Postdoctoral work on Vangl2, a protein involved in cell polarity. Cell polarity is the capability of a cell to be able to orient itself in a particular direction.

1. Alysia vandenBerg
Vangl2 functions inVangl2 functions in
non-canonical Wntnon-canonical Wnt
signaling in Femalesignaling in Female
Reproductive TractReproductive Tract
DevelopmentDevelopment

2. The role of Vangl2, a non-canonical Wnt signaling
protein, in Female Reproductive Tract development
> 273,000 deaths worldwide from cervical cancer each year> 273,000 deaths worldwide from cervical cancer each year
accounting for 9% of female cancer deathsaccounting for 9% of female cancer deaths
> 190,000 new cases of ovarian cancer worldwide each year,> 190,000 new cases of ovarian cancer worldwide each year,
accounting for around 4% of all cancers diagnosed in womenaccounting for around 4% of all cancers diagnosed in women
> 50,300 deaths from corpus uteri cancer worldwide in 2002> 50,300 deaths from corpus uteri cancer worldwide in 2002
Infertility: Most US health insurance plans do not cover infertilityInfertility: Most US health insurance plans do not cover infertility
treatments one round of in vitro fertilization costs ~ $12,440treatments one round of in vitro fertilization costs ~ $12,440

3. Epithelial-mesenchymal communication guidesEpithelial-mesenchymal communication guides
FFemaleemale RReproductiveeproductive TTract (ract (FRTFRT) development) development
anterior

4. anterior
•signal from mesenchyme
regulates epithelium
Epithelial-mesenchymal communication guidesEpithelial-mesenchymal communication guides
FFemaleemale RReproductiveeproductive TTract (ract (FRTFRT) development) development

5. anterior
•signal from epithelium
controls mesenchyme
Epithelial-mesenchymal communication guidesEpithelial-mesenchymal communication guides
FFemaleemale RReproductiveeproductive TTract (ract (FRTFRT) development) development
WntsWnts Wnt4, Wnt5a,Wnt4, Wnt5a,
Wnt7a, Wnt11Wnt7a, Wnt11

6. TCF/LEF1-dependentTCF/LEF1-dependent
transcriptiontranscription
Wnt signaling pathwayWnt signaling pathway
CanonicalCanonical
FzFz
ß-catenin
Wnt
DshDsh
ß-cateninß-catenin
ß-catenin
destruction complex
Canonical Wnt reporter activityCanonical Wnt reporter activity
Before birth- 2 weeks after birthBefore birth- 2 weeks after birth
NON-CanonicalNON-Canonical

7. TCF/LEF1-dependentTCF/LEF1-dependent
transcriptiontranscription
Wnt
FzFz
Morphological changes/Morphological changes/
Cell motilityCell motility
DshDsh
Vangl2Vangl2
Wnt signaling pathwayWnt signaling pathway
CanonicalCanonical NON-CanonicalNON-Canonical
RhoA
JNKJNK
ROCKROCK
cytoskeletoncytoskeleton
Daam1
Planar Cell Polarity,Planar Cell Polarity,
Convergent ExtensionConvergent Extension
FzFz
Wnt
DshDsh

8. TCF/LEF1-dependentTCF/LEF1-dependent
transcriptiontranscription
Morphological changes/Morphological changes/
Cell motilityCell motility
DshDsh
Wnt signaling pathwayWnt signaling pathway
CanonicalCanonical
RhoA
JNKJNK
ROCKROCK
cytoskeletoncytoskeleton
Daam1/2
Planar Cell Polarity,Planar Cell Polarity,
Convergent ExtensionConvergent Extension
FzFz
Wnt
DshDsh
NON-CanonicalNON-Canonical
Wnt
FzFz Vangl2Vangl2

9. Proximal Distal
Proper asymetric Localization is necessary for function
of PCP signaling proteins
Drosophila
ProximalDistal
FrizzledFrizzledVangl2Vangl2
actinactin
Mice

10. ReproductiveReproductive
maturitymaturity
Using the mouse to studyUsing the mouse to study
FFemaleemale RReproductiveeproductive TTract (ract (FRTFRT))
developmentdevelopment
in mice, post-natal development continues for 2 months
day60day60birth/day 0birth/day 0
ProliferationProliferation
differentiationdifferentiation

