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

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

iiDedicated to my family members

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

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

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

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

1.1 Symmetry and Asymmetry 1

1.2 Cell polarity in unicellular organisms 2

1.3 Cell polarity in embryos, cultured fibroblasts and axons 5

1.4 Egg cell polarity in specification of axis and germ line in model systems 6

1.4.1 Polarity and specification of axes during oogenesis in D melanogaster 6

1.4.2 Egg cell polarity and specification of axis in Caenorhabditis elegans (C elegans) 8

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

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 5 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

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 ∆Ngrb2-like 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

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 7 : APPENDIX 160

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) do not migrate properly and undergo cell death These results suggest that zebrafish Staufen1 and Staufen2 are required for maintenance and survival of PGCs Staufen proteins are also required for the survival of central nervous system (CNS) neurons in the zebrafish We have thus identified a new function for Staufen proteins in PGC maintenance and survival, which is distinct from its known role in germ-line

(‘t’shape, bent 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 rescued 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 morphology causes 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 and survival The studies on the Grb2 proteins suggest that they may be required for polarized behavior of PCP and EVL cell types during gastrulation movements in the zebrafish embryo

List of Figures

Fig 1.1 Intrinsic spatial cues establish sites of polarized cell growth and division 3

Fig 1.2 Patterning of the oocyte in D melanogaster 7

Fig 1.3 Cell polarity in the C elegans zygote 9

Fig 1.4 Stages of germ cell migration in zebrafish 13

Fig 1.5 Mechanisms that direct mRNA localization in cell types 17

Fig 1.6 General mRNA localization machinery 18

Fig 1.7 Localized mRNA that determines polarity of oocyte in D melanogaster 19

Fig 1.8 Localization of vg1 mRNA by Vg1 RBP and Staufen in Xenopus 21

Fig 1.9 Diversity in cell migration strategies 24

Fig 1.10 Cell movements during gastrulation in zebrafish 25

Figure.3.1 Sequence analysis of zebrafish stau2 44

Fig 3.2 Schematic of PCR based screening of ovary cDNA library through sib selection 46

Fig 3.3 Mapping of stau2 in zebrafish 48

Fig 3.4 Radiation hybrid mapping of zebrafish stau2 48

Fig 3.5 Expression of stau2 during zebrafish maternal and zygotic development 49

Fig 3.6 Northern blot analysis of stau2 expression during maternal and zygotic development 50

Fig 3.7 Temporal and spatial localization of stau2 during development of zebrafish 51

Fig 3.8 Expression and purification of Staufen ds RNA binding domains in E coli 53

Fig.3.9 Expression pattern of Stau2 protein in zebrafish oocytes by immunohistochemistry 54

Fig.3 10 mRNA binding analysis of oskar in vitro 55

Fig 3.11 GST- Stau1 ds RBD-4 fusion protein binds nanos1 and vg1 mRNA in ovary total RNA samples 56

Fig 3.12 Efficacy of splicing inhibition by staufen-related morpholinos in zebrafish 58

Fig 3.13 Germ layer patterning is not affected by disruption of Stau2 function in zebrafish 59

Fig.3.14 Zebrafish Stau2 is not required for localization of squint transcripts 61

Fig 3.15 Over-expression of Stau2 RBD5 abolishes PGCs shown by expression of vasa 62

Fig.3.16 GFP: nos1 expression in PGCs is disrupted in Staufen-disrupted embryos 65

FIG.3.17 Stau depletion by stau1 and anti-stau2 Morpholinos causes loss of germ cells 67

Fig 3.18 Stau2 is required for proper PGC migration 70

Fig 3.19 Expression of vasa is rescued by co-injection of Drosophila staufen 72

Fig 3.20 Expression of GFP nos1 is rescued by co-injection of fly staufen 73

Fig 3.21 Chimeric Fly Staufen::nos 3’ UTR is not sufficient to rescue the PGC phenotype 74

Fig 3.22 Cell death in the CNS of Stau2-depleted embryos 76

Fig 3.23 Tunel labeling in Stau2 depleted embryos during somitogenesis 77

Fig.3.24 Drosophila melanogaster staufen rescues CNS cell death in Stau2-disrupted embryos 78

Fig 4.1 Schematic representation of zebrafish ovary cDNA over-expression screen in yeast 82

Fig 4.2 Organization of the actin cytoskeleton in ∆Ngrb2-like over-expressing yeast cells 83

Fig 4.3 Organization of the microtubule cytoskeleton in ∆Ngrb2-like over-expressing yeast cells 84

Fig 4.4 Percentage of cells that show abnormal cell shape 85

Fig 4.5 Sequence analysis of Grb2 proteins 87

Fig 4.6 Expression pattern of grb2 during 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

