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

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Presentation on theme: "Lecture 3 Cell Biology of Drosophila Development Drosophila Genetics Screen for Zygotic Lethals Screen for Maternal Effect Mutants."— Presentation transcript:

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 Vitelline Micropyle

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

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

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

9 Cellular Blastoderm Hartenstein 1993

10 Blastoderm fate map Hartenstein 1993

11 Gastrulation 3 hours Hartenstein :50 h 3:40 h 4:20 h

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 OriginDifferentiation The resulting Imago Hartenstein 1993

18 Oogenesis Kalthoff 2001

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

20 Development and structure of the Cyst Ring canals Kalthoff 2001

21 Oogenesis Anterior Posterior Kalthoff 2001

22 Later stages of Oogenesis A PA A P P

23 Haploid versus Diploid Genetics Haploid genetics Diploid genetics

24 Genetic organization of Drosophila Y1st X 2nd3rd4th 1% of genome Most information on 1, 2 and 3 chromosomes

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

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

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

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 DTS91 pr cn sca/CyO virgin females X cn bw sp males EMS 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 X cn bw sp males EMS Mutagenesis Generating the random pool of mutagenized chromosomes

34 Zygotic screen F0-F1 DTS91 pr cn sca/CyO virgin females X cn bw sp males EMS F0 DTS91 pr cn sca/CyO virgin females X cn bw sp*/CyO cn bw sp*/DTS91 pr cn sca or Individual males * mutagenized chromosome Set up 10,000 crosses F1

35 Zygotic screen F0-F1 cn bw sp*/CyO cn bw sp*/DTS91 pr cn sca or Individual males Set up 10,000 crosses F1 separating the mutant chromosomes

36 Zygotic screen F1-F2 DTS91 pr cn sca/CyO virgin females X cn bw sp*/CyO cn bw sp*/DTS91 pr cn sca or Individual males * mutagenized chromosome 10,000 crosses F1 29 C for 4 days cn bw sp*/CyO Males and females F2 5,764

37 Zygotic screen F1-F2 DTS91 pr cn sca/CyO virgin females X cn bw sp*/CyO F1 cn bw sp*/CyO F2 DTS91 pr cn sca/CyO CyO/CyO DTS91 pr cn sca/ cn bw sp*

38 Zygotic screen F1-F2 DTS91 pr cn sca/CyO virgin females X cn bw sp*/CyO F1 cn bw sp*/CyO F2 DTS91 pr cn sca/CyO CyO/CyO DTS91 pr cn sca/ cn bw sp* Dead Alive

39 Zygotic screen F1-F2 DTS91 pr cn sca/CyO virgin females X cn bw sp*/CyO cn bw sp*/DTS91 pr cn sca or F1 29 C for 4 days cn bw sp*/CyO Males and females F2 F1-F2 amplifying the mutant chromosomes

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

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

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 F0F1F2F3 F0F1F2F3 Egg Maternal genome F4 Zygotic screen Maternal screen Mother Dead embryo

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 cn bw sp/ CyO DTS.513 virgins X cn bw males EMS F0

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

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

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

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

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

52 Maternal effect screen genotypes females males cn bw*/ CyO DTS.513fertile fertile cn bw*/ Fs(2) Dsterile fertile Fs(2) D/ CyO DTS.513sterile fertile F2 Intermate F3 29 C lay eggs and develop

53 Maternal effect screen F3 29 C lay eggs and develop genotypes phenotype cn bw*/ CyO DTS.513 Fs(2) D/ CyO DTS.513 cn bw*/ Fs(2) D cn bw*/ cn bw*

54 Maternal effect screen F3 29 C lay eggs and develop genotypes phenotype cn bw*/ CyO DTS.513dead Fs(2) D/ CyO DTS.513 dead cn bw*/ Fs(2) Dviable cn bw*/ cn bw*dead/viable?

55 Maternal effect screen F3 29 C lay eggs and develop genotypes phenotype cn bw*/ Fs(2) Dviable cn bw*/ cn bw*dead/viable? Of 10,842 established F3 lines 5,174 gave cn bw*/ cn bw* homozygous viable females These were tested for sterility.

56 Maternal effect screen 29 C lay eggs and develop Of 18,782 established F3 lines 7,351 gave homozygous viable females These were tested for sterility. 529 were sterile Combination of numbers from a second screen

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 mutants These 136 mutations identified 67 complementation groups. Similar screens were performed on the second and third chromosomes.


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