Can't get no dissatisfaction: fruitless love?Rotation Talk McKeown Lab Arjun Ray

Talkin Fly

gene Protein italics genealleleGene A/Gene B the

lowercase first-letter, italics uppercase first-letter, no superscript after gene name Gene or allele A on one copy of the chromosome, Gene or allele B on

or

Gene A other copy Gene B http://www.hoxfulmonsters.com/2008/10/sarah-palin-against-spending-money-of-drosophila-researc

Drosophila melanogaster - Courting behavior 2) Tapping1) Orienting 3) Singing

4) Licking

5) Attempted copulation 6) Copulation

Sokolowski et al, Nature Genetics 20

Fly Sex - Development and Behavior Pathwaysfemales males

McKeown et al, Developmental Biology 200

Fly Sex - Development and Behavior Pathways females malesFruitless -

(Fru) is an RNA pol II transcription Factor-Male fruM hypomorph-

Will court other males

-Ectopic female fruM-

Will court other females

McKeown et al, Developmental Biology 200

fruitless abnormalities not your conventional circle of life

Male chaining

fruM hypomorph males

fruM females

That s not how it works

Dickson et al, Cell 20

dissatisfaction - adult behavior Dissatisfaction (Dsf) is similar to Nuclear Hormone Type III (human) orphan receptor

http://en.wikipedia.org/wiki/File:Nuclear_Receptor_Structure.p

dissatisfaction - adult behavior Dissatisfaction (Dsf) is similar to Nuclear Hormone Type III (human) orphan receptor

dsf

-/-

phenotypes Will court other females Resists male courtship Cannot lay eggs

Will court other males Poorly effective heterosexual mating

fru hypomorph - like phenotypes

http://en.wikipedia.org/wiki/File:Nuclear_Receptor_Structure.p

dissatisfaction - adult development wildtype dsf vA5 -/-/-

dsf

Males

F-actin (phalloidin) synaptotagmi n

Larger and fewer boutons on ventral abdominal segment 5 (vA5) muscles

dsf -/- Females No uterine circumferenti al muscle innervation

M. McKeown and T. Shirangi unpublished dat

dissatisfaction - adult development wildtype dsf vA5 -/-/-

dsf

Males

F-actin (phalloidin) synaptotagmi n

Larger and fewer boutons on ventral abdominal segment 5 (vA5) muscles

UAS-GFP, fru-Gal4F-actin (phalloidin) GFP

dsf -/- Females

dsf

neuronal synapse M. McKeown and T. Shirangi unpublished dat formation

No uterine circumferenti may be al muscle involved in fru innervation

dissatisfaction - adult development wildtype dsf vA5 -/-/-

dsf

Males

F-actin (phalloidin) synaptotagmi n

Larger and fewer boutons on ventral abdominal segment 5 (vA5) muscles

UAS-GFP, fru-Gal4F-actin (phalloidin) GFP

dsf -/- Females No uterine circumferenti Is knocking down Dsf in al muscle fru cells sufficient to innervation produce fru null phenotype?

M. McKeown and T. Shirangi unpublished dat

Knocking down Dsf in fruP1 cellsGenetic Cross

Drosophila Male Courtship Behavior

ru

fruP1-gal4 splice trap (Dickson Lab) cell nucleus

Knocking down Dsf in fruP1 cellsFigure S1. Targeting Strategy

Pe Stocking r, Duda Kvitsiani, Shay Rotkopf, Lszl Tirin, and Barry J . Dickson tra e

Gal4

fru GAL4P1STOPSteps used to generate the fruGAL4 allele. The general procedure is similar to that described by Rong and Golic (2000). The major variations are the use of an eye-specific GFP reporter (GMR-GFP) that is reconstituted after excision, linearization, and reinsertion of the donor construct; and a Cre-loxP recombination step to excise this reporter after the initial targeting event. Thick red and black lines represent regions of homology in the donor construct and targeted locus, respectively. The sequenced region of the final fruGAL4 chromosome is indicated. Arrows indicate the direction of transcription; downward arrows indicate transcriptional stop signals.