11. ReproductiveReproductive
maturitymaturity
Using the mouse to studyUsing the mouse to study
FFemaleemale RReproductiveeproductive TTract (ract (FRTFRT))
developmentdevelopment
day60day60birth/day 0birth/day 0
glandsglands
formform
day 3day 3
ProliferationProliferation
differentiationdifferentiation

12. ReproductiveReproductive
maturitymaturity
Using the mouse to studyUsing the mouse to study
FFemaleemale RReproductiveeproductive TTract (ract (FRTFRT))
developmentdevelopment
day60day60day21day21birth/day 0birth/day 0
glandsglands
formform
~day15~day15day 3day 3
SmoothSmooth
musclesmuscles
formform
ProliferationProliferation
differentiationdifferentiation

13. Vangl2Vangl2
loop-tailloop-tail
S464NS464N*D255ED255E **
Hypothesis: Non-Canonical Wnt signalingHypothesis: Non-Canonical Wnt signaling
plays a role in FRT developmentplays a role in FRT development
Looptail mouse is tool for focusing on NON-canonical WNTsLooptail mouse is tool for focusing on NON-canonical WNTs
Unstable proteinUnstable protein

14. Gross morphology at birthGross morphology at birth
WT Lp mutant 2.5x mutant zoom
A B C
ovi ovi
bl
ut
ut
2mm

15. WT
Gross morphology ~8 weeksGross morphology ~8 weeks
Lp/+ heterozygous
5mm

16. wt
lp/lp
Looptail mice haveLooptail mice have
disrupted uterinedisrupted uterine
tissue organizationtissue organization
even at birtheven at birth

17. Clues that non-canonical Wnt signaling happensClues that non-canonical Wnt signaling happens
in the Uterusin the Uterus
•wnt7wnt7 mutants have changes in cell orientation (even at birth)mutants have changes in cell orientation (even at birth)
Loss of Wnts has lasting consequences upon FRT development
Development 131, 2061-2072 (2004)

18. Vangl2Vangl2 protein is concentrated at membrane of epithelial cellsprotein is concentrated at membrane of epithelial cells
Vangl2 protein localizes to the uterine epitheliumVangl2 protein localizes to the uterine epithelium
WTWT looptaillooptail
WTWT looptaillooptail
18.5 DPC18.5 DPC

19. Planar Cell Polarity defect in Looptail mice causes
mislocalization of PCP protein complexes
Is Vangl2 itself asymetrically localized?

20. 1Mo
2Mo
1Mo
2Mo
Vangl2Vangl2 protein is concentrated at apical membrane of epithelialprotein is concentrated at apical membrane of epithelial
cells and localizes to glands!!!cells and localizes to glands!!!

21. ActinActin
Prediction: Planar Cell Polarity defect in Looptail mice
causes mislocalization of PCP protein complexes
in the uterus
Morphological changes/Morphological changes/
Cell motilityCell motility
RhoA
JNKJNK
ROCKROCK
cytoskeletoncytoskeleton
Daam1
Wnt
FzFz
Vangl2Vangl2
DisheveledDisheveled

22. WT
Alexa647-phalloidin
Lp/Lp
Looptail mutants have actin polarity defect
in 18.5 DPC Uterus

23. WT
Looptail mutants have actin polarity defect
in 18.5 DPC Uterus
Lp/Lp

24. Morphological changes/Morphological changes/
Cell motilityCell motility
DshDsh
RhoA
JNKJNK
ROCKROCK
cytoskeletoncytoskeleton
Daam1
Wnt
FzFz
E-cadherinE-cadherin
Prediction: Planar Cell Polarity defect in Looptail mice
causes mislocalization of PCP protein complexes
in the uterus
•associated w/loss of cell
polarity in cancers
•involved in cell movement
Vangl2Vangl2
E-cadherinE-cadherin
B-catenin

25. E-cadherin staining reveals defect in cellular
architecture of Looptail mutant Uterui
WT 18.5 dpc Lp/Lp 18.5 dpc

26. WT Lp/Lp
E-cadherin staining reveals defect in cellular
architecture of Looptail mutant Uterui
Are there tight junction defects in Lp epithelium?Are there tight junction defects in Lp epithelium?