Fig 4.10 Patterning of neuroectoderm is affected in Grb2 or Grb2-like disrupted embryos 93 Fig 4.11 Disruption of Grb2 and Grb2-like causes defective convergence extension during

gastrulation 94 Fig 4.12 Zebrafish grb2 and grb2-like mRNA rescues the CE defects caused by disruption of Grb2 proteins 97 Fig 4.13 Grb2 proteins are required for the maintenance of cell size and shape of PCP

progenitors 101 Fig 4.14 PCP cells do not orient their cellular processes to the animal pole and lose their

polarized behavior in Grb2 and Grb2-like disrupted embryos 104 Fig 4.15 PCP cells do not move as a coherent sheet in Grb2 and Grb2-like disrupted embryos 106 Fig 4.16 Epiboly progression is delayed in Grb2 and Grb2-like disrupted embryos 108 Fig 4.17 Grb2 proteins regulate actin enrichment in the yolk cell 110 Fig 4.18 The length to width ratio (L/W) of the EVL cells is altered in Grb2 and Grb2-like disrupted embryos 112 Fig 4.19 Disruption of Grb2 proteins causes defective neuronal branching and arborization 114

CRMP Collapsin response-mediated protein

CNS Central Nervous system

DC Deep Cell

DV Dorsal Ventral

DS RBD Double Stranded RNA binding protein

EGF Epidermal Growth Factor

FGF Fibroblast Growth Factor

GST Glutathione-S-transferase

GRB2 Growth Factor Receptor Bound protein 2

GV Germinal Vesicle

HPF Hours post fertilization

KHD Kinase Homology Domain

MKLP Mitotic Kinesin Like protein

MO Morpholino

MTOC Microtubule organizing centre

Nos Nanos1

OV Otic vesicle

PABP Poly (A) binding protein

PAR Partioning defective

PARP Peri axoplasmic ribosomal plaques

PCP Pre chordal plate

PCR Polymerase chain reaction

PGC Primordial germ cell

PKC Protein-Kinase C

PUF Drosophila melanogaster Pumilio and Caenorabhditis elegans FBF

Collectively called as PUF

RACE Random amplified cDNA ends

RH Radiation hybrid

RNP Ribo-nucleoprotein

RT-PCR Reverse transcription PCR

RTK Receptor tyrosine kinase

TBD Tubulin binding domain

PUBLICATIONS

Ramasamy S, Wang H, Quach HN, Sampath K Zebrafish Staufen1 and Staufen2 are required for the survival and migration of primordial germ cells Developmental Biology 2006 Apr 15; 292:393-406

Chapter 1: INTRODUCTION

1.1 Symmetry and Asymmetry

Biological organisms appear to be asymmetric at most organizational levels This includes the asymmetric or chiral nature of organization of molecules such as proteins and nucleic acids However, the significance of chirality of the molecules in the behavior of a cell or an organism is an open question For example, the plasma membrane of the epithelial cells compartmentalizes into distinct apical and basolateral domains with characteristic lipid and protein compositions that regulate morphogenesis (Muller and Bossinger, 2003; Nelson, 2003) Apart from asymmetry, growth and division of differentiating or non-differentiating 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 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

1.2 Cell polarity in unicellular organisms

Specialized asymmetric structures in bacteria include organizing centres and asymmetrically positioned septa For instance, specific regions of the chromosomes regulate polarized behavior of unicellular bacterial

cytoskeletal-cells (Shapiro et al., 2002) In Escherichia coli asymmetric deposition of cell wall

assembly components regulates morphogenesis (De Pedro et al., 1997;

Mileykovskaya and Dowhan, 2000) Similarly, Bradyrhizobium attaches to plant

root hairs by preferential use of old poles (Loh et al., 1993) The polar pili

mediated attachment of Pseudomonas aureginosa to tracheal epithelial cells is mediated by old poles (Zoutman et al., 1991) Shigella flexneri IcsA and Listeria

monocytogenes ActA proteins located at old cell poles nucleate actin tails and

propels bacterium to penetrate host cells (Goldberg et al., 1993; Smith et al.,

1995) In Caulobacter crescentus, the asymmetric organization of signaling

proteins coordinates cell cycle progression with polar differentiation (Jensen et al.,

2002) During spore formation of Bacillus subtilis, it uses asymmetric cell

division mechanisms to produce spores that survive under harsh environmental conditions (Dworkin and Losick, 2001; van Ooij and Losick, 2003) However, the mechanisms by which bacterial cells exhibit spatial organization of complex structures are not clear

