single/double labeling) are provided. The benefit of such a TEM core facility for the ongoing research at the VIB has been demonstrated by the numerous high impact factor papers that relied on the addition of TEM-data (2011-2010): Dierendonck et al. JACS, 2011,Remijsen et al. Cell Res , 2011, Batni et al., PLoS ONE, 2011, Festjens et al., EMBO Molecular Medicine, 2011, Babiychuk et al., PNAS, 2010, Feraru et al., The Plant Cell, 2010, Curvers et al., Plant Journal, 2010, Perez Guerra et al., Plant Physiology, 2010, Skirycs et al., Plant Physiology, 2010, Mc Guire et al., J Immunol, 2010, Wirawan et al., Cell Death Dis , 2010, Guerfal et al., Microb Cell Fact, 2010.
Poster 7
BSCDB & BSM – October 22, 2011 in Antwerp De Vocht Nathalie "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
7: De Vocht Nathalie presenting author ; [email protected]
QUANTITATIVE ANALYSIS OF GLIAL CELL BEHAVIOUR FOLLOWING MESENCHYMAL STROMAL CELL IMPLANTATION IN THE CENTRAL NERVOUS SYSTEM OF IMMUNOCOMPETENT MICE.
Nathalie De Vocht (1,2), Dan Lin (3), Jelle Praet (1,2), Kristien Reekmans (1), Jasmijn Daans (1), Niel Hens (3), Patrick Pauwels (4), Zwi Berneman (1), Annemie Van der Linden (2), Peter Ponsaerts (1).
1: Laboratory of Experimental Hematology, Vaccine and Infectious Disease Institute (Vaxinfectio), University of Antwerp, Antwerp;
2: BioImaging Laboratory, University of Antwerp, Antwerp; 3: Center for Statistics, I-Biostat, University of Hasselt, Hasselt; 4: Laboratory of Pathology, University of Antwerp, Antwerp. During the past decade, many pre-clinical studies have ascribed therapeutic benefits to cell grafting as a tool to treat various traumatic and/or degenerative injuries of the central nervous system (CNS). However, currently profound knowledge regarding the cellular and/or molecular events following cell grafting in the CNS is lacking. Therefore, in this study we grafted FVB mouse-derived Luciferase/eGFP-expressing bone marrow-derived mesenchymal stromal cells (MSC) in the CNS of syngeneic immune-competent mice and monitored their survival and migration non-invasively using in vivo bioluminescence imaging (BLI) during a time period of two weeks. Optimized statistical methodology for in vivo BLI revealed no significant difference between the degree of cell survival at week 1 and 2 post-implantation. Next, in order to validate the obtained in vivo BLI results, cell grafted mouse brains were cryo-sectioned to obtain slides from the whole graft area. Fluorescence microscopic analysis and advanced image processing using TissueQuest software, i.e. image processing software that creates a flow cytometry-like representation of microscopic images, allowed for accurate determination of cell graft survival and migration. While no cell migration was observed at week 1 and 2 post-implantation, eGFP+ MSC graft survival, which was determined to be around 50%, did not show significant differences between week 1 and 2 post-implantation, thereby validating the in vivo BLI results. In addition, we determined the number and phenotype of MSC graft infiltrating microglia and MSC graft surrounding astrocytes at day 0, week 1 and week 2 post-implantation. Results clearly indicate that MSC grafts become infiltrated with Iba1+ microglia, for which around 40% display a CD11b+ and 30% display a MHCII+ activated phenotype at week 1 and 2 post-implantation, and become surrounded by GFAP+ astrocytes, indicating the development of graft-surrounding scar tissue with unknown function. In conclusion, despite the fact that the CNS has historically been considered to be immune-privileged, our data demonstrate that the CNS is not immune-ignorant to autologous cellular implants. Further research towards the cellular and molecular events following cell grafting in the CNS, including their interaction with endogenous microglia and astrocytes, will reveal the fundamental aspects of their therapeutic benefit and will help to design effective (cell-based) treatment modalities for CNS injury.
Poster 8
BSCDB & BSM – October 22, 2011 in Antwerp De Vos Winnok "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
8: De Vos Winnok presenting author ; [email protected]
REPETITIVE DISRUPTIONS OF THE NUCLEAR ENVELOPE INVOKE TEMPORARY LOSS OF CELLULAR COMPARTMENTALIZATION IN LAMINOPATHIES.
Winnok De Vos (1), Frederik Houben (2), Erik Manders (1,3), Valerie Verstraeten (2), Maurice Van Steensel (2), Carlo Marcelis (4), Arthur Van Den Wijngaard (2), Ashraf Malhas (5), David Vaux (5), Frans Ramaekers (2) and Jos Broers (2).
1: Dept. Molecular Biotechnology, Ghent University, Belgium; 2: Maastricht University Medical Center, The Netherlands; 3: Center for Advanced Microscopy, University of Amsterdam, The Netherlands; 4: Department of Clinical Genetics, Radboud University Nijmegen Medical Center, The
Netherlands; 5: Sir William Dunn School of Pathology, Oxford University, UK. The nuclear lamina provides structural support to the nucleus and has a central role in nuclear organization and gene regulation. Defects in its constituents, the lamins, lead to a class of genetic diseases collectively referred to as laminopathies. Using live cell imaging, we observed the occurrence of intermittent, non-lethal ruptures of the nuclear envelope in dermal fibroblast cultures of patients with different mutations of lamin A/C. These ruptures, which were absent in normal fibroblasts, could be mimicked by selective knockdown as well as knockout of LMNA and were accompanied by the loss of cellular compartmentalization. This was demonstrated a.o. by the efflux of nuclear regulatory proteins and complexes and resulted in downstream effects on stress-responsive gene expression. Since this phenomenon was observed in all laminopathy variants examined, albeit with varying severity, our data suggest that nuclear rupture and loss of compartmentalization may add to cellular dysfunction and disease development in various laminopathies.
Poster 9
BSCDB & BSM – October 22, 2011 in Antwerp Gervois Pascal "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
9: Gervois Pascal presenting author ; [email protected]
DENTAL PULP STEM CELLS: NEUROGENIC DIFFERENTIATION POTENTIAL AND FERUMOXIDE NANOPARTICLE LABELLING.
Pascal Gervois (1), Tom Struys (1), Annelies Bronckaers (1), Wendy Martens (1), Petra Hilkens (1) Constantinus Politis (1,2) AND Ivo Lambrichts (1).
1: Laboratory of Morphology, Biomedical Research Institute (BIOMED), Hasselt University, Diepenbeek.
2: Department of Maxillo-Facial Surgery, Hospital of East Limburg (ZOL), Genk. Dental pulp stem cells (DPSC), the mesenchymal stem cells (MSCs) found in the human dental pulp, share phenotypical and multilineage differentiation characteristics with bone marrow derived MSCs (BMMSCs). Since DPSCs are much easier to harvest in comparison to BMMSCs, it is worthwhile to consider them as a useful alternative MSC source in stem cell research. As recent studies show that MSC retain some plasticity, we evaluated the neural differentiation potential of DPSCs in vitro. In addition, DPSCs were labelled with the commercially available ferumoxide Endorem® to allow visualization with Magnetic Resonance Imaging (MRI) following engraftment. To induce neurogenic differentiation, DPSC were incubated with basis fibroblast growth factor (bFGF) and epidermal growth factor (EGF) for 21 days. Ultrastructurally, neuronal differentiated DPSC acquired a bipolar morphology and an increased metabolic activity. Vesicular transport was observed at cell-cell contact zones between differentiated cells. Immunocytochemical analysis showed that DPSCs possess baseline expression of neural associated markers. Only NeuN was found to be differentially expressed in induced DPSCs. Therefore, we propose NeuN as a marker to distinguish between undifferentiated and neurogenic differentiated DPSCs. In a second part of our study, DPSC were labelled with different concentrations of Endorem® whether or not combined with different concentrations of Poly-L-Lysine (PLL). Cell viability and intracellular iron content were determined using MTT and atomic absorption spectrophotometry (AAS) respectively. MRI, MTT, AAS and Transmission Electron Microscopy analysis showed that DPSCs could be labelled with Endorem® when combined with PLL, preserving cell viability and morphology. Furthermore, it was shown that the optimal concentration for labelling DPSCs is 15_6g/ml Endorem® combined with 0.75 _6g/ml PLL. This labelling procedure will be used in future in vivo studies where the application of DPSC as a treatment for neurological disorders will be evaluated.
