Asymmetric cell division in the drosophila embryonic neuroblast

THE NOVEL PROTEIN SPINDLE MIRANDA INTERACTS WITH INSCUTEABLE TO REGULATE DROSOPHILA NEUROBLAST ASYMMETRIC CELL DIVISION LEE SIEW CHING JOAN B. APPL SCI. (HONS.), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i ACKNOWLEDGEMENTS I would like to thank my supervisor, A/P Yang Xiaohang; for guiding me throughout the course of my project, present members of the YXH lab; Drs Wang Huashan, Lin Shuping and Yin Yijun; for helping me to troubleshoot problems that I faced, Ms Lee Chai Ling; for assisting my project by making fly food and ordering materials and members of my Thesis Advisory Commitee; A/Ps Tang Bor Luen, Li Baojie and Cai Yu; for providing me useful suggestions during my meet-ups with them. I would also like to extend my appreciation to past and present staff of the NUS Graduate School for Integrative Sciences and Engineering, Prof Ren Ee Chee and Ms Hazlina Umar; for their administrative support during my candidature. I am grateful to my parents and all my friends, for motivating me to persevere in my research. ii LIST OF ABBREVIATIONS ACD AGS A-P APC APC/C aPKC ARM ASIP Aur-A Bam Baz BCIP BMP bp Brat Casp C. elegans cDNA CIP Cnn CNS Dlg D. melanogaster DNA Dpp Dsh DTT E. coli EDTA EGFR Esg F-actin Fmi Fz GDI GFP GBB GMC GSC GST Gαi Gβ13F Gγ1 Insc IPTG JAK-STAT kb Khc-73 Asymmetric Cell Division Activator of G protein signaling Anterior-Posterior Adenomatous polyposis coli Anaphase Promoting Complex/Cyclosome atypical Protein Kinase C Armadillo Atypical PKC isotype-specific Interacting Protein Aurora A kinase Bag of marbles Bazooka 5-bromo-4-chloro-3-indoyl phosphate Bone Morphogenetic Protein base pair Brain tumour Caspase Caenorhabditis elegans complementary DNA Calf Intestinal Phosphatase Centrosomin Central Nervous System Disc large Drosophila melanogaster Deoxyribonucleic acid Decapentaplegic Dishevelled Dithiothreitol Escherichia coli Ethylenediamine tetraacetic acid Epidermal Growth Factor Receptor Escargo Filamentous actin Flamingo Frizzled Guanine nucleotide dissociation inhibitor Green fluorescent protein Glass Bottom Boat Ganglion Mother Cell Germline Stem Cell Glutathione S-transferase α subunit of heterotrimeric G protein β subunit of heterotrimeric G protein γ subunit of heterotrimeric G protein Inscuteable Isopropyl-1-thio-β-D-galactopyranoside Janus Kinase-Signal Transducer Activator of Transcription kilobase Kinesin heavy chain-73 iii LB Lgl Mira Mud Myo II NB Neur NuMA Nbl NBT PAGE PAR PBS PBT PBTW PCM PCR PDZ Pins Pk PMSF PNS Pon Pros Prox1 RGC RNA RNAi Scrib SDS Sna SOP Spim Stbm T4 PNK TEMED TTK69 TUNEL Wor Luria Bertani Lethal giant larvae Miranda Mushroom body defective Non-muscle myosin II Neuroblast Neuralized Nuclear Mitotic Apparatus protein Numb-like 4-nitro-blue-tetrazolium chloride Polyacrylamide gel electrophoresis Partitioning deficient Phosphate Buffered Saline PBS + 0.1% Triton X-100 PBS + 0.1% Tween-20 Pericentriolar Material Polymerase chain reaction PSD-95 Disc large ZO-1 Partner of Inscuteable Prickled Phenylmethylsulphonyl Fluoride Peripheral Nervous System Partner of Numb Prospero Prospero-related homeobox Radial glial cell Ribonucleic Acid RNA interference Scribbled Sodium Dodecyl Sulphate Snail Sensory Organ Precursor Spindle Miranda or CG9646 protein Strabismus T4 Polynucleotide Kinase Tetramethylethylenediamine Tramtrack p69 TdT-mediated dUTP Nick End Labeling Worniu iv LIST OF FIGURES FIGURE 1. ACD IN THE C. ELEGANS ONE-CELL ZYGOTE FIGURE 2. ACD IN THE DROSOPHILA NB 20 FIGURE 3. ACD IN THE DROSOPHILA SOP CELL 24 FIGURE 4. ACD IN THE DROSOPHILA MGSC . 