Establishment of cell polarity in zebrafish role of staufen and GRB2 in cell migration

ESTABLISHMENT OF CELL POLARITY IN ZEBRAFISH: DEFINING THE ROLE OF STAUFEN AND GRB2 IN CELL MIGRATION RAMASAMY SRINIVAS (M.Sc, University of Hyderabad, INDIA) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY TEMASEK LIFE SCIENCES LABORATORY NATIONAL UNIVERSITY OF SINGAPORE 2007 i Dedicated to my family members ii ACKNOWLEDGEMENTS I am grateful to Temasek Life Sciences Laboratory (TLL) for providing excellent scientific infrastructure, facilities, stimulating and freedom oriented scientific environment and financial support during the course of my graduate (PhD) studies. I am grateful and extremely thankful for my supervisor Dr. Karuna Sampath for providing conducive scientific environment, and patient guidance during the course of my graduate studies. Her constant scientific encouragement, insight, advice and support has helped me to sustain my interest in research. I am thankful for the thesis committee members Dr. William Chia, Dr. Suresh Jesuthasan, Dr. Sudipto Roy for their valuable guidance and effort to lead me through my graduate studies. I am grateful and thankful for the past and present Vertebrate Development group members for useful suggestions and providing me insights during the course of the study. I am especially thankful to Ms Wang Hui, Ms Helen Ngoc Bao Quach and Mr. Albert Cheong Shea Wei who has provided me excellent support and encouragement during the course of my graduate studies. I am thankful for Dr. Mohan Balasubramanian and his lab members for helping me with experiments using the fission yeast. I am thankful for the past and present iii colleagues Dr Elia Stupka, Dr. Alan Christoffels, Mr. Chen Peng, Mr. Chua Aaron, Mrs Allison Hooi Chien Soo, Ms Hamsa Srinivasan, Mr. Balamurugan Kumarasamy and Mr. Juguang for support and help in other projects for computational analysis. I am thankful for Dr. Alexander Emelyanov for suggestions for some of technical aspects in my graduate studies. I am thankful for Dr. Erez Raz for providing me reagents and suggestions and also Dr. Anne Ephrussi, Dr.Cai Yu and Dr. JA Marrs for providing me reagents. I am thankful for my senior colleagues Dr. Patrick Gilligan, Dr. Ajay Sriram and Dr. Volker Wachtler who helped me to correct the thesis and gave useful suggestions for writing. I am thankful for my friends, colleagues, fish facility members Chin Heng Goh and others, sequencing facility members, confocal facility in charge Ms Connie Er and Ms Christiana Barghazhi and other staff members for their support. I thank the Department of Biological Sciences, National University of Singapore for registering me as a part-time student and providing all facilities and support during the graduate studies. I am thankful for my friends, colleagues and all members of TLL who provided support and help. iv Finally i am thankful to all my family members, especially my parents, grand parents and in-laws who helped me to get me to this present state and finish my PhD, my wife K.S.Priya and little master Saathvik who provided me strength and support to pursue and finish my PhD. v TABLE OF CONTENTS ACKNOWLEDGEMENTS iii TABLE OF CONTENTS vi SUMMARY . x List of Figures xii List of Abbreviations . xiv List of Abbreviations . xiv Chapter 1: INTRODUCTION . 1.1 Symmetry and Asymmetry 1.2 Cell polarity in unicellular organisms . 1.3 Cell polarity in embryos, cultured fibroblasts and axons . 1.4 Egg cell polarity in specification of axis and germ line in model systems . 