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Lecture 3

Lecture 3. Cell Biology of Drosophila Development Drosophila Genetics Screen for Zygotic Lethals Screen for Maternal Effect Mutants. Lab 3 Engrailed staining. Elyse will open the teaching lab at 9:00AM Read instructions in manual and on the board in the lab

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Lecture 3

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  1. Lecture 3 • Cell Biology of Drosophila Development • Drosophila Genetics • Screen for Zygotic Lethals • Screen for Maternal Effect Mutants

  2. Lab 3 Engrailed staining Elyse will open the teaching lab at 9:00AM Read instructions in manual and on the board in the lab If you start at 9:00AM you can be finished by about 4:00PM Get yourselves organized or it will be a very long day.

  3. Next week’s reading DiNardo paper is missing a page and pages 1222 and 1223 are reversed. Can get the missing page on the web site.

  4. Cell Biology of Drosophila Development

  5. Structure of the egg Dorsal appendage Chorion Micropyle Vitelline

  6. Structure of the egg Dorsal appendage D Chorion A P V Micropyle Vitelline

  7. Syncytial blastoderm 2nd cleavage Campos-Ortega and Hartenstein 1985

  8. Syncytial blastoderm Germ-line soma division Pole cells 9th cleavage Campos-Ortega and Hartenstein 1985

  9. Cellular Blastoderm Hartenstein 1993

  10. Blastoderm fate map Hartenstein 1993

  11. Gastrulation 3 hours 2:50 h 3:40 h 4:20 h Hartenstein 1993

  12. Mesoderm and the ventral furrow Hartenstein 1993

  13. Gastrulation and germ band retraction Flybase

  14. Germ band retraction 7:20 h 9:20 h 10:20 h Hartenstein 1993

  15. Development of the Brain Hartenstein 1993

  16. The Drosophila life cycle represents the differentiation of two distinct forms: the larva and the Imago (adult). Embryogenesis: differentiation of the larva Metamorphosis: differentiation of the imago (adult) Imaginal cells are the cells of the adult or imago. Kalthoff 2001

  17. Origin and differentiation of Imaginal cells Embryonic Origin Differentiation The resulting Imago Hartenstein 1993

  18. Oogenesis Kalthoff 2001

  19. GSC niches in the Drosophila ovary and testis Yamashita, Y. M. et al. J Cell Sci 2005;118:665-672

  20. Development and structure of the Cyst Ring canals Kalthoff 2001

  21. Oogenesis Anterior Posterior Kalthoff 2001

  22. Later stages of Oogenesis A P A P A P

  23. Haploid versus Diploid Genetics Haploid genetics Diploid genetics

  24. Genetic organization of Drosophila 1% of genome Y 1st X 2nd 3rd 4th Most information on 1, 2 and 3 chromosomes

  25. Males and females XX female X/A ratio 1 XY or X0 male X/A ratio 0.5

  26. Properties of Balancer Chromosomes • Multiple inversions to suppress the recovery of recombinants. • Recessive visible markers • Dominant visible marker(s) • Second and Third balancers homozygous lethal; first chromosome balancers homozygous female sterile.

  27. Balancers

  28. Balancer chromosomes Balancer homolog Both the balancer and homolog chromosomes segregate as single mendelian units: the chromosome is a stable genetic unit.

  29. Maternal versus Zygotic genomes Sperm Paternal genome Zygote and Zygotic genome Maternal genome Oogenesis

  30. Screens for Pattern Formation Mutants Zygotic lethal screens: screens for activities expressed from the zygotic genome required for correct pattern formation. Maternal effect screens: screens for activities expressed from the maternal genome required for correct pattern formation.