27. Morphological changes/Morphological changes/
Cell motilityCell motility
RhoA
JNKJNK
ROCKROCK
cytoskeletoncytoskeleton
Daam1
Wnt
FzFz Vangl2Vangl2
Prediction: Planar Cell Polarity defect in Looptail mice
causes mislocalization of PCP protein complexes
in the uterus
ScribbleScribble
•scribblescribble mutantsmutants
mislocalize Vangl2mislocalize Vangl2
•Vangl2 IPVangl2 IP’’s with Scribs with Scrib
•4 PDZ domains4 PDZ domains
•Apical/basal polarityApical/basal polarity
•Adherins junctionsAdherins junctions
DisheveledDisheveled

28. Lp/Lp mutant uteri have increased scribble localizationLp/Lp mutant uteri have increased scribble localization
to apical domain of epitheliumto apical domain of epithelium
scribble is normally
basolaterally
restricted
WT d18.5 Lp/Lp d18.5

29. Xenografting experiment to observeXenografting experiment to observe
post-natal uterine developmentpost-natal uterine development
•Take WT & Lp/Lp mutant uterine tissue embyronic D18.5Take WT & Lp/Lp mutant uterine tissue embyronic D18.5
•Place WT tissue in right kidney capsule & Lp in leftPlace WT tissue in right kidney capsule & Lp in left
•ovarectomized Nu mouse mimics normal FRT developmentovarectomized Nu mouse mimics normal FRT development

30. Looptail mutant xenografts develop abnormallyLooptail mutant xenografts develop abnormally
(2 weeks)(2 weeks)
wild type graftwild type graft Lp/+ heterozygoteLp/+ heterozygote
gl?gl?
secretionssecretions
• abnormal epithelial morphologyabnormal epithelial morphology
• few glandsfew glands
• epitheliumepithelium
• glandsglands
• mesenchymemesenchyme
• musclemuscle
glglmesen.mesen.
muscle
muscle

31. Looptail mutant xenografts develop abnormallyLooptail mutant xenografts develop abnormally
wild type graftwild type graft Lp/+ heterozygoteLp/+ heterozygote
glands?glands?
musclemuscle
epi nuclearepi nuclear
positionposition
• abnormal epithelial morphologyabnormal epithelial morphology
• few glandsfew glands
glglmesen.mesen.
muscle
muscle
• epitheliumepithelium
• glandsglands
• mesenchymemesenchyme
• musclemuscle

32. glglmesen.mesen.
muscle
muscle
Looptail mutant xenografts develop abnormallyLooptail mutant xenografts develop abnormally
Lp mutantLp mutantwild type graftwild type graft
gl?gl?
mesen.mesen.
protrusionprotrusion
muscle
muscle
• abnormal epithelial morphologyabnormal epithelial morphology
• few glandsfew glands
• big lumenbig lumen
• "protrusions""protrusions"
• epitheliumepithelium
• glandsglands
• mesenchymemesenchyme
• musclemuscle

33. glglmesen.mesen.
muscle
muscle
Looptail mutant xenografts develop abnormallyLooptail mutant xenografts develop abnormally
Lp mutantLp mutantwild type graftwild type graft
• abnormal epithelial morphologyabnormal epithelial morphology
• few glandsfew glands
• big lumenbig lumen
• "protrusions""protrusions"
• epitheliumepithelium
• glandsglands
• mesenchymemesenchyme
• musclemuscle

34. Looptail mutant xenografts develop abnormallyLooptail mutant xenografts develop abnormally
Lp mutantLp mutantwild type graftwild type graft
•Lp have many abnormal epithelial cellsLp have many abnormal epithelial cells
•very high # of pseudo-stratified epithelial cellsvery high # of pseudo-stratified epithelial cells

