he roles of hedgehog signalling in drosophila postembryonic brain development

  THE ROLES OF HEDGEHOG SIGNALLING IN DROSOPHILA POSTEMBRYONIC BRAIN DEVELOPMENT CHAI PHING CHIAN A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY TEMASEK LIFE SCIENCES LABORATORY NATIONAL UNIVERSITY OF SINGAPORE 2011 Acknowledgement This work was carried out in the laboratory of Prof. William Chia, and Dr. Cai Yu, at the Temasek Life Sciences Laboratory, Singapore. Firstly, I thank Bill for giving me the opportunity to work in his lab, and giving me the freedom to explore and experiment. I will cherish his immense scientific insight, encouragement, as well as his extreme patience with my insufficiencies. I thank him for his guidance and supervision throughout all the years of my PhD. I am equally thankful to Dr. Cai Yu, who is a mentor as well as a good friend. I am indebted to him for his selfless assistance, and patient guidance in times of need, as well as being extremely tolerant of my nonsense at times. I also thank him for sharing the knowledge of stem cell development through numerous discussions, especially in the final year after Bill retired as a scientist. Without their constant supports, this work would not be possible. Several people contributed to the completion of this work. I am very grateful to Gu Yi, an attachment student from Fudan University, for her technical support in the live imaging of neuroblasts. I would also like to thank Wang Liwei, an ex-technician in Dr. Cai Yu’s lab for his assistance in preparing Maxiprep of the constructs, as well as to generate some antibodies needed for the experiments. I am very thankful to the members of my thesis committee, namely Prof. Mohan Balasubramaniam, and Dr. Yang Xiaohang for their constructive critiques of this work during the committee meetings. I also thank all the former members of Bill’s lab who shared their knowledge and reagents with me, in particular Dr. Gregory Somers, Dr. Rita Sousa-Nunes, Dr. Wang Hongyan, Dr. Sergey Prokopenko, Dr Marita Buescher. Special thanks to Simi and Sarada for being cool labmates and great friends through the up and down periods. I am extremely grateful to the Drosophila community for generously sharing the antibodies, fly stocks and protocol, especially to Philip Ingham, Joan Hooper, Yuh-Nung Jan, Tabata Tetsuya, Thomas Kornberg, Matthew Scott, Konrad Basler, Chris Doe, Steve Cohen, Jin Jiang, Isabel Guerrero, Bruno Bello, Alex Gould, Ward Odenwald, Yang Xiaohang, DSHB and Bloomington Stock Center. I’m very thankful to Bill, Cai Yu and Sarada who made critical comments and spent their valuable time to proof-read my manuscripts and thesis. Thanks are due to TLL facilities and staffs for prompt technical supports. I am also grateful to Temasek Life Sciences Laboratory and Singapore Millennium Foundation for their financial support. I owe my deepest gratitude and appreciation to family, especially to my parents for their unfailing support, encouragement and love through 31 years of my life and in the years to come. Many thanks to all my friends, in and out of the lab, for the wonderful moments spent together. I thank all the kind souls, be it acquaintances or strangers whom I have encountered in my life. With their little touches of kindness and compassion, I learn to appreciate life in its fullness. Lastly, I would like to extend my sincere thankfulness to millions or perhaps billions of the forgotten tiny heroes, who suffered and sacrificed their lives in the name of science, an extremely noble feat that neither I nor mankind could ever repay.   i Table of Content ACKNOWLEDGEMENT . I  TABLE OF CONTENT II  OVERALL SUMMARY IV  LIST OF FIGURES . VII  ABBREVIATIONS IX  CHAPTER 1:  INTRODUCTION . 1  1.1.  DROSOPHILA MELANOGASTER AS A MODEL ORGANISM . 1  1.2.  DROSOPHILA LIFE CYCLE . 2  1.2.1.  Drosophila embryogenesis and post-embryonic development . 4  1.3.  NEUROGENESIS IN DROSOPHILA MELANOGASTER 6  1.4.  ASYMMETRIC DIVISION OF THE NB 10  1.4.1.  Establishment of polarity in the NB . 13  1.4.2.  Segregation of cell fate determinants . 15  1.4.3.  Roles of cell cycle regulators . 20  1.4.4.  Protein phosphatases . 24  1.4.5.  Spindle orientation . 25  1.4.6.  Cell size asymmetry 27  1.5.  TEMPORAL REGULATION OF THE NBS 28  1.5.1.  The roles of Grh . 32  1.6.  HEDGEHOG SIGNALLING . 33  1.6.1.  Hh interacting partners 36  1.6.2.  Hh signalling during neurogenesis. . 38  1.7.  PERSPECTIVE 40  CHAPTER 2:  MATERIALS AND METHODS 41  2.1.  