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Genetic Mechanisms Behind Cell Specification in the Drosophila CNS

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Linköping University Medical Dissertations No. 1157 Genetic mechanisms behind cell specification in the Drosophila CNS Magnus Baumgardt Department of Clinical and Experimental Medicine Linköping University, Sweden Linköping 2009 During the course of the research underlying this thesis, Magnus Baumgardt was enrolled in Forum Scientium, a multidisciplinary doctoral programme at Linköping University, Sweden. Magnus Baumgardt, 2009 Cover picture/illustration: An Apterous cluster, stained for Apterous (green), Nplp1 (red), and FMRFa (blue). Published articles have been reprinted with the permission of the copyright holder. Printed in Sweden by LiU-Tryck, Linköping, Sweden, 2009 ISBN 978-91-7393-483-1 ISSN 0345-0082 “Science is built up of facts, as a house is built of stones; but an accumulation of facts is no more a science than a heap of stones is a house.” Henri Poincaré, Science and Hypothesis, 1905 CONTENTS LIST OF PAPERS ..................................................................................... 1 ABBREVIATIONS .................................................................................... 3 ABSTRACT .............................................................................................. 5 INTRODUCTION ..................................................................................... 7 The fundamental questions of developmental neurobiology .............. 7 Evolution, structure and function of the CNS ..................................... 9 The cells of the nervous system: neurons and glia ........................................... 9 Nervous system architecture ............................................................................ 10 The evolution of the nervous system ................................................................ 12 The CNS of Drosophila melanogaster .............................................................. 12 The development of the CNS .............................................................14 Axes formation and the specification of the neuroectoderm........................... 15 Patterning of the neuroectoderm ..................................................................... 18 Lineage in the Drosophila CNS ....................................................................... 29 Lineage in the vertebrate CNS......................................................................... 36 The Drosophila temporal gene cascade .......................................................... 40 Temporal specification of vertebrate neural progenitors ............................... 47 Terminal cell fate specification ....................................................................... 50 The Ap neuron model ...................................................................... 58 AIMS ..................................................................................................... 69 MATERIALS AND METHODS ................................................................. 71 RESULTS AND DISCUSSION ................................................................. 73 Paper I............................................................................................. 73 Paper II ............................................................................................ 77 Paper III .......................................................................................... 80 Paper IV .......................................................................................... 85 IDENTIFIED MECHANISMS ................................................................. 89 FUTURE CHALLENGES ........................................................................ 93 ACKNOWLEDGEMENTS ....................................................................... 95 REFERENCES ....................................................................................... 97 List of papers LIST OF PAPERS This thesis is based upon the following papers, which are referred to in the text by their roman numerals: PAPER I Baumgardt M, Miguel-Aliaga I, Karlsson D, Ekman H, and Thor S. (2007). Specification of Neuronal Identities by Feedforward Combinatorial Coding. PLoS Biology 5(2):e37. PAPER II Baumgardt M, Karlsson D, Terriente J, Díaz-Benjumea FJ, and Thor S. Neuronal Subtype Specification within a Lineage by Opposing Temporal Feed-Forward Loops. Cell, in press. PAPER III Karlsson D, Baumgardt M, and Thor S. Segment-specific Neuronal Sub-type Specification by the Integration of Anteroposterior and Temporal Cues. Submitted. PAPER IV Benito-Sipos J, Estacio A, Moris M, Baumgardt M, Thor S, and Díaz-Benjumea FJ. A genetic cascade involving the genes klumfuss, nab and castor specifies the abdominal leucokinergic neurons in the Drosophila CNS. Manuscript. 1 2 Abbreviations ABBREVIATIONS CNS Central nervous system VNC Ventral nerve cord NB Neuroblast GMC Ganglion mother cell A-P Anterior-Posterior D-V Dorsal-Ventral HD Homeodomain LIM-HD LIM-homeodomain bHLH Basic helix-loop-helix ISN Intersegmental nerve SN Segmental nerve TN Transverse nerve MMC Medial motor column LMC Lateral motor column PGC Pre-ganglionic chain FFL Feed-forward loop 3 4 Abstract ABSTRACT The human central nervous system (CNS) contains a daunting number of cells and tremendous cellular diversity. A fundamental challenge of developmental neurobiology is to address the questions of how so many different types of neurons and glia can be generated at the precise time and place, making precisely the right connections. Resolving this issue involves dissecting the elaborate genetic networks that act within neurons and glia, as well as in the neural progenitor cells that generates them, to specify their identities. My PhD project has involved addressing a number of unresolved issues pertaining to how neural progenitor cells are specified to generate different types of neurons and glial cells in different temporal and spatial domains, and also how these early temporal and spatial cues are integrated to activate late cell fate determinants, which act in post-mitotic neural cells to activate distinct batteries of terminal differentiation genes. Analyzing the development of a specific Drosophila melanogaster (Drosophila) CNS stem cell – the neuroblast 5-6 (NB5-6) – we have identified several novel mechanisms of cell fate specification in the Drosophila CNS. We find that, within this lineage, the differential specification of a group of sequentially generated neurons – the Ap cluster neurons – is critically dependent upon the simultaneous triggering of two opposing feed-forward loops (FFLs) within the neuroblast. The first FFL involves cell fate determinants and progresses within the post-mitotic neurons to establish a highly specific combinatorial code of regulators, which activates a distinct battery of terminal differentiation genes. The second loop, which progresses in the neuroblast, involves temporal and sub-temporal genes that together oppose the progression of the first FFL. This leads to the establishment of an alternative code of regulators in late-born Ap cluster neurons, whereby alternative cell fates are specified. Furthermore, we find that the generation and specification of the Ap cluster neurons is modulated along the neuraxis by two different mechanisms. In abdominal segments, Hox genes of the Bithorax cluster integrates with Pbx/Meis factors to instruct NB5-6 to leave the cell cycle before the Ap cluster neurons are generated. In brain segments, Ap cluster neuron equivalents are generated, but improperly specified due to the absence of the proper Hox and temporal code. Additionally, in thoracic segments we find that the specification of the Ap cluster neurons is critically dependent upon the integration of the Hox, Pbx/Meis, and the temporal genes, in the activation of the critical cell fate determinant FFL. We speculate that the developmental principles of (i) feed-forward combinatorial coding; (ii) simultaneously triggered yet opposing feed-forward loops; and (iii) integration of different Hox, Pbx/Meis, and temporal factors, at different axial levels to control inter-segmental differences in lineage progression and specification; might be used widely throughout the animal kingdom to generate cell type diversity in the CNS. 5 6 Introduction INTRODUCTION The fundamental questions of developmental neurobiology Developmental biology is the study of the processes by which a multicellular organism develops from one cell, into an adult. However, most multicellular organisms continue development, in a sense, even as adults, as old or injured cells constantly need to be replaced by new ones, making also these processes an intricate part of the field of developmental biology. In essence developmental biologists aim to answer the same questions, and solve the same problems, as nature itself had to solve some 2 billion years ago as true multicellularity evolved. This transition posed several challenges for the ancient organisms. The most fundamental of these challenges was how the initial cell mass of the organism - commonly only one cell, a zygote – could propagate in such a way that the cells it generated became differentially specified to perform various specialized functions within the organism, while their proliferation and integration was still controlled in a precise enough manner to allow for a fully functional final structure. In no other area of developmental biology are the questions of how cells are precisely generated, specified, and integrated, as challenging as in the study of the animal central nervous system (CNS).
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