UC Davis GREAT Program Final Presentation

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The Final Presentation Slides of UC Davis, GREAT Program

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UC Davis GREAT Program Final Presentation

  1. 1. How CRE effects dsx expression 
 to establish and modify 
 sexual dimorphic traits" Presenter: Peng, Yi-Hung" PI: Artyom Copp"♀ ♂
  2. 2. Background"of Drosophila’s Sexual Dimorphism and dsx (doublesex gene)"
  3. 3. Sexual Dimorphism"The differences between Male and Female
 (性別二型性)"
  4. 4. Model Organism:
Drosophila melanogaster" A kind of fruit flies"
  5. 5. ♂ Sex Comb (性梳)" The sexual Dimorphic trait of Male D. melanogaster"
  6. 6. ♀ No Sex Comb"
  7. 7. Sex Comb is a good example of 
 Sexual Dimorphism! What causes this?!
  8. 8. dsx!Drosophila double sex gene"
  9. 9. XY XX ♂ Sxl tra ♀ Sxl tra fru M fru dsx M F dsx dsx (double sex gene) "establishes and modifies sexual dimorphic traits of Drosophila melanogaster" Greenspan et al. Annual Review of Genetics 2000
  10. 10. ♂ ♀ dsx M dsx F Male" Origin" Female"dsx s role"in D. melanogaster is to break downintralocus sexual conflict in the other." Tanaka el al PLOS Biology Reviews 2011
  11. 11. How"dsx break down "intralocus sexual conflict?"
  12. 12. Family of!DM domain 
 genes! Share a common DNA binding domain! dsx is in the family of DM domain genes" DM domain genes share the same characteristic DM DNA binding site; dsxM and dsxF both share a common binding domain"
  13. 13. Common DNA binding domain
 DSX F & DSX M"DSX F/M! Bind, Control, 
 Expression or Repression! 5’! 3’! Gene’s binding site for DSX!A gene must gain a binding site for dsx to be expressed sex specifically."
  14. 14. On the other hand…" The DSX (protein product of dsx gene) is 
 Not expressed in all cells" DSX Expression Region!Robinett el al PLOS Biology Reviews 2010
  15. 15. Something else determined the expression of dsx!
  16. 16. CRE" cis Regulatory Element 
of Drosophila double sex gene"
  17. 17. CREs for dsx gene! dsx gene!5’! 3’! + CRE enhance dsx gene expression! DSX is produced! Common binding domain
 DSX F/M! Dsx F & Dsx M! Bind, Control, 
 Expression or Repression! Roles of CREs 
 (Cis Regulatory Elements)" 5’! 3’! Gene’s binding site for dsx!
  18. 18. Other"Drosophila species’ dsx expression"
  19. 19. D. willistoniD. virilis (pic D,hydei)The sexually dimorphic structure of the sex combsdiffer greatly over a "Short evolutionary time" Kopp, EVOLUTION & DEVELOPMENT 2012
  20. 20. Kopp, EVOLUTION & DEVELOPMENT 2012
  21. 21. melanogaster dsx expression is correlated with sex comb "not only in Drosophila melanogaster "but also with these closely related species." Kopp, EVOLUTION & DEVELOPMENT 2012
  22. 22. This raises the question," Does dsx simply break down interlocus sexual conflict……" " Or does it also play a role in regulation within a sex in determining the optimal spatial, temporal, and level of expression?"
  23. 23. To know the answer, we need to" Compare the CREs of dsx between species of Drosophila"
  24. 24. Under the model of the sex comb, there areTwo paths:" Enhancer Bashing" Establish the minimal CRE within D. mel" to see if there is any CRE inside these introns " Testing of the homologous regions" Establish the approximate region with species within the melanogaster and obscura species groups."
  25. 25. dsx gene"Exons (Coding Regions in dsx)! Introns (Non-Coding Regions in dsx)! Enhancer Bashing" to see if there is any CRE inside these introns "
  26. 26. 1" 2"3" 4"
  27. 27. Methods"Of Enhancer Bashing (Looking for CRE) "
  28. 28. dsx gene"Exons (Coding Regions in dsx)! Introns (Non-Coding Regions in dsx)! Intron 1" PCR! Add Gal4 
