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:

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

21. Oogenesis
Anterior
Posterior
Kalthoff 2001

22. Later stages of OogenesisP A P A P

23. Haploid versus 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
DTS91 pr cn sca/CyO virgin females X cn bw sp males
DTS91: dominant temperature sensitive mutation
Dies at 29 C.
CyO second chromosome balancer
cn 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

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

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

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

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

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

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

40. Zygotic screen F2-F3 cn bw sp*/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 F3 started the testing cn bw sp*/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
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
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
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

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

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

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

55. Maternal effect screen
29 C lay eggs and develop F3
genotypes phenotype
cn bw*/ Fs(2) D viable
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
Combination of numbers from a second screen
Of 18,782 established F3 lines
7,351 gave homozygous viable females
These 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 mutants
These 136 mutations identified 67 complementation groups.
Similar screens were performed on the second and third chromosomes.