Role of Vangl2 in Female Reproductive Tract Development, 2008

Alysia vandenBerg

Vangl2 functions in Vangl2 functions in non-canonical Wnt non-canonical Wnt signaling in Female signaling in Female Reproductive Tract Reproductive Tract

DevelopmentDevelopment

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 yearaccounting for 9% of female cancer deathsaccounting for 9% of female cancer deaths

> 190,000 new cases of ovarian cancer worldwide each > 190,000 new cases of ovarian cancer worldwide each year, accounting for around 4% of all cancers diagnosed in year, accounting for around 4% of all cancers diagnosed in womenwomen

> 50,300 deaths from corpus uteri cancer worldwide in > 50,300 deaths from corpus uteri cancer worldwide in 20022002

Infertility: Most US health insurance plans do not cover Infertility: Most US health insurance plans do not cover infertility treatments one round of in vitro fertilization infertility treatments one round of in vitro fertilization costs ~ $12,440costs ~ $12,440

Epithelial-mesenchymal communication Epithelial-mesenchymal communication guides guides FFemale emale RReproductive eproductive TTract (ract (FRTFRT) )

developmentdevelopment

anterior

anterior

•signal from mesenchyme regulates epithelium

Epithelial-mesenchymal communication Epithelial-mesenchymal communication guides guides FFemale emale RReproductive eproductive TTract (ract (FRTFRT) )

developmentdevelopment

anterior

•signal from epithelium controls mesenchyme

Epithelial-mesenchymal communication Epithelial-mesenchymal communication guides guides FFemale emale RReproductive eproductive TTract (ract (FRTFRT) )

developmentdevelopment

WntWntss

Wnt4, Wnt5a,Wnt4, Wnt5a,Wnt7a, Wnt11Wnt7a, Wnt11

TCF/LEF1-dependentTCF/LEF1-dependenttranscriptiontranscription

Wnt signaling pathwayWnt signaling pathway CanonicalCanonical

FzFz

ß-catenin

Wnt

DshDsh

ß-cateninß-catenin

ß-catenindestruction complex

Canonical Wnt reporter Canonical Wnt reporter activity Before birth- 2 weeks activity Before birth- 2 weeks

after birthafter birth

NON-CanonicalNON-Canonical

TCF/LEF1-dependentTCF/LEF1-dependenttranscriptiontranscription

WntFzFz

Morphological Morphological changes/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 Extension Convergent Extension

FzFzWnt

DshDsh

TCF/LEF1-dependentTCF/LEF1-dependenttranscriptiontranscription

Morphological Morphological changes/changes/

Cell motilityCell motility

DshDsh

Wnt signaling pathwayWnt signaling pathway CanonicalCanonical

RhoA

JNKJNK

ROCKROCK

cytoskeletoncytoskeleton

Daam1/2

Planar Cell Polarity,Planar Cell Polarity, Convergent Extension Convergent Extension

FzFzWnt

DshDsh

NON-CanonicalNON-Canonical Wnt

FzFz Vangl2Vangl2

Proximal Distal

Proper asymetric Localization is necessary for function

of PCP signaling proteins

Drosophila

Prox

imal

Dis

tal

FrizzledFrizzledVangl2Vangl2

actinactin

Mice

Reproductive Reproductive maturitymaturity

Using the mouse to studyUsing the mouse to studyFFemale emale RReproductive eproductive TTract (ract (FRTFRT) )

developmentdevelopment

in mice, post-natal development continues for 2 months

day60day60birth/day 0birth/day 0

Proliferation Proliferation differentiationdifferentiation

Reproductive Reproductive maturitymaturity

Using the mouse to studyUsing the mouse to studyFFemale emale RReproductive eproductive TTract (ract (FRTFRT) )

developmentdevelopment

day60day60birth/day 0birth/day 0

glandsglandsformform

day 3day 3

Proliferation Proliferation differentiationdifferentiation

Reproductive Reproductive maturitymaturity

Using the mouse to studyUsing the mouse to studyFFemale emale RReproductive eproductive TTract (ract (FRTFRT) )