Fig 1.1 Intrinsic spatial cues establish sites of polarized cell growth and division

A) Haploid budding yeast grows new buds adjacent to the previous sites of cell

division marked by the septin ring and the landmark protein Bud10p (blue)

B) Diploid budding yeast grow new buds adjacent to the site of previous cell

growth (mother cell) and at the site of previous cell division (daughter cell), as marked by Bud8p (red) and Bud9p (green)

C) Fission yeast grows at previous sites of cell growth (blue) first and then begins

to grow at the previous cell division sites (green)

D) Microtubules (green), through deposition of a landmark protein Tea1p (pink),

at the tips, regulate fission yeast growth

E) The medial site of cell division in fission yeast is marked by the nucleus

through the landmark protein Mid1p (orange)

In fission (Schizosaccahromyces pombe) and budding (Saccahromyces cervisiae)

yeast cells polarized growth is observed during budding or fission and mating (Chang and Peter, 2003; Madden and Snyder, 1998) (Fig 1.1 A-E) A site in the cell surface is chosen as a landmark to assemble components depending on intrinsic and extrinsic spatial cues (Fig 1.1 A) (Drubin, 1991; Pringle et al., 1995) GTPase proteins such as Cdc42 are activated near the landmark site followed by cytoskeletal organization In budding yeast, asymmetric growth is restricted to the daughter bud (Fig 1.1 A, B) Mutant studies show that Bud3P, 4P and Integrin membrane protein Bud10P regulate axial budding pattern, a landmark of polarized behavior (Harkins et al., 2001; Taheri et al., 2000; Zahner et al., 1996) The patches of polarity proteins, rings of Septins and actin delineate the site and the bud begins to grow at the tip in a polarized manner (Fig 1.1 A) Budding yeast cells segregate ash1 mRNA to the daughter cells for specification of cell fate during mating type switch (Amon, 1996; Bobola et al., 1996; Long et al., 1997) However, fission yeast cells grow at previous sites of cell growth initially and then grow at the previous cell division sites (Fig 1.1 C) Numerous classes of mutants have defective genes that encode for Tea or Ban class proteins, show orb (round), branched, bent and Y shaped morphology, an indication of loss of cell polarity (Verde et al., 1995) These mutants show defective organization of the actin and microtubule cytoskeleton During interphase, cytoplasmic microtubule filaments extend along the long axis of the cell, and filaments depolymerize to form an intranuclear spindle during interphase-mitosis transition (Hagan and Hyams, 1988; Marks et al., 1986) Tea1p is a plus end microtubule binding protein that

regulates microtubule organization and hence the maintenance of the antipodal growth behavior Tea4p, as a novel SH3 domain protein serves as bridge between Tea1p and cortical actin 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 is marked by the nucleus through the landmark protein Mid1p (Bahler et al., 1998; Chang and Nurse, 1996) (Fig 1.1 E)

1.3 Cell polarity in embryos, cultured fibroblasts and axons

During embryonic development, polarized epithelial cells are formed either during cleavage or from the mesenchymal cells (Muller and Bossinger, 2003) The basal domain of the early blastomeres differs from the apical domain due to the asymmetric presence of adhesion and tight junction proteins (Kuraishi and Osanai, 1989) The asymmetric distribution of Protein Kinase C (aPKC) and

the Ephrin receptors establish cell polarity in mouse and Xenopus (Fleming et al.,

2000a; Fleming et al., 2000b; Winning et al., 2001) In zebrafish embryos, epithelial polarity is determined by microtubule arrays with the growing cleavage

furrows (Jesuthasan and Strähle, 1997) Mutants like nebel show defective

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

binding proteins such as Tau, Collapsin response mediated protein-2 (CRMP-2) and Mitotic kinesin like protein (MKLP-1) regulate axon or dendrite morphology (Baas and Buster, 2004; Fukata et al., 2002) However, it is not clear how the differences between the apical and basal domains (spatial segregation of these proteins) are regulated

1.4 Egg cell polarity in specification of axis and germ line in model systems

The asymmetrical localization of maternal determinants establishes the sperm entry site and this determination results in specification of anterior-posterior (A/P), dorsal-ventral (D/V) and left-right (L/R) axes in most of the organisms

1.4.1 Polarity and specification of axes during oogenesis in D melanogaster

In D melanogaster, the position of the oocyte nucleus initiates local

signaling from the oocyte to the adjacent epithelial follicle cells that control the

A/P and D/V axes specification The A/P polarity in the D melanogaster egg is

established by localization of oskar mRNA to the posterior and bicoid mRNA to the anterior (Dubowy and Macdonald, 1998; St Johnston, 1995) (Fig 1.2 B, C)

