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

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.

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.

Cell Biology of Drosophila Development

Structure of the egg

Dorsal appendage

Chorion

VitellineMicropyle

Structure of the egg

Dorsal appendage

Chorion

VitellineMicropyle

D

V

A P

Syncytial blastoderm

2nd cleavage

Campos-Ortega and Hartenstein 1985

Syncytial blastoderm

9th cleavage

Campos-Ortega and Hartenstein 1985

Pole cells

Germ-line soma division

Cellular Blastoderm

Hartenstein 1993

Blastoderm fate map

Hartenstein 1993

Gastrulation 3 hours

Hartenstein 1993

2:50 h

3:40 h

4:20 h

Mesoderm and the ventral furrow

Hartenstein 1993

QuickTime™ and aYUV420 codec decompressor

are needed to see this picture.

QuickTime™ and aYUV420 codec decompressor

are needed to see this picture.

Gastrulation and germ band retraction

Flybase

Germ band retraction

7:20 h

9:20 h

10:20 h

Hartenstein 1993

Development of the Brain

Hartenstein 1993

The Drosophila life cycle represents the differentiation of two distinct forms: the larva and the Imago (adult).

Embryogenesis: differentiation of the larva

Metamorphosis:differentiation ofthe imago (adult)

Imaginal cells are the cellsof the adult or imago.

Kalthoff 2001

Origin and differentiation of Imaginal cells

Embryonic Origin Differentiation

The resulting Imago

Hartenstein 1993

Oogenesis

Kalthoff 2001

Yamashita, Y. M. et al. J Cell Sci 2005;118:665-672

GSC niches in the Drosophila ovary and testis

Development and structure of the Cyst

Ring canals

Kalthoff 2001

Oogenesis

Anterior

Posterior

Kalthoff 2001

Later stages of Oogenesis

A

PA

A

P

P

Haploid versus Diploid Genetics

Haploidgenetics

Diploidgenetics

Genetic organization of Drosophila

Y 1stX

2nd 3rd 4th

1% of genome

Most information on 1, 2 and 3 chromosomes

Males and females

XY or X0 maleX/A ratio 0.5

XX femaleX/A ratio 1

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.

Balancers

chromosome First Second Third

name FMBasc

CyOSM

TM

Dominantmarkers

Bar Curly StubbleSerrateUltrabithorax

Balancer chromosomes

Balancer homolog

Both the balancer and homolog chromosomes segregateas single mendelian units: the chromosome is a stable geneticunit.

Maternal versus Zygotic genomes

Maternal genomeOogenesis

Zygote and Zygoticgenome

SpermPaternal genome

Screens for Pattern Formation Mutants

Zygotic lethal screens: screens for activities expressedfrom the zygotic genome required for correct pattern formation.

Maternal effect screens: screens for activities expressed from the maternal genome required for correct pattern formation.

Basic steps of a screen

1 Mutagenesis

2 Separating the mutant chromosomes

3 Amplifying the mutant chromosomes

4 Testing for mutant phenotypes

Zygotic screenF0

DTS91 pr cn sca/CyO virgin females X cn bw sp males

EMS

DTS91: dominant temperature sensitive mutationDies at 29 C.

CyO second chromosome balancer

cn bw mutant combination results in white eyes

Zygotic screenF0

X cn bw sp males

EMSMutagenesis

Generating the random pool of mutagenized chromosomes

Zygotic screenF0-F1

DTS91 pr cn sca/CyO virgin females X cn bw sp males

EMS

F0

DTS91 pr cn sca/CyO virgin females Xcn bw sp*/CyO

cn bw sp*/DTS91 pr cn sca

or

Individual males

* mutagenized chromosomeSet up 10,000 crosses

F1

Zygotic screenF0-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

Zygotic screenF1-F2

DTS91 pr cn sca/CyO virgin females Xcn 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 femalesF2

5,764

Zygotic screenF1-F2

DTS91 pr cn sca/CyO virgin females X cn bw sp*/CyOF1

cn bw sp*/CyOF2

DTS91 pr cn sca/CyO CyO/CyODTS91 pr cn sca/ cn bw sp*

Zygotic screenF1-F2

DTS91 pr cn sca/CyO virgin females X cn bw sp*/CyOF1

cn bw sp*/CyOF2

DTS91 pr cn sca/CyO CyO/CyODTS91 pr cn sca/ cn bw sp*

Dead Dead Dead

Alive

Zygotic screenF1-F2

DTS91 pr cn sca/CyO virgin females Xcn bw sp*/CyO

cn bw sp*/DTS91 pr cn sca

orF1

29 C for 4 days

cn bw sp*/CyO

Males and femalesF2

F1-F2 amplifying the mutant chromosomes

Zygotic screenF2-F3

* mutagenized chromosome

cn bw sp*/CyO

Males and femalesF2

5,764

F3 Screen for vials that have no white eyed flies.

4,217

Zygotic screenF2-F3

* mutagenized chromosome

cn bw sp*/CyO

Males and femalesF2

5,764

F3 Screen for vials that have no white eyed flies.

4,217

F3 started the testing

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

Complementation analysis

The 272 mutants identified 61 complementationgroups (genes).

Maternal effect screens

F0 F1 F2 F3

F0 F1 F2 F3

Egg

Maternalgenome

F4

Zygotic screen

Maternal screenMother

Dead embryo

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.

Maternal effect screen

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

EMSF0

Maternal effect screen

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

EMS

F1cn bw*/ CyO DTS.513 virgins

Collect individual virgin female flies

Maternal effect screen

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

EMS

F1cn bw*/ CyO DTS.513 virgins X Fs(2) D/ CyO DTS.513

Dominant female sterile

males

Maternal effect screen

F1cn 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

Maternal effect screen

F1cn bw*/ CyO DTS.513 virgins X Fs(2) D/ CyO DTS.513

males

genotypes females males

cn bw*/ CyO DTS.513 fertile

cn bw*/ Fs(2) D sterile

Fs(2) D/ CyO DTS.513 sterile

F2

Maternal effect screen

F1cn bw*/ CyO DTS.513 virgins X Fs(2) D/ CyO DTS.513

males

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

F2

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

F2

Intermate

F3

29 C lay eggs and develop

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*

Maternal effect screen

F3

29 C lay eggs and develop

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?

Maternal effect screen

F3

29 C lay eggs and develop

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 femalesThese were tested for sterility.

Maternal effect screen29 C lay eggs and develop

Of 18,782 established F3 lines

7,351 gave homozygous viable femalesThese were tested for sterility.

529 were sterile

Combination of numbers from a second screen

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.