Supervisor:(Prof.(Cédric(BLANPAIN,(MD,(PhD ( (

Academic(year(2015/2016 (( ( ( ( (((( Giuseppe(CHIAPPARO,(MA(

Supervisor:(Prof.(Cédric(BLANPAIN,(MD,(PhD ( (

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!MECHANISMS!REGULATING!CARDIOVASCULAR!

PROGENITOR!SPECIFICATION!AND!DIFFERENTIATION!

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PhD(academic(degree(in(Biomedical(Sciences(

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Université(Libre(de(Bruxelles( ! Faculty(of(Medicine( (

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R EMERCIEMENTS !

! Cette! thèse! de! doctorat! représente! une! étape! importante! de! ma! vie,! durant!

laquelle! j’ai! appris! et! grandi,! tant! scientifiquement! que! personnellement.! Dans! cette!

première!section,!je!tiens!à!remercier!toutes!les!personnes!qui!ont!joué!un!rôle!essentiel!

au!cours!de!ce!travail.!

!!!!!!!!!!!!En! premier! lieu,! je! tiens! à! remercier! Cédric! Blanpain! pour! m’avoir! accueilli! et!

encadré! au! sein! de! son! laboratoire.! Merci! Cédric! pour! ton! enthousiasme,! ton! esprit!

scientifique! et! tes! connaissances,! qui! m’ont! encouragé! et! motivé! au! cours! de! mon!

parcours.!Merci!de!m’avoir!accordé!une!liberté!me!permettant!!tant!de!développer!mon!

esprit! critique! et! d’initiative! que! d’approfondir! mes! connaissances! scientifiques.! Ton!

enseignement!et!tes!conseils!ont!fondamentalement!contribué!à!l’aboutissement!de!ce!

travail.!

!!!!!!!!!!!!Je!remercie!grandement!Antoine!Bondue!avec!qui!j’ai!eu!le!privilège!de!travailler!

durant!le!début!de!mon!doctorat.!Merci!Tony!pour!ton!entrain,!ta!bonne!humeur!et!tes!

conseils,!scientifiques!et!moins!scientifiques,!qui!m’ont!toujours!aidé!dans!mon!travail.!

Tu! t’es! souvent! montré! présent! et! à! l’écoute,! et! ton! départ! en! clinique,! même! si!

certainement!et!justement!motivé,!a!laissé!un!énorme!vide!dans!le!groupe!cardio.!Merci!

cependant!d’avoir!su!rester!attentif!et!à!l’écoute,!même!à!distance,!autant!que!faire!se!

peut.!

!!!!!!!!!!!!Je!tiens!à!remercier!tout!particulièrement!le!groupe!«!cardio!»!avec!qui!j’ai!sans!

doutes!eu!le!plus!de!partage!scientifique!durant!ces!années.!Merci!Miss!Samira!Chabab!

pour!tes!précieux!coups!de!mains,!tes!conseils!éclairés,!tes!questions!scientifiquement!

pertinentes,! et! aussi! pour! tous! les! potins! partagés.! Tu! as! souvent! été! un! appui! et! une!

confidente! de! qualité.! Merci! Fabienne! Lescroart! (alias! Queen! cardio)! pour! ton! écoute,!

tes!conseils,!mais!aussi!tous!les!précieux!coups!de!mains!que!tu!m’as!donnés!durant!la!

seconde!partie!de!ce!projet.!Tu!as!été!un!soutien!essentiel!au!cours!de!la!finalisation!de!

mon!travail.!I!would!like!to!greatly!thank!Xionghui!for!all!the!excellent!advices!that!he!

gave!to!me!in!the!field!of!molecular!biology.!All!your!advices!have!profoundly!helped!me!

to!quickly!learn!the!important!technics!I!needed.!

Catherine! Paulissen,! tu! as! contribué! à! pratiquement! toutes! mes! expériences!

durant! ma! thèse,! que! ce! soit! en! amont! des! manips! ou! dans! l’obtention! finale! de! mes!

résultats.! Ton! aide! a! simplement! été! fondamentale! dans! la! grande! majorité! de! mes!

manips!au!cours!de!ma!thèse,!surtout!lorsque!je!t’appelais!en!urgence!sur!ton!portable!

en!période!de!rush...!Merci!Kate!!!

Qu’en!aurait/il!été!sans!la!présence!de!voisins!de!bureau!exceptionnels!?!Je!tiens!à!

remercier,!pour!leurs!inputs!particuliers!durant!mon!doctorat,!pour!leur!sérieux!et!leur!

humour,! pour! leurs! conseils! et! leurs! blagues! infaillibles,! pour! leurs! styles! et! leur!

fraicheur,! mes! amis! Soufiane! Boumahdi,! Farida! Benhadou,! Agnieszka! Checinska! et!