MOLECULAR BIOLOGY 41  2.1.1.  Recombinant DNA methods . 41  2.1.2.  Cloning strategies 41  2.1.3.  Strains and growth conditions . 43  2.1.4.  Heat shock transformation of E. coli . 43  2.1.5.  Plasmid DNA preparation . 43  2.1.6.  Genomic DNA Preparation 44  2.1.7.  RNA probe preparation 45  2.1.8.  List of primers used 46  2.2.  IMMUNOHISTOCHEMISTRY AND IMAGING 49  2.2.1.  Frequently used reagents and buffers 49  2.2.2.  Antibodies . 49  2.2.3.  Drosophila strains 50    ii 2.2.4.  Clonal analysis . 50  2.2.5.  Fixing and staining for Drosophila larval brains 53  2.2.6.  Live imaging . 53  2.2.7.  BrdU incorporation 54  2.2.8.  TUNEL assay . 54  2.2.9.  In situ hybridization . 55  2.3.  TRANSFECTION OF S2 CELLS 56  2.4.  CHROMATIN IMMUNOPRECIPITATION (CHIP) . 56  2.4.1.  Quantitative PCR . 56  CHAPTER 3:  THE ROLES OF HH SIGNALLING IN THE POSTEMBRYONIC LARVAL BRAIN . 57  3.1.  3.2.  3.3.  3.4.  3.5.  3.6.  3.7.  3.8.  HEDGEHOG SIGNALLING REGULATES THE PROLIFERATION OF THE POSTEMBRYONIC NB . 57  HH SIGNALLING CONTROLS NB PROLIFERATION BUT NOT NEURONAL DIFFERENTIATION . 65  HH SIGNALLING FUNCTIONS THROUGH THE CANONICAL PATHWAY IN THE NBS . 70  HIGH LEVEL OF HH PATHWAY SIGNALLING IS NECESSARY TO INHIBIT NB PROLIFERATION AND INDUCE CELL CYCLE EXIT . 72  NBS ARE HH SIGNAL RECEIVING CELLS . 76  HH SIGNALLING PROMOTES CELL CYCLE EXIT OF THE POSTEMBRYONIC NBS . 81  AN EARLY TRANSIENT PULSE OF CAS EXPRESSION IS REQUIRED FOR THE LATER HH EXPRESSION IN GMCS 87  CAS IS LIKELY TO INTERACT DIRECTLY WITH HH GENOMIC REGION 92  CHAPTER 4:  HH SIGNALLING AND ASYMMETRIC DIVISION 95  4.1.  4.2.  PROS IS ESSENTIAL FOR HH INDUCED CELL CYCLE EXIT 95  THE ROLES OF PROTEIN PHOSPHATASES IN MODULATING HH SIGNALLING . 101  CHAPTER 5:  DISCUSSION . 106  5.1.  5.2.  5.3.  5.4.  5.5.  HH ACTS AT SHORT RANGE IN THE LARVAL BRAIN . 106  HIGH LEVEL OF HH SIGNALLING IS NECESSARY TO TRIGGER NB CELL CYCLE EXIT 107  TEMPORAL REGULATION OF HH SIGNALLING . 111  DOWNSTREAM TARGETS OF HH 115  HH SIGNALLING PROVIDES A LINK BETWEEN NB ASYMMETRY AND THE TEMPORAL SERIES . 117  REFERENCES . 119    iii Overall summary The development of every multi-cellular organism from a single fertilized embryo is a spectacular process which requires tight spatial-temporal coordination of gene expression not only to enable growth, but at the same time to ensure proper body patterning, differentiation and morphogenesis that give rise to tissues, organs and anatomy within a functionally competent organism. It has been known for decades that all the cells within an organism carry identical DNA information through perpetual rounds of DNA replication and cell division. But the questions being: (i) how is cellular diversity generated? and (ii) how does the intrinsic development program of the organism determine the cell types and the ultimate number of cells needed? The Drosophila central nervous system (CNS) offers an excellent model for experimental analysis of such developmental processes. In the CNS, each neuroblast (NB) lineage is generated from a NB that undergoes multiple rounds of asymmetric cell division to produce two different cell types, namely the self-renewing NB, as well as the ganglion mother cell (GMC) which divides and differentiates into neurons and/or glial cells. However, asymmetric cell division alone is insufficient to generate functionally diverse neuronal progeny. In order to specify differential neuronal identity within a single lineage, the NB undergoes ordered transition of gene expression such that the neurons or glial cells born into each distinct temporally defined window will adopt a different fate. Studies in the past have identified the major components of this temporal cascade, but there are still many questions yet to be answered due to the complexity of this system. The work described in this thesis, uses the Drosophila postembryonic brain to gain some insight into these processes, with the major emphasis being placed on termination of the NB cell cycle at the end   iv of neurogenesis. The results are presented in two chapters, followed by a general discussion. Chapter deals with roles of Hedgehog (Hh) signalling in regulating the proliferation of Drosophila postembryonic NB. I described how aberration of the Hh signalling pathway within the NB can alter the cell fate, and the proliferative capacity of the NB and its progeny. In addition, I found that Hh ligand is expressed in a temporally regulated fashion by the NBs and the new