Poster 10
BSCDB & BSM – October 22, 2011 in Antwerp Hick Anne-Christine "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
10: Hick Anne-Christine presenting author ; [email protected]
EPITHELIAL-ENDOTHELIAL CROSS-TALK DURING THYROID FOLLICLE FORMATION.
Anne-Christine Hick, Céline Forez, Pierre J. Courtoy and Christophe E. Pierreux.
Université Catholique de Louvain, de Duve Institute, CELL Unit, Brussels. The thyroid is an endocrine gland composed of many spheroid structures called follicles. These consist of a monolayer of polarized epithelial cells whose apical pole delimits a lumen and the basal pole is closely apposed to a dense capillary network. The association of epithelial follicles with capillaries forms the angio-follicular units and allows the delivery of thyroid hormones into the circulation. During embryonic development, the thyroid primordium emerges from the endoderm as a mass of epithelial cells, which later reorganize into polarized monolayers. To understand formation of the thyroid angio-follicular units, we asked 2 questions: (i) when and how are endothelial cells recruited to the developing thyroid and (ii) are endothelial cells important for epithelial folliculogenesis? Using multiplex immunolabelling on tissue sections and whole-mount preparations, we showed that endothelial cells are closely associated with epithelial cells as soon as they emerge from the endoderm and that endothelial density increases with thyroid development. In situ experiments showed a strong expression of VEGF-A by thyroid epithelial cells and of its receptor (VEGFR2) by endothelial cells. This suggests that the recruitment of endothelial cells and their close association with epithelial cells could be due to the VEGFA/R2 pathway. The functional role of endothelial cells was studied ex vivo and in vivo. We set up an original ex vivo culture system of microdissected thyroid explants that faithfully reproduce thyroid follicle formation and differentiation. Treatment of these explants with VEGFR2 inhibitors causes a dramatic reduction of blood vessel density and this was accompanied by impaired epithelial folliculogenesis. To confirm these results in vivo, we inactivated Vegfa specifically in the thyroid. Whole-mount immunolabeling, in situ hybridization and RT-qPCR demonstrated that vascular density was reduced in Vegfa KO thyroid. Furthemore, epithelial folliculogenesis was impaired: instead of forming open follicles, the Vegfa KO epithelial cells failed to acquire apico-basal polarity and remained grouped as a mass of cells with numerous small lumens. In conclusion, our data suggest that epithelial production of VEGF-A by thyroid progenitors recruits and determines the spatial organization of endothelial cells that, in turn, control epithelial cell polarization and their three-dimensional organization into follicles.
Poster 11
BSCDB & BSM – October 22, 2011 in Antwerp Hilkens Petra "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
11: Hilkens Petra presenting author ; [email protected]
THE ANGIOGENIC PROPERTIES AND ENDOTHELIAL DIFFERENTIATION POTENTIAL OF HUMAN DENTAL PULP STEM CELLS
Petra Hilkens (1), Annelies Bronckaers (1), Wendy Martens (1), Constantinus Politis (1,2) and Ivo Lambrichts (1).
1: Laboratory of functional morphology, Biomedical research Institute, Hasselt University, B-3590 Diepenbeek.
2: Department of Maxillo-Facial Surgery, Ziekenhuis Oost-Limburg (ZOL), B-3600 Genk. Angiogenesis plays an important role in wound healing and tissue engineering, but also in diseases such as stroke and myocardial infarction (MI). Since the traditional revascularization therapies and the currently tested stem cell therapies do not meet the expectations, this study proposes human dental pulp stem cells (hDPSC) as a potential tool for vascular regeneration. Recent studies have indicated that hDPSC might have angiogenic properties. However, a more elaborate angiogenic profiling of these stem cells is required before any therapeutic application is possible. The goal of this study is to elucidate the angiogenic properties and the endothelial differentiation potential of hDPSC. The angiogenic secretion profile of hDPSC was identified by means of RT-PCR and ELISA. In order to determine the biological effects of hDPSC on endothelial cells (EC), different in vitro models of angiogenesis were performed with hDPSC conditioned medium, such as an MTT assay (for proliferation), a transwell assay and a wound healing assay (for migration) and a hypoxia assay (for survival). After incubating hDPSC with different induction media for several periods of time, their endothelial differentiation potential was determined by means of FACS and transmission electron microscopy (TEM). hDPSC express pro-angiogenic as well as anti-angiogenic factors at mRNA and protein level, such as VEGF, IL-8, endostatin and PAI-1. Furthermore, the hDPSC conditioned medium significantly increased EC migration, as shown by the transwell and wound healing assay. However, the addition of neutralizing antibodies against VEGF and IL-8 did not significantly decrease EC migration, suggesting the participation of other angiogenic factors in the migration process. There was also no significant effect of hDPSC conditioned medium on EC proliferation and survival. In terms of endothelial differentiation, FACS indicated no increased endothelial marker expression in the tested conditions. TEM analysis showed no straightforward results. However, there were minor morphological changes suggestive of an endothelial differentiation potential. These results suggest that hDPSC have a predominant paracrine angiogenic effect in vitro, in particular on EC migration. In the future, the in vitro models of angiogenesis need to be performed with a human endothelial cell line in order to rule out potential species-related differences. Since hDPSC cannot differentiate into endothelial cells under the given circumstances, an optimization of the applied differentiation protocols is also required in order to further determine the endothelial differentiation potential of hDPSC. If these stem cells are able to regenerate and/or to contribute to vascular networks, this will have great therapeutic potential, not only in terms of pulp regeneration, but also as a cell-based therapy for stroke and MI patients.
Poster 12
BSCDB & BSM – October 22, 2011 in Antwerp Lembrechts Robrecht "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
12: Lembrechts Robrecht presenting author ; [email protected]
UNRAVELING MECHANOSENSORY TRANSDUCTION IN THE PULMONARY NEUROEPITHELIAL BODY MICROENVIRONMENT: A MULTILABEL IMMUNOHISTOCHEMICAL AND HIGH RESOLUTION CONFOCAL CALCIUM IMAGING STUDY.
Robrecht Lembrechts, Inge Brouns, Kathy Schnorbusch, Isabel Pintelon, Jean-Pierre Timmermans and Dirk Adriaensen.
Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, B-2020 Antwerp.