 Intron 2" (a Transcription Factor gene) 
 Intron 3.1" into different introns 
 Intron 3.2" of dsx! Intron 3.3" Gal4! Intron 3.4" Intron 3.5" Intron 4" Create Different 
 Intron 5" intron - Gal4 Line 
 D. melanogaster "
  29. 29. UAS-GFP! X! Different 
Collect Virgin
 intron - Gal4!Female Only! ♀ ♂ Collect F1 (First offspring generation) Only!
  30. 30. F1 (First offspring generation) Genotype"5’! Chromosome from Gal4 Line! 3’! Different intron position of dsx! Gal4! X!5’! Chromosome from UAS-GFP Line! 3’! UAS! GFP gene! *GFP: Green fluorescent protein (綠螢光蛋白)
  31. 31. F1 (First offspring generation) Genotype" Different intron 
 position of dsx! Activate!5’! With CRE" + 3’! Different intron position of dsx! Gal4! Produce Gal4! Bind and Gal4" activate UAS!5’! 3’! UAS! GFP gene! Activate! +
  32. 32. Crossing Lines"of intron-Gal4 line and UAS-GFP line"
  33. 33. Collection: White Prepupal Collect 2 developmental stages: "5 hr. & 24 hr." Pupal"Primary Dissection and Fixation Add Paraformaldehyde
for Fixation"
  34. 34. Dissection Dissecting 2 developmental stages: "5 hr. & 24 hr." Dissection Work! 5 hr Pupals Leg! 24 hr Pupals Leg!
  35. 35. Slides Making"Different Gender, Developmental Stage andDifferent Lines! Polylysine slide!
  36. 36. Results"of Enhancer Bashing (Looking for CRE) "
  37. 37. Exons (Coding Regions in dsx)! Introns (Non-Coding Regions in dsx)! dsx gene" Previous Work 
 by Gavin Rice! Enhancer Bashing 
 by Peng, Yi-Hung![79]! [51]! [64]! [12A]! [27c]! [12]! [ 8 ]!Enhancer Bashing"to see if there is any CRE inside these introns "
  38. 38. dsx gene" dsx intron 3.1 
 (first 5 kb)" Male, 24 hours" Antibody GFP" Staining"
  39. 39. dsx gene"dsx intron 3.1 
the 0-3 kb!dsx intron 3.1 
the 2-5 kb!
  40. 40. dsx gene"dsx intron 3.1 
the 0-3 kb!dsx intron 3.1 
the 2-5 kb! Male, 36 hours" Antibody GFP" Staining"
  41. 41. dsx gene"dsx intron 3.1 
the 0-3 kb!dsx intron 3.1 
the 2-5 kb! Male, 36 hours" Antibody GFP" Staining"
  42. 42. dsx intron 3.1 
the 0-3 kb! So we are sure that there is a " CRE in dsx intron 3.1 the 0-3kb"
  43. 43. How about the other introns?"
  44. 44. doublesex and the Evolution of Sexual Dimorphism5 hr 
 PLoS Biology | www.plosbiology.org 3 August 2011 | Volume 9 | Issue 8 | e1001131Developemental Stage"Previous study suggested that thedsx Do express in this stage."
  45. 45. We got nothing in "5hr Developmental Stage!So, we look into 24 hr 
 Developmental Stage……! !
  46. 46. dsx gene" [79]! [51]! [64]! [12A]! [27c]! [12]! [ 8 ]! ti 1" ti 1" dsx intron 2 
 ti 2" ti 2" ti 1" Male, 24 hours" ti 2" (Previous) Video" GFP" Antibody Staining"
  47. 47. ti 1" ti 1"dsx intron 2 
 ti 1"Female, 24 hours" ti 2" ti 2" ti 2" (Previous) Video" GFP" Antibody Staining"
  48. 48. dsx intron 2 
 Male and Female, 24 hours"GFP" ti 1" ti 2" ti 3" ti 4" ti 5"Video" ti 1" ti 2" ti 3" ti 4" ti 5"
  49. 49. CRE in dsx intron 2?" Different Pattern of Expression and Monomorphic characteristic “might” due to the effect of" Shadow Enhancer"
  50. 50. Future Work"of Enhancer Bashing (Looking for CRE) "
  51. 51. ti 1" ti 1" New 
 Antibody ti 1" ti 1" Staining for Reference"ti 2" ti 2" ti 1" ti 2" ti 2" ti 2" (Previous) Video" GFP" Video" GFP" Antibody Staining" ♂ ♀ Antibody Staining"
  52. 52. dsx gene" Previous Work 
 by Gavin Rice! Enhancer Bashing 
 by Peng, Yi-Hung! [79]! [51]! [64]! [12A]! [27c]! …… …… …… …… [12]! …… [ 8 ]! dsx must also exist in in 5hr places ! No CRE was found other Stage! Future Work!Further Enhancer Bashing"
  53. 53. Artyom Kopp" Gavin Rice"Acknowledgements" Olga 
 Barmina"
  54. 54. Thank you"

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