UNIVERSITY OF FRIBOURG DEPARTMENT OF BIOLOGY NEUROGENETICS, AUTUMN 2014

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1 UNIVERSITY OF FRIBOURG DEPARTMENT OF BIOLOGY NEUROGENETICS, AUTUMN 2014 BEFRI Master Programme, BL.0117, 2 hr/week, 3 ECTS Organizer: Dr Boris Egger (boris.egger@unifr.ch) Lecturers: Prof. Simon Sprecher Prof. Dominique Glauser Dr Soeren Diegelmann Prof. Claire Jacob Prof. Smita Saxena Dr Boris Egger FACULTÉ DES SCIENCES DÉPARTEMENT DU BIOLOGIE INSTITUTE DE ZOOLOGY MATHEMATISCH- NATURWISSENSCHAFTLICHE FAKULTÄT DEPARTEMENT BIOLOGIE

2 1. INTRODUCTION TO NEUROGENETICS 1.1. Aims of this course 1.2. History of neurogenetics 1.3. Definition 1.4. Instrumental vs. analytical neurogenetics 1.5 Neurogenetic model systems Caenorhabditis elegans Drosophila melanogaster Vertebrate models 1. INTRODUCTION TO NEUROGENETICS 1.1. Aims of this course This lecture course provides a framework of neurogenetics, the discipline that seeks to analyze the relations between the genome, neurons, neural networks and behavior. Chapters 1-2 aim to provide an essential overview on the field neurogenetics, distinct animal model systems and the basis of embryonic development in Drosophila. Chapters 3, 4 and 6 give insights in the neural and eye development of Drosophila. Chapter 5 gives an overview about basic genetic and imaging techniques but also highlights the newest developments in this field. Chapter 7 looks at the genetics of learning and memory. Chapter 8 and 9 is dedicated to behaviour in C. elegans. Chapters focus on neural development and neurogenetic disease models in mammalian systems. Finally, chapter 13 describes the Drosophila brain as model for tumourigenesis and metastasis History of neurogenetics (origins at about 1970): - Locomotor mutants of mice (R.L. Sidman & others) - Behavioral mutants of Drosophila melanogaster (Seymour Benzer, California Institute of Technology) - Neurological mutants of Caenorhabditis elegans (Sydney Brenner, MRC Cambridge, Nobel Prize 2002) : Isolation of many neurological and behavioral mutants in Drosophila, C. elegans and in the mouse today: Molecular genetic analysis of many neural genes; detection of homologous neural genes in Drosophila, C. elegans and vertebrates - Today: Whole genome sequences are available for many model organisms, which allow genomic screens for developmental or behavioral mutants 1.3. Definition Neurogenetics deals with the relations between the genome and the nervous system. The term neurogenetics is used in at least three different ways: 2

3 Instrumental ( applied ): Use of neural mutants or genetic techniques as tools for analyzing structural or functional aspects of the nervous system. Examples: application of enhancer trap lines as neuron-type specific markers; genetic surgery provided by a homeotic transformation of appendages. Analytical: Attempts to explain the molecular mechanisms of genes that affect the nervous system and to elucidate their interactions. It tries to understand how far the nervous system is genetically programmed and whether true neural genes exist, i.e., genes whose activity is restricted to the nervous system. Generally speaking, analytical neurogenetics seeks to elucidate the relations between genes, neurons, the nervous system and behavior. Medical: Investigates the genetic background of human neurogenetic disorders (e.g. Lesch-Nyhan syndrome or Alzheimer s Disease) 1.4. Instrumental vs. analytical neurogenetics Example: Homeotic mutants in Drosophila modify the identity of specific body segments, which may lead, for example, to a replacement of antennae by legs. Question: where do the sensory neurons from such a transformed appendage project in the brain: to antennal centers or to leg centers? Homeotic mutants thus allow one to analyze how the axons of sensory neurons find their correct targets in the brain. To analyze the relations between genes and neural phenotypes it is important to know whether true neural genes indeed exist (which are exclusively expressed in the nervous system). It was argued, for example, that neural phenotypes are generated by a hierarchical set of genes that also affect many non-neural tissues (Stent, 1981). Examples: - Homeotic genes are not neural genes per se, yet profoundly affect neuronal connectivity - Abnormal visual pathway in albinotic mammals (see below) Normal visual pathway in mammals: The vertebrate retina is topographically represented in the brain. In mammals, retinal ganglion cells project to the first association area, the lateral geniculate nucleus (LGN) in a topographical manner: The two layers A and A1 receive corresponding projections of the contralateral visual field from both the ipsilateral and the contralateral eye. Secondary fibers extend from the LGN in parallel to the area 17 of the visual cortex. Thus, LGN and area 17 both have a binocular representation of the contralateral visual field ( basis of stereoscopic vision). Abnormal visual pathway in albinotic mammals: In albinos (e.g. Siamese cats) A1 receives additional fibers from the contralateral eye (aba1), but less fibers from the ipsilateral eye. Mirror symmetric positions in A1 and aba1 are in register. In the LGN-cortex projection, the continuity is maintained, i.e., only a small part of area 17 gets binocular information (Shatz & LeVay, 1979). Stereoscopic vision is impaired. 3