Neuroepithelial bodies (NEBs) are morphologically well-defined airway receptors composed of densely innervated groups of neuroendocrine cells that are shielded from the airway lumen by Clara-like cells, and together compose the so-called NEB microenvironment. Because NEBs receive at least two different populations of myelinated vagal afferents with intraepithelial nerve terminals, we strongly suggest that NEBs are the morphological counterparts of at least a subpopulation of the electrophysiologically characterized vagal mechanosensory airway receptors. This study was designed to find evidence for NEBs as airway mechanotransducers, by combination of high resolution confocal live cell Ca2+ imaging and multilabel immunohistochemistry. Hypo-osmotic solutions cause cell swelling that will likely increase pressure in the tight junction sealed airway epithelium and more specifically in the NEB microenvironment. Hypo-osmotic stimulation may, therefore, be an interesting approach to study mechanosensitivity in the NEB microenvironment. Mouse lung vibratome slices (120 µm thick) were used for confocal live cell imaging of pulmonary NEBs in an imaging chamber perfused with a physiological solution (290 mOsm/ kg H2O). In these slices, we are able to visualize NEBs by loading with the styryl pyridinium dye 4-Di-2-ASP, and Ca2+ mediated cell activation by loading with the intracellular Ca2+ indicator Fluo-4. Short term (30s) exchange of the perfusion of the lung slices with a mildly hypo-osmotic solution (230 mOsm/ kg H2O) resulted in a fast, reversible and reproducible Ca2+ rise in NEB cells. Osmomechanical activation of NEB cells gives rise to a typical delayed activation of Clara-like cells. As recently demonstrated in our lab, the latter is mediated by the release of ATP from activated NEB cells, which in turn binds to P2Y2-ATP receptors expressed on Clara-like cells. Because the osmomechanical activation of NEB cells seems to be dependent on extracellular Ca2+, we investigated the expression of Ca2+ permeable osmo- and mechanosensitive TRP channels on NEB cells. Immunofluorescence of mouse lung cryosections showed the expression of TRPC5, a Ca2+ permeable hypo-osmotically and mechanically gated channel, on the apical pole of NEB cells. The hypo-osmotic activation of NEB cells in lung slices was prevented by GsMTx-4 (5 microM), a toxin that blocks TRPC5. Gadolinium (500 microM), reported to activate TRPC5 channels, evoked a transient Ca2+ rise in NEB cells. In conclusion, NEB cells revealed a pathway for hypo-osmotic/mechanical activation via TRPC5, resulting in an intracellular Ca2+ rise and exocytosis of ATP. These data confirm the potential role of NEBs as airway mechanotransducers, able to activate the NEB-associated P2X2/3 ATP receptor expressing myelinated vagal afferents upon mechanical stimulation. Support: IWT fellowship SB/81162 (RL) FWO grant G.0081.08 (DA, IB) and G.0589.11 (DA, JPT) UA grant GOA BOF 2007 (DA)
Poster 13
BSCDB & BSM – October 22, 2011 in Antwerp Martens Wendy "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
13: Martens Wendy presenting author ; [email protected]
HUMAN DENTAL PULP STEM CELLS ARE ABLE TO DIFFERENTIATE TOWARDS PERIPHERAL GLIAL CELLS.
Wendy Martens (1), Annelies Bronckaers (1), Tom Struys (1), Constantinus Politis (1,2) and Ivo Lambrichts (1).
1: Laboratory of functional morphology, Biomedical Research Institute, Hasselt University, B-3590 Diepenbeek;
2: Department of Maxillo-Facial Surgery, Ziekenhuis Oost-Limburg (ZOL), B-3600 Genk. Surgical procedures, traumas or accidents can often result in peripheral nerve injury. Numerous techniques have been developed during the last several years, as treatment for peripheral nerve injury, but the results are far from the desired outcome. Recently, several groups have demonstrated the possible transdifferentiation of mesenchymal stem cells into Schwann-like cells having a positive effect on neurons and neurite outgrowth. In the present study, human dental pulp stem cells are induced to undergo peripheral glial cell differentiation by means of growth media supplemented with different (growth) factors. After 21 days in culture, the differentiation of DPSC towards Schwann cells was analyzed. Via immunocytochemistry, the expression of Schwann cell markers GFAP, S100, and NGFRp75 was evaluated. After differentiation, the expression of GFAP, p75 and S100 was observed together with a decreased expression of nestin and Stro-1. Via transmission electron microscopical analysis, differentiated cells showed a Schwann cell-like morphology. Differentiated DPSC displayed a spindle shaped bipolar morphology with numerous organelles spread throughout the cell cytoplasm. In addition, the production and secretion of striated collagen fibers into the extracellular matrix was observed suggesting the formation of an endoneurium around the cell. In addition, the production of neurotrophic factors was analyzed via a cytokine array. DPSC and differentiated DPSC produced and secreted several (neuro)trophic factors (BDNF, GDNF, NCAM-1, NT-3 and TGF-Beta1) which promoted the survival and neurite outgrowth in dorsal root ganglia cultures, with differentiated DPSC yielding a significantly better effect than naive DPSC. The results of this study showed that DPSC are able to undergo differentiation towards peripheral glial cells, like Schwann cells. Therefore, DPSC can be good candidates for cell-based therapies as treatment for peripheral nerve injuries.
Poster 14
BSCDB & BSM – October 22, 2011 in Antwerp Munck Sebastian "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
14: Munck Sebastian presenting author ; [email protected]
SUGGESTING A LANDMARK BASED COORDINATE SYSTEM FOR MEASURING CELLULAR ORGANIZATION.
Sebastian Munck and Peter Baatsen.
Light Microscopy and Imaging Network LiMoNe & EM core facility, VIB Department of Molecular and Developmental Genetics, K.U.Leuven, Campus Gasthuisberg, Herestraat 49, box 602, 3000 Leuven.
Cell surface proteins transduce dynamic extracellular signals to the cell interior (Spiller et al., 2010). To orchestrate the different signaling networks the plasma membrane forming the limiting border of the cell is compartmentalized. Lipid components of the membrane form inhomogeneities through selective interactions, so called nano- and microdomains. These nanodomains are thus forming signaling platforms (for review see (Simons and Gerl, 2010) for example). Consequently the plasma membrane is a self organizing organelle with strong interdependencies of its constituents. While the functional compartmentalization of a eukaryotic cell is reflected by the differential occurrence of proteins in their compartments, we have no good means to measure this specific structural organization. The recent advent of super-resolution techniques has shown an unprecedented precision ((Schermelleh et al., 2010)). However this precision raises even more the question about quantification and how to handle the images acquired. The inherent problem of microscopical images of cells is that they are difficult to align. Interestingly, at the tissue level recently several drosophila brains have been morphed together to create valid maps ((Jenett et al., 2006)). At the cellular level micro-patterned surfaces have shown to be a successful strategy for some cells (Schauer et al., 2010). Here we advocate our new strategies to measure the structural organization of cells and more specific protein distributions on the plasma membrane. The key to the realization is directionality, generalization and accommodate the 3-dimensional nature of the sample. With this we believe to have a general strategy applicable to different modes of microscopy. Jenett, A., Schindelin, J. E. and Heisenberg, M. (2006). The Virtual Insect Brain protocol: creating and comparing standardized neuroanatomy. BMC Bioinformatics 7, 544. Schauer, K., Duong, T., Bleakley, K., Bardin, S., Bornens, M. and Goud, B. (2010). Probabilistic density maps to study global endomembrane organization. Nat Methods 7, 560-566. Schermelleh, L., Heintzmann, R. and Leonhardt, H. (2010). A guide to super-resolution fluorescence microscopy. J Cell Biol 190, 165-175. Simons, K. and Gerl, M. J. (2010). Revitalizing membrane rafts: new tools and insights. Nat Rev Mol Cell Biol 11, 688-699. Spiller, D. G., Wood, C. D., Rand, D. A. and White, M. R. (2010). Measurement of single-cell dynamics. Nature 465, 736-745.
Poster 15
BSCDB & BSM – October 22, 2011 in Antwerp Notelaers Kristof "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
15: Notelaers Kristof presenting author ; [email protected]
DYNAMICS AND ORGANIZATION OF ALPHA3-CONTAINING GLYCINE RECEPTORS USING SINGLE MOLECULE DETECTION.
Kristof Notelaers (1,2), Susana Rocha (2), Nick Smisdom (1), Daniel Janssen (1), Jochen C. Meier (3), Jean-Michel Rigo (1), Johan Hofkens (2) and Marcel Ameloot (1).
1: Biomedical Research Institute, Hasselt University and School of Life Sciences, transnational University Limburg, Agoralaan building C, 3590 Diepenbeek;
2: Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, B-3001 Heverlee;
3: RNA Editing and Hyperexcitability Disorders Helmholtz Group, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Strasse 10, 13092 Berlin, Germany.