1.4.1 Polarity and specification of axes during oogenesis in D. melanogaster 1.4.2 Egg cell polarity and specification of axis in Caenorhabditis elegans (C. elegans) . 1.4.3 Egg cell polarity and specification of axis in vertebrates 10 1.4.4 Egg cell polarity in germ line specification . 11 1.5 Mechanisms that generate cell polarity 15 1.5.1 RNA localization and translational regulation in establishment of cell polarity 16 1.6 RNA binding proteins in mRNA localization . 22 1.7 Function of Staufen proteins in mRNA localization and mechanisms that establishment of cell polarity . 22 1.8 Cell polarity: an attribute to cell migration and cell movement . 24 Chapter 2: MATERIALS AND METHODS 29 2.1 Maintenance of zebrafish embryo and larval cultures 29 2.1.1 Zebrafish embryonic and larval cultures 29 2.2 Manipulation of zebrafish embryos and adults 29 2.2.1 Obtaining mature oocytes from adult zebrafish females . 29 2.2.2 Fin clipping of adult zebrafish . 30 2.2.3 Microinjection of morpholino, mRNA and peptides in zebrafish embryos30 2.3 Staining techniques . 32 2.3.1 Whole mount in situ hybridization 32 2.3.2 Generation of Stau2 antibody in zebrafish . 33 2.3.3 Immunohistochemistry on whole embryos 34 2.3.4 Immunohistochemistry on sections 34 vi 2.3.5 Actin and nuclear staining . 35 2.3.6 TUNEL Labeling . 35 2.4 Molecular Biology and Biochemistry techniques . 35 2.4.1 Manipulation of DNA, RNA and protein 35 2.4.2 Reagents for embryo injections . 36 2.4.3 Radiation hybrid mapping 38 2.4.4 Northern Blotting . 38 2.4 Labeling of mRNA 39 2.4.6 Labeling of Antisense RNA probes . 39 2.5 Biochemistry techniques . 40 2.5.1 Expression of recombinant fusion proteins 40 2.5.2 RNA binding assays . 40 2.6 Protocols in Schizosacharomyces pombe 41 2.6.1 Media and cell culture 41 2.6.2 Antibody and phalloidin staining . 41 2.6.3 Fluorescence microscopy of fixed samples . 41 Chapter 3: Zebrafish Staufen proteins are required for survival and migration of primordial germ cells . 43 3.1 Identification, cloning and sequence analysis of zebrafish staufen homologs . 43 3.2 Isolation of zebrafish staufen homologs 45 3.3 Radiation hybrid (RH) mapping of staufen-related genes in zebrafish . 47 3.4 Expression and localization of staufen in zebrafish . 49 3.4.1 Expression of stau2 during maternal and zygotic development of zebrafish . 50 3.5 Localization of stau1 and stau2 transcripts during development of zebrafish . 51 3.5.1 Stau2 protein is localized during oocyte development in zebrafish 52 3.6 RNA binding analysis by zebrafish Stau proteins . 54 3.6.1 Zebrafish Staufen dsRNA binding domains bind mRNA in vitro . 55 3.6.2 Zebrafish Staufen dsRBD-4 binds nanos1 and vg1 mRNA in ovary total RNA 57 3.7 Functions of Staufen proteins in zebrafish . 57 3.7.1 Specificity of dominant negative peptides to deplete Staufen in zebrafish 57 3.7.2 Specificity of antisense morpholinos to deplete Staufen in zebrafish . 58 3.8 Disruption of Stau function does not affect germ layer patterning 59 3.9 Staufen proteins are not required for localization of maternal squint mRNA . 61 3.10 Stau function is required for primordial germ cell survival and migration . 62 vii 3.10.1 Disruption of stau function by dominant negative peptides 62 3.10.2 Expression of nanos1 in PGCs is lost in Staufen-disrupted embryos 63 3.11 Depletion of Staufen by morpholinos 66 3.12 Zebrafish Staufen proteins are essential for proper migration of PGCs. 69 3.13 Fly staufen mRNA is required for survival of PGCs . 