born GMCs. Further analysis using immuno-fluorescence, in situ hybridization and live imaging showed that Hh is a regulator of the temporal series as activation of this signalling pathway can downregulate the last known component of the temporal series, Grainyhead (Grh). Downregulation of Grh in the central brain and thoracic neuroblasts is a natural process required for NB cell cycle exit in the early pupal stage. In addition, Hh functions downstream of Castor (Cas), and its expression is directly regulated by the binding of Cas to its promoter sequences. Chapter shows how the Hh signalling pathway impinges on the asymmetric division apparatus to control cell cycle exit in the NB. Hh signalling pathway interacts genetically with protein phosphatase (PP4), an essential component of the asymmetric division pathway. Modulation of the Hh pathway can abrogate the asymmetric division defects seen in mutants for PP4. Indeed, PP4 had been identified as the phosphatase for Smoothened, reinforcing the view that it can function to finetune the strength of Hh signalling. Chapter summarises these studies and discusses two different aspects of Hh signalling pathway in the development of postembryonic neuroblast: 1) its roles as a   v regulator of the temporal series, and 2) its potential function as the link between the temporal series and the asymmetric division pathway.   vi List of Figures Figure 1.1:  The life cycle of Drosophila melanogaster. . 3  Figure 1.2:  Neuroectoderm specification and NB formation. . 7  Figure 1.3:  Asymmetric division of NBs. . 10  Figure 1.4:  Summary of the key players in NB asymmetric division. 14  Figure 1.5:  Temporal series progression in the Type I NB. 31  Figure 1.6:  Hh signalling pathway in Drosophila. 35  Figure 3.1:   Hedgehog signalling affects the proliferation of NBs. . 61  Figure 3.2:  Hedgehog signalling reduces the proliferation of NBs but does not lead to cell death . 64  Figure 3.3:   Hh signalling pathway is required to control NB proliferation but not neuronal differentiation. . 68  Figure 3.4:  All cells in smoIA3 mutant clone express neuronal marker in adult brain. 69  Figure 3.5:  Hh functions through its canonical pathway in the NBs 71  Figure 3.6:   NB proliferation is only affected with the induction of high level of Hh signalling. . 74  Figure 3.7:  hh transcripts are detected in the NBs and GMCs. . 77  Figure 3.8:   Hh expression in the larval brain shows a temporal dependence. 79  Figure 3.9:  hhAC mutant NB over-proliferated to produce a large clone when induced at 24 hr ALH . 81  Figure 3.10:  ptc mutant NBs have reduced cell size. 82  Figure 3.11:  Hh signalling induces cell cycle exit in the NBs via downregulation of Grh . 84  Figure 3.12:  The developmental timing of NB cell cycle exit depends on Hh signalling . 86    vii Figure 3.13:  Hh signalling interacts with NB temporal cascade component. . 88  Figure 3.14:  hh functions downstream of cas. 91  Figure 3.15:  Cas binds physically to the hh genomic region. . 94  Figure 4.1:  Excess Hedgehog signalling only affects the localization of Mira/Pros complex 96  Figure 4.2:  Excessive Pros expression causes mis-regulation of Mira. 98  Figure 4.3:  The phenotype of ptcS2 clones can be suppressed by removing one copy of pros. 100  Figure 4.4:  Hh signalling acts as a functional link between the temporal cascade and the asymmetric division machinery. . 103  Figure 4.5:  Components of Hh signalling show slight genetic interaction with the catalytic subunit of PP2A. 105    viii Abbreviations Hh Hedgehog TGF-β Transforming growth factor-beta RNA Ribonucleic acid UAS Upstream activation sequence NB Neuroblast GMC Ganglion mother cell CNS Central nervous system VNE Ventral neuroectoderm E(SPL)-C enhancer of split complex AS-C achaete-scute complex Vnd Ventral nervous system condensation defective EGFR Epidermal growth factor receptor GC Ganglion cell Insc Inscuteable Par Partition defective Baz Bazooka DaPKC Drosophila atypical protein kinase C Pins Partner of Insc Loco Locomotion defective Pros Prospero Brat Brain tumor Pon Partner of Numb Mira Miranda PP4 Protein phosphatase PP2A Protein phosphatase 2A PTB Phosphotyrosine binding protein   ix Boone, J.Q., and Doe, C.Q. 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Cell 127, 409-422.     136 [...]