27 FIGURE 5. A MODEL FOR MITOTIC SPINDLE ORIENTATION AND ASYMMETRIC CELL DIVISION DURING MAMMALIAN NEUROGENESIS 31 FIGURE 6. MISLOCALIZATION OF MIRA AND DEFECTIVE SPINDLE ORIENTATION IS OBSERVED FROM RNAI EXPERIMENTS 57 FIGURE 7. GENERATION OF D964667 AND D964680 BY IMPRECISE MOBILIZATION OF PELEMENT INSERTION LINE EY(2)06560. . 58 FIGURE 8. RNA EXPRESSION PATTERNS OF SPIM 59 FIGURE 9. MIRA AND NUMB ARE LOCALIZED NORMALLY IN MITOTIC NBS IN SPIM MUTANT EMBRYOS 60 FIGURE 10. MIRA AND NUMB ARE MISLOCALIZED IN NBS UNDERGOING METAPHASE IN 22 SPIM INSC (NM) EMBRYOS . 62 FIGURE 11. IN SPIM INSC22 (NM) METAPHASE NBS, MIRA IS LOCALIZED AT THE MITOTIC SPINDLE AND RECRUITS PROS TO THE SPINDLE 63 FIGURE 12. SPIM INSC22 (NM) EMBRYOS DO NOT EXHIBIT HIGHER LEVELS OF APOPTOTIC CELL DEATH AS COMPARED TO WILD-TYPE EMBRYOS 65\ FIGURE 13. LGL IS ABSENT IN METAPHASE NBS IN SPIM INSC22 (NM) EMBRYOS. . 67 FIGURE 14. MYOSIN II IS ABSENT IN METAPHASE NBS IN SPIM INSC22 (NM) EMBRYOS 68 FIGURE 15. F-ACTIN IS NOT UNIFORMLY DISTRIBUTED AROUND THE CORTEX OF 22 METAPHASE NBS IN SPIM INSC (NM) EMBRYOS . 70 FIGURE 16. APKC IS ABSENT IN METAPHASE NBS OF SPIM INSC22 (NM) EMBRYOS 71 FIGURE 17. PINS IS ABSENT IN METAPHASE NBS OF SPIM INSC22 (NM) EMBRYOS . 73 FIGURE 18. SUMMARY OF THE GENETIC INTERACTION OF SPIM WITH INSC AND THE CONCOMITANT EFFECT ON ASYMMETRIC PROTEIN LOCALIZATION. . 88 v TABLE OF CONTENTS ACKNOWLEDGEMENTS .ii LIST OF ABBREVIATIONS . iii LIST OF FIGURES . v SUMMARY . x CHAPTER INTRODUCTION ASYMMETRIC CELL DIVISION 1.1 ASYMMETRIC CELL DIVISION IN CAENORHABDITIS ELEGANS 1.1.1 Establishment and Maintenance of the Anterior-Posterior (A-P) axis 1.1.2 Asymmetric spindle positioning 1.1.3 Asymmetrically segregated proteins . 1.2 ASYMMETRIC CELL DIVISION IN DROSOPHILA MELANOGASTER 1.2.1 Drosophila Neuroblasts . 1.2.1.1 Establishment and maintenance of NB apicobasal polarity . 10 1.2.1.2 Coordination of mitotic spindle orientation with cortical polarity 11 1.2.1.3 Spindle asymmetry and differential cell size . 12 1.2.1.4 Centrosome asymmetry . 13 1.2.1.5 Mechanisms of segregating cell fate determinants 13 1.2.1.6 Cell cycle regulators and asymmetric protein localization 16 1.2.1.7 Role of cell fate determinants 18 1.2.1.8 Asymmetric cell division and tumour suppression 19 1.2.2 Drosophila Sensory Organ Precursors (SOP) . 20 1.2.2.1 Establishment of SOP planar polarity and Numb segregation 21 1.2.2.2 1.2.3 Directional signaling and cell fate difference 22 Drosophila Germline Stem Cells 25 vi 1.3 ASYMMETRIC CELL DIVISION IN VERTEBRATES . 28 1.3.1 Neural progenitor cells 28 1.3.1.1 Mitotic spindle orientation and modes of cell division 28 1.3.1.2 Segregating cell fate determinants . 29 1.3.2 Epithelial cells . 31 CHAPTER MATERIALS AND MeTHODS 2.1 MOLECULAR WORK 34 2.1.1 Recombinant DNA methods . 34 2.1.2 Strains and growth conditions . 34 2.1.3 Preparation of competent E. coli cells for heat-shock transformation 35 2.1.4 Cloning strategy and heat-shock transformation . 35 2.1.5 PCR reaction . 36 2.1.6 Plasmid DNA preparation . 37 2.1.7 Protein analysis . 38 2.1.8 Generation of polyclonal antibody 39 2.2 FLY WORK . 40 2.2.1 Embryo fixing . 40 2.2.2 Antibody staining 41 2.2.3 Antibodies . 42 2.2.4 In situ Cell Death Detection 42 2.2.5 Confocal analysis and image processing . 43 2.2.6 Whole embryo RNA in-situ hybridization 43 2.2.7 Double-stranded RNA interference . 45 2.2.8 Single fly genomic DNA extraction 46 2.2.9 Mobilization of EP element . 47 vii 2.2.10 Generation of double mutants of insc22 and CG9646 . 47 2.2.11 Removal of maternal contribution of CG9646 in embryos . 48 2.3 Fly stocks used 49 2.4 Primers used . 50 CHAPTER RESULTS 3.1 BACKGROUND 53 3.2 RESULTS . 