4 What is the cause of this neural defect? genetic defect at albino locus (gene coding for dopa-oxidase) block of melanin synthesis (fur pigmentation; eye pigment) pigmented region in eye stalk missing?? impairment of axon growth?? direction of growing axons in optic chiasm modified abnormal visual representation in LGN and cortex impaired stereoscopic vision This example shows that the activity of a non-neural gene can induce a cascade of events that finally leads to specific structural and functional defects in the nervous system. It was used as an argument to suggest that neurogenesis may be due to complex tissue interactions with limited genetic programming (Stent, 1981). This hypothesis is partially supported by recent evidence, showing that many genes fulfill different tasks during different periods of development, within and outside the nervous system. Nevertheless, many neural-specific genes have been described ( chapter 3). In summary, there may be two extremes of genes affecting the nervous system: - Genes with a broad spectum of expression inside and outside the nervous system (often active early in development) - Neural-specific genes (usually active late in development) 1.5. Neurogenetic model systems Only a small number of neurogenetic model systems have emerged, because such systems should meet a variety of criteria: small genome size, simple genetics, powerful genetic and molecular techniques available, simple breeding, short generation time, large number of progeny, small body size, limited number of neurons, cellular identification, interesting behavioral repertoire, relevance for higher nervous systems, relevance for medical neurogenetics Caenorhabditis elegans - Size of haploid genome (entirely cloned and sequenced): kb; genes - 6 chromosomes (5A; 1X). Sex determination: 5AA + XX: hermaphrodite; 5AA + XO: male; mating of hermaphrodite with male allows crossing of mutant strains; self-fertilization of hermaphrodites simplifies generation of homozygous strains - Generation time: only 52 hr (25 C) - Cooling and deep-freezing possible (simplifies stock keeping) 4

5 - Size 1 mm: breeding in high numbers possible - Constant cell numbers (e.g. hermaphrodite: 959 cells; 302 neurons): electron microscopic identification of all synapses (in sections) - Only 150 functionally different cell types (among them 118 types of neurons) - Transparency: Lineage of all cells analyzed in living animals (Nomarski optics) - Laser cell ablation - Many neurological mutants known - Limited behavioral repertoire Drosophila melanogaster - Genome (entirely cloned and sequenced): kb; genes; 3 autosomes, X/Y sex chromosomes - Classical and molecular genetics (reviewed by Rubin, 1988; Greenspan, 1996) - Transposable elements (e.g. P-elements): for enhancer trap technique - Polytene chromosomes: characteristic pattern of 102 major bands allows easy identification of chromosomal deletions and rearrangements, as well as mapping of cloned genes and P element insertions - In males meiotic cross-over is suppressed - Generation time: 10 days (25 C) - Many neurons (or neuroblast clones) identifiable - In terms of complexity half-way between unicellular organisms and humans (Drosophila: neurons, humans: neurons) - Many non-genetic manipulations possible (sensory physiology, pharmacology, developmental physiology, in vitro culture, immunocytochemistry) - Large behavioral repertoire. Many behavioral assays available - More relevant for higher nervous systems than C. elegans Vertebrate models Zebrafish (Danio rerio) - Genome (entirely cloned and sequenced): kb (1.5 Gb) - Subset of large, identifiable neurons (Mauthner neurons) - Major relevance for higher nervous systems with respect to developmental genetics - Excellent model organism for live cell imaging - Good for behavioral studies Chicken (Gallus gallus) - Genome (entirely cloned and sequenced): kb (1.0 Gb) - Amniote and therefore easy to manipulate (electroporation in ovo) - used for studies on axon guidance and neuronal cell type specification 5

