Determining the cause of defective
reproductive tract development and
sterility in retained mutant female
Drosophila melanogaster
Jennifer Coppersmith
Dr. Barbara Taylor
Department of Zoology
Oregon State University
“Why can’t a woman be more like a man?...
Why can’t a woman behave like a man?” –
Rex Harrison, My fair lady (1964)
What makes males and females look and
behave differently?
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♂
♂
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♂
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How do reproductive tracts
develop in females and how
does improper development
affect the sterility and behavior
of that species.
Why Fruit Flies?
Easy maintenance
Fast life cycle
Entire genome is well known
Model Organism
Mutations
How Mutations are Created
P-transposable elements (P-elements): are DNA
sequences, which can insert randomly into chromosomes
Requires enzyme transposase for removal and reinsertion.
Can cause
mutations that
allow us to make
genomic clones
to study the
expression
pattern of that
gene
P-element
Normal Gene
Insertion
Disrupted Gene
retained Gene
Development 132, 155-164 (2005) Published by The Company of Biologist 2005
P-Element
Strong mutations, prevents the
gene from transcription,
therefore no expression.
Results in embryonic lethal
Weak mutations, a single
amino acid change (missense).
Adults viable, but female sterile
with abnormalities in courtship
behavior.
Female Reproductive System
Genital Disc
Keisman, E.L., Christiansen, A.E.,
and Baker, B.S. (2001).
Stages and development of the
reproductive system with in the
female fruit fly
Ovaries
Oviducts
Uterus
Volker Hartenstein, published by Cold Spring Harbor Laboratory Press, 1993
Determine the cause of sterility in retn mutants
How is the development of the
reproductive tract different in the retn
mutants compared to wild type?
Methods and materials
Dissect out reproductive tract
or genital disc, then stain for
expression pattern using
immunohistochemistry.
Dissection
After staining, analyze and
compare normal vs. mutant
using CCD Camera to
capture images.
Analyze
Method (cont’d)
Immunohistochemistry
Useful in determining
pattern of expression
of genes
Fluorescent/staining tag
Secondary
antibody
Primary
antibody
CELL
Tissue
antigen
Gene Expression
retnDac
Hth
Abd-B
Genital
Disc
Reproductive difference between Wild
Type and mutant genotypes
Wild Type
retn-
dac-
AbdB0569
Fertility
Fertile
Not fertile
-----
Not Fertile
Courtship /
Copulation
Normal
Abnormal
------
Abnormal
Reproductive
tract
Normal
Abnormal Abnormal
structure
structure
Marker
Staining
Fas III +
Abd B +
Dac +
Fas III +/Abd B +
Dac +
-------------
Normal
Fas III +/ Abd B +
-----
Comparing Wild Type vs. retn Mutant
Average Surface Area Oviduct
Ovaries
800
700
Mcd8 CSA
R044/dri8 z2426/dri8 R044
Ovaries
Surface Area
600
Oviduct
500
400
300
200
100
0
1
2
3
4
5
Oviduct
Genotype
Uterus
Normal (Wild Type) fruit
fly reproductive tract
Uterus
Retn mutant fruit fly
reproductive tract
Fas III staining
Wild type
B0569
Calyx
retn-
Abd-
Diagram of oviducts and egg laying
retn-
Wild Type
EGG
Calyx
Abd-B0569
Genetic Crosses
retn+
retn+
XX+
retn+
X
retnretn+
retn-
(punnett squares)
X+
X+
Genetic interaction?
Fertile and
normal
reproductive
tract
retnX+
X-
X-
retn-
X-
Sterile and
abnormal
reproductive
tract
Determining possible Genetic interactions
retndri8/
dac4
Fertility
Courtship /
Copulation
Reproductive
tract
retnR044 /dac retnR044/
rk364
Hth
retnR044
/Abd-B05659
Fertile Low Fertility Fertile
Normal
Normal
Normal
Fertile
Normal
Normal
Normal
Normal
Normal
Summery of Data
•Has an abnormal
reproductive structure and
Fas III staining
•Suggest that Abd B and Dac
are not regulated by retn
•Possible genetic interaction
with Dac rk364
Determine the cause of sterility in retn mutants
Can we recreate this oviduct
abnormality and sterility by removing retn
function by using selective RNA interfere
in the female reproductive tract or through
genetic interaction studies?
retn RNAi target the retn transcription, and
destroys it.
Future Work
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Perform more Fas III staining to verify results
Waiting for flies to emerge from crosses to
further test sterility and observe possible
defects in reproductive tract
Setting up crosses with RNAi’s to recreate
Sterility and abnormal reproductive tracts.
Dr. Barbara Taylor (department of Zoology)
Howard Hughes Medical Institute
Dr. Kevin Ahern (department of
biochemistry/biophysics)
Everyone working in the Taylor Lab!
"Time's fun when you're having flies."
-- Kermit the Frog'