Tatiana!!Revenco.!

Ce! parcours! n’aurait! certainement! pas! été! aussi! enrichissant! sans! les! différents!

partages! et! rencontres! parmi! les! membres! du! laboratoire.! Merci! à! Peggy,! Gaelle,! Alex,!

Ben,!Khalil!et!Federico!pour!les!conseils,!discussions!(scientifiques!ou!non)!partagées,!et!

pour,!à!certains!moments,!avoir!été!un!exemple!professionnel!pour!moi.!Merci!aussi!à!

Andrea,!May!Yin,!Sophie,!Marielle,!Maud,!Mariaceleste,!Aline,!J/C,!Ozzy,!Raj,!…!pour!avoir!

été!des!collègues!si!sympas!et!pour!avoir!contribué,!de!près!ou!de!loin,!à!l’aboutissement!

de!mon!travail.!

Enfin,! ma! plus! grande! reconnaissance! revient! à! mon! épouse! exceptionnelle!

Letizia,!qui!au!cours!de!ces!années!m’a!toujours!encouragé,!et!a!patiemment!supporté!

mes! week/ends! et! mes! nuits! au! labo.! Merci! d’avoir! toujours! cherché! à! m’apporter! le!

meilleur,!et!d’avoir!été!mon!soutien!sans!faille!quand!j’en!avais!besoin.!Sans!toi,!rien!de!

tout! ceci! n’aurait! été! possible.! Je! veux! également! remercier! ma! fille! Loïse! pour!

l’immense!bonheur!et!l’énergie!qu’elle!m’apporte!tous!les!jours.!Merci!aussi!à!ma!belle/

famille,! tout! particulièrement! à! mes! beaux/parents! pour! leur! énorme! gentillesse! et!

soutien,!surtout!pendant!la!finalisation!de!ma!thèse.!Merci!de!m’avoir!apporté!toujours!

plus! de! ce! que! j’espérais.! Je! remercie! également! Esther,! ma! sœur! chérie,! ainsi! que! sa!

famille!pour!m’avoir!encouragé!et!soutenu!tout!au!long!de!mon!parcours.!Merci!à!mes!

amis!Nathanaël,!Fabrizio,!Biagio,!Alex,!et!tous!les!autres!qui!m’ont!aidé!à!me!construire!

tout!au!long!de!ma!vie.!

Je!dédie!cette!thèse!aux!deux!personnes!les!plus!aimantes!et!généreuses!que!j’ai!

pu!connaître!et!qui!s’en!sont!allées!beaucoup!trop!tôt:!mes!parents,!Moïse!Chiapparo!et!

Giuseppina! Schembri.! Leurs! encouragements! inconditionnels! m'ont! toujours! soutenu!

durant!mes!études.!

Ce!travail!a!pu!être!réalisé!grâce!au!financement!FRIA/FNRS.!

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!

Above&all&else,&guard&your&heart,&

for&it&is&the&wellspring&of&life…&

Proverbs!4:23

! Content!

1.!SUMMARY!...!1!

2.!INTRODUCTION!...!3!

2.1.!!At!the!heart!of!development!and!life!...!3 !

2.2.!Cardiogenesis:!a!multi:lineage!contribution!...!3 !

2.2.1.!Embryonic!origin!of!the!heart:!from!the!primitive!streak!to!the!cardiovascular!cell! lineages!...!4 !

2.2.2.The!first!and!the!second!heart!fields!...!5 !

2.2.3.!The!neural!crest!contribution!...!6 !

2.2.4.!The!epicardial!lineage!...!7 !

2.2.5.!Conclusion!...!8 !

2.3.!Cardiac!specification!and!morphogenetic!cell!movements!occur!concomitantly!during!early! embryonic!development!...!9 !

2.3.1.!Epithelial!to!mesenchymal!transition!...!9 !

2.3.1.1.&EMT&allows&to&the&prospective&cardiac&mesoderm&to&leave&the&PS&...&9 ! 2.3.1.2.&Transcriptional&regulation&of&EMT&...&10 ! 2.3.2.!Actin!cytoskeletal!remodelling!...!11 !

2.3.3.!Extrinsic!controls!of!early!mesoderm!migration!...!11 !

2.3.3.1.&FGF&signalling&and&cardiac&mesoderm&migration&...&11 ! i.#Fgfr1#mediated#EMT#in#early#mesodermal#cells#...#11 !

ii.#FGF8#and#FGF4##are#essential#for#early#mesoderm#migration#...#12 !