55 3.2.1 Defects in Miranda mislocalization and spindle orientation are observed in RNAi experiments in insc mutant background 55 3.2.2 Generation of genetic mutants of CG9646 (Spim) 57 3.2.3 CG9646 RNA is expressed in neuroblasts 58 3.2.4 Mira and Numb are localized normally in spim mutant NBs 59 3.2.5 Mira and Numb are mislocalized in spim insc (nm) mutant NBs . 60 3.2.6 Mira recruits Pros and is associated with the mitotic spindle . 61 3.2.7 Embryos deficient in spim and insc are not undergoing elevated levels of apoptotic cell death 62 3.2.8 Lgl is absent in spim insc (nm) metaphase NBs 64 3.2.9 Non-muscle myosin II is absent in spim insc (nm) metaphase NBs . 66 3.2.10 NBs deficient in spim and insc exhibit aberrant F-actin localization 69 3.2.11 Apical crescents of Pins are absent in NBs deficient in spim and insc . 69 CHAPTER DISCUSSION 4. DISCUSSION . 75 4.1 Spim is regulated by the Sna family transcription factors and may be involved in telophase rescue of cell fate determinants in insc NBs 75 4.2 The Mira mislocalization phenotype in genetic mutants of spim insc differs from that obtained from spim knockdown in insc embryos 77 4.3 Spim is a novel protein and may interact with CG9986 and synapsin . 78 viii 4.4 Spim RNA is expressed in NBs 79 4.5 Maternal dosage of Spim masks the phenotype of spim insc mutants 80 4.6 Basal localization of Mira/Pros in NBs requires genetic interaction between Spim and Insc 81 4.7 Pins expression in NBs by metaphase requires genetic interaction of Spim and Insc 84 4.8 Normal cell cycle progression in mitotic NBs requires genetic interaction between Spim and Insc 85 4.9 Normal embryonic development requires the genetic interaction between Spim and Insc 87 CHAPTER BIBLIOGRAPHY 5. BIBLIOGRAPHY 90 ix SUMMARY Drosophila CG9646 protein, named as Spindle Miranda (Spim), is a novel coiled-coil protein that was isolated from a microarray screen for potential effectors of telophase rescue in insc mutant background. Both Spim and Insc are positively regulated by the Snail family of transcription factors. Single mutants of spim not exhibit any defects in development or asymmetric division. Only spim insc double mutant embryos manifest abnormalities like delayed development and severe mislocalization of cell fate determinants in NBs; Mira/Pros associates with the mitotic spindle. These findings strongly support the existence of a genetic interaction between Spim and Insc in the NB, as Insc is specifically expressed in NBs. The genetic interaction between Spim and Insc is involved in targeting Mira/Pros to the basal cortex in mitotic NBs, possibly by stabilizing the actin cytoskeleton and interacting with Lgl and non-muscle Myosin II. 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Neuron 48: 539-45. 107 [...]... where the actomyosin network is non-contractile Thereafter, the PAR-3–PAR-6– PKC-3-containing domain retracts further, whereas the PAR-2-containing domain expands reciprocally As a result, at the end of the first cell cycle, PAR-3–PAR-6– PKC-3 covers the anterior and PAR-2 covers the posterior half of the embryo (Cuenca et al., 2003) The anterior and posterior cortical domains are maintained as distinct... restricting the localization of germline-specific proteins such as SKN-1, PAL1, MEX-1, POS-1 and the Zn-finger protein PIE-1 to the posterior end of the zygote (Seydoux et al., 1996) In the absence of MEX-5 and MEX-6, germ-line specific proteins, which are normally only inherited by the P1 cell, are expressed in both daughter cells 7 Chapter 1 Introduction Figure 1 ACD in the C elegans one -cell zygote The. .. 