利用模式动物果蝇 研究神经发育和神经疾病

1. 利用模式动物果蝇研究神经发育和神经疾病 概述: 1，果蝇在生命科学研究中的历史贡献 2，来源于果蝇研究中的重大技术突破 3，果蝇在现代医学研究中的应用 专题: 以果蝇为模型研究重大神经疾病的分子遗传机制

2. Why work with flies? • Short life cycle (~10 days) • Highly prolific (>1000 offsprings / female) • Economical • Harmless to humans and environment • Century long accumulation of genetic tools Practically, flies are an excellent model system

4. Drosophila has a minimum set of chromosomes

5. 成虫 4d 胚胎 1d 蛹 10hrs 1.5d 1d 2d 幼虫 ~10 days at 250C

6. Most Commonly Used Mutants wildtype, WT white yellow Curly Bar ebony

7. Basic Genetics Concepts Mutants--lots and lots of various mutants Mutations--loss-of-function and gain-of-function mutations Mutagen--chemical, physical and biological mutagens Mutagenesis--generation of various mutants Mosaics--mutant tissues or cells in otherwise wildtype individual Epistasis--gene interaction in terms of phenotypes Genotype versus phenotype Forward genetics versus Reverse Genetics

8. Genetics Versus Biochemistry Biochemistry: the scientific study of the chemistry of living cells, tissues, organs and organisms; in vitro structural and functional study of purified living components Genetics: the study of the patterns of inheritance of specific traits; understanding the in vivo functions of a gene through mutants Interdisciplinary approach to study biological processes Protein: 蛋白、蛋白质 Mutation/mutants: 突变、突变体

9. The Nobel Prize in Physiology or Medicine 1933 For his discoveries concerning the role played by the chromosome in heredity Morgan, T.H. Science 32: 120-122 (1910) Sex limited inheritance in Drosophila From Columbia University to California Institute of Technology

10. Morgan and His Three Extremely Gifted Students Alfred H. Sturtevant Calvin B. Bridges Hermann J. Muller

11. A. H. Sturtevant

12. The Nobel prize in Physiology or Medicine, 1946 For the discover of the production of mutations by means of X-ray irradiation Hermann Joseph Muller, 1890-1967 Indiana University, Bloomington, IN, USA Before his discovery, mutants were largely from spontaneous mutations!! It revolutionized animal and plant breeding, offered novel insights into irradiation damage and cancer biology

13. Ed B. Lewis, 1918-2004 Nobel Prize Winner in Physiology or Medicine, 1995 Lewis, E.B. (1978) A Gene Complex Controlling Segmentation in Drosophila. Nature 276, 565-570

15. Homeotic gene expression--colinearity All transcription factors

16. The Nobel Prize in Physiology or Medicine, 1995 For their discoveries concerning the genetic control of early embryonic development Edward B. Lewis Christiane Nusslein-Volhard Eric F. Wieschaus 77 52 48 Nuesslein-Volhard, C., Wieschaus, E. (1980). Mutations Affecting Segment Number and Polarity in Drosophila. Nature 287, 795-801 Some of these genes may be responsible for spontaneous abortions and congenital malformations

17. Walter Gehring, University of Basel, Switzerland Those who succeed… …are the most hard-working and persistent. EMBL, Heidelberg

18. A Turning Point in the Historyof Developmental Genetics The October 30, 1980 cover “Mutations Affecting Segment Number and Polarity in Drosophila”

19. Classical F3 Screen for Recessive Mutations EMS propagation Self-cross Balanced homozygotes: Dia as larvae Balanced mutant stock: viable Homozygous for mutations: Screen embryos for phenotypes Tedious and time-consuming: single crosses, three generations

20. Mutations and Genes Identified from the Screen WT wingless staufen

21. 150 development-regulating genes that affect gross morphology in Drosophila stimulated the search for development genes in other systems (nematode and mouse) virtually all the genes involved in development of Drosophila are represented also in vertebrates, conserved over 700 million years of evolution

22. The Uniqueness of the Nobel Prize Screen 1, Screen for phenotypes in embryo instead of adult (1965-1980) 2, the first screen to find mutations affecting a given process Developmental features crucial for the success of the screen Larval cuticle: an excellent readout of embryo patterning Embryogenesis is very fast, housekeeping proteins are provided from the egg. Only the patterning genes are specifically identified.

