1. Tramtrack69 regulates the expansion of
cellular tubes in the Drosophila ovary
through Mirror and Paxillin
Nathaniel Peters
Berg Lab
University of Washington
2. Cellular tubes are the plumbing for
organs and tissues in multicellular
organisms
During development, cells undergo
morphogenesis (Greek - ‘the beginning
of shape’) to create tubes.
Errors in tube morphogenesis can
result in birth defects, such as spina
bifida, or even death.
3. Tube morphogenesis requires 3 events
Tube patterning
Tube formation
Tube expansion
Adapted from Lubarsky
and Krasnow, 2003
4. In the Drosophila ovary, egg chambers
develop through 14 stages of
oogenesis
Adapted from Zartmanet al., 2010
Dorsal
Anterior Posterior
Ventral
DAs
S6 S11
S7 S8 S9 S10B
Mature Egg
Dorsal respiratory appendages (DAs) of the mature
egg help the embryo to breathe. They are molded
from the insides of cellular tubes during oogenesis
5. DA-tube morphogenesis a useful
model for tube morphogenesis
Adapted from Zartmanet al., 2010
Dorsal
Dorsal
Anterior Posterior
Appendage
Ventral
S6 S11 (DA)Tubes
S7 S8 S9 S10B
Patterning Tube formation Tube expansion
S10B S11 S12 S13 S14
DM = Dorsal midline DA Tube Cells DA Tube
6. The tramtrackgene encodes two
transcription factors:
Tramtrack69 &Tramtrack88 Adapted from
French et al., 2003
tramtrack
Boxes = exons Shaded Boxes = coding exons
Lines = introns 1a, 1b, & 1c = alternative transcription start sites
Only Tramtrack69 is required for DA-tube
expansion during oogenesis
7. The twin peaks (twk) mutation
disrupts expression of Tramtrack69
specifically during oogenesis, when it is
needed for DA-tube expansion
twin peaks mutation
(P element
insertion) BTB Zinc Finger
tramtrack Domain Domains
Adapted from
French et al., 2003
8. Loss of Tramtrack69 in twkdisrupts DA-
tube expansion (S13-14),
forming stunted DAs
DM = Dorsal midline DA Tube Cells DA Tube
9. My Question:
What are the Tramtrack69 effectors required
for epithelial tube expansion in the ovary?
10. Let me now introduce you to my
experimental approach for
identifying and characterizing
TTK69-regulated genes with roles
in DA-tube expansion
11. First, I dissected ovaries (AffymetrixDrosophila 2.0
microarrays,
from wild type and twk SAM analysis q ≤ 0.05)
and extracted the mRNA.
Using microarrays, I then
compared mRNA levels
(gene expression) to
identify differentially
expressed genes in twk.
~1000 genes were differentially expressed in twk. I
selected a subset of these genes for further
expression and functional analysis.
12. Our lab has developed a protocol for
fluorescence in situ hybridization (FISH)
in the Drosophila ovary, allowing us to
precisely visualize mRNA expression
with a confocal microscope
broad mRNA (FISH)
DNA (DAPI stain)
13. Using this technique, I validated my
microarrays and identified genes
with expression in the DA-tube cells
(like above)
14. RNAi disrupts mRNA By expressing RNAi
translation for an against differentially
individual gene and can expressed genes in twk, I
be used to test whether a distinguished genes with
gene has a functional role functional roles in DA-
in a given process tube expansion
15. Now let me tell you about two genes I
identified with these approaches:
Paxillinencodes a regulator of cell
migration and adhesion
mirror encodes a transcription factor
with known roles in patterning
Neither of these genes have described
roles in tube expansion
17. Our lab has also developed a protocol
for dual FISH:Immunostaining in the
Drosophila ovary, to simultaneously
visualize gene expression (mRNA) and
protein. We can use this to label DA-
tubes, with a reporter protein, and
confirm that PaxillinmRNA is expressed
in DA-tube cells.
18. Indeed, Paxillinis expressed in DA-tube
cells (rhomboid-lacZ); expression peaks
(S12) prior to DA-tube expansion (S13+)
19. Additionally, PaxillinRNAi disrupts
DA-tube expansion and indicates that
Paxillinand twk interact
+/+ +/twk
DA Defects: N = % Normal M = % Moderate S = % Severe
20. So, Paxillinis very interesting and
appears to play a role in DA-tube
expansion downstream of
Tramtrack69.
Now, what about mirror?
21. mirror expresses in DA-tube cells prior
to Paxillin(S10B)and during S13-14
S10B
Tramtrack69
WT twk regulates
mirror-mRNA DNA mirror
expression
S14
WT twk
22. mirror RNAi disrupts DA tube
expansion, resembling twk, without
affecting embryonic patterning
(arrowheads) or viability (not shown)
DA Defects: N = % Normal M = % Moderate S = % Severe
23. The level of mirrorexpression during
late oogenesis regulates the degree of
DA-tube expansion Gain of Function)
(GOF means
30°
(stronger)
25°
(weaker)
DA Defects: N = % Normal M = % Moderate S = % Severe
24. Since mirror regulates tube expansion
and is expressed before Paxillin, could
Mirror regulate Paxillinexpression?
S11 S12
Control
(WT)
PaxillinFISH PaxillinFISH
mirror
RNAi
Yes!
PaxillinFISH PaxillinFISH
25. If Paxillinis downstream of mirror, can
over-expression of Paxillinsuppress the
mirror RNAi phenotype?
Yes!
DA Defects: N = % Normal M = % Moderate S = % Severe
26. Tramtrack69 regulates DA-tube
expansion in the Drosphilaovary.
So far, I have identified several of the
Tramtrack69 effectors, mirror and
Paxillin, which interact and play
important roles in DA-tube expansion.
I will continue to identify more TTK69
effectors and determine how they fit
into this network.
27. Rachael French Graduate Committee
The Berg Lab Michael Boyle David Raible Stephen Hauschka
Celeste Berg Scott Kerr
Barbara WakimotoValeriVasioukhin
Faith Hassinger
Sandra Zimmerman
Robert Matlock
Fly Stocks
Center for Dan Kiehart
Array Technologies HanneleRuohola-Baker
Rob Hall Thank You! Helen McNeil
Kurt Hardesty Susan Parkhurst
TrudiSchüpbach
KeckMicroscopy Resource
Center &Stock Centers
Greg Martin DGRC, NIG-Fly, VDRC,BSC