1.2.1.3 Spindle asymmetry and differential cell size In NB divisions, the mitotic spindle is initially symmetric, with the metaphase plate placed at the centre of the cell At anaphase, the apical aster enlarges and the basal aster shrinks as spindle microtubules elongate at the apical side and shorten at the basal side, generating an asymmetric spindle This results in a shift of the cleavage plane and the. .. target vesicles to the plasma membrane (Lehman et al., 1999) Therefore, Lgl might similarly promote the delivery of Pon and Mira to the cell membrane in D melanogaster neuroblasts 1.2.1.6 Cell cycle regulators and asymmetric protein localization Asymmetric localization of proteins is linked to cell cycle progression The cell cycle regulator Cdc2 functions in the embryonic CNS by maintaining the correct localization... when implanted in adult hosts (Castellanos et al., 2008) Figure 2 ACD in the Drosophila NB The components of the apical complex interact with each other to establish polarity, position the spindle and localize cell fate determinants to the basal daughter cell The cell- fate determinants and their adaptors are inherited by the basal cell The figure is adapted from Zhong and Chia (2008) 1.2.2 Drosophila Sensory... amount of Numb to inhibit Notch in either daughter cell, can explain the extensive proliferation in polo mutations (Wang et al., 2007) The mitotic regulator anaphase-promoting complex/cyclosome (APC/C) also functions during asymmetric cell division to basally locate the adaptor protein Mira and its cargo proteins, the cell fate determinants Pros and Brat in the mitotic NB Mutations in different APC/C... by removing cortical PAR-2 1.1.2 Asymmetric spindle positioning In the one -cell zygote, the centrally-located mitotic spindle is displaced asymmetrically as it elongates during anaphase, becoming positioned closer to the posterior cortex by the end of mitosis Experiments in which the spindle was severed in live specimens using a laser microbeam revealed that pulling forces act on the two spindle poles,... blocking endocytosis does not prevent Notch signalling in Drosophila (Seugnet et al., 1997), and numb mutants that lack domains that are essential for binding with endocytic proteins, including α-adaptin, can nevertheless specify cell fate in the nervous system (Tang, 2005) Thus, Numb can specify cell fate independently of endocytosis The E3 ubiquitin ligase Neur present in the pIIb daughter cell ubiquitinates... positioned at the interface of the GSC and the hub cells Upon division the mother centrosome is retained in the GSC while the newly formed centrosome is inherited by the gonialblast (Yamashita et al., 2007) The mother centrosome has more PCM than the daughter centrosome In mutants for Centrosomin (Cnn), which is required for microtubule nucleation and centrosome anchoring to astral microtubules, the position...Chapter 1 Introduction Chapter 1 Introduction 1 Chapter 1 1 Introduction ASYMMETRIC CELL DIVISION Asymmetric cell division (ACD) is a highly conserved mechanism to generate cellular diversity during development in multi-cellular organisms and is also an attractive means for stem cells to balance the competing needs of self-renewal and differentiation During ACD, the mother cell divides to generate . ACD IN THE DROSOPHILA NB 20 FIGURE 3. ACD IN THE DROSOPHILA SOP CELL 24 FIGURE 4. ACD IN THE DROSOPHILA MGSC 27 FIGURE 5. A MODEL FOR MITOTIC SPINDLE ORIENTATION AND ASYMMETRIC CELL DIVISION. of cell fate determinants in NBs; Mira/Pros associates with the mitotic spindle. These findings strongly support the existence of a genetic interaction between Spim and Insc in the NB, as Insc. expressed in NBs. The genetic interaction between Spim and Insc is involved in targeting Mira/Pros to the basal cortex in mitotic NBs, possibly by stabilizing the actin cytoskeleton and interacting