The mobility and spatial organization of membrane proteins are important factors in their interaction capabilities and physiological roles. Our model is the strychnine-sensitive glycine receptor (GlyR), known to function as a chloride permeable ligand gated ion channel and to mediate synaptic inhibition. Due to a possible involvement in the pathophysiology of temporal lobe epilepsy, the different properties of the GlyRs containing alpha3L and K subunit isoforms are being investigated. Previous characterizations of homomeric receptors consisting of these isoforms have shown a difference in their electrophysiological properties and their membrane distribution observed by diffraction-limited fluorescence microscopy. To understand the motives behind these isoform-related differences, single molecule fluorescence techniques are being used to study the membrane behavior of the GlyRs in transfected HEK 293T cells. This includes studying the diffusion properties in living cells using single molecule tracking (SMT) and investigating the spatial distribution in the membrane of fixated cells using direct stochastic optical reconstruction microscopy (dSTORM). For both techniques the GlyRs are stained using a primary antibody directly labeled with Alexa Fluor dyes. Our results confirm the difference in membrane behavior between the isoforms when separately expressed in the cell membrane. The nanoscale topography images from the dSTORM experiments confirm that alpha3L GlyRs are clustered, while the alpha3K GlyRs are more uniformly spread. The analysis of short range apparent diffusion coefficients by SMT reveals a broad distribution with a higher average diffusion coefficient for the K-isoform. Deviation from normal diffusion is also quantified and results show a mild deviation for the GlyR alpha3K and a strong deviation for the GlyR alpha3L. Future investigation will attempt to resolve the cause of this anomalous diffusion.
Poster 16
BSCDB & BSM – October 22, 2011 in Antwerp Paesen Rik "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
16: Paesen Rik presenting author ; [email protected]
APPLICATIONS OF POLARIZATION SECOND HARMONIC GENERATION MICROSCOPY.
Rik Paesen and Marcel Ameloot.
Biomedical Research Institute, Hasselt University and School of Life Sciences, Agoralaan building C, B-3590 Diepenbeek.
A crucial issue in tissue engineering is to control stem cell behaviour to obtain desired lineage and growth yielding a functional tissue. We propose a new technique to study cell-matrix interactions on a microscopic level using complementary label-free imaging techniques such as two-photon excitation autofluorescence (TPEF) and second harmonic generation (SHG). The two techniques provide a way to discriminate the cell from a collagen type I containing hydrogel matrix (TPEF), and to study cell-induced deformation and forces in the hydrogel matrix itself (SHG), both without the addition of exogenous dyes. With the help of polarization-SHG (P-SHG) and the underlying SHG-theory, the proposed technique will reveal extensive quantitative 3D structural and interactional information including cell induced forces. To evaluate our P-SHG approach and set-up, pilot experiments have been performed. Estimation of the helical pitch angle -- a sub-diffraction limit property -- of the SH-Generating molecule amylopectin (starch) was done by analyzing the SHG intensity variations for various polarization directions of the incident beam. A helical pitch angle of 47.5 degrees was obtained which is comparable to the value of 44.85 +/- 1.99 degrees reported by others using the same technique (Zhou et al., Journal of Structural Biology, 2010).
Poster 17
BSCDB & BSM – October 22, 2011 in Antwerp Praet Jelle "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
17: Praet Jelle presenting author ; [email protected]
COMPARATIVE ANALYSIS OF CELLULAR GRAFT BEHAVIOR IN VIVO REVEALS CELL TYPE-ASSOCIATED AND INFLAMMATION-INDUCED DIFFERENCES IN SURVIVAL, MIGRATION AND IMMUNOGENICITY.
Jelle Praet (1,2,3), Kristien Reekmans (1,2), Nathalie De Vocht (1,2,3), Irene Bergwerf (1,2), Bart Tambuyzer (1,2), Jasmijn Daans (1,2), Herman Goossens (2), Patrick Pauwels (4), Zwi Berneman (1,2), Annemie Van der Linden (3) and Peter Ponsaerts (1,2).
1: Laboratory of Experimental Hematology, University of Antwerp, Antwerp; 2: Vaccine and Infectious Disease Institute (Vaxinfectio), University of Antwerp, Antwerp; 3: BioImaging Laboratory, University of Antwerp, Antwerp; 4: Laboratory of Pathology, University of Antwerp, Antwerp. Although the neuro-protective and -regenerative effects of cell transplantation in the central nervous system (CNS) have been demonstrated, most pre-clinical cell therapy studies report mainly on clinical observations, while currently little is known regarding the actual in vivo fate of grafted cell populations. In this study, we transplanted neural stem cells (NSC), bone marrow mononuclear cells (BMMNC), dendritic cells (DC), mouse embryonic fibroblasts (MEF) and splecocytes (SPLEEN) into the CNS of healthy C57BL/6 mice and into the demyelinated CNS of C57BL/6 mice that received a 4-week Cuprizone-supplemented diet. For each experimental group, extensive histological analysis was performed at week 2 post-grafting in order to: (i) quantify cell graft migration, survival, and toxicity, and (ii) determine and characterize endogenous neuro-immune responses against the different cell types grafted. While MEF, NSC and SPLEEN grafts did not significantly migrate from the injection site in both healthy and demyelinated CNS, the significant migration observed for BMMNC and DC grafts was associated with substantial toxicity to both healthy and demyelinated CNS tissue. In addition, none of the grafted cell populations were able to contribute to the natural remyelination process occurring following arrest of Cuprizone-supplemented diet. Moreover, no endogenous remyelination was observed at the site of cell injection in demyelinated CNS, while MEF, BMMNC, DC and SPLEEN grafts, but not NSC grafts, induced substantial damage to the myelin structure of the graft site in the healthy CNS. Furthermore, we investigated whether and how the CNS innate immune system interacted with the different cell types grafted. NSC, BMMNC and SPLEEN grafts became highly infiltrated with microglia and astrocytes, both in healthy and in the demyelinated CNS. MSC grafts however, became highly infiltrated by microglia, but encapsulated by astrocytes. DC grafts in contrast, due to their extreme toxicity, were mainly surrounded by an endogenous astrocytic scar tissue. In summary, despite many previously published observations that cell grafting in the CNS can contribute to tissue regeneration, we warrant that further research should be undertaken to understand - and eventually control - cell graft induced tissue damage and activation of the CNS innate immune system.
Poster 18
BSCDB & BSM – October 22, 2011 in Antwerp Reekmans Kristien "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
18: Reekmans Kristien presenting author ; [email protected]
CLINICAL POTENTIAL OF INTRAVENOUS NEURAL STEM CELL DELIVERY FOR TREATMENT OF NEURO-INFLAMMATORY DISEASE IN MICE ?
Kristien Reekmans (1), Jelle Praet (1), Nathalie De Vocht (1), Bart Tambuyzer (1), Irene Bergwerf (1), Jasmijn Daans (1), Veerle Baeckelandt (2), Greetje Vanhoutte (3), Herman Goossens (4), Philippe Jorens (5), Dirk Ysebaert (6), Shyama Chatterjee (7), Patrick Pauwels (7), Eric Van Marck (7), Zwi Berneman (1), Annemie Van der Linden (3) and Peter Ponsaerts (1).
1: Laboratory of Experimental Hematology, University of Antwerp, Antwerp; 2: Laboratory of Neurobiology & Gene Therapy, Catholic University Leuven, Leuven; 3: BioImaging Lab, University of Antwerp, Antwerp; 4: Vaccine 1 & Infectious Disease Institute, Vaxinfectio, University of Antwerp, Antwerp; 5: Clinical Pharmacotherapy, University of Antwerp, Antwerp; 6: Laboratory of Experimental Surgery, University of Antwerp, Antwerp; 7: Laboratory of Pathology, University of Antwerp, Antwerp. While NSC are widely expected to become a therapeutic agent for treatment of severe injuries to the CNS, currently there are only few detailed preclinical studies linking cell fate with experimental outcome. In this study, we aimed to validate whether intravenous (IV) administration of allogeneic NSC can improve EAE, a well-established animal model for human MS. For this, we cultured adherently growing luciferase-expressing NSC (NSC-Luc), which displayed a uniform morphology and expression profile of membrane and intracellular markers, and which displayed an in vitro differentiation potential into neurons and astrocytes. Following labelling with green fluorescent MPIO particles (f-MPIO-NSC-Luc) or lentiviral transduction with the eGFP reporter gene (NSC-Luc/eGFP), cell implantation experiments demonstrated the intrinsic survival capacity of adherently cultured NSC in the CNS of syngeneic mice, as analysed by real-time BLI, MRI and histological analysis. Next, EAE was induced in C57BL/6 mice followed by IV administration of NSC-Luc/eGFP with or without daily immunosuppressive therapy (Cyclosporine A, CsA). During a follow-up period of 20 days, a clinical benefit could be attributed solely to CsA treatment. Histological analysis demonstrated the absence of NSC-Luc/eGFP at sites of neuro-inflammation. In order to investigate the absence of therapeutic potential, BLI biodistribution analysis of IV administered NSC-Luc/eGFP revealed cell retention in lung capillaries as early as 1-minute post-injection, resulting in massive inflammation and apoptosis in lung tissue. In summary, we conclude that IV administration of NSC currently has limited or no therapeutic potential for neuro-inflammatory disease in mice, and presumably also for human MS. However, given the fact that grafted NSC have an intrinsic survival capacity in the CNS, their therapeutic exploitation should be further investigated, and – in contrast to several other reports - will most likely be highly complex.
