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 Micropyle Vitelline

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

Syncytial blastoderm 2nd cleavage Campos-Ortega and Hartenstein 1985

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

Cellular Blastoderm Hartenstein 1993

Blastoderm fate map Hartenstein 1993

Gastrulation 3 hours 2:50 h 3:40 h 4:20 h Hartenstein 1993

Mesoderm and the ventral furrow Hartenstein 1993

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 of the imago (adult) Imaginal cells are the cells of the adult or imago. Kalthoff 2001

Origin and differentiation of Imaginal cells Embryonic Origin Differentiation The resulting Imago Hartenstein 1993

Oogenesis Kalthoff 2001

GSC niches in the Drosophila ovary and testis Yamashita, Y. M. et al. J Cell Sci 2005;118:665-672

Development and structure of the Cyst Ring canals Kalthoff 2001

Oogenesis Anterior Posterior Kalthoff 2001

Later stages of Oogenesis P A P A P

Haploid versus Diploid Genetics

Genetic organization of Drosophila 1% of genome Y 1st X 2nd 3rd 4th Most information on 1, 2 and 3 chromosomes

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

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

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

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

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.

Basic steps of a screen 1 Mutagenesis 2 Separating the mutant chromosomes 3 Amplifying the mutant chromosomes 4 Testing for mutant phenotypes

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

Mutagenesis Zygotic screen F0 EMS X cn bw sp males Generating the random pool of mutagenized chromosomes

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

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

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

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

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

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

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

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

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 complementation groups (genes).

Maternal effect screens Zygotic screen F0 F1 F2 F3 Dead embryo Maternal screen Mother Egg F0 F1 F2 F3 F4 Maternal genome

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

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

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

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

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

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

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

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*

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?

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.

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

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.