developmentdevelopment

day60day60day21day21birth/day 0birth/day 0

glandsglandsformform

~day15~day15day 3day 3

Smooth Smooth muscles muscles

formformProliferation Proliferation

differentiationdifferentiation

Vangl2Vangl2

loop-tailloop-tail S464NS464N*D255ED255E **

Hypothesis: Non-Canonical Wnt signaling Hypothesis: Non-Canonical Wnt signaling plays a role in FRT developmentplays a role in FRT development

Looptail mouse is tool for focusing on NON-canonical Looptail mouse is tool for focusing on NON-canonical WNTsWNTs

Unstable proteinUnstable protein

Gross morphology at birthGross morphology at birth

WT Lp mutant 2.5x mutant zoom

A B C

ovi ovi

bl

ut

ut

2mm

WT

Gross morphology ~8 weeksGross morphology ~8 weeks

Lp/+ heterozygous

5mm

wt

lp/lp

Looptail mice Looptail mice have disrupted have disrupted uterine tissue uterine tissue organizationorganizationeven at birtheven at birth

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 mutants have changes in cell orientation (even at birth)birth)Loss of Wnts has lasting consequences upon FRT development

Development 131, 2061-2072 (2004)

Vangl2Vangl2 protein is concentrated at membrane of protein is concentrated at membrane of epithelial cellsepithelial cells

Vangl2 protein localizes to the uterine Vangl2 protein localizes to the uterine epitheliumepithelium

WTWT looptaillooptail

WTWT looptaillooptail

18.5 DPC18.5 DPC

Planar Cell Polarity defect in Looptail mice causes mislocalization of PCP protein

complexesIs Vangl2 itself asymetrically localized?

1Mo

2Mo

1Mo

2Mo

Vangl2Vangl2 protein is concentrated at apical membrane of protein is concentrated at apical membrane of epithelial cells and localizes to glands!!!epithelial cells and localizes to glands!!!

ActinActin

Prediction: Planar Cell Polarity defect in Looptail mice causes mislocalization of PCP

protein complexes in the uterus

Morphological Morphological changes/changes/

Cell motilityCell motility

RhoA

JNKJNK

ROCKROCK

cytoskeletoncytoskeleton

Daam1

WntFzFz

Vangl2Vangl2

DisheveledDisheveled

WT Alexa647-phalloidin

Lp/Lp

Looptail mutants have actin polarity defect in 18.5 DPC Uterus

WT

Looptail mutants have actin polarity defect in 18.5 DPC Uterus

Lp/Lp

Morphological Morphological changes/changes/

Cell motilityCell motility

DshDsh

RhoA

JNKJNK

ROCKROCK

cytoskeletoncytoskeleton

Daam1

WntFzFz

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

E-cadherin staining reveals defect in cellular architecture of Looptail mutant Uterui

WT 18.5 dpc Lp/Lp 18.5 dpc

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?

Morphological Morphological changes/changes/

Cell motilityCell motility

RhoA

JNKJNK

ROCKROCK

cytoskeletoncytoskeleton

Daam1

WntFzFz Vangl2Vangl2

Prediction: Planar Cell Polarity defect in Looptail mice causes mislocalization of PCP

protein complexes in the uterus

ScribbleScribble•scribble scribble mutants mutants 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

Lp/Lp mutant uteri have increased scribble Lp/Lp mutant uteri have increased scribble localization to apical domain of epitheliumlocalization to apical domain of epithelium

scribble is normally

basolaterally restricted

WT d18.5 Lp/Lp d18.5

Xenografting experiment to observeXenografting experiment to observe post-natal uterine developmentpost-natal uterine development