Poster 19
BSCDB & BSM – October 22, 2011 in Antwerp Schnorbusch Kathy "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
19: Schnorbusch Kathy presenting author ; [email protected]
TOWARDS AN OPTIMISED LUNG SLICE MODEL FOR CONFOCAL LIVE CELL IMAGING OF THE PULMONARY NEUROEPITHELIAL BODY MICROENVIRONMENT USING GAD-GFP MICE.
Kathy Schnorbusch, Inge Brouns, Robrecht Lembrechts, Isabel Pintelon, Jean-Pierre Timmermans and Dirk Adriaensen.
Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Groenenborgerlaan 171, BE-2020 Antwerp.
A few years ago, we established an ex vivo lung slice model to perform confocal molecular live cell imaging (LCI) of neuroepithelial bodies (NEBs) in mice. These pulmonary NEBs are sensory receptor structures composed of neuroendocrine cell groups that are extensively innervated, and together with Clara-like cells make up the NEB microenvironment. Since NEBs are dispersed in the airway epithelium, LCI of NEBs was only possible after time-consuming vital staining with the styryl pyridinium dye 4-Di-2-ASP. Recently, it has been shown that in monkey lungs gamma-aminobutyric acid (GABA) and its synthesizing enzyme glutamic acid decarboxylase (GAD) are expressed in NEB cells. Since GABAergic structures are frequently studied using GAD67-green fluorescent protein (GFP) mice, the aim of the present study was to unravel a GABAergic signalling system in mouse pulmonary NEBs, enabling the future use of GAD67-GFP mice for confocal LCI. Immunofluorescence, combined with spinning disk confocal microscopy, of lung cryostat sections of prenatal (gestational day 17-20), three-week-old and adult mice, revealed that in mouse lungs GAD was expressed in pulmonary NEB cells. Immunohistochemical staining with a marker for NEBs on cryostat sections of GAD67-GFP mice, taught us that all pulmonary NEB cells express GFP. For confocal LCI, ex vivo vibratome lung slices of GAD67-GFP mice could be directly loaded with fluorescent functional probes, without the necessity of prior live visualization of NEBs. As a proof of principal, the red-fluorescent styryl pyridinium dye 4-Di-2-ASP was applied as an indicator for the mitochondrial membrane potential. Since activation of cells is often accompanied by changes in intracellular calcium concentration [Ca2+]i, the use of a red fluorescent calcium indicator was optimized. This model will certainly boost future functional studies using our ex vivo lung slice model for functional confocal LCI of mouse NEBs. Moreover, since the pulmonary NEB microenvironment is nowadays regarded as a potential stem cell niche, identification of a GABAergic signalling mechanism opens up new perspectives in unravelling the role of pulmonary NEBs in health and disease. Support: IWT fellowship SB/81162 (RL) FWO grants G.0081.08 (DA, IB) and G.0589.11 (DA, JPT) UA grant GOA BOF 2007 (DA)
Poster 20
BSCDB & BSM – October 22, 2011 in Antwerp Smisdom Nick "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
20: Smisdom Nick presenting author ; [email protected]
DIFFUSION AND AGGREGATION STATE OF GLYCINE RECEPTOR ALPHA3 SPLICE VARIANTS PROBED USING ENSEMBLE MICROFLUORIMETRIC TECHNIQUES.
Nick Smisdom (1), Kristof Notelaers (1,2), Susana Rocha (2), Daniel Janssen (1), Johan Hofkens (2), Jochen C. Meier (3), Jean-Michel Rigo (1) and Marcel Ameloot (1).
1: Biomedical Research Institute, Hasselt University and School of Life Sciences, transnational University Limburg, Agoralaan building C, 3590 Diepenbeek;
2: Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, B-3001 Heverlee;
3: RNA Editing and Hyperexcitability Disorders Helmholtz Group, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Strasse 10, 13092 Berlin, Germany.
The glycine receptor (GlyR) mediates a significant amount of the fast inhibitory neurotransmission in the mature central nervous system. Clustering of GlyRs at the postsynaptic membrane is essential to obtain efficient transmission. This is achieved through interaction with gephyrin, a cytoplasmic anchoring protein. Post-transcriptional processing of GlyR alpha3, one of the isoforms of the GlyR, is associated with pathophysiological aspects of temporal lobe epilepsy (TLE). Two splice variants, GlyR alpha3 L and K, exhibit a differential expression in the hippocampus of TLE patients. Interestingly, both isoforms display also a different clustering behavior: GlyR alpha3 L is the clustering isoform, while GlyR alpha3 K exhibits a diffuse distribution. The clustering of GlyR alpha3 L appears to be gephyrin-independent. The feasibility to study and quantify the diffusion and aggregation state of these receptors using ensemble microfluorimetric techniques is explored using GlyRs expressed in HEK 293T. Several complementary microfluorimetric techniques are combined, including fluorescence recovery after photobleaching (FRAP), image correlation spectroscopy (ICS), raster image correlation spectroscopy (RICS) and temporal image correlation spectroscopy (TICS). The added value of these techniques in addition to ongoing single molecule studies in our research group are discussed.
Poster 21
BSCDB & BSM – October 22, 2011 in Antwerp Swinnen Nina "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
21: Swinnen Nina presenting author ; [email protected]
MICROGLIA IN THE EMBRYONIC CORTEX.
Nina Swinnen (1,2), Bert Brône (1), Pascal Legendre (2) and Jean-Michel Rigo (1).
1: Universiteit Hasselt, BIOMED Institute, Cell Physiology group, Agoralaan (gebouw C), B-3590 Diepenbeek;
2: INSERM, U952, CNRS UMR 7224, Université Paris 06, Paris, France. Microglia are known as the macrophages of the central nervous system (CNS). These cells are derived from primitive myeloid progenitor cells that arise in in the yolk sac before embryonic day (E) 8. In the mouse, the start of microglial colonization (E9.5) just precedes the beginning of a moment with increased activity in the embryonic cortex, the period of neurogenesis. By their production of growth factors, it has been suggested that microglia can influence axonal growth and synaptogenesis. The aim of this study is to investigate the colonization and distribution of the microglial cells in the embryonic murine neocortex and to determine their activation stages. Transgenic C57BL/6 CX3CR1+/eGFP mouse embryos (E12.5-15.5) were isolated at the desired age. Coronal sections (10µm) were stained for ionized calcium binding adaptor molecule 1 (Iba-1), CD68, CD11b. As activation markers we used Galectin-3/Mac-2 and major histocompatibility complex class II (MHC II). Radial glial cell fibers and blood vessels were identified with antibodies for nestin and Von Willebrand factor respectively. Images were analyzed with ImageJ and statistics was performed with Prism, p-values < 0.05 were considered significant. The cell density and number of microglia increase as the embryo ages. This is mainly due to invasion of microglia into the cortex, since only a small percentage of the microglia present in the cortex is actively proliferating. Microglial cells are also present in the lateral ventricle and at the pial surface; it has been shown that microglia can enter the nervous parenchyma from these sites. The orientation of the protruding ramifications of microglia present in the parenchyma suggests that the cells migrate along radial glial fibers to reach their final position; confocal images confirm this contact. Approximately half of the microglia present in the cortex made contact with or were lining a blood vessel, suggesting a possible additional route of migration or a supportive role.