... essential for determining the localization of the basal components, whereas the Pins-Gαi cassette predominates in controlling the spindle orientation along the apical-basal axis of the NB (Izumi et al., 2004; Wang and Chia, 2005) Although these two signalling cassettes appear to serve distinct roles with respect to the asymmetric segregation of cell fate determinants, they function redundantly in processes... inhibition The NB then enlarges and delaminates basally into the embryo (B) Schematic diagram depicting lateral inhibition involving Notch, Delta and the proneural genes The binding of Delta to Notch represses proneural genes and stabilizes the epidermalizing signal within the signal receiving cell (presumptive epidermal cell) The feedback loop ensures continuous expression of proneural genes within... epidermalizing signal in the receiving cells As such, the prospective NB that has initiated neurogenesis will inhibit the surrounding cells from adopting a neural fate while reinforcing the neural decision within itself Similarly, the surrounding cells that have received the epidermalizing signal through Notch-Delta interaction will stabilize their epidermal decision by suppressing the proneural proteins Indeed,... transition occurs at approximately 48-60 hours after hatching The three larval instars can be distinguished by their spiracles, increasing number of “teeth” of the mouth hooks, and the form of the pharyngeal bars (Ashburner et al., 2005) Over the course of larval development, the larva burrows in the medium and feeds continuously, leading to rapid growth in body size and surface area With a behavioral change... that the cleavage plane is orthogonal to the apical-basal polarity axis Adapted from Chia et al., 2008 In the VNE, NBs inherit the Par proteins from the neuroectodermal epithelial cells and localize them to the apical stalk which is in transient contact with the neuroepithelium during NB delamination Hence the Par protein complex appears to be the first component to be assembled at the apical cortex of. .. and reinforcement of the neural pathway in a single cell with a higher concentration of proneural gene products than other cells within the same proneural cluster As a result, the increasing concentration of proneural proteins triggers the transcription of Delta which encodes the epidermalizing signal molecule   8 CHAPTER 1 Introduction Delta then binds to and activates its receptor Notch on the adjacent... pupation), it leaves the medium and starts to wander on the wall of the culture vial (Godoyherrera et al., 1984) At the end of L3 stage, the body of the larva shortens and the larval skin hardens and darkens to form a puparium Metamorphosis occurs within the puparium and causes the development of imaginal discs into adult organs and appendages within a period of approximately 105 hours As the adult fly emerges... domains 9, B, 1, 5, and 2 of the procephalic ectoderm The mitotic domains are assigned numbers to indicate the temporal sequence of the clusters of cells which undergo locally synchronized mitosis during the 14th mitotic cycle during embryogenesis (Foe, 1989) Unlike the VNE, there are several modes of NB formation from the PNE that are related to their mitotic domain of origin For example, domain B... 1 Introduction The generation of cellular diversity is essential for the development of the CNS during which a single NB generates a vast number of neuronal cell types with distinct functions (Pearson and Doe, 2004) In general, there are two mechanisms deployed during development to generate cellular diversity – intrinsic and extrinsic mechanisms (Hawkins and Garriga, 1998) Extrinsic mechanisms involve... of protein complexes on the opposite poles of the NB cortex Apart from that, the orientation of the mitotic spindle must be positioned such that the plane of division is perpendicular to the polar distribution of cell fate determinants to ensure their differential inheritance into the daughter cells (Bilder, 2001; Broadus and Doe, 1997; Schober et al., 1999; Wodarz et al., 1999) The establishment of . with roles of Hedgehog (Hh) signalling in regulating the proliferation of Drosophila postembryonic NB. I described how aberration of the Hh signalling pathway within the NB can alter the cell.  THE ROLES OF HEDGEHOG SIGNALLING IN DROSOPHILA POSTEMBRYONIC BRAIN DEVELOPMENT CHAI PHING CHIAN A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY. lateral inhibition. The NB then enlarges and delaminates basally into the embryo. (B) Schematic diagram depicting lateral inhibition involving Notch, Delta and the proneural genes. The binding of