Transcript PowerPoint - Oregon State University

Using RNA
interference mosaics
to map retained
mutant phenotypes
HHMI Internship
Megan Kelly
www.berkeley.edu/.../07/images/fruitflies.jpg
Mentor:
Dr. Barbara Taylor
Dept. of Zoology
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
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.
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
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
Goal
Map the location of cells responsible for
female sterility by comparing retn flies to retn
RNA-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
= retn expression location
Wildtype(CSA)
retained(retn)
X
Mosaic
Creating mosaics
UAS retn-RNAi
hthgal4
UAS
gal4 gene
homothorax gene
X
retn-RNAi gene
retn-RNAi I
retn-RNAi III
retn-RNAi ARID
Gal4 protein
gal4 gene
homothorax gene
retn-RNAi gene
Normal
homothorax
expression
•homothorax
•hthgal4
•UASretn-RNAi
Normal retn
expression
•retn
expressing
Loss of retn
function
•retn
expressing
•homothorax
•UASretn-RNAi
•hthgal4
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%20fli
es/fly%20laying%20egg.jpg
•Physical makeup of the
reproductive tract through
dissections
http://flybase.net/reports/FBim0000078.html
Behavioral test
Latency to courtship
Wildtype latency to courtship
average: 1-2 minutes
Latency to copulation
Wildtype latency to copulation
average:2-5 minutes
Copulation duration
Wildtype copulation duration
average 18-30 minutes
Behavioral Results:
Latency to courtship
90
70
60
50
40
30
20
10
o
tn
/c
y
re
tn
tn
/re
re
ht
hg
ht
al
hg
4/
al
+
4/
R
N
A
iA
R
ID
ht
hg
al
4/
R
N
A
ht
iI
hg
al
4/
R
N
A
iI
II
iI
II/
+
R
N
A
iI
/+
N
A
iA
R
N
A
R
R
ID
/+
SA
0
C
Time (in seconds)
80
retn
retn
mutant control
Mosaic controls
Mosaics
Analysis of variance concluded no behavioral difference due to genotype
Behavioral results
Latency to Copulation
900
700
600
500
400
300
200
100
cy
o
n/
re
t
re
tn
n/
4/
R
hg
al
ht
re
t
NA
iI
II
NA
iI
4/
R
ht
hg
al
NA
iA
RI
D
4/
R
4/
+
hg
al
ht
Mosaic controls
ht
hg
al
RN
A
iI
/+
RN
A
iI
/+
ID
iA
R
RN
A
II/
+
0
CS
A
Time (in seconds)
800
retn
retn
mutant control
Mosaics
Analysis of variance concluded no behavioral difference due to genotype
Behavioral test results
Copulation duration
Time (in minutes)
30
25
20
15
10
5
Mosaic controls
ht
ht
hg
hg
al
al
4/
4/
+
R
N
A
iA
ht
R
ID
hg
al
4/
R
ht
N
A
hg
iI
al
4/
R
N
A
iI
II
re
tn
/re
tn
re
tn
/c
yo
iI
II/
+
R
N
A
iI
/+
R
N
A
/+
ID
R
N
A
iA
R
C
SA
0
retn
mutant
retn
control
Mosaics
Analysis of variance concluded no behavioral difference due to genotype
Fertility results
120
Eggs laid
18
24
Number counted
100
80
18
20
20
60
40
20
23
19
20
22
23
0
Mosaic controls
Mosaics
retn
retn
mutant control
Mosaics
Reproductive tract
Wildtype
retn mutant
retn mutant
Mosaic
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
comparative work in retn
reproductive tract
Continue
•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
www.anatomy.unimelb.edu.au/.../life_cycle.jpg
Acknowledgments
Howard Hughes Medical Institute
Dr. Barbara Taylor (Department of Zoology)
Dr. Kevin Ahern (Department of
Biochemistry/Biophysics)
Taylor Lab