Can't get no dissatisfaction: fruitless love?

Rotation Talk McKeown Lab Arjun Ray

Talkin’ Fly

gene Protein italics geneallele
Gene 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

Courting behavior 2) Tapping 1) Orienting 3) ‘Singing’ 4) Licking 5) Attempted copulation 6) Copulation Sokolowski et al.Drosophila melanogaster . Nature Genetics 20 .

Development and Behavior Pathways females males McKeown et al. Developmental Biology 200 .Fly Sex .

Developmental Biology 200 .Fly Sex .Development and Behavior Pathways females males Fruitless - (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.

Cell 20 .fruitless abnormalities – not your conventional circle of life… Male chaining fruM hypomorph males fruM females That ’ s not how it works Dickson et al.

org/wiki/File:Nuclear_Receptor_Structure.dissatisfaction .p .wikipedia.adult behavior • Dissatisfaction (Dsf) is similar to Nuclear Hormone Type III (human) orphan receptor • – http://en.

org/wiki/File:Nuclear_Receptor_Structure.wikipedia.like phenotypes http://en.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 .

dissatisfaction .Females – No uterine circumferenti al muscle innervation M.adult development wildtype dsf vA5 -/-/- • dsf Males F-actin (phalloidin) synaptotagmi n •  • – Larger and fewer boutons on ventral abdominal segment 5 (vA5) muscles • dsf -/. McKeown and T. Shirangi unpublished dat .

dissatisfaction . fru-Gal4 F-actin (phalloidin) GFP • dsf -/.Females dsf neuronal synapse M. McKeown and T. Shirangi unpublished dat formation • – No uterine circumferenti may be al muscle involved in fru innervation .adult development wildtype dsf vA5 -/-/- • dsf Males F-actin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.

McKeown and T. fru-Gal4 F-actin (phalloidin) GFP • dsf -/. Shirangi unpublished dat .dissatisfaction .adult development wildtype dsf vA5 -/-/- • dsf Males F-actin (phalloidin) synaptotagmi n • – Larger and fewer boutons on ventral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.Females – No uterine circumferenti Is knocking down Dsf in al muscle fru cells sufficient to innervation produce fru null • phenotype? M.

Knocking down Dsf in fruP1 cells Genetic Cross .

Thick red and black lines represent regions of homology in the donor construct and targeted locus. Targeting Strategy Pe Stocking r. The sequenced region of the final fruGAL4 chromosome is indicated. and reinsertion of the donor construct. Duda Kvitsiani. and Barry J . Shay Rotkopf. respectively. Dickson tra e Gal4 fru GAL4 P1 STOP Steps used to generate the fruGAL4 allele. The general procedure is similar to that described by Rong and Golic (2000). downward arrows indicate transcriptional stop signals. and a Cre-loxP recombination step to excise this reporter after the initial targeting event. Dickson et al. The major variations are the use of an eye-specific GFP reporter (GMR-GFP) that is reconstituted after excision. László Tirián. linearization. Arrows indicate the direction of transcription. Cell 20 .Drosophila Male Courtship Behavior ru fruP1-gal4 – splice trap (Dickson Lab) cell nucleus • Knocking down Dsf in fruP1 cells Figure S1.

and reinsertion of the donor construct. The major variations are the use of an eye-specific GFP reporter (GMR-GFP) that is reconstituted after excision. respectively.RNAi Dickson et al. and Barry J . downward arrows indicate transcriptional stop signals. The general procedure is similar to that described by Rong and Golic (2000). Dickson tra e Gal4 fru GAL4 P1 STOP Steps used to generate the fruGAL4 allele. linearization. UAS dsf . Cell 20 . Thick red and black lines represent regions of homology in the donor construct and targeted locus. László Tirián. Arrows indicate the direction of transcription. The sequenced region of the final fruGAL4 chromosome is indicated. Shay Rotkopf. Targeting Strategy Pe Stocking r. and a Cre-loxP recombination step to excise this reporter after the initial targeting event. Duda Kvitsiani.Drosophila Male Courtship Behavior fruP1-gal4 – splice trap (Dickson Endogenous Lab) fru expression • Knocking down Dsf in fruP1 cells Figure S1.

Duda Kvitsiani. Thick red and black lines represent regions of homology in the donor construct and targeted locus. downward arrows indicate transcriptional stop signals. Shay Rotkopf. The major variations are the use of an eye-specific GFP reporter (GMR-GFP) that is reconstituted after excision. and reinsertion of the donor construct. The sequenced region of the final fruGAL4 chromosome is indicated. UAS dsf . Dickson tra e Endogenous fru expression Gal4 fru GAL4 P1 STOP Steps used to generate the fruGAL4 allele. and a Cre-loxP recombination step to excise this reporter after the initial targeting event.Drosophila Male Courtship Behavior Knocking down Dsf in fruP1 cells Figure S1. Cell 20 . László Tirián. Arrows indicate the direction of transcription. The general procedure is similar to that described by Rong and Golic (2000).RNAi Dickson et al. and Barry J . Targeting Strategy Pe Stocking r. linearization. respectively.

Cell 20 . and reinsertion of the donor construct. and Barry J . The general procedure is similar to that described by Rong and Golic (2000). Thick red and black lines represent regions of homology in the donor construct and targeted locus. The major variations are the use of an eye-specific GFP reporter (GMR-GFP) that is reconstituted after excision. Dickson tra e Endogenous fru expression Gal4 fru GAL4 P1 STOP Steps used to generate the fruGAL4 allele. downward arrows indicate transcriptional stop signals. The sequenced region of the final fruGAL4 chromosome is indicated. Duda Kvitsiani. and a Cre-loxP recombination step to excise this reporter after the initial targeting event. Targeting Strategy Pe Stocking r. linearization. Dsf is knocked down in fruP1 cells Dsf UAS dsf . Arrows indicate the direction of transcription.Drosophila Male Courtship Behavior Knocking down Dsf in fruP1 cells Figure S1. respectively. Shay Rotkopf.RNAi Dickson et al. László Tirián.

* = fruP1-Gal4/dsf-RNAi Courtship Experiment – dsfRNAi extends copulation time mating scheme regular mating *male x female male x *female *male x *female n= 4 4 3 3 ID A B C D + T min 10 naïve males 10 naïve 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 .

20 . Neurobiol. J.Severe fru hypomorphs exhibit similar extended copulation times Conclusion : Knockdown of Dsf in fruP1 cells may partially recapitulate a fru hypomorph phenotypeHall et al.

Future Directions What about same-sex courting of fru hypomorph?   Modification Benefit baseline 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 .RNAi control or low and even CO 2 usage density control helpfu mutiple RNAis l for behavi drive Dicer or locali drive UAS -GFP zation costain with NMJ factors .dsf .

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

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