During early oogenesis of D melanogaster, Gurken (Grk), a Transforming growth

factor alpha (TGF-α) type protein secreted from the egg, signals to Torpedo an EGF receptor in overlying follicle cells at posterior end of the oocyte (Neuman-Silberberg and Schupbach, 1993) (Fig 1.2 A) Gurken-Torpedo signaling is involved in establishment of the A/P axis of the follicle cells and induces them to send signals to the oocyte for re-polarization of microtubules

Fig 1.2 Patterning of the oocyte in D melanogaster

A) The microtubule cytoskeleton is repolarized by Grk signaling at stage 6–7 of

B) Localization of bicoid and oskar mRNA at stage 9 egg chamber Bicoid mRNA

and oskar mRNA are transported through the ring canals into the oocyte Bicoid mRNA localizes at the anterior cortex of the oocyte, while oskar mRNA and

Staufen protein are transported by Kinesin from minus ends of the MT towards the posterior pole

C) Localization of anterior and posterior determinants in the egg, bicoid mRNA

(blue) is anchored at the anterior pole while oskar and nanos mRNAs as well as Oskar and Nanos proteins are anchored at the posterior pole

The re-polarization of microtubules by gurken is essential for localization of the maternal mRNAs within oocyte at stage 6-7 (Gonzalez-Reyes et al., 1995; Gonzalez-Reyes and St Johnston, 1998) In response to the reverse signals from the follicle cells to the oocyte, the posterior Microtubule organizing centre (MTOC) disorganizes and the microtubule polymers nucleate from the anterior

and lateral cortices of the oocyte (Fig 1.2 A, B) Maternal bicoid transcription is regulated by the maternal transcription factor Serendipity delta and its translation

is inhibited in the posterior by Nanos (Gavis and Lehmann, 1994, Payre et al., 1994) Oskar mRNA localization to the posterior is crucial for the germ cell specification and formation (Lehmann and Nusslein-Volhard, 1986; Ephrussi et al., 1991; Ephrussi and Lehmann, 1992) Nanos mRNA is localized to the pole plasm, a specialized cytoplasm later incorporated into pole cells, the precursors of the fly's reproductive system (Wang et al., 1994; Wang and Lehmann, 1991) (Fig 1.2 C) Thus, several transcripts and protein gradients in the egg determine and

establish the specification of axes and germ line in D melanogaster

1.4.2 Egg cell polarity and specification of axis in Caenorhabditis elegans (C elegans)

The A/P polarity in C elegans embryos is determined by the sperm and

leads to the asymmetric localization of PAR (partitioning defective) proteins

which in turn control spindle positioning (Fig 1.3 A-C) ( Hird and White, 1993, Goldstein and Hird, 1996 )

Fig 1.3 Cell polarity in the C elegans zygote

A) Shows the cortex of C elegans zygote, which is partitioned in to distinct

anterior, and posterior regions PAR-3, PAR-6 and the atypical protein kinase C isoform PKC-3 (red) localize in the anterior cortex, whereas PAR-1 and PAR-2 (green) are restricted to the posterior cortex The mitotic spindle positioned closer

to the posterior pole generates larger anterior and a smaller posterior cell after cytokinesis Aster microtubules that originate from the spindle poles reach the cortex Microtubules are shown in blue and the metaphase chromosomes are shown in yellow

B) Wild-type C elegans zygote stained with anti-PAR-6 (green) and

anti-α-tubulin (red) antibodies

C) Wild-type C elegans zygote stained with anti-PAR-1 (green) and

anti-α-tubulin (red) antibodies

The A/P polarity and the cytoplasmic flow or the localization of PAR proteins occurs without sperm pronucleus (Sadler and Shakes, 2000) The machinery include Cdc-42 and G-protein signaling which maintains the aster induced early

cell polarity during C elegans development (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 protein PAR-2 occupy the posterior half (Boyd et al., 1996; Guo and Kemphues, 1995) The conserved mechanism of PAR proteins in polarity iterates the importance to understand parallel polarity mechanisms in various cell types and organisms

