ASYMMETRIC CELL DIVISION IN THE REGULATION OF NEURAL STEM CELL SELFRENEWAL IN DROSOPHILA MELANOGASTER CHANG KAI CHEN B.Sc (Hons), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY TEMASEK LIFE SCIENCES LABORATORY NATIONAL UNIVERSITY OF SINGAPORE 2009 ACKNOWLEDGEMENTS I would like to express my heartfelt thanks to my supervisor Prof William Chia for his guidance, patience and support during the course of my PhD study I would also like to extend my sincere thanks and gratitude to a/P Wang Hongyan who went beyond her call of duty to supervise and guide me in the last year of my PhD study Her insightful suggestions and critical comments have been instrumental in shaping this work to its present form I am extremely grateful to Dr Greg Somers for his invaluable help and guidance for the initial part of the Zif and PP2A work Many thanks to Dr Rita Sousa-Nunes for giving me the zif mutant to work on, to Dr Wang Cheng for being a great collaborator for the PP2A project, and to my Ph.D advisory committee, A/P David Ng, A/P Sudipto Roy and a/P Toshie Kai for their suggestions during the committee meetings My sincere gratitude goes to Liu Ming for being a friend, and for his very kind assistance with microinjection for the generation of some of the zif transgenic lines I would also like to thank Dr Gisela Garcia-Alvarez for her contribution to the kinase assays in this work In addition, I would like to acknowledge Jacqueline Chin, Wong Jian Xiang and Soon Swee Beng for their technical assistance I also thank all the past and present members of Bill Chia lab and Wang Hongyan lab as well as the TLL fly community who have generously shared reagents with me at various stages of this work A very big thank you goes to dear friends like Shvetha, Vera, Maddy, Xiaodong, Ai Khim, Dawn, Kenneth, Kris and Charissa for their support during the trying times of my study I also thank my Toh Yi Caregroup for being understanding and supportive while I was busy with the writing of my thesis My most sincere thanks and gratitude go to my dearest friend Chin Fern, Coach Jo-Ann, Pastor Benjamin and Pauline for being my constant source of encouragement, support and wise counsel at various critical points of my PhD study Most importantly, I would like to extend my deepest thanks to my everso-supportive family - Dad, Mom, and my two sisters, Kai-Tirng and Kai Chirng I thank them for loving me the way they - for always being there to listen, to help, and to provide the emotional support that I need, especially during the toughest times I especially thank beloved Kai-Tirng for her love, patience, incessant support and prayers Without my loving family, I will not be where I am today Finally, my most heartfelt thanks and gratitude go to Mark Chong for faithfully supporting me in more ways than I can ask for I am deeply touched and extremely grateful for his love, patience, thoughtfulness and unyielding encouragement I thank him for the laughter and the joy that he has brought into my life, and for being the most faithful prayer warrior I’ve ever had Above all else, I am forever grateful to Jesus Christ, my Lord and Savior No amount of words can fully express my gratitude for His faithfulness, His unfailing love and His abundant grace toward me Kai Chen Dec2009 i TABLE OF CONTENTS ACKNOWLEDGEMENTS i TABLE OF CONTENTS ii ABBREVIATIONS vi LIST OF FIGURES AND TABLES ix SUMMARY xii CHAPTER 1: INTRODUCTION 1.1 Drosophila as a model system 1.2 Stem cell in development 1.3 Stem cells in Drosophila neurogenesis 1.4 Asymmetric cell division in Drosophila neural stem cell 1.4.1 Setting up neuroblast polarity 1.4.2 Mitotic spindle orientation 11 1.4.3 Asymmetric localization and segregation of cell fate determinants 13 1.4.3.1 Adaptor proteins required for asymmetric localization of cell fate determinants 16 1.5 Stem cells and cancer – the cancer stem cell hypothesis 20 1.6 Link between failure in stem cell asymmetric division and tumor formation 21 1.7 Drosophila Stem Cell self-renewal and Tumor suppression 24 1.7.1 Tumor growth induced by altered stem cell division 26 1.7.2 Mitotic Spindle orientation and tumor suppression 28 1.7.3 Tumor growth induced by impaired terminal differentiation 29 1.8 Cell cycle genes regulate asymmetric division and act as tumor suppressors 30 1.9 Protein phosphatases, asymmetric division and tumor suppression 33 ii 1.10 Objectives 36 