71 3.14 Role of zebrafish Staufen in neuronal development 75 3.14.1 Zebrafish Staufen proteins are essential for the survival of central nervous system (CNS) neurons 79 Chapter 4: Grb2 and Grb2-like are required for convergent-extension movements during gastrulation in zebrafish 81 4.1 Identification of genes that influence cell polarity in zebrafish 81 4.2 The Microtubule cytoskeleton is affected upon over-expression of ∆Ngrb2like in yeast cells 83 4.3 Sequence analysis and cloning of zebrafish grb2 and grb2-like . 88 4.4 Expression and localization of grb2 transcripts . 89 4.5 Functions of Grb2 proteins in zebrafish . 89 4.5.1 Knockdown of grb2 and grb2-like by antisense morpholino 89 4.6 Grb2 and Grb2-like are required for convergence and extension cell movement . 90 4.7 Interference of Grb2 and Grb2-like by mutant mRNA does not affect germ layer patterning . 92 4.7.1 Interference of Grb2 and Grb2-like by mutant mRNA affects patterning of the neuroectoderm . 93 4.8 Grb2 proteins are necessary for normal convergence extension (CE) movements during gastrulation . 94 4.8.1 Injection of full-length grb2 or grb2-like mRNA rescues the CE cell movement defects caused by disruption of Grb2 proteins 99 4.9 Grb2 proteins are required for cellular morphogenesis during gastrulation . 100 4.9.1 Grb2 proteins are necessary for maintenance of the cell size and shape of pre-chordal plate progenitors during gastrulation . 102 4.10 Grb2 proteins are essential for polarized cellular behavior of PCP progenitors . 103 4.10.1 Axial mesendodermal cells (PCP) show defective cell movement upon disruption of Grb2 and Grb2-like proteins 105 4.11 Disruption of Grb2 proteins delays epiboly movements . 108 4.12 Disruption of Grb2 proteins affects actin enrichment in the yolk cell and the EVL margin during epiboly . 109 viii 4.13 The cell size and shape of EVL layer is affected in Grb2 and Grb2-like disrupted embryos 112 4.14 Grb2 and Grb2-like are required for neuronal arborization and branching . 113 Chapter 5: DISCUSSION 115 5.1 Functions of Staufen proteins in PGC development 115 5.1.1 Implications of Staufen proteins in mRNA binding and localization 115 5.1.2 Are Staufen proteins required for specification of axis and patterning? . 116 5.1.3 Disruption and depletion of Staufen proteins by dominant negative peptides and morpholinos . 117 5.1.4 Zebrafish Staufen proteins mediate proper migration and survival of PGCs . 118 5.1.5 Zebrafish Staufen proteins are required for survival of central nervous system neurons 120 5.2 A functional over-expression screen for identification of genes that influence cell polarity in zebrafish . 120 5.2.1 Grb2 proteins are required for patterning the neuroectoderm . 121 5.2.2 Function of Grb2 proteins in convergent-extension cell movements 122 5.2.3 Grb2 proteins are necessary for cellular morphogenesis and movement of PCP cells . 122 5.2.4 Grb2 proteins are required for cellular morphogenesis and cell movement in epiboly 123 5.3 Conclusion . 124 Chapter 6: REFERENCES 126 Chapter : APPENDIX . 160 ix SUMMARY I have investigated the mechanisms by which proteins establish cell polarity in zebrafish for my thesis study. Cell polarity is partly dependent on asymmetric localization of transcripts and proteins. Staufen is an RNA-binding protein, required for the correct localization of many transcripts and is required for specification of the anterior-posterior axis and the germ line in Drosophila melanogaster (D. melanogaster). In order to study the role of Staufen proteins during zygotic development of zebrafish, I isolated and characterized stau1 and stau2 cDNAs. The function of stau1 and stau2 was tested by using antisense morpholino-mediated knockdown and dominant negative peptide interference studies. Staufen proteins are not required for patterning of the germ layers during zygotic development. Interference of Staufen by morpholinos or disruption of function of Staufen proteins by dominant negative peptides abolishes expression of germ-cell specific transcripts vasa and nanos1. In the absence of Staufen proteins, primordial germ cells (PGCs) not migrate properly and undergo cell death. 