1.4.3 Egg cell polarity and specification of axis in vertebrates

In Xenopus laevis (Xenopus) the segregation of cytoplasmic determinants

in the egg or zygote establish A/V axis (Hill and Strome, 1990; Moon and Kimelman, 1998) Twenty mRNAs have been identified that are localized using two major pathways to distinct but overlapping domains within the vegetal cortex (Elinson et al., 1993; Forristall et al., 1995; Kloc and Etkin, 1995) Xcat2 and xdazl, are transcripts of the germ plasm component, localize during the early pathway through mitochondrial cloud (MC) or Balbiani body (Heasman et al., 1984) The late pathway is characterized by localized RNAs such as vg1 and vegT involved in somatic cell fates (Joseph and Melton, 1998; Zhang et al., 1998, Kloc

et al., 1998) In contrast to frog embryos, the early development of the mouse embryo is highly regulative where isolated blastomeres change their

developmental fate (Zernicka-Goetz, 2006) However, some reports suggest the presence or absence of predetermination and on the whole, polarity in the mouse preimplantation embryo is controversial (Gardner, 2007; Hiiragi et al., 2006; Hiiragi and Solter, 2006) The mature zebrafish oocyte is radially symmetrical about the A/V axis and no D/V polarity is evident during oogenesis (Mizuno et al., 1999; Ober and Schulte-Merker, 1999) The dorsal determinant is initially located

at the vegetal pole which translocates along microtubules to the future dorsal side before the first cleavage division occurs (Holley and Ferguson, 1997; Jesuthasan and Stahle, 1997; Moon and Kimelman, 1998; Schier and Talbot, 2005; Strahle and Jesuthasan, 1993; Sumoy et al., 1999; Weaver and Kimelman, 2004) Studies from Gore and colleagues suggest that zebrafish dorsal axis is apparent as early as the 4 cell stage as marked by localization of squint transcripts to two blastomeres (Gore et al., 2005) Thus, localization of molecules during maternal and early development patterns the embryo

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 Volhard 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 ribosomal rich structures (polar granules rich in RNA and protein), are required for germ plasm

(Nusslein-assembly in D melanogaster (Ephrussi et al., 1991; Ephrussi and Lehmann, 1992;

Hay et al., 1988; Kobayashi et al., 1993; Lasko and Ashburner, 1988; Smith et al., 1992) However, in mouse the mode of germ cell specification is inductive event between the extra embryonic ectoderm and epiblast (Extavour and Akam, 2003)

The extracellular signaling molecules such as BMP4, BMP8b and BMP2 in mice are important for germ cell formation (Lawson et al., 1999; Ying et al., 2000) In

C elegans and Xenopus germ cell specification requires specialized germ plasm

characterized as large electron-dense particles containing RNA and protein (called

as Nuage or Polar granules or ‘P’ granules) (Andre and Roulier, 1957; Eddy 1974; Knaut et al., 2000; Kobayashi et al., 1993; Mahowald, 2001; Mahowald et al., 1976) In zebrafish, these granules are present at the 4-cell stage in indented furrows where germ cell specific transcripts are localized (Knaut et al., 2000) Vasa and Nanos that are localized to early blastomere furrows, have no effect on PGC formation, however the migration of these cells are disrupted (Olsen et al., 1997; Yoon et al., 1997; Muller et al., 2002; Weidinger et al., 2003; Yoon et al.,

1997 Koprunner et al., 2001) Recent studies also show separate pathways of RNA recruitment that lead to compartmentalization of the zebrafish germ plasm (Theusch et al., 2006) The mechanism by which primordial germ cell fate is maintained until they migrate to the gonadal anlagen is not understood well

1.4.4.1 Primordial germ cell migration

In D melanogaster germ cell migration is active during crossing of the

midgut epithelium Ultrastructural studies showed that germ cells pass through

gaps that form from apical junctions In mutants like serpent (srp) or huckebein

(hKb), germ cells are incapable of migrating across the gap (Callaini et al., 1995;

Jaglarz and Howard, 1994; Moore et al., 1998; Rongo et al., 1997) In Hmgcr Hydroxy methyl glutaryl Co-enzyme A reductase) mutant embryos, germ cells fail

(3-to migrate (3-toward the mesoderm and remain associated with the dorsal region of the posterior midgut (Van Doren et al., 1998)

Fig 1.4 Stages of germ cell migration in zebrafish

The left side of each panel shows the onset of each step during PGC migration and the right panel shows the respective views at specific hour post fertilization The bottom right of each panel shows genes required

A) The PGCs (blue and yellow) are arranged in four clusters and start their

migration towards the dorsal side of the embryo

B) The PGCs align with the anterior and lateral mesoderm at 80% epiboly

C) At two-somite stage, the PGCs migrate close to the somites 1–3, which are

intermediate targets

D) They start migrating towards the final targets at 8–10 somites from the

intermediate targets

E) At 24 hpf, the PGCs stop migration and coalesce with their somatic

counterparts to form the gonads

In chick embryos, the PGCs use the vascular system as a vehicle to transport them