Poster 22
BSCDB & BSM – October 22, 2011 in Antwerp Uyttebroek Leen "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
22: Uyttebroek Leen presenting author ; [email protected]
EXPRESSION AND DISTRIBUTION OF NEUROTRANSMITTERS AND NEUROPEPTIDES DURING THE DEVELOPMENT OF THE ENTERIC NERVOUS SYSTEM IN ZEBRAFISH, COMPARED WITH A MUTANT MODEL FOR HIRSCHSPRUNG’S DISEASE.
Leen Uyttebroek (1), Iain T. Shepherd (2), Guy Hubens (1), Jean-Pierre Timmermans (3), and Luc Van Nassauw (1,3)
1: Laboratory of Human Anatomy and Embryology, Faculty of Medicine, University of Antwerp, B-2020 Antwerp, 2: Department of Biology, Emory University, Atlanta, United States of America, 3: Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, B-2020 Antwerp.
In the last decade, zebrafish emerged as a leading model organism in experimental research, including studies of congenital gastrointestinal diseases like Hirschsprung‟s disease (HD). Hirschsprung‟s disease (HD) is a congenital disorder of the enteric nervous system characterized by aganglionosis along a variable portion of the intestine. A recently developed mutant zebrafish, lessen, expressing HD characteristics, is suggested to be an experimental model to unravel underlying developmental mechanisms for HD. This study aims to compare the neurochemical content of enteric neurons between wild-type zebrafish and the mutant lessen to further validate this mutant as a suitable model for HD. Multiple immunofluorescence staining was used to detect previously characterized neurochemical markers from 3 to 6 days post-fertilization (dpf) in the proximal (PI), mid (MI) and distal intestine (DI) of both wild-type and mutant animals. In both strains, the total number of enteric neurons increased in time. In mutants, the number of enteric neurons was significantly reduced in DI (even absent at 3dpf) and MI, but less in PI at each embryonic stage. The proportion of nitrergic neurons was significantly reduced in all regions at 3 dpf, but nearly unaffected in PI and MI at 4 and 5dpf. Although there was a decrease, nitrergic neurons constitute a major neuronal subpopulation in both strains. In mutants, serotonin, calretinin and calbindin showed a delayed expression and a decrease in both number and proportion at all points of time and in each intestinal region. In both strains, galanin, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) were only detected on nerve fibers. Moreover, VIP and PACAP were completely colocalized. In wild-type zebrafish, VIP/PACAP-positive and galanin-positive fibers were present at all points of time and in each region of the developing intestine. However in mutants, only a few immunoreactive fibers were present at 3 dpf in the PI, while at 4 dpf immunostained fibers appeared in the MI and at 5 dpf in the DI, showing a delayed expression of galanin, VIP and PACAP in mutants. The present study shows abnormalities in the number and relative frequency of neurons expressing various neurochemical markers at each embryonic stage. These results are similar to data obtained in the intestine proximal to the aganglionic segment and the aganglionic segment of the ls mutant mice, an experimental HD model, indicating that the zebrafish mutant lessen is a suitable model to study HD.
Poster 23
BSCDB & BSM – October 22, 2011 in Antwerp van Hengel Jolanda "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
23: van Hengel Jolanda presenting author ; [email protected]
LOSS OF αT-CATENIN ALTERS THE HYBRID ADHERING JUNCTIONS IN THE HEART LEADING TO DILATED CARDIOMYOPATHY.
Jolanda van Hengel (1,2), Jifen Li (3), Steven Goossens (1,2), Erhe Gao (3), Lan Cheng (3), Koen Tyberghein (1,2), Xiying Shang (3), Riet De Rycke (1,2), Frans van Roy (1,2) and Glenn L. Radice (3)
1: Department of Molecular Biomedical Research, VIB, 9052 Ghent; 2: Department of Biomedical Molecular Biology, Ghent University, 9052 Ghent; 3: Center for Translational Medicine, Department of Medicine, Thomas Jefferson
University, Philadelphia PA, USA. Members of the cadherin family of cell adhesion molecules are located in the adherens junction. They interact homophilically to mediate strong cell-cell adhesion and play a key role in the maintenance of tissue structure. AlphaT-catenin is a newly identified cadherin-associated protein that mediates linkage of the cadherin–catenin complex to the actin cytoskeleton. In the mammalian heart, mixed-type junctional structures (areae compositae) are present at the intercalated discs (ICD). We observed alphaT-catenin localized in this novel type of junction, where it associates with endogenous plakophilin-2. To investigate the function of alphaT-catenin in the heart, a murine loss-of-function model was generated. AlphaT-catenin-null mice are viable but exhibit progressive cardiomyopathy. It has been reported that suppression of PKP-2 activity in cultured cardiomyocytes leads to reduction in Cx43, which affects gap junctional communication. We observed reduced expression of PKP-2 in the ICD of alphaT-catenin-null myocardium. Furthermore, Cx43 was reduced at the ICD, and its co-localization with N-cadherin was also reduced. This indicates that alphaT-catenin might act synergistically with desmosomal PKP-2 to stabilize gap junctions at the area composita. This gap junction remodeling was associated with an increase in the incidence of ventricular arrhythmias in alphaT-catenin knockout mice subjected to acute ischemia. The human alphaT-catenin gene, CTNNA3, has been mapped to chromosome 10q21, a region linked to arrhythmogenic right ventricular cardiomyopathy (ARVC). Though genetic screening has not detected an association between CTNNA3 mutations and ARVC to date, CTNNA3 is considered a candidate cause of this disease.
Poster 24
BSCDB & BSM – October 22, 2011 in Antwerp Van Hove Inge "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
24: Van Hove Inge presenting author ; [email protected]
ABERRANT CEREBELLAR DEVELOPMENT IN MMP-3 DEFICIENT MICE.
Inge Van Hove, Mieke Verslegers, Tom Buyens, Kim Lemmens and Lieve Moons.
Research Group Neural Circuit Development & Regeneration, Katholieke Universiteit Leuven, Naamsestraat 61, B-3000 Leuven.
Cell-cell and cell-extracellular matrix (ECM) interactions are necessary for neuronal patterning and brain wiring during development. Matrix metalloproteinases (MMPs) are proteolytic enzymes which exert, through cleavage of ECM substrates, receptors, cytokines, cell adhesion molecules etc., beneficial as well as detrimental functions in the physiological and/or pathological brain. This study focuses on the involvement of MMP-3 during postnatal development of the mouse cerebellar cortex, which serves as an ideal model to study processes like neuronal proliferation, migration and synaptogenesis. MMP-3 mRNA expression levels increase during cerebellar development and peak at the end of the second postnatal week. MMP-3 protein expression and activity are, at that time, clearly localized in Purkinje cells & Golgi cells and to a lesser extent in the interneurons of the molecular layer (ML) and in the internal granular layer (IGL) neurons. Despite a normal overall cerebellar architecture, detailed morphometric investigations revealed an increased external granular layer (EGL) thickness in MMP-3-/- cerebella from postnatal day 8 (P8) on. BrdU pulse studies combined with TAG-1 and P27 immunostainings were performed to investigate possible GC proliferation and/or migration deficits and revealed a stalling of GCs between P6 and P8 in the deeper EGL, resulting in an increased deeper EGL area in MMP-3-/- mice. Ex vivo explant studies showed prolonged parallel fibers and an increased number of migrated GCs. This protracted GC tangential migration resulted in a delayed GC radial migration. No overt abnormalities were found in GC apoptosis, in Bergmann glia morphology or in PC number, alignment and soma diameter in the absence of MMP-3. However, PC dendrite formation was found to be impaired, resulting in a reduced primary dendrite length and tree size, lasting until adulthood. Of note, the delayed GC radial migration and the abnormal PC dendritic development resulted in an impaired interneuron migration and maturation, a retarded inhibitory & excitatory neuronal synaptogenesis on PCs and a sustained GC proliferation rate at later stages during the MMP-3-/- cerebellar development. Importantly, the observed anatomical developmental alterations were accompanied by deficits in motor behaviour in MMP-3-/- mice. Our results clearly suggest that MMP-3 is necessary for a proper development of the cerebellar cortex.