6 African clawed frogs (Xenopus laevis) and (Xenopus tropicalis) -Genome of X. tropicalis (entirely cloned and sequenced) kb (1.7 Gb) -Xenopus oocytes are very large cells and easy to culture and to inject -used for first nuclear transfer experiments (cloning) -mostly used for developmental studies Mouse (Mus musculus) - Mouse genome (entirely cloned and sequenced): kb (2.5 Gb) - Major mammalian model systems (e.g. human developmental genetics, neurogenetic disorders) - Advanced molecular and genetic tools are available (ie. homologous recombination, brainbow ) - Many mouse derived neuronal cell culture systems are available 2. GENETICS OF EMBRYOGENESIS IN DROSOPHILA 2.1. Introduction 2.2. Restriction of the potential in developing cells: formation of compartments 2.3. The parasegment as a construction unit 2.4. Commitment of cells to parasegments 2.5. Segment polarity genes 2.6. Selector genes 2. GENETICS OF EMBRYOGENESIS IN DROSOPHILA 2.1. Introduction Apart from C.elegans, the genetic control of development of a multicellular organism is perhaps best understood in Drosophila. This chapter will focus on how the genomic information of the zygote is used to build an entire organism. Early steps of these processes (segmentation and allocation of segmental identity) are treated in the course in developmental biology. Flies, like other insects are divided into morphologically distinct body segments: - 3 preoral (supraoesophageal) head segments - 3 postoral (suboesophageal) head segments - 3 thoracic segments (pro-, meso-, meta-) - 8 abdominal segments 6

7 (Lawrence, 1992) Each of these, except the preoral segments, consists of an anterior ( a ) and a posterior ( p ) compartment. However, as shown later, the basic construction units are parasegments (PS), rather than segments. A PS consists of the p compartment of a particular body segment and the a compartment of the following segment. For example, PS 6 consists of the p compartment of the metathoracic segment and the a compartment of the first abdominal segment. Embryonic pattern formation begins already during oogenesis, by the activation of maternal genes. Their products are deposited in the egg in a precise pattern and establish a system of coordinates, consisting of several independent components: (1) an anteroposterior gradient of the bicoid protein, (2) a posterior system for abdomen formation depending on the nanos gene, (3) a system for defining head and tail, depending on activation of the torso receptor protein at both ends of the embryo, and (4) a dorsoventral system that is based on activation of a receptor protein Toll along the ventral midline. These four maternal systems are then interpreted by a first group of zygotic genes, the gap genes (e.g. hunchback, Krüppel, knirps). Each of the four maternal gradients is read out as several narrower bands of zygotic gene products with initially overlapping boundaries. The different gap gene bands apparently control a next set of pair rule genes (e.g. fushi tarazu (ftz: not enough segments ), even-skipped (eve)), which are expressed in every second segment. 7

8 2.3. The parasegment (PS) as a construction unit There is good evidence that PS rather than segments are the construction units of the insect ( 3.1.). Arguments: - Parasegmental grooves are the first signs of metamerization (=segmentation) - The metameric chain starts in the presumptive head region with a p compartment and ends in the abdomen with an a compartment - The first commitment of embryonic cells is toward PS, which become later on divided into compartments - Selector genes are expressed in a PS pattern (bithorax & Antennapedia complex) 2.4. Commitment of cells to parasegments The expression pattern of the pair rule genes ftz and eve strongly suggest that they are involved in the commitment of cells to PS. Initially the ftz and eve expression stripes are broad and overlapping. However, when they reach their highest concentration, the anterior expression borders become sharp which activates the engrailed (en) gene in all cells of future p compartments. This process seems to establish PS borders, and thus to commit cells to PS. In contrast to ftz and eve, en is the first gene that remains permanently expressed until adulthood. Maturation of ftz (red) and eve (grey) stripes and allocation of compartments. Parasegment boundaries are delineated by anterior boundaries of stripes; later, the anteriormost cells begin to express engrailed (black dots) (Lawrence, 1992). 8

9 2.5. Segment polarity genes Segment polarity mutants (wingless, armadillo, hedgehog) change the pattern and polarity of every segment, as visualized by the pattern of denticles in the ventral abdomen of the larva. In the wildtype, denticles are polarized and are restricted almost entirely to a compartments, whereas in wingless and related mutants the whole ventral abdomen is covered by denticles that show weak polarity. wingless expression depends on en activity, and vice versa. Understanding the function of segment polarity genes is difficult, because their products are very diverse (cell junction proteins, extracellular proteins, transmembrane proteins). Basically, they could be involved in shaping the segmental pattern, such as: - Maturation of en expression - Establishment of PS borders as a long-lived feature - Establishment of a segmental gradient of positional information and polarity 2.6. Selector genes The role of homeotic selector genes (bithorax complex: BX-C; Antennapedia complex: ANT-C) is also treated here only marginally ( courses in Developmental & Molecular Biology ). In short, once a compartment has been founded by a subset of cells, the segmental identity of the compartment has to be established. This is accomplished by homeotic selector genes, which assure that cells in a specific compartment become different from cells in a homologous compartment of another segment. Loss or overexpression of homeotic genes in particular segments leads to homeosis, the transformation of segments or appendages by homologous structures of other segments. Two spectacular regulatory mutations of the Ultrabithorax (Ubx) gene illustrate the role of selector genes. bithorax (bx) and postbithorax (pbx) transform the a and p compartments of the metathoracic segment, respectively, into homologous mesothoracic compartments, leading to a transformation of halteres into second wings. Interestingly, SCO-induced Ubx clones in the haltere show that (1) Ubx is cell autonomous, and suggest that (2) there is a common ground plan in wings and halteres, e.g., by a system of coordinates. Positions would be read in the same way in both appendages, but the interpretation depends on Ubx. Similarly as en, Ubx seems also to influence cell affinities. 9

