Presentation on theme: "Lecture 3 Cell Biology of Drosophila Development Drosophila Genetics"— Presentation transcript:
1 Lecture 3 Cell Biology of Drosophila Development Drosophila Genetics Screen for Zygotic LethalsScreen for Maternal Effect Mutants
2 Lab 3 Engrailed staining Elyse will open the teaching lab at 9:00AMRead instructions in manual and on the board in the labIf you start at 9:00AM you can be finished by about 4:00PMGet yourselves organized or it will be a very long day.
3 Next week’s readingDiNardo paper is missing a page and pages 1222 and 1223 are reversed. Can get the missing page on the web site.
16 The Drosophila life cycle represents the differentiation of two distinct forms: the larva and the Imago (adult).Embryogenesis: differentiationof the larvaMetamorphosis:differentiation ofthe imago (adult)Imaginal cells are the cellsof the adult or imago.Kalthoff 2001
17 Origin and differentiation of Imaginal cells Embryonic OriginDifferentiationThe resulting ImagoHartenstein 1993
24 Genetic organization of Drosophila 1% of genomeY1stX2nd3rd4thMost information on 1, 2 and 3 chromosomes
25 Males and femalesXX femaleX/A ratio 1XY or X0 maleX/A ratio 0.5
26 Properties of Balancer Chromosomes Multiple inversions to suppress the recovery of recombinants.Recessive visible markersDominant visible marker(s)Second and Third balancers homozygous lethal; first chromosome balancers homozygous female sterile.
28 Balancer chromosomesBalancerhomologBoth the balancer and homolog chromosomes segregateas single mendelian units: the chromosome is a stable geneticunit.
29 Maternal versus Zygotic genomes SpermPaternal genomeZygote and ZygoticgenomeMaternal genomeOogenesis
30 Screens for Pattern Formation Mutants Zygotic lethal screens: screens for activities expressedfrom the zygotic genome required for correctpattern formation.Maternal effect screens: screens for activitiesexpressed from the maternal genome required forcorrect pattern formation.
31 Basic steps of a screen 1 Mutagenesis 2 Separating the mutant chromosomes3 Amplifying the mutant chromosomes4 Testing for mutant phenotypes
32 Zygotic screenF0EMSDTS91 pr cn sca/CyO virgin females X cn bw sp malesDTS91: dominant temperature sensitive mutationDies at 29 C.CyO second chromosome balancercn bw mutant combination results in white eyes
33 Mutagenesis Zygotic screen F0 EMS X cn bw sp males Generating the random pool of mutagenized chromosomes
38 Dead Dead Dead Alive Zygotic screen F1-F2 CyO/CyO cn bw sp*/CyO F1 DTS91 pr cn sca/CyO virgin females XCyO/CyODTS91 pr cn sca/CyODTS91 pr cn sca/ cn bw sp*DeadDeadDeadF2cn bw sp*/CyOAlive
39 Zygotic screen F1-F2 F1-F2 amplifying the mutant chromosomes cn bw sp*/CyOF1DTS91 pr cn sca/CyO virgin females Xorcn bw sp*/DTS91 pr cn scaF1-F2 amplifying themutant chromosomes29 C for 4 dayscn bw sp*/CyOF2Males and females
40 Zygotic screen F2-F3 cn bw sp*/CyO 5,764 F2 Males and females Screen for vials thathave no white eyedflies.F34,217* mutagenized chromosome
41 Zygotic screen F2-F3 F3 started the testing cn bw sp*/CyO 5,764 F2 Males and femalesF3 started the testingScreen for vials thathave no white eyedflies.F34,217* mutagenized chromosome
42 F3 tests The 4,217 F3 flies were allowed to lay eggs CyO/CyO larvae die after hatchingScreen for >25% unhatched eggs2,843 lines had > 25% unhatched eggsTest for cuticle defects272 lines had distinct cuticular changes
43 Complementation analysis The 272 mutants identified 61 complementationgroups (genes).
54 Maternal effect screen 29 C lay eggs and developF3genotypes phenotypecn bw*/ CyO DTS.513 deadFs(2) D/ CyO DTS.513 deadcn bw*/ Fs(2) D viablecn bw*/ cn bw* dead/viable?
55 Maternal effect screen 29 C lay eggs and developF3genotypes phenotypecn bw*/ Fs(2) D viablecn bw*/ cn bw* dead/viable?Of 10,842 established F3 lines5,174 gave cn bw*/ cn bw* homozygous viable femalesThese were tested for sterility.
56 Maternal effect screen 29 C lay eggs and developCombination of numbers from a second screenOf 18,782 established F3 lines7,351 gave homozygous viable femalesThese were tested for sterility.529 were sterile
57 Maternal effect screen Of these 529 mutants 136 laid normal looking eggs that did not develop normally.We are looking at the eggs in the F4 generations.Maternal effect mutantsThese 136 mutations identified 67 complementation groups.Similar screens were performed on the second and third chromosomes.