M267 January 2008 Larry Zipursky Lecture 1 Fly Genetics and Signaling During Development

1. M267 January 2008 Larry Zipursky Lecture 1 Fly Genetics and Signaling During Development

2. Principle Signaling Pathways:Ancient pathways regulating development > 650 million years old Receptor tyrosine kinase Receptor serine threonine kinase Wnt (ligands) Hedgehog (ligands) The Notch pathway GPCRs Nuclear hormone receptors

3. Central Issues in Signaling Signals: Short and Long Range 2. Receptor: How does binding lead to intracellular changes? 3. Transmission of signal to the nucleus

4. Intercellular Signaling in Drosophila: The Power of Fly Genetics to Dissect Signaling Pathways 1. Discovery of signaling pathways through genetic screens: Unbiased approaches to identify developmental pathways 2. Identification of signaling and signal receiving cells: Genetic mosaic techniques 3. Ligand and receptor modifications and interactions 4. Dissecting signal transduction assays: Sensitized genetic screens. 5. How signals regulate patterns: Induction, lateral inhibition, and gradients

5. Fly as a Model System Sophisticated classical genetics: many screening strategies, analytical tools. Easy cloning of mutation-defined genes: excellent correlation between physical and genetic maps. Short generation time: ~10 days. Sophisticated expression systems: transgene technologies. Multicellular biology: Development: Embryo plan, Signaling pathways Behavior: membrane excitability, learning/memory. Recently developed gene knock out via homologous recombination. 7. RNAi technology in cells, embryos and transgenic animals 8. Complete genome sequence: • 177/289 human disease genes in the fly. • 68% of cancer genes have fly homologs. • 2758 strict human/fly orthologues 9. Centralized data base: www.flybase.org

6. The Fly Life Cycle

8. Genetics in an “egg shell” 1. Chromosomes: X=1, 2, 3, 4 % genome 20 40 40 a little X 2 3 4 L R L R 2. Genotype: Recessive Lower case italics, typically three letters Allele, superscript e.g., sevenlessLY3 =sevLY3 Dominant First letter uppercase, italics, three letters e.g., Ultrabithorax =Ubx Protein First letter uppercase, no italics e.g. Sevenless =Sev

9. Genetics in an “egg shell” (cont.) Usually 4th is left off unless mutation of interest is on 4th. Genotype of fly: cn, bw sev Ubx e.g., ; ; sev cn, bw ri, e If everything is wild type except for one gene, then only designate mutant chromosome over wild type. Ubx e.g., +

10. 3. Typical cross: cn, bw ri, e sev + + + ; ; X ; ; sev + + cn, bw ri, e cn, bw ri, e cn, bw ri, e sev sev ; ; ; ; + + + + + • 4. Meiotic recombination: only in , no recombination. • Balancer chromosomes suppress meiotic recombination. • Multiple chromosome inversions, dominant marker. • Meiotic recombination results in inviable gametes. • 5. Flybase (www.flybase.com) • Comprehensive data base of Drosophila reagents including gene annotation, mutant strains, cDNAs, expression patterns etc. Ease of correlated physical and genetic maps

11. Screens for Recessive Mutations on Autosomes Mutagenesis Scheme to Identify Genes Affecting Early Embryogenesis EMS En masse X Several per vial X Single fly per vial or CONTINUED…

12. Mate at permissive temperature. Grow at 29° to kill all flies carrying DTS-91. Dump parents. No need to select virgin flies for interse mating, as all are heterozygous for mutant second chromosome. X If vials do not contain flies that are cnbwsp*/cnbwsp*, then lethal mutation(s) have been induced on second chromosome. If vials contain flies that are cnbwsp*/cnbwsp*, then lethal mutation(s) have not been induced. Analyze dead embryos

13. cn = cinnabar eye color mutants. bw = brown eye color mutants. cnbw eyes are white. sp = dark body color. In(2LR)O,Cy = balancer chromosome. Multiple inversions to suppress recombination. Cy indicates “curly;” wings curl upwards rather than being straight. DTS-91 = dominant temperature-sensitive mutation. Homozygotes die at room temperature.

14. How do inversions suppress recombination? A B C D E AA BCDB E C D E A B D C E A B C D E Paracentric inversion A B D C E Homologous recombination A B C D E viable gametes A B C E C D E A B D A B D C E viable gametes

15. How do inversions suppress recombination? (con’t) A B C D E Pericentric inversion A D C B E Homologous recombination only viable gametes A B C D E A D C B E A B C D A E B C D E

16. Three Approaches/Techniques to be Covered 1. Construction of transgenic flies using P elements. Interruption cassette technology: Using the yeast FRT/FLP recombination system in the fly. The Gal4/UAS system: Using the yeast transcription system in the fly.

17. Constructing Transgenic Flies Gene identification: Rescue of mutant phenotypes with genomic or cDNAs. To generate flies in which genes are expressed at the wrong place and/or time during development. 3. To generate markers for studying development and function . 4. To generate genetically mosaic animals with high efficiency for both analysis and screening.

18. P- element mediated DNA Transformation Spradling and Rubin (1982) Science, 218, 341 Rubin and Spradling (1982) Science, 218, 348

19. P- element mediated DNA Transformation (con’t)

20. The FRT/FLP System Golic and Lindquist (1989) Cell 59, 499-509

21. Interruption Cassette Technology Struhl and Basler (1993) Cell 73, 427 Basler and Struhl (1994) Nature 368, 208 Fly carries another transgene with the heat shock promoter fused to a cDNA encoding FLP

23. Ectopic Expression of hedgehog Gene in the Developing Eye Imaginal Disc Proliferation

24. The Gal4/UAS System (Fischer et al, 1988) (Brand and Perrimon, 1993)

25. Gal4/UAS Gene Expression System Chromosomal DNA Enhancer Gal4 hsp70 Gene X UAS TATA Protein X

26. Source of Enhancer - Gal 4 1. Transgene containing characterized enhancer fused to Gal 4 2. Random insertion of Gal 4 using P-element hopping: “Parasitize endogenous enhancers”

27. Source of UAS Target Sequences 1. Transgene containing UAS sequences placed upstream of cDNA 2. Random insertion of UAS using P-element to drive expression of neighboring genes (referred to as EP lines)

28. E132 Enhancer Trap Line Expression Seen Using UAS-LacZ Halder et al. (1995) Science 267 1788. Eye Wing Leg

29. Antenna to Eye Wing to Eye Antenna to Eye Leg to Eye