23. The Nobel Prize in Physiology or Medicine, 1995 For their discoveries concerning the genetic control of early embryonic development Edward B. Lewis Christiane Nusslein-Volhard Eric F. Wieschaus Determined to understand a biological process and be persistent! We were young and foolish, and it was worth trying

24. Thomas Hunt Morgan Medal Genetics Society of America Medal George W. Beadle Medal 1983 1995 7 Nobel Prize Winners 1958 2003 1995 2003 2003

25. Thomas Hunt Morgan Medal Genetics Society of America Medal George W. Beadle Medal 12 recipients

26. Michael Ashburner Gene regulation: chromosomal puffs Books: most popular books Genome: successful fly genome projects Database: most comprehensive flybase Department of Genetics Cambridge University

27. David S. Hogness: The 2003 Thomas Hunt Morgan Medal Department of Biochemistry, Stanford University David Hogness and son, Peter

28. David S. Hogness: The 2003 Thomas Hunt Morgan Medal Department of Biochemistry, Stanford University application of mol. biol. to fly genetics 1, produced first genomic library of any organism 2, Established the method of “positional cloning” or “map-based cloning” 3, Establsihed “colony hybridization”, 1975 4, First person to practice “genomics” and “functional genomics” 5, Discovered “Goldberg-Hogness box”, now known as the TATA box, 6, Trained many well-known scientists, eg., Gerry Rubin, Richard Mann

29. Two Most Active Players in the Fly Community in the Last Twenty Years Gerry M. Rubin UC Berkeley Vice President of the HHMI Allan C. Spradling, Director of the Department of Embryology of the Carnegie Institute Rubin, G.M., Spradling, A.C. Genetic transformation of Drosophila with transposable element vectors. Science, 1982 218(4570):348—353 Prominent leaders in the Drosophila Genome Project Gerry: the Berkeley Drosophila Genome Project, sequencing of genomic clones, EST clones and mapping of P element insertions. The genome sequence of Drosophila melanogaster. Science2000 287(5461):2185--2195 Allan: Genetic screens to mutate the genome by P elements, aiming at having a P insertion in each of the 15,000 genes. The BDGP gene disruption project: single transposon insertions associated with 40% of Drosophila genes.

30. 模式动物果蝇在生命科学和医学研究中的应用 1，果蝇在生命科学研究中的历史贡献 2，来源于果蝇研究中的重大技术突破 (P Insertion; UAS-GAL4; FRT-FLP) 3，果蝇在现代医学研究中的应用

31. Landmarks in Fly Genetics • Elucidation of early development by systematic genetic screens, 1980; Nobel Prize in 1995 • Germline transformation of fruit flies, 1983 • Completion of Genome Sequencing in 2000, fly as a model for human diseases • Mutagenesis by P element insertion, each gene has a insertion • UAS-GAL4 system to manipulate gene expression • FRT-FLP system to make mosaic clones

35. Gene Expression Manipulation UAS-GAL4 System Derived From Yeast Brand, A.H., Perrimon, N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. DEVELOPMENT 1993 118(2):401--415 Rorth, P. A modular misexpression screen in Drosophila detecting tissue-specific phenotypes. PNAS 1996 93(22):12418--12422 Rorth, P., Szabo, K., Bailey, A., Laverty, T., Rehm, J., Rubin, G., Weigmann, K., Milan, M., Benes, V., Ansorge, W., Cohen, S. Systematic gain-of-function genetics in Drosophila. DEVELOPMENT 1998 125(6):1049--1057

36. GAL4–UAS System for Directed Gene Expression GAL4: a transcription factor UAS: upstream activating sequence

37. Chromosomal Recomb. —Mosaic Analyses FRT: a specific DNA sequence FRT-FLP System FLP: flippase or FLP recombinase 1, Golic KG, Lindquist S. The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome. CELL1989 59:499--509 2, Golic, K.G. Site-specific recombination between homologous chromosomes in Drosophila. SCIENCE 1991 252:958--961 3, Xu, T., Rubin, G.M. Analysis of genetic mosaics in developing and adult Drosophila tissues. DEVELOPMENT 1993 117(4):1223--1237 4, Lee, T., Luo, L.Mosaic analysis with a repressible neurotechnique cell marker for studies of gene function in neuronal morphogenesis. NEURON 1999 22(3):451--461

38. Mosaic Analyses—Powerful Genetic Toolconditional knockout Daughter cells DNA duplication No recombination 偷梁换柱 Recombination induced by yeast derived Flipase and FRT sequences

39. Advanced Mosaic Analysis--MARCM Lee T & Luo L, Neuron, 1999 Mutant, no label classical WT, labeled Mutant, labeled MARCM WT, no label

40. Single Brain Neuron When FMRP Expression is Altered WT Mutant Overexpression

41. 模式动物果蝇在生命科学和医学研究中的应用 1，果蝇在生命科学研究中的历史贡献 2，果蝇研究中的重大技术突破 3，果蝇在现代医学研究中的应用

42. 果蝇在医学研究中的应用 1, Infection and immunology 2, Cancer biology and signal transduction 3, Neurobiology: ion channels, circadian rhythm, learning and memory 4, Neurological diseases: neurodegenerative disease and mental retardation

43. A Good Model to Study Cancer Biology

44. A Good Model to Study Cancer Biology

45. Fly Models of Neurodegenerative Diseases

46. Drosophila Eye is an Ideal System to Study Neurodegeneration

47. 小结

48. Fly Community as a Close Family • You have to come to afly lab to learn fly genetics. We have a well kept fly researcher phylogeny • The best database for a model organism: http://flybase.bio.indiana.edu • A well maintained stock center and genomic resources center • Easy and willing to share reagents privately: stocks, antibodies, constructs etc. • A fly community is established in China!

49. Fly Albums