Using RNA Using RNA interference interference

mosaics to map mosaics to map retainedretained mutant mutant

phenotypesphenotypesHHMI InternshipMegan Kelly

Mentor:Dr. Barbara Taylor

Dept. of Zoology

www.berkeley.edu/.../07/images/fruitflies.jpg

BackgroundBackgroundHow do genes How do genes work in creating work in creating body parts and body parts and controlling controlling function?function?

I focused onI focused onanswering this answering this questionquestionfor the female for the female fruitflyfruitflyreproductive reproductive tracttract

Ovaries

Oviducts

Uterus

http://flybase.net/static_pages/imagebrowser/imagebrowser10.html

One method for analysisOne method for analysis

Using mutation in general to Using mutation in general to disrupt gene expression causing disrupt gene expression causing

mutant phenotypemutant phenotype

Changing gene expression in only Changing gene expression in only specific cells to locate mutant specific cells to locate mutant

phenotypesphenotypes

I altered (I altered (retainedretained) gene ) gene expression in cells of the female expression in cells of the female

and found that in resulting and found that in resulting phenotypes no eggs were laid - a phenotypes no eggs were laid - a

result of retention.result of retention.

BackgroundBackgroundretained(retn) gene in fruit flies plays role in

female reproductive tract development

Strong mutation, prevents the gene

from transcription, therefore no

expression. Results in embryonic lethal

Weak mutation, (missense). Adults

viable, results in female sterility, and

abnormalities in courtship behavior.

HypothesisHypothesis

Female sterility is Female sterility is due todue to

a retained mutanta retained mutant

phenotype in thephenotype in the

reproductive tractreproductive tract

If true, females If true, females should beshould be

able to mate but notable to mate but not

release an egg from release an egg from the ovariesthe ovaries

Wildtype reproductive tract

http://flybase.net/reports/FBim0000078.html

Map the location of cells responsible for female sterility by comparing retn flies to retn RNA-

interference mosaics

GoalGoal

Mosaics:

Groups of cells in the body vary in gene expression

Ex: Calico cat has variation in pigment due to genotypic variation in skin cells

http://tigerpixie.com/tigerpixieart/HalloweenaSM.jpg

= retn expression location

X

retained(retn)

Wildtype(CSA)

Mosaic

X retn-RNAi gene

UAS retn-RNAi

UAS

Creating mosaics

retn-RNAi I

retn-RNAi ARID

retn-RNAi III

gal4 gene

hthgal4

homothorax gene

Gal4 protein

homothorax gene

gal4 gene

retn-RNAi gene

•homothorax•hthgal4

Normal homothorax expression

•UASretn-RNAi

Normal retn expression

•retn expressing

Loss of retn function •UASretn-RNAi

•retn expressing

•homothorax

•hthgal4

Comparing Comparing retnretn mutants to our mutants to our RNAi mosaics in three ways:RNAi mosaics in three ways:

http://www.csus.edu/indiv/h/hollandb/Pictures/fruit%20flies/fly%20laying%20egg.jpg

http://www.pnas.org/misc/archive072803.shtml

http://flybase.net/reports/FBim0000078.html

•Behavior before Behavior before and during and during copulationcopulation

•FertiliFertilityty

•Physical makeup of Physical makeup of the reproductive the reproductive tract through tract through dissectionsdissections

Behavioral testBehavioral test

Wildtype latency to Wildtype latency to courtship average: 1-courtship average: 1-

2 minutes2 minutes

Wildtype copulation Wildtype copulation duration average 18-duration average 18-

30 minutes30 minutes

Wildtype latency to Wildtype latency to copulation average:2-5 copulation average:2-5

minutesminutes

Copulation duration

Latency to copulation

Latency to courtship

Behavioral Results:

Latency to courtship

0

10

20

30

40

50

60

70

80

90

Tim

e (

in s

eco

nd

s)

Wild

type

Mosaic controls Mosaics

retn mutan

t

retn contr

ol

Analysis of variance concluded no behavioral difference due to genotype

Behavioral resultsBehavioral results

Analysis of variance concluded no behavioral difference due to genotype

Latency to Copulation

0

100

200

300

400

500

600

700

800

900

Tim

e (i

n s

eco

nd

s)

Mosaic controls

Mosaics

retn mutan

t

retn contr

ol

Wild

type

Behavioral test resultsBehavioral test results

Analysis of variance concluded no behavioral difference due to genotype

Copulation duration

0

5

10

15

20

25

30

Tim

e (

in m

inu

tes

)

Wild

type

Mosaic controls

Mosaics

retn mutan

t

retn contr

ol

0

20

40

60

80

100

120

Nu

mb

er

cou

nte

d

Eggs laid

Fertility resultsFertility results

18

20 20

23

18

19

20

22

23

24

Wild

type

RNAi ARID

/+

RNAi I/+

RNAi III/

+ht

hgal

4/+

hthg

al4/

RN

Ai A

RID

hthg

al4/

RN

Ai I

hthg

al4/

RN

Ai I

IIre

tn/r

etn

retn

/cyo

Mosaic controls Mosaics

retn mutant

retn control

Mosaics

Wildtype

retn mutant

Reproductive tract

retn mutant

Mosaic

ConclusionConclusion

Results gear us toward Results gear us toward understanding that sterility in understanding that sterility in mosaic is due to another phenotypic mosaic is due to another phenotypic mutation resulting from mutation resulting from retn retn degredationdegredation

retn retn mutants may be sterile due to mutants may be sterile due to another product of another product of retnretn degredation degredation and not fully because of the loss of and not fully because of the loss of the common oviductthe common oviduct

Future WorkFuture Work

Continue comparative work in Continue comparative work in retn retn reproductive tractreproductive tract

•Use a different driver than Use a different driver than Gal4 to increase strength of Gal4 to increase strength of sterilitysterility

Localize Localize retnretn degradation to the degradation to the nervous system and not the nervous system and not the reproductive tractreproductive tract

One step closer to understanding the One step closer to understanding the process of cell expressionprocess of cell expression

www.anatomy.unimelb.edu.au/.../life_cycle.jpg

AcknowledgmentsAcknowledgments

Howard Hughes Medical Institute

Dr. Barbara Taylor (Department of Zoology)

Dr. Kevin Ahern (Department of

Biochemistry/Biophysics)

Taylor Lab