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Chapter : APPENDIX Antibody staining buffers PBS (1x) 1x PBDT solution TBST solution NaCl 136.9 mM 1xPBST with 1%DMSO X TBST KCl 2.7 mM 155 mM NaCl KH2PO4 1.5 mM 10 mM Tris-HCl pH Na2 hPO4 7.7 mM (7.4) Adjust pH to 7.4 0.1% NP-40 1x PBST (1x PBS + 0.1% Tween20) Danieus Solution 160 Stock solution (500 ml) For 250 ml of Full Strength Solution ml NaCl (50x) NaCl - 84.75g (50X) 2.5ml KCl KCl - 2.6g (100x) 2.5 ml MgSO4.7H2O MgSO4.7H2O - 4.95g (100x) 2.5 ml Ca(NO3)2 Ca(NO3)2 - 7.35g (100x) 2.5 ml HEPES HEPES - 59.5g (100x) Egg water 0.03% sea salt powder Genomic DNA lysis buffer 50 mM Tris-HCl pH 8.2 100 mM NaCl mM EDTA and 100 µg/ml proteinase K Insitu hybridization buffers HybB HybA MAB 25 ml formamide 10 ml HybB, 20µl t-RNA 600 ml sterile 1.25 ml 20x SSC (DEPC (50mg/ml), 10 µl heparin water treated), 0.5 ml Triton-X-100 (50mg/ml) 100 ml 1M mixed and adjusted to pH 5.5 200 µl sonicated salmon maleic acid with HCl sperm DNA (10mg/ml) 10 ml 5M NaCl NTMT solution (Stratagene) adjust pH to 7.5 1M Tris HCl pH 9.5 (4ml) Roche blocking 1M MgCl2 (2.2ml) reagent 5M NaCl (0.8ml) 1.25g in 50ml Triton-X-100 (400 µl) MAB and heat at 161 1M levamisole (40 µl) 68oC to dissolve sterile water (40ml) and adjust pH to 7.5 with NaOH Northern Blotting buffers X FA gel running 10 X FA gel buffer buffer 200 mM MOPS (3-[N- 100 ml 10X FA gel buffer morpholino] propane sulfonic 20 ml 37% (12.3 M acid (free acid)) formaldehyde) 50 mM sodium acetate 880ml sterile RNase free 10 mM EDTA and adjust pH to water 7.0 with NaOH Loading dye (0.25 % (w/v) Bromophenol blue 162 Native protein Lysis RNA binding assay Buffer buffers 50 mM NaH2PO4 Binding buffer 97.9 sterile water 50 mMNa2 hPO4 50 mM Tris pH 7.5 2.1 ml M Tris-HCl pH 300 mM NaCl 150 mM NaCl (9.0) 0.1% TritonX-100 20 mM KCl 0.2 M EDTA mM MgCl2 100 μM PMSF mM EGTA Tricaine solution 400mg Tricaine mM DTT 0.05% NP40 0.125 mg/ml BSA 40 units/ml RNasin Wash buffer Binding buffer + 0.5 M urea 163 [...]... Exploiting commonalities of cellular mechanisms involved in cellular symmetry, asymmetry or signaling themes in cellular asymmetry of various cell types or organisms may improve our understanding of cell polarity in a global perspective This thesis addresses the function of two proteins namely Staufen and Grb2, in controlling the migration of cells 1 1.2 Cell polarity in unicellular organisms Specialized... 1.4.4 Egg cell polarity in germ line specification A number of posterior group genes including vasa, nanos and oskar are involved in formation of polar granules that specify the germ line (NussleinVolhard et al., 1987) Accumulation of vasa and nanos requires the function of Oskar protein (Lehmann and Nusslein-Volhard, 1991) Oskar and the RNA binding proteins Vasa, Tudor and Aubergeine including the large... delay in epiboly in Grb2 disrupted embryos Due to improper cell movements during gastrulation, the axis is shortened in Grb2 mutant mRNA injected embryos These results suggest that Grb2 proteins regulate cell movements by modulating cell shape In conclusion, my analysis