35. Looptail mutant xenografts develop abnormallyLooptail mutant xenografts develop abnormally
Lp mutantLp mutant
•Lp have many abnormal epithelial cellsLp have many abnormal epithelial cells
•very high # of pseudo-stratified epithelial cellsvery high # of pseudo-stratified epithelial cells
Lp mutantLp mutantWTWT

36. Xenografts 2
weeks
WTWT Lp graftLp graft
•E-cadherin
•Actin
•nuclei

37. Xenografts 2 weeks- Oil Red stain
WT Lp heterozygote Lp mutant

38. Loss of Wnts affects uterine developmentLoss of Wnts affects uterine development
wnt7awnt7a mutants are sterilemutants are sterile
Wnt7a-/-Wnt7a-/-WTWT
uterine cytological changes at 2 months :uterine cytological changes at 2 months :
•Expanded and stratified epithelial layerExpanded and stratified epithelial layer
•Expanded smooth muscle layerExpanded smooth muscle layer
•No glandsNo glands
uterine cytological changes at 2 months :uterine cytological changes at 2 months :
•Expanded and stratified epithelial layerExpanded and stratified epithelial layer
•Expanded smooth muscle layerExpanded smooth muscle layer
•No glandsNo glands
Wnt7a +/-Wnt7a +/-
uterine cytological changes at 2 months :uterine cytological changes at 2 months :
•Expanded and stratified epithelial layerExpanded and stratified epithelial layer
•Expanded smooth muscle layerExpanded smooth muscle layer
•No glandsNo glands
•Less Wnt7a, more glands formLess Wnt7a, more glands form
Development. 1998
Aug;125(16):3201-11.

39. Wnt signaling appears to be involved in gland formationWnt signaling appears to be involved in gland formation
Mathias Mericskay

40. Morphological changes/Morphological changes/
Cell motilityCell motility
Wnt
Fz1Fz1
Vangl2 functions in FRT development
Vangl2 protein is apically enriched
and localizes to glandular membranes
Vangl2Vangl2

41. ScribbleScribble
Morphological changes/Morphological changes/
Cell motilityCell motility
Wnt
Fz1Fz1
Lp mutants have defects in actin polarization
and scribble protein distribution
Vangl2Vangl2
DshDsh
RhoA
JNKJNK
ROCKROCK
cytoskeletoncytoskeleton
Daam1
β-catenin

42. Morphological changes/Morphological changes/
Cell motilityCell motility
DshDsh
RhoA
JNKJNK
ROCKROCK
cytoskeletoncytoskeleton
Daam1
Wnt
Fz1Fz1
FUTURE STUDIES: tying it together
Vangl2Vangl2
ScribbleScribble
LGL2LGL2
aPKCaPKC
β-catenin
Adapted from
Annu.Rev.Cell Dev. Biol
2006.22:207-235
EarlyEarly
polaritypolarity
crumbs3crumbs3
Par3Par3

43. Morphological changes/Morphological changes/
Cell motilityCell motility
DshDsh
RhoA
JNKJNK
ROCKROCK
cytoskeletoncytoskeleton
Daam1
Wnt
Fz1Fz1
Vangl2Vangl2
ScribbleScribble
LGL2LGL2
aPKCaPKC
Cdc42Cdc42
β-catenin
Adapted from
Annu.Rev.Cell Dev. Biol
2006.22:207-235
VASPVASP
viniculinviniculin
CytoskeletalCytoskeletal
modulatorsmodulators
EarlyEarly
polaritypolarity
Par3Par3
crumbs3crumbs3
FUTURE STUDIES: tying it together

44. Morphological changes/Morphological changes/
Cell motilityCell motility
DshDsh
RhoA
JNKJNK
ROCKROCK
cytoskeletoncytoskeleton
Daam1
Wnt
Fz1Fz1
FUTURE STUDIES: Epithelial tight junction defects?
Vangl2Vangl2
TightjunctionTightjunction
ScribbleScribble
??
LGL2LGL2
Zo-1Zo-1
Zo-3Zo-3
Zo-2Zo-2
OccludinOccludin
claudinclaudin
JAM
Par6Par6 Par3Par3
PATJPATJ
PALS1PALS1
aPKCaPKC
Cdc42Cdc42
crumbs3crumbs3
CadherinsCadherins
β-catenin
Adapted from
Annu.Rev.Cell Dev. Biol
2006.22:207-235
VASPVASP
viniculinviniculin
CytoskeletalCytoskeletal
modulatorsmodulators
EarlyEarly
polaritypolarity
actin
actin