CHAPTER 2: MATERIALS AND METHODS 38 Molecular Biology 38 2.1 2.1.1 Recombinant DNA methods 38 2.1.2 Bacterial host strains and growth conditions 38 2.1.3 Cloning strategies 39 2.1.4 Transformation of E coli cells 40 2.1.4.1 Preparation of competent cells for heatshock transformation 40 2.1.4.2 Heat shock transformation of E coli 40 2.1.4.3 Preparation of competent cells for electroporation 41 2.1.4.4 Electroporation transformation of E coli 41 2.1.5 Plasmid DNA preparations 42 2.1.6 Isolation of total genomic DNA from adult flies 42 2.1.7 Reverse Transcription (RT)-PCR 43 2.1.7.1 Isolation of total RNA 43 2.1.7.2 First strand cDNA synthesis 43 2.1.7.3 PCR reaction after RT 44 2.1.8 2.2 Site-directed mutagenesis 45 Cell Culture 45 2.2.1 Production of double-stranded RNA (dsRNA) 45 2.2.2 Cell culture, dsRNA and drug treatment 47 2.3 Biochemistry 47 2.3.1 Frequently used buffers and solutions 47 2.3.2 PAGE and Western transfer of protein samples 48 2.3.3 Immunological detection of proteins and antibodies used 49 2.3.4 Generation of anti-Zif polyclonal antibody 49 2.3.5 Fusion protein expression 50 2.3.6 Two-dimensional PAGE 51 iii 2.3.7 Chromatin Immunoprecipitation (ChIP) Assay 51 2.3.8 Luciferase Assay 54 2.3.9 In vitro Kinase Assay 54 2.4 Immunohistochemistry and microscopy 55 2.4.1 Frequently used reagents and buffers 55 2.4.2 Antibodies 55 2.4.3 Fixing and staining of Drosophila larval brains 56 2.4.4 Neuroblast quantification and brain orientation 57 2.4.5 BrdU labeling 57 2.4.6 Spindle orientation quantification 57 2.5 Fly Genetics 58 2.5.1 Fly stocks and growth conditions used in this study 58 2.5.2 Generation of positively labeled neuroblast MARCM clones 59 CHAPTER 3: A NOVEL ZINC FINGER PROTEIN NEGATIVELY REGULATES aPKC EXPRESSION TO INHIBIT EXCESS SELF-RENEWAL OF DROSOPHILA NEURAL STEM CELLS 60 3.1 Introduction 60 3.2 Results 62 3.2.1 Identification of a novel zinc-finger protein with a role in regulating neuroblast asymmetry 62 3.2.2 Disruption of zif leads to excess neuroblasts in larval brain clones 64 3.2.3 Subcellular localization of endogenous Zif 68 3.2.4 Zif transgene fully rescues defects in zif mutant neuroblasts 70 3.2.5 Asymmetric localization of aPKC and Numb/Pon requires Zif function70 3.2.6 Zif represses aPKC transcription and downregulates aPKC protein expression 76 3.2.7 Zif directly represses aPKC transcription to inhibit excess neuroblast self-renewal 78 iv 3.2.8 aPKC phosphorylates Zif 82 3.2.9 Nuclear localization of Zif depends on its phosphorylation state 84 3.2.10 Non-phosphorylatable form of Zif inhibits excess neuroblast selfrenewal 3.3 89 Discussion 91 3.3.1 Role of Zif in neuroblast self-renewal and neuroblast asymmetry 91 3.3.2 Zif is the first identified transcription factor to regulate neuroblast self-renewal through direct transcriptional repression of aPKC 93 3.3.3 aPKC phosphorylates Zif to regulate its nuclear localization, thereby modulating activity of Zif as a transcriptional repressor of aPKC 94 3.3.4 Reciprocal repression between aPKC and Zif in neuroblast asymmetric division and neuroblast self-renewal 96 CHAPTER 4: PROTEIN PHOSPHATASE 2A REGULATES SELFRENEWAL OF DROSOPHILA NEURAL STEM CELLS 98 4.1 Introduction 98 4.2 Results 98 4.2.1 Microtubule Star (Mts) is a novel brain tumor suppressor in Drosophila 99 4.2.2 PP2A can inhibit excess self-renewal of neuroblasts 102 4.2.3 PP2A regulates asymmetric protein localizations as well as mitotic spindle orientation 107 4.2.4 PP2A and Polo enhance Numb phosphorylation and asymmetric localization 110 4.2.5 PP2A acts in Polo/Numb pathway to inhibit neuroblast overgrowth 117 Discussion 119 CHAPTER 5: CONCLUSION AND PERSPECTIVES 122 REFERENCES 128 v ABBREVIATIONS aa a/P A/P aPKC APS Ase ATP Aur-A Baz bp Brat BSA C elegans amino acid Assistant Professor Associate Professor Atypical protein kinase C ammonium persulphate Asense Adenosine 5’ Triphosphate Aurora-A Bazooka basepairs Brain tumor Bovine serum albumin Caenorhabditis elegans CaCl2 cDNA ChIP CIP CNN CNS CNS Cy3 Cyc DEPC DGRC Dlg DM DM DNA dNTP Dpn DSHB dsRNA DTT E coli ECL EDTA Calcium chloride complementary DNA chromatin immunoprecipitation calf intestinal phosphatase Centrosomin Central nervous system Central nervous system Cyanine conjugated cyclin Diethyl Pyrocarbonate Drosophila Genomics Resource Center Disc large dorsomedial dorsomedial Deoxyribonucleic acid deoxynucleotide