Poster 25
BSCDB & BSM – October 22, 2011 in Antwerp Van Nassauw Luc "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
25: Van Nassauw Luc presenting author ; [email protected]
THE EFFECT OF INTESTINAL INFLAMMATION ON THE EXPRESSION AND DISTRIBUTION OF MAS-RELATED GENE RECEPTORS IN THE MURINE ILEUM.
Leela Rani Avula (1), Luc Van Nassauw (1, 2), Roeland Buckinx (1), Katrien Alpaerts (1), Dirk Adriaensen (1), Herman Favoreel (3), Eric Cox (3) and Jean-Pierre Timmermans (1).
1: Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Antwerp;
2: Faculty of Medicine, Department ASTARC, Laboratory of Human Anatomy and Embryology, University of Antwerp, Antwerp;
3: Department of Virology, Parasitology and Immunology, Laboratory of Immunology, Ghent University, Ghent.
Introduction: Mas-related gene receptors (Mrgs) constitute a complex family of orphan GPCRs, some of which are predominantly expressed in spinal sensory neurons. Some Mrgs are suggested to have an important role in regulating inflammatory responses to IgE-independent mast cell activation, as well as in the neuroimmune communication. In a previous study, we have already demonstrated that intestinal inflammation negatively affects the expression of some Mrgs, i.e., MrgE and MrgF, in the murine ileum. Aims & Methods: The lack of data concerning other Mrgs in the gastrointestinal tract, in both healthy and inflamed conditions, led us to investigate the expression and distribution of these Mrgs in the non-inflamed and Schistosoma mansoni-infected murine ileum. Immunohistochemical analyses were performed on cryosections and whole-mounts of the non-inflamed and inflamed ileum using custom-made polyclonal antisera directed against MrgA4, MrgB2, MrgB8 and MrgB10, as well as a commercially available antiserum directed against MrgD. Real-time RT-PCR was performed on the non-inflamed and inflamed ileum to detect and quantify the expression levels of MrgA4, MrgB2, MrgB8, MrgB10 and MrgD mRNAs. Results: In the non-inflamed ileum, immunohistochemistry revealed no MrgB10 or MrgD immunoreactivity, whereas moderate MrgA4, MrgB2 and MrgB8 immunoreactivities were detected in some neuronal somata in both enteric plexuses. In the inflamed ileum, MrgA4, MrgB2, MrgB8, MrgB10 and MrgD immunoreactivities were observed in a significantly increased number of neuronal somata in both enteric plexuses (p-value < 0.05), as well as in nerve fibres present in the tunica muscularis and in the lamina propria. Colocalisation studies using antibodies directed against neurochemical markers demonstrated that MrgA4-, MrgB2-, MrgB8-, MrgB10- and MrgD-expressing neurons were predominantly intrinsic sensory neurons. In the inflamed ileum, MrgB10 and MrgD immunoreactivities were also detected in mucosal mast cells (MMCs). These results were corroborated by real-time RT-PCR which demonstrated the upregulation of MrgA4, MrgB2, MrgB8, MrgB10 and MrgD mRNAs in the inflamed ileum. Conclusions: Intestinal schistosomiasis positively influences the expression of MrgA4, MrgB2, MrgB8, MrgB10 and MrgD in neurons and nerve fibres, as well of MrgB10 and MrgD in MMCs, indicating the involvement of Mrgs in neuronal and mast cell responses during intestinal inflammation. Future work should aim at elucidating the (patho)physiological role of Mrgs in the murine ileum. This study was supported by the FWO-grant G.0179.08N.
Poster 26
BSCDB & BSM – October 22, 2011 in Antwerp Van Peer Els "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
26: Van Peer Els presenting author ; [email protected]
ONTOGENY OF DRUG TRANSPORT AND METABOLISM IN THE PIG SMALL INTESTINE: A PRELIMINARY INVESTIGATION.
Els Van Peer, Maartje De Vos, Véronique Huygelen, Sofie Willemen, Chris Van Ginneken and Steven Van Cruchten.
Laboratory of Applied Veterinary Morphology, Department of Veterinary Sciences, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, Antwerp University, Universiteitsplein 1, 2610 Wilrijk.
Introduction: Drug efflux transporters and metabolising enzymes play a pivotal role in the pharmacokinetics of most drugs. Their presence and activity in the small intestine influence oral bioavailability and as such they may determine the clinical efficacy and toxicity of orally ingested drugs. The information on the ontogeny of drug transporters and metabolising enzymes, such as CYP3A, is limited in animals despite the fact that, from a safety perspective, juvenile toxicity studies may be required in one or more species prior to the start of clinical trials in children. (Mini)pigs are increasingly used as non-rodent species in toxicity studies and they also seem to be a good model for human oral absorption of drugs due to their similarities in gastrointestinal morphology and physiology. The aim of this preliminary investigation was to assess the expression of the drug efflux transporter P-glycoprotein and the CYP3A subfamily in the small intestine of late gestational pig foetuses and juvenile pigs. Methods: Pig foetuses (n = 5 90-115 days of gestation) and piglets of Day 0 (n = 5), 3 (n = 6), 10 (n = 4) and 28 days of age (n = 3) were obtained from Large White x Landrace pregnant sows at the slaughterhouse and after natural delivery at a local farm, respectively. The piglets were anaesthetised by an intraperitoneal injection of sodium pentobarbital, followed by exsanguination. The small intestine (middle part of the jejunum) was dissected, rinsed, formalin-fixed and embedded in paraffin. A mouse anti-human monoclonal antibody (Ab C219, Abcam, ab3364, Cambridge) was used for immunohistochemical evaluation of P-glycoprotein, whereas rabbit anti-human polyclonal antibodies were used for CYP3A4 (ab1254, Millipore, Billerica) and CYP3A7 (ab10323, Millipore, Billerica). Results: The at term piglets (day 0 of age) and the piglets at 3, 10 and 28 days of age showed a clear staining of P-glycoprotein at the apical surface of the intestinal villi. There was no staining visible in the crypts. P-glycoprotein was absent in the small intestine of the pig foetuses. These results are similar to man, in which expression of P-glycoprotein in the small intestine occurs at birth and is also absent prenatally. Regarding CYP3A, both isoforms were present in the cytoplasma of the villous enterocytes of all age groups. This seems in contrast to human data, where CYP3A7 is known to be the fetal and neonatal isoform and CYP3A4 the adult isoform. However, cross-reactivity of the antibodies to both isoforms can be the reason for this apparent discrepancy. Conclusion: These preliminary results suggest that the ontogeny of P-glycoprotein in the small intestine is similar in pigs compared to humans. However, more elaborate data are needed to confirm these results. Regarding the ontogeny of CYP3A, no distinction between CYP3A7 and CYP3A4 could be made. Transcriptomics can be used to unravel the different CYP3A isoforms in pig small intestine.
Poster 27
BSCDB & BSM – October 22, 2011 in Antwerp Verstraelen Peter "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
27: Verstraelen Peter presenting author ; [email protected]
FUNCTIONAL AND MORPHOLOGICAL VISUALIZATION OF IN VITRO NEURONAL NETWORK MATURATION.
Peter Verstraelen (1), Isabel Pintelon (1), Rony Nuydens (2), Sofie Thys (1), Inge Keuleers (1), Frans Cornelissen (2), Christoph Tombeur (2), Theo Meert (2) and Jean-Pierre Timmermans (1).
1: Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, B-2020 Antwerp;
2: Johnson & Johnson Pharmaceutical Research and Development, Neuroscience Department, B-2340 Beerse.