to the region of the gonad In mouse, germ cells do not rely on contacts between themselves and move from their site of induction toward the primitive streak and finally enter the posterior endoderm, where they spread along the endoderm (Anderson et al., 2000; (Godin et al., 1990; Molyneaux et al., 2001) Each zebrafish gonad requires the alignment of two clusters of germ cells The somatic cues bring together clusters of PGCs on either side of the zebrafish embryo to specific intermediate targets before they migrate to their final destination (Koprunner et al., 2001; Weidinger et al., 2003; Weidinger et al., 1999; Weidinger

et al., 2002) The PGCs appear to align along the lateral and ventral borders of the trunk mesoderm, as well as along the border between the trunk and head mesoderm (Raz, 2003; Raz and Hopkins, 2002) (Fig 1.4 B, C) During early somitogenesis, they migrate towards two lateral positions at the anteroposterior level of the first somites and form two PGC clusters (Raz, 2003; Raz and Hopkins, 2002) (Fig 1.4 D) During late segmentation stages the PGC clusters migrate further posterior to reach the level of the 8th somite where their final target may reside which are the cells comprising the somatic portion of the gonad (Weidinger

et al., 1999; Weidinger et al., 2002; Yoon et al., 1997) (Fig 1.4 E) The signals that direct the migrating of PGCs was identified in embryos bearing mutations that affect the development of specific somatic structures (Weidinger et al., 1999; Weidinger et al., 2002) Specific genetic and morpholino screens identified

candidates that are required for guidance (extracellular cue and its receptor) (Topczewski et al., 2001) In vertebrates ENU-based screens identified CXCR4b,

a seven transmembrane G-protein coupled chemokine receptor required for PGC migration (Chong et al., 2001; Doitsidou et al., 2002; Knaut et al., 2003) The ligand for CXCR-4b is Sdf-1a for guidance of PGCs The map of migration of PGCs is similar to domains of Sdf-1a expression pattern in the zebrafish embryo suggesting it as an attractive cue (Doitsidou et al., 2002; Weidinger et al., 1999; Weidinger et al., 2002) Over-expression of Sdf-1a results in ectopic PGCs where Sdf-1a is mis-expressed suggesting that CXCR-4b and Sdf-1a as proteins that are important in guidance of PGCs (Doitsidou et al., 2002) The migration and survival of germ cells are coupled The challenge ahead is to identify molecular basis of these polarizing cues to influence the migration behavior of PGCs

1.5 Mechanisms that generate cell polarity

In D melanogaster, blastoderm, mRNAs are transported apically by motor

proteins such as dynein in a microtubule-dependent manner (Tekotte and Davis, 2002; Wilkie and Davis, 2001) For example the phosphorylation of PAR-1 by PKC-3 excludes the localization of PAR-1 to the anterior cortex (Cuenca et al., 2003; Munro et al., 2004) Bazooka class of proteins that regulate PAR is phosphorylated to prevent its localization to the posterior cortex, suggesting a

mutual phosphorylation-dependent exclusion event in D melanogaster oocyte

polarity (Cox et al., 2001; Huynh et al., 2001) Asymmetric cell division in neuroblasts is preceded by localization of Prospero to an asymmetric cortical domain in the neural progenitor which is distinct from their behavior in GMC, where they are found in the nucleus (Schuldt et al., 1998) This shows that Prospero proteins may be important for regulation of mitotic potential of the

GMCs In D melanogaster, Sec15 exocyst complex protein is required for

specific vesicle trafficking during cell fate determination of sensory organ precursor formation (Jafar-Nejad et al., 2005) Similar to mammalian epithelial

cells, depletion analysis of Par-3, aPKC, PatJ and PalS1 proteins in C elegans

showed that these proteins were crucial for establishment of the tight junctions (Margolis and Borg, 2005) Reports show that MTOCs are reoriented and this organization is dependent of Cdc-42 and Dyenin motor protein in fibroblasts (Etienne-Manneville and Hall, 2001; Etienne-Manneville and Hall, 2003) The other pathway requires the Myotonic dystrophy kinase related Cdc-42 binding kinase (MRCK), myosin and actin acting downstream of Cdc-42 (Gomes et al., 2005) Studies also support the role for Growth factor receptor bound protein

(GRB2) adaptor signaling in N-Wasp-mediated actin polymerization in vitro

(Boerner et al., 2003; Carlier et al., 2000) In zebrafish, non cell-autonomous activity of STAT3 signaling in gastrula organizer cells controls the polarity of neighboring cells through Dishevelled-RhoA signaling in the Wnt-planar cellpolarity (Wnt-PCP) pathway (Miyagi et al., 2004) Mouse and frog models for neural tube closure birth defects, indicate that Van Gogh-like-2 (Vangl2, also known as Strabismus) and other components of planar cell polarity (PCP)

signaling control neurulation

1.5.1 RNA localization and translational regulation in establishment of cell polarity

RNAs have been found localized in several cell types and embryos in specific domains to perform specific functions (Bashirullah et al., 1998; Jansen and Kiebler, 2005; Kloc et al., 2002; Palacios and St Johnston, 2001) mRNA localization mechanisms include several means as shown in (Fig 1.5)

