GFP" Staining"

1. How CRE effects dsx expression
to establish and modify
sexual dimorphic traits"
Presenter: Peng, Yi-Hung"
PI: Artyom Copp"
♀
♂

2. Background"
of Drosophila’s Sexual Dimorphism
and dsx (doublesex gene)"

3. Sexual Dimorphism"
The differences between Male and Female
(性別二型性)"

4. Model Organism:
Drosophila melanogaster"
A kind of fruit flies"

5. ♂
Sex Comb (性梳)"
The sexual Dimorphic trait of
Male D. melanogaster"

6. ♀
No Sex Comb"

7. Sex Comb is a good example of
Sexual Dimorphism!
What causes this?!

8. dsx!
Drosophila double sex gene"

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. ♂
♀
dsx M
dsx F
Male" Origin" Female"
dsx s role"
in D. melanogaster is to break down
intralocus sexual conflict in the other."
Tanaka el al PLOS Biology Reviews 2011

11. How"dsx break down "
intralocus sexual conflict?"

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. 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. 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. Something else determined
the expression of dsx!

16. CRE"
cis Regulatory Element
of Drosophila double sex gene"

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!

19. Other"
Drosophila species’ dsx expression"

20. D. willistoni
D. virilis (pic D,hydei)
The sexually dimorphic structure of the sex combs
differ greatly over a "
Short evolutionary time"
Kopp, EVOLUTION & DEVELOPMENT 2012

21. Kopp, EVOLUTION & DEVELOPMENT 2012

22. melanogaster
dsx expression is correlated with sex comb "
not only in Drosophila melanogaster "
but also with these closely related species."
Kopp, EVOLUTION & DEVELOPMENT 2012

23. 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?"

24. To know the answer, we need to"
Compare the CREs of dsx
between species of Drosophila"

25. Under the model of the sex comb, there are
Two 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."

26. 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 "

27. 1" 2"
3" 4"

28. Methods"
Of Enhancer Bashing (Looking for CRE) "

29. 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 "

30. UAS-GFP! X! Different
Collect Virgin intron - Gal4!
Female Only!
♀
♂
Collect F1 (First offspring generation) Only!

31. 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)

32. 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! +

33. Crossing Lines"
of intron-Gal4 line and UAS-GFP line"

34. Collection: White Prepupal
Collect 2 developmental stages: "
5 hr. & 24 hr."
Pupal"
Primary Dissection and Fixation
Add Paraformaldehyde
for Fixation"

35. Dissection
Dissecting 2 developmental stages: "
5 hr. & 24 hr."
Dissection Work! 5 hr Pupal's Leg! 24 hr Pupal's Leg!

36. Slides Making"
Different Gender, Developmental Stage and
Different Lines!
Polylysine slide!

38. Results"
of Enhancer Bashing (Looking for CRE) "

39. 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 "

40. dsx gene"
dsx intron 3.1
(first 5 kb)"
Male, 24 hours"
Antibody
GFP"
Staining"

41. dsx gene"
dsx intron 3.1
the 0-3 kb!
dsx intron 3.1
the 2-5 kb!

42. dsx gene"
dsx intron 3.1
the 0-3 kb!
dsx intron 3.1
the 2-5 kb!
Male, 36 hours"
Antibody
GFP"
Staining"

43. dsx gene"
dsx intron 3.1
the 0-3 kb!
dsx intron 3.1
the 2-5 kb!
Male, 36 hours"
Antibody
GFP"
Staining"

44. 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"

45. How about the other introns?"

46. doublesex and the Evolution of Sexual Dimorphism
5 hr PLoS Biology | www.plosbiology.org 3 August 2011 | Volume 9 | Issue 8 | e1001131
Developemental Stage"
Previous study suggested that the
dsx Do express in this stage."

47. We got nothing in "
5hr Developmental Stage!
So, we look into 24 hr
Developmental Stage……!
!

48. 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"

49. ti 1" ti 1"
dsx intron 2 ti 1"
Female, 24 hours" ti 2"
ti 2" ti 2" (Previous)
Video" GFP" Antibody Staining"

50. 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"

51. CRE in dsx intron 2?"
Different Pattern of Expression and
Monomorphic characteristic
“might” due to the effect of"
Shadow Enhancer"

52. Future Work"
of Enhancer Bashing (Looking for CRE) "

53. 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"

54. 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"

55. Artyom Kopp" Gavin Rice"
Acknowledgements"
Olga
Barmina"

56. Thank you"