Over the last decade, a lot of attention has been drawn to the maturation of neuronal networks, both in vivo and in vitro, as certain mental disorders (e.g. schizophrenia) are thought to be initiated during early neuronal maturation. Using in vivo EEG recordings on schizophrenic patients, it became clear that the synchronized, oscillatory network activity is disrupted. Also, when primary neuronal cultures are grown in vitro, spontaneous electrical activity develops which tends to synchronize over time as the neurites grow and the synapses mature. In order to set up a valid in vitro model to study certain aspects of the pathophysiology of schizophrenia, it is very important to first characterize the maturation of neuronal cultures under control conditions. Therefore, we created a panel of three different functional or morphological techniques to describe the maturation of primary hippocampal cultures, grown in 96-well plates. First, through immunocytochemical staining for neuronal class III beta-tubulin and the synaptic protein synaptophysin 1 and consequent image analysis, we are able to quantify both neurite outgrowth and synapse density in a range from 4 until 21 days after plating. Second, through staining with the lipophilic dye DiI and high-resolution confocal imaging, we can determine the dendritic spine density in 7 to 21 day old cultures. These dendritic spines are probably the most plastic part of the neuron and therefore very essential in the context of neuronal network maturation. The third technique involves the functional imaging of neuronal network activity. By live cell imaging using Fluo-4 and a confocal spinning disk microscope, we are able to observe calcium waves that occur in response to the depolarization of neurons after a burst of action potentials. From five until seven days in culture, the examined parameters e.g. percentage active neurons, frequency of synchronized bursts and correlation score, show a marked increase, reminiscent of the maturation of the network. Moreover, the network activity can be modulated both acutely (e.g. through the addition of forskolin or TTX) or chronically (e.g. with neutralizing antibodies against Nerve Growth Factor). Briefly, by implementing one or more of the three techniques presented here on primary hippocampal cultures, a very good assessment can be made regarding the maturation status of the neuronal network under investigation. As the cultures are reproducibly grown in 96-well plates and the image analyses can be performed semi-automatically, this system can eventually be translated into a fully automated high-content screening assay, crucial for the screening of possible target proteins involved in different developmental CNS disorders.
Poster 28
BSCDB & BSM – October 22, 2011 in Antwerp Willemen Sofie "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
28: Willemen Sofie presenting author ; [email protected]
THE INTESTINAL DISTRIBUTION OF GHRELIN CELLS DURING THE PERINATAL PERIOD OF RUNTS AND NORMAL WEIGHT PIGLETS.
Sofie Willemen, Maartje De Vos, Véronique Huygelen, Els Van Peer, Steven Van Cruchten and Chris Van Ginneken.
Laboratory of Applied Veterinary Morphology, Department of Veterinary Sciences, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk.
Introduction: Ghrelin is an orexigenic peptide that stimulates growth hormone and gastric acid secretion as well as gastrointestinal (GI) motility. Ghrelin cells (GC) are present in the intestinal mucosa of fetal and postnatal rodents and humans. Their prenatal presence indicates a role in development. This is substantiated by the presence of ghrelin in the fetal circulation and an increased (fetal) plasma level in case of intrauterine growth retardation (IUGR) in human fetuses. Since porcine intestinal development, physiology and morphology are highly comparable to those in men we wanted to examine the distribution of GC in the perinatal porcine intestinal tract. Furthermore, this study compared the intestinal distribution of GC in IUGR piglets with normal weight (NW) littermates. Material & Methods: Fetal pigs from the 3rd trimester of gestation were gathered from a local slaughterhouse. Postnatal pigs from day 0 and day 3 were collected from a local farm. The intestinal tract from these pigs was immediately removed after euthanasia. Each piglet was designated IUGR or NW according to body weight and ponderal index and as such each group (fetal pigs, postnatal pigs from day 0 and day 3) contained 5 pairs of piglets (age and gender-matched). The intestinal samples were fixated, paraffin embedded and processed for immunohistochemistry visualizing GC. The distribution of GC was semi-quantitatively described, and these data was statistically evaluated in order to detect age, regional and birth weight related differences. Results & Discussion: This study demonstrates that GC are present throughout the porcine intestinal tract both pre- and postnatally. The cells were scattered in the epithelia of crypts and villi. Similar to human and rodent intestine, 2 types of GC were found: round opened type cells, which are in contact with the lumen and triangular closed type cells. Moreover, similar as in men and in the adult pig, the density of GC decreased from the small to large intestine. Hence, this study demonstrates that the intestinal distribution of GC during the porcine perinatal period is comparable to its distribution in the adult porcine intestine. This study also suggests that the distribution of GC in the intestine is not affected by birth weight. The latter apparently contrasts with previous findings in man where higher plasma ghrelin levels are encountered in case of IUGR. However a clear-cut relationship between plasma levels and tissue distribution of GC in the intestine is not yet documented in part because gastric ghrelin is the major source for circulating ghrelin. Thus in order to elucidate this apparent contrast, serum and stomach ghrelin levels will be determined in the different age and weight groups.
List of participants
BSCDB & BSM – October 22, 2011 in Antwerp List of participants "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
LIST OF PARTICIPANTS
ADRIAENSEN Dirk [email protected]
ALPAERTS Katrien [email protected]
AMELOOT Marcel [email protected]
ASSELBERGH Bob [email protected]
AVULA Leela Rani [email protected]
BAATSEN Peter [email protected]
BELLEFROID Eric [email protected]
BOEYKENS Nele [email protected]
BOLLET-QUIVOGNE Frederic [email protected]
BOUZIN Caroline [email protected]
BRAECKMANS Kevin [email protected]
BRONCKAERS Annelies [email protected]
BROUNS Inge [email protected]
BUCKINX Roeland [email protected]
CHEN chang [email protected]
DE CLERCQ Luc [email protected]
DE CLERCQ Ben [email protected]
DE RIJCK Dominique [email protected]
DE RYCKE Riet [email protected]
DE VOCHT Nathalie [email protected]
DE VOS Winnok [email protected]
DUGGAL Galbha [email protected]
GAUBLOMME Djoere [email protected]
GERVOIS Pascal [email protected]
GUÉRIN Chris [email protected]
HAASE Günther [email protected]
HARRISSON Fernand [email protected]
HICK Anne-Christine [email protected]
HILKENS Petra [email protected]
HOORNAERT Chloe [email protected]
JANSSENS Els [email protected]
KEULEERS Inge [email protected]
KOCKELBERG Stef [email protected]
LAMBRICHTS Ivo [email protected]
List of participants
BSCDB & BSM – October 22, 2011 in Antwerp List of participants "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
LEMBRECHTS Robrecht [email protected]
LIPPENS Saskia [email protected]
LU Yuechao [email protected]
MARTENS Wendy [email protected]
MOENECLAEY Liliane [email protected]
MOERS Carine [email protected]
MORAES Gustavo [email protected]
MUNCK Sebastian [email protected]
NEUPANE Jitesh [email protected]
NOTELAERS Kristof [email protected]
NUYDENS Rony [email protected]
NYSTEN Bernard [email protected]
PAESEN Rik [email protected]
PIERREUX Christophe [email protected]
PINTELON Isabel [email protected]
PONSAERTS Peter [email protected]
PRAET Jelle [email protected]
RAES Martine [email protected]
REEKMANS Kristien [email protected]
RIGO Jean-Michel [email protected]
RIGORT Alexander [email protected]
SADZOT Catherine [email protected]
SANEN Kathleen [email protected]
SCHNORBUSCH Kathy [email protected]
SHEPHERD Iain [email protected]
SMISDOM Nick [email protected]
STRUYS Tom [email protected]
SVENSSON Lieve [email protected]
SWINNEN Nina [email protected]
THYS Sofie [email protected]
TIMMERMANS Jean-Pierre [email protected]
UYTTEBROEK Leen [email protected]
VAN CAMPENHOUT Claude [email protected]
VAN CRUCHTEN Steven [email protected]
VAN DYCK christophe [email protected]
List of participants
BSCDB & BSM – October 22, 2011 in Antwerp List of participants "ADVANCED MICROSCOPY MEETS CELL AND DEVELOPMENTAL BIOLOGY"
VAN HENGEL Jolanda [email protected]
VAN HOVE Inge [email protected]
VAN NASSAUW Luc [email protected]
VAN PEER Els [email protected]
VAN ROY Frans [email protected]
VANDERWINDEN Jean-Marie [email protected]
VANLANGENAKKER Nele [email protected]
VERBUEKEN Evy [email protected]
VERSTRAELEN Peter [email protected]
VERVOORT Paul [email protected]
WALKER Rebecca [email protected]
WANET Anaïs [email protected]
WILLEMEN Sofie [email protected]
WILLEMS Peter [email protected]
ZWIJSEN An [email protected]