  31. Basic steps of a screen 1 Mutagenesis 2 Separating the mutant chromosomes 3 Amplifying the mutant chromosomes 4 Testing for mutant phenotypes

  32. Zygotic screen F0 EMS DTS91pr cn sca/CyO virgin females X cn bwsp males DTS91: dominant temperature sensitive mutation Dies at 29 C. CyO second chromosome balancer cn bw mutant combination results in white eyes

  33. Zygotic screen F0 EMS Mutagenesis X cn bwsp males Generating the random pool of mutagenized chromosomes

  34. Zygotic screen F0-F1 EMS F0 DTS91pr cn sca/CyO virgin females X cn bwsp males cn bwsp*/CyO F1 DTS91pr cn sca/CyO virgin females X or cn bwsp*/DTS91pr cn sca Individual males Set up 10,000 crosses * mutagenized chromosome

  35. Zygotic screen F0-F1 cn bwsp*/CyO F1 separating the mutant chromosomes or cn bwsp*/DTS91pr cn sca Individual males Set up 10,000 crosses

  36. Zygotic screen F1-F2 10,000 crosses cn bwsp*/CyO F1 DTS91pr cn sca/CyO virgin females X or cn bwsp*/DTS91pr cn sca Individual males 29 C for 4 days cn bwsp*/CyO 5,764 F2 Males and females * mutagenized chromosome

  37. Zygotic screen F1-F2 cn bwsp*/CyO F1 DTS91pr cn sca/CyO virgin females X CyO/CyO DTS91pr cn sca/CyO DTS91pr cn sca/ cn bwsp* F2 cn bwsp*/CyO

  38. Zygotic screen F1-F2 cn bwsp*/CyO F1 DTS91pr cn sca/CyO virgin females X CyO/CyO DTS91pr cn sca/CyO DTS91pr cn sca/ cn bwsp* Dead Dead Dead F2 cn bwsp*/CyO Alive

  39. Zygotic screen F1-F2 cn bwsp*/CyO F1 DTS91pr cn sca/CyO virgin females X or cn bwsp*/DTS91pr cn sca F1-F2 amplifying the mutant chromosomes 29 C for 4 days cn bwsp*/CyO F2 Males and females

  40. Zygotic screen F2-F3 cn bwsp*/CyO 5,764 F2 Males and females Screen for vials that have no white eyed flies. F3 4,217 * mutagenized chromosome

  41. Zygotic screen F2-F3 cn bwsp*/CyO 5,764 F2 Males and females F3 started the testing Screen for vials that have no white eyed flies. F3 4,217 * mutagenized chromosome

  42. F3 tests The 4,217 F3 flies were allowed to lay eggs CyO/CyO larvae die after hatching Screen for >25% unhatched eggs 2,843 lines had > 25% unhatched eggs Test for cuticle defects 272 lines had distinct cuticular changes

  43. Complementation analysis The 272 mutants identified 61 complementation groups (genes).

  44. Maternal effect screens Zygotic screen F0 F1 F2 F3 Dead embryo Maternal screen Mother Egg F0 F1 F2 F3 F4 Maternal genome

  45. Maternal effect screen Trudi Schupbach screen on the second chromosome. Beautiful design: avoids having to sort the flies and collect virgins once the mutagenized chromosomes have been separated.

  46. Maternal effect screen F0 EMS cn bw sp/ CyO DTS.513 virgins X cn bw males

  47. Maternal effect screen EMS cn bw sp/ CyO DTS.513 virgins X cn bw males F1 cn bw*/ CyO DTS.513 virgins Collect individual virgin female flies

  48. Maternal effect screen EMS cn bw sp/ CyO DTS.513 virgins X cn bw males F1 cn bw*/ CyO DTS.513 virgins X Fs(2) D/ CyO DTS.513 males Dominant female sterile

  49. Maternal effect screen F1 cn bw*/ CyO DTS.513 virgins X Fs(2) D/ CyO DTS.513 males F2 genotypes females males cn bw*/ CyO DTS.513 cn bw*/ Fs(2) D Fs(2) D/CyO DTS.513

  50. Maternal effect screen F1 cn bw*/ CyO DTS.513 virgins X Fs(2) D/ CyO DTS.513 males F2 genotypes females males cn bw*/ CyO DTS.513 fertile cn bw*/ Fs(2) D sterile Fs(2) D/CyO DTS.513 sterile

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