45. Morphological changes/Morphological changes/
Cell motilityCell motility
DshDsh
RhoA
JNKJNK
ROCKROCK
cytoskeletoncytoskeleton
Daam1
Wnt
Fz1Fz1
FUTURE STUDIES: Epithelial tight junction defects?
Vangl2Vangl2
TightjunctionTightjunction
ScribbleScribble
??
LGL2LGL2
Zo-1Zo-1
Zo-3Zo-3
Zo-2Zo-2
OccludinOccludin
claudinclaudin
JAM
Par6Par6 Par3Par3
PATJPATJ
PALS1PALS1
aPKCaPKC
Cdc42Cdc42
crumbs3crumbs3
CadherinsCadherins
β-catenin
Adapted from
Annu.Rev.Cell Dev. Biol
2006.22:207-235
VASPVASP
viniculinviniculin
CytoskeletalCytoskeletal
modulatorsmodulators
EarlyEarly
polaritypolarity
Scribble required for
basolateral exocytosis
Loss of beta-catenin fcn
associated lack of cadherin
actin-binding fucntion
•MAGI-3 linked with
RhoA activation
•In Lp heart 
ROCK  RhoA
actin
LGL2 knockdown-
abnormal cell aggregates
fill lumen in MDCK cells
& have abnormal actin
polarization
RAC1

46. Morphological changes/Morphological changes/
Cell motilityCell motility
DshDsh
RhoA
JNKJNK
ROCKROCK
cytoskeletoncytoskeleton
Daam1
Wnt
Fz1Fz1
FUTURE STUDIES: Epithelial tight junction defects?
Vangl2Vangl2
TightjunctionTightjunction
ScribbleScribble
??
LGL2LGL2
Zo-1Zo-1
Zo-3Zo-3
Zo-2Zo-2
OccludinOccludin
claudinclaudin
JAM
Par6Par6 Par3Par3
PATJPATJ
PALS1PALS1
aPKCaPKC
Cdc42Cdc42
crumbs3crumbs3
CadherinsCadherins
β-catenin
Adapted from
Annu.Rev.Cell Dev. Biol
2006.22:207-235
VASPVASP
viniculinviniculin
CytoskeletalCytoskeletal
modulatorsmodulators
EarlyEarly
polaritypolarity

47. Future ExperimentsFuture Experiments
• Full analysis of WT Vangl2 protein and mRNA localization
• Repeat looptail grafting
• In situs of looptail grafts: wnts, other PCP genes
* Does disruption of Vangl2 change wnt signaling in
either mesenchyme or epithelium?
* Examine gene expression of bona-fide Wnt7 targets
using tissue recombination experiments
• vangl2 mutant epithelium with WT mesenchyme
• WT epithelium with vangl2 mesenchyme
Description of Wild type Vangl2Description of Wild type Vangl2
Confirm looptail mutant analysisConfirm looptail mutant analysis
Are wnts (canonical signaling) affected inAre wnts (canonical signaling) affected in looptaillooptail mutantsmutants
Functional analysis of Vangl2Functional analysis of Vangl2

48. Acknowledgements
NYC Group Lab
Risheng Ma
Martina Schwarzkopf
Former Members
Mathias Mericskay
Cary Miller
Luca Carta
Claudia Cosmineau
Anna Pavlova
Maria Arufe
Cellular Imagery
PITIE-SALPETRIERE
Collaborators
Mirielle Montcouquiol
Matthew Kelley
Daniel Dufort
Phillipe Gros
Sergei Sokol & Lab
MSSM shared
microscopy facility
And many brave mice
Sassoon Lab
Ara Parlakian
Gisela Garcia
Giovanna Marazzi
Iman Gomaa
Kathryn Mitchell
Katia Castrillo
Nathalie Didier
Vanessa Besson
U787 members