10 (Lawrence, 1992) The three main players in the commitment of thoracic and abdominal segments are the genes Ubx, abdominal-a (abd-a) and Abdominal-B (Abd-B). They all encode homeobox proteins that bind DNA. Their phenotypes suggest several important points: - The 3 genes work in particular domains: Ubx from PS 5 backwards, abd-a from PS 7 back, Abd-B from PS 10 back - Genes of the BX-C are expressed in PS, not segments - Combinatorial effects between Ubx, abd-a & Abd-B - In the absence of all 3 gene products, mesothoracic segments (corresponding to PS 4) are formed all over, apparently a genetic ground state of a segment The coincidence of expression boundaries with PS borders may be due to ftz and eve expression. However, several issues complicate this simple model, such as unequal expression of the genes in their domains, unstable expression borders, complex spatial regulation of BX-C genes, complex splicing products, phenotypic suppression of gene products expressed in anterior parts by posterior gene products. Also, it is not yet understood how a single protein can produce a change from one structure to another. Downstream regulatory genes of the BX-C that are involved in tissue specification are to be expected. Possible candidates are proneural genes of the achaete-scute complex. Finally, it is not known how the specific expression of BX-C genes along the a/p axis is regulated. 10

11 LITERATURE Ashburner M (1989) Drosophila. A laboratory handbook & manual. CSH Lab. Pr., Cold Spring Harbor NY Ashburner M, Novitski E & Wright TRF, eds. ( ) The genetics and biology of Drosophila. Several Vols. Acad. Pr., New York Bate M & Martinez Arias A (1993) The development of Drosophila melanogaster pp, CSH Lab. Pr., Cold Spring Harbor NY Blair SS (2003) Genetic techniques for studying Drosophila development. Development 130: Bryant P (1993) The polar coordinate model goes molecular. Science 259: Campos-Ortega JA & Hartenstein V (1985) The embryonic development of Drosophila melanogaster. Springer, Heidelberg French V, Bryant PJ & Bryant SV (1976) Pattern regulation in epimorphic fields. Science 193: Greenspan RJ (1996) Fly Pushing: The theory and practice of Drosophila genetics. CSH Lab. Pr., Cold Spring Harbor NY Hartenstein V (1993) Atlas of Drosophila development. CSH Lab. Pr., Cold Spring Harbor NY Lawrence PA (1992) The making of a fly. Blackwell Scientific Publ., Oxford Löhr R, Godenschwege T, Buchner E & Prokop A (2002) Compartmentalization of central neurons in Drosophila: a new strategy of mosaic analysis reveals localization of presynaptic sites to specific segments of neurites. J Neurosci 22: Meinhardt H (1983) Cell determination boundaries as organizing regions for secondary embryonic fields. Dev Biol 96: Nellen D, Burke R, Struhl G & Basler K (1996) Direct and long-range action of a DPP morphogen gradient. Cell 85: Postlethwait JH & Schneiderman HA (1971) Pattern formation and determination in the antenna of the homoeotic mutant Antennapedia of Drosophila melanogaster. Dev Biol 25: Ransom R, ed (1982) A handbook of Drosophila development. Elsevier Biomedical Pr., Amsterdam Roberts DB, ed (1986) Drosophila. A practical approach. IRL Pr., Oxford Seyffert W et al, eds (1998) Lehrbuch der Genetik. Gustav Fischer, Stuttgart Shatz CJ & LeVay S (1979) Siamese cat: altered connections of visual cortex. Science 204: Stent GS (1981) Strength and weakness of the genetic approach to the development of the nervous system. Annu Rev Neurosci 4: Technau GM (1986) Lineage analysis of transplanted individual cells in embryos of Drosophila melanogaster. I. The method. Roux s Arch Dev Biol 195: Wolpert L (2007) Principles of Development. Oxford University Press 11