McKeown Rotation Talk
Transcript of McKeown Rotation Talk
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M. McKeown and T. Shirangi unpublished dat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neuronal synapse M. McKeown and T. Shirangi unpublished dat formation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