of the Staufen protein did not reveal a role in establishment of cell polarity, but uncovered novel functions in PGC migration, maintenance... (Gotta and Ahringer, 2001) In contrast to flies, polarity in C elegans starts after fertilization and requires the actin cytoskeleton and the PAR proteins PAR3 and PAR6, two PDZ domain proteins and a atypical protein kinaseC PKC-3 that form a complex in the anterior half of the zygote (Etemad-Moghadam et al., 1995; Tabuse and Miwa, 1993) (Fig 1.3 B, C) The serine threonine kinase PAR-1 and the ring finger... development of zebrafish 88 Fig 4.7 Schematic representation of Grb2 or Grb2- like proteins showing the SH2 and SH3 domains 90 Fig 4.8 Grb2 and Grb2- like are required for convergence and extension cell movements 91 Fig 4.9 Germ layer patterning is not affected in Grb2 or Grb2- like disrupted embryos 92 xii Fig 4.10 Patterning of neuroectoderm is affected in Grb2 or Grb2- like disrupted... nondifferentiating cells function due to symmetry For example in Schizosaccharomyces pombe (S pombe), Rho-Gap regulates cell size and diameter by ensuring proper distribution of Formin-3P and actin during cell growth (Das et al., 2007) The asymmetric or symmetric positional information of transcripts or proteins are crucial for establishing cell polarity in several unicellular and multicellular organisms Exploiting... by co-injection with wild type grb2 or grb2- like mRNA Disruption of Grb2 proteins causes rounded cell shape of enveloping layer (EVL) and the prechordal plate (PCP) cells Actin accumulation is abolished in the yolk and the PCP cells, affecting morphogenesis of the EVL or the PCP The PCP cells do not undergo normal convergence and extension cell movements in Grb2 disrupted embryos The rounded EVL cell. .. nucleating protein Formin3p (Martin and Chang, 2006; Martin et al., 2005) (Fig 1.1 D) Actin filaments deposit growth machinery through vesicles and polarity of the growth zones in the cell tips are determined by microtubule polymers and its associated proteins (Martin et al., 2005; Mata and Nurse, 1997; Verde et al., 1995) Other examples of polarity include the medial site of cell division in fission yeast... organization of Integrin, ß-catenin, Cadherin proteins and hence malfunction in early cleavage of blastomeres, an example of mispolarized epithelium (Pelegri et al., 1999) For example in migrating fibroblast cells, ß-actin mRNA is localized to the leading edge Similarly during wound healing there is local activation of PI3 Kinase (Lawrence and Singer, 1986, Haugh et al., 2000) Similarly, microtubule 5 binding... shape) defects Grb2, a SH3-SH2-SH3 domain containing protein, was one of the candidates identified from the screen Knockdown of grb2 by morpholinos showed no phenotype Over-expression of mutant grb2 or grb2- like mRNA in zebrafish embryo causes defects in convergent-extension and epiboly cell movements during gastrulation The convergent-extension defects caused by the interference of Grb2 proteins can be . i ESTABLISHMENT OF CELL POLARITY IN ZEBRAFISH: DEFINING THE ROLE OF STAUFEN AND GRB2 IN CELL MIGRATION RAMASAMY SRINIVAS (M.Sc, University of Hyderabad, INDIA) . understanding of cell polarity in a global perspective. This thesis addresses the function of two proteins namely Staufen and Grb2, in controlling the migration of cells. 2 1.2 Cell polarity in. regulation in establishment of cell polarity 16 1.6 RNA binding proteins in mRNA localization 22 1.7 Function of Staufen proteins in mRNA localization and mechanisms that establishment of cell polarity