triphosphate Deadpan Developmental Studies Hybridoma Bank double-stranded ribonucleic acid 1, 4-Dithio-DL-threitol Escherichia coli Enhanced Chemiluminescence Ethylenediaminetetraacetic acid Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’tetraacetic acid Embryonic Lethal Abnormal Vision ethlymethane sulfonate Falafel FLP recombinase recombination target grams guanine-nucleotide-dissociation inhibitor Green fluorescent protein Ganglion mother cell EGTA ELAV EMS Flfl FRT g GDI GFP GMC vi GST HCl hr IEF IgG Insc IPG IPTG Kb KCl Khc73 Kv L3 LB LiCl Loco M Glutathione-S-Transferase Hydrochloric acid hour Isoelectric focusing Immunoglobulin Inscuteable immobilized pH gradient Isopropyl-β-thiogalactopyranoside Kilobase potassium chloride kinesin heavy chain 73 Kilovolts third-instar larval Luria bertani Lithium chloride Locomotion defects Molar MgCl2 mins Mira ml mM MOPs MTOC Mts Mud NB Magnesium chloride minutes Miranda millilitre millimolar 4-Morpholinepropanesulphonic acid microtubule organizing center Microtubule star Mushroom body defective Neuroblast N-terminal OD PAGE PAN Par PBS PCM PCR PH3 Pins PON PP2A Pp4 Pros RNA RNAi RT RT-PCR S SDS secs Amino (NH2) terminal optical density Polyacrylamide gel electrophoresis Posterior Asense-Negative partitioning defective Phosphate Buffered Saline Pericentriolar material Polymerase Chain Reaction phospho-histone H3 Partner of Inscuteable Partner of Numb Protein phosphatase 2A Protein phosphatase Prospero Ribonucleic acid RNA interference room temperature real-time polymerase chain reaction Serine Sodium Dodecyl Sulphate seconds vii SOP stau TE TEMED Tris Tws UAS V Wdb β-Gal μg μl μM 2-D PAGE Sensory organ precursor staufen Tris EDTA N, N, N’, N’ tetramethylethylene diamine Tris (hydroxymethyl) aminomethane Twins Upstream Activator Sequence Volts Widerborst β-Galactosidase microgram microlitre micromolar two-dimensional polyacrylamide gel electrophoresis viii List of Figures and Table LIST OF FIGURES AND TABLES FIGURES Chapter PAGE Figure Life cycle of Drosophila melanogaster Figure Regulation of stem cell division Figure Asymmetric neural stem cell division in Drosophila embryo Figure Postembryonic neuroblast development Figure Key players in neuroblast asymmetric division Figure Overgrowth of mutant brain tissues implanted into adult 23 hosts Figure Models of the origin of Drosophila stem-cell derived 25 tumors Figure Proposed mechanism where activation of Aur-A leads 32 to the phosphorylation of Numb Protein phosphatase 2A is a heterotrimeric complex 34 Figure 10 Mosaic Analysis with a Repressible Cell Marker 61 Figure 11 Disruption of a novel gene causes ectopic Miranda 62 Figure Chapter persistence Figure 12 Schematic of zif locus and depiction of molecular 1L15 lesions in zif , zif 2L745 and zif 63 2L497 Figure 13 Zif inhibits excess neuroblast self-renewal 65 Figure 14 Zif acts to suppress neuroblast self-renewal and 66 promote neuronal differentiation Figure 15 Zif inhibits excess neuroblast self-renewal in both Ase- 67 positive non-DM and Ase-negative DM neuroblast lineages Figure 16 Polyclonal antibody against full-length Zif specifically 68 recognizes Zif Figure 17 Subcellular localization of Zif in third instar larval brain 69 recapitulated by an inducible Zif transgene ix Conclusion and Perspectives CHAPTER CONCLUSION AND PERSPECTIVES For many years, Drosophila neuroblasts have proved to be a remarkably fruitful model system for investigating stem cell symmetry breaking The study of stem cell asymmetric division in Drosophila embryonic neuroblasts has brought about a period of rapid progress in identifying many central players in asymmetric cell divisions In the recent years, Drosophila larval brain neuroblasts have also emerged as a novel model for the study of stem cell self-renewal and tumor suppression as a growing body of evidence strongly suggest that defects in asymmetric cell division can upset homeostasis of stem cell self-renewal causing neural stem cells to turn into tumor-initiating cells that recapitulate several hallmarks typical of mammalian tumors The advent of new methods for self-renewal assays such as MARCM (Lee and Luo 1999), made it possible to generate single mutant neuroblast clones that lack a particular gene in a heterozygous (phenotypically) wild-type background Such mosaic clonal analyses made it easier to ascertain the effects