Fig 1.5 Mechanisms that direct mRNA localization in cell types

Directional transport mediated by mRNP complexes through cytoskeleton

Random cytoplasmic diffusion aided by cytoplasmic flow or cyclosis

Vectorial export mechanisms by which transcripts are spatially exported to one side of cell and elimination mechanisms by which mislocalized transcripts are destabilized by cellular factors

1.5.1.1 Mechanisms of mRNA localization

Although the knowledge of the components of mRNP complexes is increasing, the present list does not completely explain how RNA-protein complex is loaded and translocated from one cytoplasmic domain to another domain

Fig 1.6 General mRNA localization machinery

The process of transporting mRNA is mediated by several factors that are transacting along with the translocation machinery The transacting proteins are RNA binding proteins, Poly-A-binding proteins, other mRNA binding factors that aid in conferring specificity, adaptor protein that links ribonucleo protein complex (RNP) with motor protein that translocates through the cytoskeleton to specific cytoplasmic domains

In budding yeast, ash1 mRNA is transported along actin filaments by a type V myosin, She1p/Myo4p, which is a part of an RNP complex, termed the locasome (Beach et al., 1999; Bertrand et al., 1998) The zip code sequences of the ß-actin mRNA immediately downstream of the stop codon recruit ZBP1 proteins by interacting with its K-homology (KH) domains, along actin filaments to the leading edge during migration of fibroblasts (Kislauskis et al., 1994; Ross et al., 1997; Sundell and Singer, 1991; Job and Eberwine, 2001) For example Myelin basic protein (MBP) mRNA is targeted to the myelin membranes of

oligodendrocyte cell processes by hnRNPA2 RNA binding protein in

microtubules through kinesin motor (Ainger et al., 1997; Hoek et al., 1998) In D

melanogaster, bicoid mRNA is localized and anchored to the anterior of the

oocyte by Swallow and Staufen proteins (Fig 1.7) The posteriorly localized nanos mRNA which is transported through microtubule polymers, is stable, anchored and protected by the actin cytoskeleton (Forrest and Gavis, 2003) (Fig 1.7) Nanos mRNA contains several stem loop structures within its 3’ UTR, that is recognized

by Smaug and oskar for proper localization required for localization (Ephrussi et al., 1991)

Fig 1.7 Localized mRNA that determines polarity of oocyte in D melanogaster

Transcripts that are transported from the nurse cells are localized to anterior and posterior ooplasmic domains The blue arrows depict the anterior, posterior, dorsal and ventral regions of the oocyte

The first picture depicts, bicoid (green) mRNP complex containing Staufen (yellow) and Swallow (red) proteins traverse from the + end direction to the – end

of microtubules to the anterior of the oocyte using unknown motor protein (yellow) Bicoid mRNA (red) is anchored and maintained at the anterior cortex by Staufen (yellow)

The second picture depicts, oskar (red) mRNP complex containing Exon junction complex proteins (EJC) (blue), Staufen (yellow) traverse towards the posterior of the oocyte from the – end to the + end of microtubules using kinesin (black and Indigo) as motor

The third picture depicts the diffusion and cytoplasmic events that localize nanos mRNA to the posterior, Cortical actin in blue anchors nanos mRNA to the posterior

The oskar 3’ UTR is recognized by exon junction complex proteins (EJC) suggesting a role for mRNA splicing in localization of oskar mRNA localization (Hachet and Ephrussi, 2004)(Fig 1.7)

Fig 1.8 Localization of vg1 mRNA by Vg1 RBP and Staufen in Xenopus

Transport of vg1 mRNA (red) to vegetal cortex in Xenopus oocytes, Vg1 RNA

binding protein (RBP) (indigo) with Staufen (yellow) is loaded into microtubules (red) using adaptor proteins and motor proteins Kinesin I and II Vg1 mRNA is anchored in the vegetal cortex by actin cytoskeleton

In Xenopus, vg1 mRNA zipcode sequences (Mowry and Melton, 1992) interacts

with Vg1-RBP/Vera protein (Fig 1.8) (Deshler et al., 1998; Deshler et al., 1997; Havin et al., 1998; Schwartz et al., 1992) through its KH domains (Git and Standart, 2002) and mediate association with microtubules (Elisha et al., 1995) The translocation of vg1 mRNA along microtubules (Yisraeli et al., 1990) may involve kinesin motors and the Staufen protein (Allison et al., 2004; Betley et al., 2004; Yoon and Mowry, 2004) In zebrafish, several candidate mRNAs that may involve in patterning the embryo are localized in the oocyte, but it is not clear how they are achieved

