Cell Biology of Drosophila Development

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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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Lab 3 Engrailed staining
Elyse will open the teaching lab at 900AM Read
instructions in manual and on the board in the
lab If you start at 900AM you can be finished
by about 400PM Get yourselves organized or it
will be a very long day.
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Next weeks reading
DiNardo paper is missing a page and pages 1222
and 1223 are reversed. Can get the missing page
on the web site.
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Cell Biology of Drosophila Development
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Structure of the egg
Dorsal appendage
Chorion
Micropyle
Vitelline
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Structure of the egg
Dorsal appendage
D
Chorion
A
P
V
Micropyle
Vitelline
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Syncytial blastoderm
2nd cleavage
Campos-Ortega and Hartenstein 1985
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Syncytial blastoderm
Germ-line soma division
Pole cells
9th cleavage
Campos-Ortega and Hartenstein 1985
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Cellular Blastoderm
Hartenstein 1993
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Blastoderm fate map
Hartenstein 1993
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Gastrulation 3 hours
250 h
340 h
420 h
Hartenstein 1993
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Mesoderm and the ventral furrow
Hartenstein 1993
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Gastrulation and germ band retraction
Flybase
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Germ band retraction
720 h
920 h
1020 h
Hartenstein 1993
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Development of the Brain
Hartenstein 1993
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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
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Origin and differentiation of Imaginal cells
Embryonic Origin
Differentiation
The resulting Imago
Hartenstein 1993
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Oogenesis
Kalthoff 2001
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GSC niches in the Drosophila ovary and testis
Yamashita, Y. M. et al. J Cell Sci
2005118665-672
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Development and structure of the Cyst
Ring canals
Kalthoff 2001
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Oogenesis
Anterior
Posterior
Kalthoff 2001
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Later stages of Oogenesis
A
P
P
A
A
P
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Haploid versus Diploid Genetics
Haploid genetics
Diploid genetics
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Genetic organization of Drosophila
1 of genome
Y
1st X
2nd
3rd
4th
Most information on 1, 2 and 3 chromosomes
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Males and females
XX female X/A ratio 1
XY or X0 male X/A ratio 0.5
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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.

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Balancers
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Balancer chromosomes
Balancer
homolog
Both the balancer and homolog chromosomes
segregate as single mendelian units the
chromosome is a stable genetic unit.
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Maternal versus Zygotic genomes
Sperm Paternal genome
Zygote and Zygotic genome
Maternal genome Oogenesis
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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.
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Basic steps of a screen
1 Mutagenesis 2 Separating the mutant
chromosomes 3 Amplifying the mutant
chromosomes 4 Testing for mutant phenotypes
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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
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Zygotic screen F0
EMS
Mutagenesis
X cn bw sp males
Generating the random pool of mutagenized
chromosomes
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Zygotic screen F0-F1
EMS
DTS91 pr cn sca/CyO virgin females X cn bw sp
males
F0
cn bw sp/CyO
DTS91 pr cn sca/CyO virgin females X
F1
or
cn bw sp/DTS91 pr cn sca
Individual males
Set up 10,000 crosses
mutagenized chromosome
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Zygotic screen F0-F1
cn bw sp/CyO
F1 separating the mutant chromosomes
or
cn bw sp/DTS91 pr cn sca
Individual males
Set up 10,000 crosses
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Zygotic screen F1-F2
10,000 crosses
cn bw sp/CyO
DTS91 pr cn sca/CyO virgin females X
F1
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
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Zygotic screen F1-F2
cn bw sp/CyO
DTS91 pr cn sca/CyO virgin females X
F1
CyO/CyO
DTS91 pr cn sca/CyO
DTS91 pr cn sca/ cn bw sp
F2
cn bw sp/CyO
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Zygotic screen F1-F2
cn bw sp/CyO
DTS91 pr cn sca/CyO virgin females X
F1
CyO/CyO
DTS91 pr cn sca/CyO
DTS91 pr cn sca/ cn bw sp
Dead
Dead
Dead
F2
cn bw sp/CyO
Alive
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Zygotic screen F1-F2
cn bw sp/CyO
DTS91 pr cn sca/CyO virgin females X
F1
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
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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
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Zygotic screen F2-F3
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
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F3 tests
The 4,217 F3 flies were allowed to lay eggs
CyO/CyO larvae die after hatching Screen
for gt25 unhatched eggs
2,843 lines had gt 25 unhatched eggs Test for
cuticle defects 272 lines had distinct cuticular
changes
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Complementation analysis
The 272 mutants identified 61 complementation grou
ps (genes).
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Maternal effect screens
Zygotic screen
F0
F1
F2
F3
Dead embryo
Maternal screen
Mother
Egg
F0
F1
F2
F3
F4
Maternal genome
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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.
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Maternal effect screen
F0
EMS
cn bw sp/ CyO DTS.513 virgins X cn bw males
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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
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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
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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
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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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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 fertile cn bw/ Fs(2)
D sterile fertile Fs(2) D/ CyO
DTS.513 sterile fertile
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Maternal effect screen
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
Intermate
29 C lay eggs and develop
F3
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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
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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?
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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.
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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
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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.