of specific gene mutation on neuroblast self-renewal by comparing stem cell numbers with and without the activity of candidate self-renewal regulators In fact, many of the widely conserved cellular polarity proteins (e.g lgl and dlg) known to regulate asymmetric cell division were discovered to act as tumor suppressors during neuroblast self-renewal through such new techniques Consistent with the link between asymmetric cell division and tumor formation, molecular genetic data from the study of Drosophila neural stem 122 Conclusion and Perspectives cell together with results from mammalian studies provided evidence to show that mechanisms regulating stem cell self-renewal and tumor suppression via asymmetric cell division are evolutionarily conserved These studies show that breakdown of asymmetry in dividing Drosophila neuroblast stem cells leads to symmetric, proliferative divisions and hence impaired differentiation (Rolls, Albertson et al 2003; Caussinus and Gonzalez 2005)l(Lee, Robinson et al 2006) In addition, impaired basal targeting and defective cell fate determination during asymmetric neuroblast division in Drosophila lead to the formation of malignant neoplasm due to excessive numbers of overproliferating mutant progenitor cells (Caussinus and Gonzalez 2005; Bello, Reichert et al 2006; Betschinger, Mechtler et al 2006; Lee, Wilkinson et al 2006) Interestingly, it has been observed that only a small subset of the cells in a human tumor when transplanted into immuno-compromised mice can reinitiate tumor formation Data from these recent studies also demonstrated that these so-called ‘cancer stem-cells’ cells may cause leukemia, and solid tumors of the breast and brain (Lapidot, Sirard et al 1994; Bonnet and Dick 1997; Al-Hajj, Wicha et al 2003; Singh, Clarke et al 2003; Al-Hajj and Clarke 2004) lending further support to the notion that failure of the otherwise tightly regulated self-renewing capacities of either stem cells or progenitor cells can result in neoplasm Though it is true that more advanced tumors usually lack polarity, it is unclear at present whether there is a direct causal link between loss of cell polarity and tumor initiation in humans Nonetheless, data from Drosophila clearly demonstrate that impaired asymmetric cell division, and in turn errors in the process of normal differentiation can be initiating events in the formation of malignant tumors 123 Conclusion and Perspectives Despite tremendous progress in the field, mechanistic understanding of how these regulators of asymmetric cell division exert their function in the self-renewal of neuroblasts remain elusive Results from my Ph.D work on the two novel players in asymmetric cell division of Drosophila neuroblasts certainly reveal new perspectives to our understanding of the underlying mechanisms that controls stem cell self-renewal, progenitor differentiation, and shed light on the link between tumor suppression and asymmetric cell division Chapter outlines a novel zinc-finger protein, Zif, which suppresses excess neuroblast self-renewal primarily through modulating aPKC expression and/or localization When Zif function is compromised, not only is neuroblast polarity disrupted, aPKC transcript and protein levels are also upregulated Since aPKC is a potent positive regulator that enhances neuroblast self-renewal, it is no surprise that zif mutant exhibit neuroblast overgrowth phenotype In fact, recent data implicate the PAR-aPKC complex in human carcinogenesis Gene amplification and elevated constitutive activity of PKC-ι, one of two human aPKC homologs, was detected in ovarian, lung and colon cancer, suggesting the PKC-ι may be an oncogene (Murray, Jamieson et al 2004; Eder, Sui et al 2005; Regala, Weems et al 2005; Regala, Weems et al 2005) Not unexpectedly, tumors with elevated levels of aPKC had lost epithelial polarity, which is consistent with the overexpression phenotype of a constitutively active form of aPKC in Drosophila epithelia (Eder, Sui et al 2005; Lee, Robinson et al 2006) Using ChIP and Luciferase assays, I also showed that Zif is able to negatively regulate aPKC expression through direct transcriptional repression To the best of our knowledge, this is the first time that a transcription factor is reported to inhibit self-renewing capacity of neuroblasts in the Drosophila larval brain by direct transcriptional regulation of