1.6 RNA binding proteins in mRNA localization

Double stranded RNA binding (ds RBD) proteins interact with as minimum of 11 bp of mRNA that is known to form secondary and tertiary structures However, it remains unclear how the stem-loop or secondary/tertiary structures in the UTR or the coding sequence of localizing mRNAs are recognized

by the dsRBD domains The ds RBD domains interact with A-form of double helical RNAs, which differs from typical ds DNA helical forms Several structural studies show that, the 2’OH group of the RNA helix is important for interaction Evidences from viral dsRBD protein PKR suggest a role for autophosphorylation

of EIFα, after binding to its dsRNA target (Galabru and Hovanessian, 1987; Thomis and Samuel, 1995) Vera, an RNA binding protein, localizes vg1 mRNA

to the vegetal cortex in Xenopus (Deshler et al., 1998) The vera homolog of chick

is zbp ZBP is required for the localization of ß-actin mRNA Similar to ZBP,

Vera binds sequences in the vg1 localization element and is responsible for vegetal localization of vg1 Staufen is also an RNA binding protein that plays multiple roles in different cell types

1.7 Function of Staufen proteins in mRNA localization and mechanisms that establishment of cell polarity

Staufen is required for the localization of maternal determinants such as oskar mRNA to the posterior and bicoid to the anterior in developing oocytes of

D melanogaster (St Johnston et al., 1991) Staufen protein is also involved in

translational regulation of posterior group transcript oskar in pole plasm formation

and germ line specification in D melanogaster (Micklem et al., 2000) Staufen

protein has five conserved dsRBD domains and a tubulin-binding domain (TBD)

(Micklem et al., 2000; Wickham et al., 1999) RNA binding domain dsRBD-2 is required for microtubule-dependent localization of oskar mRNA, whereas dsRBD-5 functions in translational regulation of oskar mRNA (Micklem et al., 2000) The dsRBDs-1, -3, and -4 bind double-stranded RNA invitro but RBDs-2

and -5 do not bind (Micklem et al., 2000) During zygotic development in D

melanogaster, the asymmetric localization of prospero RNA during neuroblast

division is dependent on Staufen protein (Broadus et al., 1998; Li et al., 1997;

Micklem et al., 2000) Stau is required for localization of pros RNA but not of Pros protein (Broadus et al., 1998) In mouse, staufen1 and staufen2 are expressed

in germ cells during oogenesis and embryogenesis (Saunders et al., 2000) Human Staufen 2 protein is found in the somatodendritic compartment and mainly expressed in the brain (Duchaine et al., 2002) Stau particles in RNA containing granules co-localize with microtubules in dendrites of hippocampal neurons, suggesting its role in neuronal mRNA localization responsible for spatially restricted elicitation of neurons (Kohrmann et al., 1999) Studies show that Staufen protein in neuronal RNA granules play important role in dendritic mRNA

transport (Kim and Kim, 2006) In Xenopus, Staufen proteins localizes to the

vegetal cortex in a complex containing Vg1 RNA and Kinesin Disruption of Staufen function blocks localization of Vg1 RNA (Allison et al., 2004; Yoon and Mowry, 2004) Mammalian Staufen protein recruits UPF1 proteins to 3’ UTR of ARF-1 mRNA to mediate nonsense-mediated mRNA decay (Kim et al., 2005) Recent studies show that Staufen proteins regulate the expression of physiological transcripts and metabolic pathways by mRNA decay based mechanisms (Kim et al., 2007) Though several mechanisms about Staufen in mRNA localization are

studied, it is also important to understand how these mechanisms communicate to establish cell polarity

1.8 Cell polarity: an attribute to cell migration and cell movement

Migratory strategies in several cells are similar to amoeboid mode of

migration (Fig 1.9 A) This pattern of migration is seen in Dictyostelium amoebae,

immune cells, leukocytes and the cancer cells (Fig 1.9 A) (Condeelis et al., 2005; Wolf et al., 2003)

Fig 1.9 Diversity in cell migration strategies

A) Polarized amoeboid migrating cells, with clear leading edge that change their

shape and direction

B) Mesenchymal migrating cells are spindle-shaped and show elongated morphology C) Collectively migrating cells in a cluster of cells that are

connected by adherens junctions (blue lines) These cells migrate as a sheet and move through the matrix