Tramtrack69 regulates the expansion of cellular tubes in the Drosophila ovary       through Mirror and Paxillin           ...
Cellular tubes are the plumbing for organs and tissues in multicellular              organisms During development, cells u...
Tube morphogenesis requires 3 events          Tube patterning          Tube formation          Tube expansion             ...
In the Drosophila ovary, egg chambers     develop through 14 stages of              oogenesis Adapted from Zartmanet al., ...
DA-tube morphogenesis a useful        model for tube morphogenesisAdapted from Zartmanet al., 2010                        ...
The tramtrackgene encodes two        transcription factors:    Tramtrack69 &Tramtrack88                                   ...
The twin peaks (twk) mutation  disrupts expression of Tramtrack69specifically during oogenesis, when it is    needed for D...
Loss of Tramtrack69 in twkdisrupts DA-       tube expansion (S13-14),          forming stunted DAs     DM = Dorsal midline...
My Question:What are the Tramtrack69 effectors required for epithelial tube expansion in the ovary?
Let me now introduce you to my    experimental approach for  identifying and characterizingTTK69-regulated genes with role...
First, I dissected ovaries   (AffymetrixDrosophila 2.0                                    microarrays, from wild type and ...
Our lab has developed a protocol forfluorescence in situ hybridization (FISH) in the Drosophila ovary, allowing us to  pre...
Using this technique, I validated my microarrays and identified geneswith expression in the DA-tube cells             (lik...
RNAi disrupts mRNA        By expressing RNAi    translation for an      against differentially individual gene and can exp...
Now let me tell you about two genes I identified with these approaches:  Paxillinencodes a regulator of cell       migrati...
Paxillinexpresses in DA-tube cells and is       downstream of Tramtrack69  WT        Paxillin-mRNA (FISH)   DNA  twk
Our lab has also developed a protocol  for dual FISH:Immunostaining in the  Drosophila ovary, to simultaneously visualize ...
Indeed, Paxillinis expressed in DA-tubecells (rhomboid-lacZ); expression peaks(S12) prior to DA-tube expansion (S13+)
Additionally, PaxillinRNAi disrupts DA-tube expansion and indicates that       Paxillinand twk interact+/+                ...
So, Paxillinis very interesting andappears to play a role in DA-tube    expansion downstream of           Tramtrack69.   N...
mirror expresses in DA-tube cells prior to Paxillin(S10B)and during S13-14  S10B                                    Tramtr...
mirror RNAi disrupts DA tube expansion, resembling twk, without   affecting embryonic patterning(arrowheads) or viability ...
The level of mirrorexpression during  late oogenesis regulates the degree of           DA-tube expansion Gain of Function)...
Since mirror regulates tube expansionand is expressed before Paxillin, could  Mirror regulate Paxillinexpression?         ...
If Paxillinis downstream of mirror, canover-expression of Paxillinsuppress the        mirror RNAi phenotype?              ...
Tramtrack69 regulates DA-tube    expansion in the Drosphilaovary. So far, I have identified several of the   Tramtrack69 e...
Rachael French                Graduate CommitteeThe Berg Lab          Michael Boyle            David Raible       Stephen ...
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  1. 1. Tramtrack69 regulates the expansion of cellular tubes in the Drosophila ovary through Mirror and Paxillin Nathaniel Peters Berg Lab University of Washington
  2. 2. Cellular tubes are the plumbing for organs and tissues in multicellular organisms During development, cells undergomorphogenesis (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. 3. Tube morphogenesis requires 3 events Tube patterning Tube formation Tube expansion Adapted from Lubarsky and Krasnow, 2003
  4. 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 EggDorsal respiratory appendages (DAs) of the matureegg help the embryo to breathe. They are moldedfrom the insides of cellular tubes during oogenesis
  5. 5. DA-tube morphogenesis a useful model for tube morphogenesisAdapted from Zartmanet al., 2010 Dorsal Dorsal Anterior Posterior Appendage VentralS6 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. 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 sitesOnly Tramtrack69 is required for DA-tube expansion during oogenesis
  7. 7. The twin peaks (twk) mutation disrupts expression of Tramtrack69specifically 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. 8. Loss of Tramtrack69 in twkdisrupts DA- tube expansion (S13-14), forming stunted DAs DM = Dorsal midline DA Tube Cells DA Tube
  9. 9. My Question:What are the Tramtrack69 effectors required for epithelial tube expansion in the ovary?
  10. 10. Let me now introduce you to my experimental approach for identifying and characterizingTTK69-regulated genes with roles in DA-tube expansion
  11. 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 differentiallyexpressed genes in twk.~1000 genes were differentially expressed in twk. I selected a subset of these genes for further expression and functional analysis.
  12. 12. Our lab has developed a protocol forfluorescence 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. 13. Using this technique, I validated my microarrays and identified geneswith expression in the DA-tube cells (like above)
  14. 14. RNAi disrupts mRNA By expressing RNAi translation for an against differentially individual gene and can expressed genes in twk, Ibe used to test whether a distinguished genes withgene has a functional role functional roles in DA- in a given process tube expansion
  15. 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 patterningNeither of these genes have described roles in tube expansion
  16. 16. Paxillinexpresses in DA-tube cells and is downstream of Tramtrack69 WT Paxillin-mRNA (FISH) DNA twk
  17. 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, andconfirm that PaxillinmRNA is expressed in DA-tube cells.
  18. 18. Indeed, Paxillinis expressed in DA-tubecells (rhomboid-lacZ); expression peaks(S12) prior to DA-tube expansion (S13+)
  19. 19. Additionally, PaxillinRNAi disrupts DA-tube expansion and indicates that Paxillinand twk interact+/+ +/twkDA Defects: N = % Normal M = % Moderate S = % Severe
  20. 20. So, Paxillinis very interesting andappears to play a role in DA-tube expansion downstream of Tramtrack69. Now, what about mirror?
  21. 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. 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. 23. The level of mirrorexpression during late oogenesis regulates the degree of DA-tube expansion Gain of Function) (GOF means30°(stronger)25°(weaker) DA Defects: N = % Normal M = % Moderate S = % Severe
  24. 24. Since mirror regulates tube expansionand is expressed before Paxillin, could Mirror regulate Paxillinexpression? S11 S12 Control (WT) PaxillinFISH PaxillinFISH mirror RNAi Yes! PaxillinFISH PaxillinFISH
  25. 25. If Paxillinis downstream of mirror, canover-expression of Paxillinsuppress the mirror RNAi phenotype? Yes!DA Defects: N = % Normal M = % Moderate S = % Severe
  26. 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 playimportant roles in DA-tube expansion. I will continue to identify more TTK69 effectors and determine how they fit into this network.
  27. 27. Rachael French Graduate CommitteeThe 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 KiehartArray 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

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