aPKC 124 Conclusion and Perspectives levels It has been shown previously that neuroblasts express a temporal series of transcription factors during embryonic and larval stages (Isshiki, Pearson et al 2001; Maurange, Cheng et al 2008) During pupal stages neuroblasts shrink and eventually undergo symmetric division leading to terminal differentiation However, when certain larval transcription factors of the neuroblast clock are missing (e.g seven up), this does not happen, and neuroblasts continue to proliferate into adulthood It could be that defects in asymmetric cell division perturb the neuroblast clock, causing stem cells to become tumorous It will be interesting to find out if Zif or its targets, possibly genes that are involved in both asymmetric cell division and stem cell selfrenewal, are regulated in a temporal manner Given the importance of aPKC as a central component of the polarity machinery and its vital role as a positive regulator of neuroblast self-renewal, it is with pleasant surprise that I further discovered that aPKC is able to phosphorylate Zif directly More intriguingly, this phosphorylation of Zif by aPKC is able to inhibit nuclear localization of Zif As a transcriptional repressor, exclusion from the nucleus would effectively inactivate Zif, making it non-functional in the modulation of aPKC expression, and hence its role in the regulation of neuroblast self-renewal Hence, the proposed reciprocal interaction between aPKC and Zif in the regulation of neuroblast asymmetric division and neuroblast self-renewal adds a new mechanism to the complex framework of regulatory pathways that orchestrates robust aPKC polarity for its normal function It will be interesting to investigate whether this reciprocal interaction between aPKC and Zif is conserved in other systems (e.g epithelial cells, ovarian follicle cells and photoreceptor cells) where aPKC also plays a pivotal role in establishing polarity Taking a more global view, a genome wide microarray analysis combined with a biochemical search for common binding 125 Conclusion and Perspectives partners of Zif and aPKC may shed light on the regulatory mechanisms that modulate this reciprocal interaction Chapter elaborates on a novel role of the serine/threoninephosphatase PP2A in the control of neuroblast self-renewal Though mammalian PP2A has been shown to participate in malignant transformation by regulating multiple pathways (Li, Scuderi et al 2002; Westermarck and Hahn 2008) as a tumor suppressor, this is the first time that PP2A is demonstrated to be involved in the link between asymmetric cell division and the regulation of neural stem cell homeostasis To limit neuroblast self-renewal, PP2A is not just involved in the regulation of asymmetric protein localization but also plays a role in mitotic spindle orientation – both are key aspects of proper asymmetric cell division (outlined in Chapter Introduction) We also discovered that when PP2A function is compromised, aPKC protein levels increase and Numb hyperphosphorylation is dramatically reduced These PP2A-dependent defects in asymmetric division of mitotic neuroblast resemble that of polo loss-of-function mutants Polo is a kinase that phosphorylates and thereby activates the cell fate determinant Numb Prompted to investigate the possible link between Polo and PP2A, we first discover that in polo loss-of-function mutant, hyperphosphorylation of Numb is also dramatically reduced Consistent with our prediction, we subsequently discovered that indeed, in the absence of PP2A, both Polo protein and transcript levels are dramatically reduced These results suggest that PP2A regulates Numb activity by promoting Polo expression Based on these and previous findings, we proposed a novel pathway in which PP2A acts upstream of Polo and Numb to block excessive neuroblast self-renewal It will be interesting to identify potential substrates of PP2A (possibly novel transcription factors) that are able to promote polo expression and control 126 Conclusion and Perspectives neural stem cell self-renewal Given the structural diversity of PP2A, and its involvement in a myriad of cellular and developmental processes, it is of utmost interest to find out whether this function of PP2A in neural stem cell self-renewal is conserved in other stem cell systems 127 References 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