•Take WT & Lp/Lp mutant uterine tissue embyronic Take WT & Lp/Lp mutant uterine tissue embyronic D18.5D18.5•Place WT tissue in right kidney capsule & Lp in Place WT tissue in right kidney capsule & Lp in leftleft•ovarectomized Nu mouse mimics normal FRT ovarectomized Nu mouse mimics normal FRT developmentdevelopment

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 abnormal epithelial morphologymorphology• few glandsfew glands

• epitheliumepithelium• glandsglands• mesenchymemesenchyme• musclemuscle

glglmesen.mesen.

muscle

muscle

Looptail mutant xenografts develop abnormallyLooptail mutant xenografts develop abnormally

wild type graftwild type graft Lp/+ heterozygoteLp/+ heterozygote

glands?glands?musclemuscle

epi nuclear epi nuclear positionposition

• abnormal epithelial abnormal epithelial morphologymorphology• few glandsfew glands

glglmesen.mesen.

muscle

muscle

• epitheliumepithelium• glandsglands• mesenchymemesenchyme• musclemuscle

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

musclemuscle

• abnormal epithelial abnormal epithelial morphologymorphology• few glandsfew glands• big lumenbig lumen• "protrusions""protrusions"

• epitheliumepithelium• glandsglands• mesenchymemesenchyme• musclemuscle

glglmesen.mesen.

muscle

muscle

Looptail mutant xenografts develop abnormallyLooptail mutant xenografts develop abnormally

Lp mutantLp mutantwild type graftwild type graft

• abnormal epithelial abnormal epithelial morphologymorphology• few glandsfew glands• big lumenbig lumen• "protrusions""protrusions"

• epitheliumepithelium• glandsglands• mesenchymemesenchyme• musclemuscle

Looptail mutant xenografts develop abnormallyLooptail mutant xenografts develop abnormallyLp mutantLp mutantwild type graftwild type graft

•Lp have many abnormal epithelial cellsLp have many abnormal epithelial cells•very high # of pseudo-stratified epithelial very high # of pseudo-stratified epithelial cellscells

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 very high # of pseudo-stratified epithelial cellscells

Lp mutantLp mutantWTWT

Xenografts 2 weeks

WTWT Lp graftLp graft

•E-cadherin•Actin•nuclei

Xenografts 2 weeks- Oil Red stain

WT Lp heterozygote Lp mutant

Loss of Wnts affects uterine developmentLoss of Wnts affects uterine developmentwnt7awnt7a mutants are sterile mutants 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 uterine cytological changes at 2 months :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 uterine cytological changes at 2 months :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.

Wnt signaling appears to be involved in gland Wnt signaling appears to be involved in gland formationformation

