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Using RNA interference mosaics to map retained mutant phenotypes HHMI Internship Megan Kelly Mentor: Dr. Barbara Taylor Dept. of Zoology www.berkeley.edu/.../07/images/fruitflies.jpg Background How do genes work in creating body parts and controlling function? I focused on

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slide1

Using RNA interference mosaics to map retained mutant phenotypes

HHMI Internship

Megan Kelly

Mentor:

Dr. Barbara Taylor

Dept. of Zoology

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

background
Background

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

I focused on

answering this question

for the female fruitfly

reproductive tract

Ovaries

Oviducts

Uterus

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

one method for analysis
One method for analysis

Using mutation in general to disrupt gene expression causing mutant phenotype

Changing gene expression in only specific cells to locate mutant phenotypes

I altered (retained) gene expression in cells of the female and found that in resulting phenotypes no eggs were laid - a result of retention.

background4
Background

retained(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.

hypothesis
Hypothesis

Female sterility is due to

a retained mutant

phenotype in the

reproductive tract

If true, females should be

able to mate but not

release an egg from the ovaries

Wildtype reproductive tract

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

slide6

Goal

Map the location of cells responsible for female sterility by comparing retn flies to retnRNA-interference mosaics

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

slide7

Wildtype(CSA)

retained(retn)

Mosaic

= retn expression location

X

slide8

UAS retn-RNAi

hthgal4

UAS

X

gal4 gene

homothorax gene

retn-RNAi gene

Gal4 protein

gal4 gene

homothorax gene

retn-RNAi gene

Creating mosaics

retn-RNAi I

retn-RNAi III

retn-RNAi ARID

slide9

Normal homothorax expression

  • homothorax
  • hthgal4
  • UASretn-RNAi

Normal retn expression

  • retn expressing
  • retn expressing
  • homothorax

Loss of retn function

  • UASretn-RNAi
  • hthgal4
comparing retn mutants to our rnai mosaics in three ways
Comparing retn mutants to our RNAi mosaics in three ways:
  • Behavior before and during copulation
  • Fertility

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

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

  • Physical makeup of the reproductive tract through dissections

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

behavioral test

Latency to courtship

Latency to copulation

Copulation duration

Behavioral test

Wildtype latency to courtship average: 1-2 minutes

Wildtype latency to copulation average:2-5 minutes

Wildtype copulation duration average 18-30 minutes

slide12

Behavioral Results:

Wildtype

retn mutant

retn control

Mosaic controls

Mosaics

Analysis of variance concluded no behavioral difference due to genotype

behavioral results
Behavioral results

Wildtype

retn mutant

retn control

Mosaic controls

Mosaics

Analysis of variance concluded no behavioral difference due to genotype

slide14

Behavioral test results

Wildtype

retn mutant

retn control

Mosaic controls

Mosaics

Analysis of variance concluded no behavioral difference due to genotype

fertility results

18

24

18

20

20

20

19

23

22

retn/cyo

retn/retn

23

hthgal4/RNAi I

hthgal4/RNAi III

Wildtype

RNAi ARID/+

RNAi I/+

hthgal4/+

hthgal4/RNAi ARID

RNAi III/+

retn mutant

retn control

Mosaic controls

Mosaics

Mosaics

Fertility results
slide16

Reproductive tract

Wildtype

retn mutant

conclusion
Conclusion
  • Results gear us toward understanding that sterility in mosaic is due to another phenotypic mutation resulting from retn degredation
  • retn mutants may be sterile due to another product of retn degredation and not fully because of the loss of the common oviduct
future work
Future Work
  • Continue comparative work in retn reproductive tract
  • Use a different driver than Gal4 to increase strength of sterility
  • Localize retn degradation to the nervous system and not the reproductive tract
one step closer to understanding the process of cell expression
One step closer to understanding the process of cell expression

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

acknowledgments
Acknowledgments

Howard Hughes Medical Institute

Dr. Barbara Taylor (Department of Zoology)

Dr. Kevin Ahern (Department of Biochemistry/Biophysics)

Taylor Lab