Dickson et al, Cell 20

Drosophila Male Courtship Behavior

fruP1-gal4 splice trap (Dickson Endogenous Lab) fru expression

Knocking down Dsf in fruP1 cellsFigure S1. Targeting Strategy

Pe Stocking r, Duda Kvitsiani, Shay Rotkopf, Lszl Tirin, and Barry J . Dickson tra e

Gal4

fru GAL4P1STOPSteps used to generate the fruGAL4 allele. The general procedure is similar to that described by Rong and Golic (2000). The major variations are the use of an eye-specific GFP reporter (GMR-GFP) that is reconstituted after excision, linearization, and reinsertion of the donor construct; and a Cre-loxP recombination step to excise this reporter after the initial targeting event. Thick red and black lines represent regions of homology in the donor construct and targeted locus, respectively. The sequenced region of the final fruGAL4 chromosome is indicated. Arrows indicate the direction of transcription; downward arrows indicate transcriptional stop signals.

UAS

dsf - RNAi

Dickson et al, Cell 20

Drosophila Male Courtship Behavior

Knocking down Dsf in fruP1 cellsFigure S1. Targeting Strategy

Pe Stocking r, Duda Kvitsiani, Shay Rotkopf, Lszl Tirin, and Barry J . Dickson tra e

Endogenous fru expression Gal4

fru GAL4P1STOPSteps used to generate the fruGAL4 allele. The general procedure is similar to that described by Rong and Golic (2000). The major variations are the use of an eye-specific GFP reporter (GMR-GFP) that is reconstituted after excision, linearization, and reinsertion of the donor construct; and a Cre-loxP recombination step to excise this reporter after the initial targeting event. Thick red and black lines represent regions of homology in the donor construct and targeted locus, respectively. The sequenced region of the final fruGAL4 chromosome is indicated. Arrows indicate the direction of transcription; downward arrows indicate transcriptional stop signals.

UAS

dsf - RNAi

Dickson et al, Cell 20

Drosophila Male Courtship Behavior

Knocking down Dsf in fruP1 cellsFigure S1. Targeting Strategy

Pe Stocking r, Duda Kvitsiani, Shay Rotkopf, Lszl Tirin, and Barry J . Dickson tra e

Endogenous fru expression Gal4

fru GAL4P1STOPSteps used to generate the fruGAL4 allele. The general procedure is similar to that described by Rong and Golic (2000). The major variations are the use of an eye-specific GFP reporter (GMR-GFP) that is reconstituted after excision, linearization, and reinsertion of the donor construct; and a Cre-loxP recombination step to excise this reporter after the initial targeting event. Thick red and black lines represent regions of homology in the donor construct and targeted locus, respectively. The sequenced region of the final fruGAL4 chromosome is indicated. Arrows indicate the direction of transcription; downward arrows indicate transcriptional stop signals.

Dsf is knocked down in fruP1 cellsDsf

UAS

dsf - RNAi

Dickson et al, Cell 20

* = fruP1-Gal4/dsf-RNAi

Courtship Experiment dsfRNAi extends copulation timemating scheme regular mating *male x female male x *female *male x *female n= 4 4 3 3

ID A B C D

+T min10 nave males 10 nave females

How many?

Mutant males appear to stay in copulation longer than expected compared to WT

*

* WT males may have suffered from excess CO2, reducing

Severe fru hypomorphs exhibit similar extended copulation times

Conclusion : Knockdown of Dsf in fruP1 cells may partially recapitulate a fru hypomorph phenotypeHall et al, J. Neurobiol. 20

Future DirectionsWhat about same-sex courting of fru hypomorph?

Modification

Benefitbaseline mating effect of this genotype baseline mating effect of this genotype avoid CO 2 negative effect on activity remove crowding effects from development increase completeness of RNAi knockdown Dicer is not well expressed in all fly tissues but is necessary for RNAi labels fruP1 cells where dsf is being knocked down look at how distribution / relative quantities are affected

necess fruP1 - Gal4 control ary for behavi UAS - dsf - RNAi control orlow and even CO 2 usage density control

helpfu mutiple RNAis l for behavi drive Dicer or locali drive UAS -GFP zationcostain with NMJ factors

Thanks to

Dr. Michael McKeown McKeown Lab Kathleen Yan Aron Gyuris Juliana Guarente Feifei Ding Dr. Kristi Wharton Wharton Lab Reenan Lab Dr. Barbaraout, piano fly play them Taylor, OSU http://worldphotocollections.blogspot.com/2009/12/adventures-of-mr-fly-funny-photos.h Dr. Troy Shirangi, Truman lab