Mathias Mericskay

Morphological Morphological changes/changes/

Cell motilityCell motility

WntFz1Fz1

Vangl2 functions in FRT developmentVangl2 protein is apically enriched

and localizes to glandular membranesVangl2Vangl2

ScribbleScribble

Morphological Morphological changes/changes/

Cell motilityCell motility

WntFz1Fz1

Lp mutants have defects in actin polarizationand scribble protein distribution

Vangl2Vangl2

DshDsh

RhoA

JNKJNK

ROCKROCK

cytoskeletoncytoskeleton

Daam1-catenin

Morphological Morphological changes/changes/

Cell motilityCell motility

DshDsh

RhoA

JNKJNK

ROCKROCK

cytoskeletoncytoskeleton

Daam1

WntFz1Fz1

FUTURE STUDIES: tying it together

Vangl2Vangl2

ScribbleScribble

LGL2LGL2

aPKCaPKC

-catenin

Adapted fromAnnu.Rev.Cell Dev. Biol

2006.22:207-235

Early Early polaritypolarity

crumbs3crumbs3

Par3Par3

Morphological Morphological changes/changes/

Cell motilityCell motility

DshDsh

RhoA

JNKJNK

ROCKROCK

cytoskeletoncytoskeleton

Daam1

WntFz1Fz1

Vangl2Vangl2

ScribbleScribble

LGL2LGL2

aPKCaPKC

Cdc42Cdc42

-catenin

Adapted fromAnnu.Rev.Cell Dev. Biol

2006.22:207-235

VASPVASPviniculinviniculin

CytoskeletalCytoskeletalmodulatorsmodulators

Early Early polaritypolarity

Par3Par3

crumbs3crumbs3

FUTURE STUDIES: tying it together

Morphological Morphological changes/changes/

Cell motilityCell motility

DshDsh

RhoA

JNKJNK

ROCKROCK

cytoskeletoncytoskeleton

Daam1

WntFz1Fz1

FUTURE STUDIES: Epithelial tight junction defects?

Vangl2Vangl2 Tight junction

Tight junction ScribbleScribble ??

LGL2LGL2

Zo-1Zo-1

Zo-3Zo-3

Zo-2Zo-2OccludinOccludin

claudinclaudin

JAM

Par6Par6 Par3Par3

PATJPATJ

PALS1PALS1

aPKCaPKC

Cdc42Cdc42 crumbs3crumbs3

CadherinsCadherins

-catenin

Adapted fromAnnu.Rev.Cell Dev. Biol

2006.22:207-235

VASPVASPviniculinviniculin

CytoskeletalCytoskeletalmodulatorsmodulators

Early Early polaritypolarity

actinactin

Morphological Morphological changes/changes/

Cell motilityCell motility

DshDsh

RhoA

JNKJNK

ROCKROCK

cytoskeletoncytoskeleton

Daam1

WntFz1Fz1

FUTURE STUDIES: Epithelial tight junction defects?

Vangl2Vangl2 Tight junction

Tight junction ScribbleScribble ??

LGL2LGL2

Zo-1Zo-1

Zo-3Zo-3

Zo-2Zo-2OccludinOccludin

claudinclaudin

JAM

Par6Par6 Par3Par3

PATJPATJ

PALS1PALS1

aPKCaPKC

Cdc42Cdc42 crumbs3crumbs3

CadherinsCadherins

-catenin

Adapted fromAnnu.Rev.Cell Dev. Biol

2006.22:207-235

VASPVASPviniculinviniculin

CytoskeletalCytoskeletalmodulatorsmodulators

Early Early 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 polarizationRAC1

Morphological Morphological changes/changes/

Cell motilityCell motility

DshDsh

RhoA

JNKJNK

ROCKROCK

cytoskeletoncytoskeleton

Daam1

WntFz1Fz1

FUTURE STUDIES: Epithelial tight junction defects?

Vangl2Vangl2 Tight junction

Tight junction ScribbleScribble ??

LGL2LGL2

Zo-1Zo-1

Zo-3Zo-3

Zo-2Zo-2OccludinOccludin

claudinclaudin

JAM

Par6Par6 Par3Par3

PATJPATJ

PALS1PALS1

aPKCaPKC

Cdc42Cdc42 crumbs3crumbs3

CadherinsCadherins

-catenin

Adapted fromAnnu.Rev.Cell Dev. Biol

2006.22:207-235

VASPVASPviniculinviniculin

CytoskeletalCytoskeletalmodulatorsmodulators

Early Early polaritypolarity

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 analysisAre wnts (canonical signaling) affected in Are wnts (canonical signaling) affected in looptaillooptail mutantsmutants

Functional analysis of Vangl2Functional analysis of Vangl2

Acknowledgements

NYC Group LabRisheng MaMartina Schwarzkopf

Former MembersMathias MericskayCary MillerLuca CartaClaudia CosmineauAnna PavlovaMaria Arufe

Cellular ImageryPITIE-SALPETRIERE

CollaboratorsMirielle Montcouquiol Matthew KelleyDaniel DufortPhillipe GrosSergei Sokol & Lab

MSSM shared microscopy facility

And many brave mice

Sassoon LabAra ParlakianGisela GarciaGiovanna MarazziIman GomaaKathryn MitchellKatia CastrilloNathalie DidierVanessa Besson

U787 members