2.3.3.2.&Wnt&signalling&roles&during&early&cardiac&morphogenesis&...&12 ! i.#Canonical#and#noncanonical#Wnt#signalling#during#early#cardiac#mesoderm#development#...#13 !

ii.#Convergent#and#extension#movements#and#Wnt/PCP#signalling#...#13 !

2.3.3.3.&Bone&morphogenetic&proteins&as&negative&regulators&of&CE&movement&in&zebrafish&...&14 ! 2.3.4.!Conclusion!...!15 !

2.4.!Molecular!control!of!early!cardiovascular!development!...!16 !

2.4.1.!An!ancestral!network!of!regulatory!genes!...!16 !

2.4.2.!Mesp!genes!...!18 !

2.4.2.1.&Mesp&genes&identification&...&18 !

2.4.2.2.&Mesp&genes&expressions&during&mice&embryonic&development&...&19 !

2.4.2.3.&Mesp1&is&the&earliest&sign&of&cardiovascular&development&in&mice&...&19 !

2.4.2.4.&Mesp1&and&Mesp2&present&a&functional&redundancy&during&early&mesoderm&

development&...&20 !

2.4.2.5.&Mesp1&acts&as&a&key&regulator&of&cardiovascular&lineage&commitment&...&21 !

2.4.2.6.&A&cellular&autonomous&requirement&of&Mesp1&and&Mesp2&for&cardiovascular&lineage&

commitment&...&22 !

2.4.2.7.&Mesp1&promotes&the&expression&of&the&cardiovascular&gene&network&...&22 !

2.4.2.8.&Mesp1&mediates&EMT&through&regulation&of&EMT&related&transcription&factors&...&23 !

2.4.2.9.&Mesp&emerges&during&evolution&with&chordate&specification&...&24 !

2.4.2.10.&Conclusion&...&24 !

2.5.!Embryonic!stem!cells!differentiation:!a!model!to!study!early!cardiovascular!development!...!25 !

2.5.1.!ES!cells!differentiation:!from!blastocyst!to!embryoid!body!...!25 !

2.5.2.!Extrinsic!signals!regulate!cardiovascular!lineage!commitment!within!EBs!...!25 !

2.5.3.!Identification!and!isolation!of!different!cardiovascular!progenitors!...!26 !

2.5.4.!Mesp1!as!potential!intrinsic!inducer!of!the!specification!of!the!earliest!CPs!...!28 !

3.!AIMS!OF!THE!WORK!...!30!

4.!RESULTS!–!PART!I!–!ARTICLE!1!...!32!

5.!RESULTS!–!PART!II!–!ARTICLE!2!(IN!PREPARATION)!...!49!

6.!DISCUSSION!...!93!

6.1.!Mesp1:expressing!CPs!reside!at!the!top!of!the!cellular!hierarchy!leading!to!cardiovascular! differentiation!...!93 !

6.2.!Differentiation!potential!of!Mesp1:CPs!in!vitro!...!94 !

6.3.!Identification!of!the!cell!surface!markers!Flk1,!PDGFRa!and!CXCR4!...!95 !

6.4.!Heterogeneity!within!the!Mesp1:CPs!...!96 !

6.5.!Mesp2!promotes!CPs!specification!...!97 !

6.6.!Mesp2!does!not!recapitulate!the!pro:migratory!functions!of!Mesp1!...!97 !

6.7.!Mesp1/RasGRP3/ERK!axis!is!required!for!early!CP!migration!...!98 !

6.8.!Pk1!is!important!for!polarized!cell!migration!of!CPs!...!99 !

7.!PERSPECTIVES!...!100!

7.1.!Is!the!early!temporal!fate!restriction!of!Mesp1:CPs!present!in!vitro?!...!100 !

7.2.!Clinical!importance!of!Mesp1!CPs!...!100 !

7.3.!Which!co:factors!modulate!Mesp1!functions!in!early!vs!late!Mesp1:CPs!in!vivo!(and!possibly!in! vitro)!...!102 !

7.4.!Does!the!epigenetic!landscape!of!CPs!specify!different!Mesp1!functions?!...!102 !

7.5.!How!the!RasGRP3/ERK!axis!modulates!the!migration!of!the!early!Mesp1:CPs!in!vivo?!...!103 !

7.6.!How!Pk1!mediates!its!effect!in!polarized!cell!migration!within!Mesp1!cells?!...!104 !

8.!REFERENCES!...!105!

9.!ABBREVIATIONS!...!114!

!

! !

1.#Summary!

! During!embryonic!development!and!embryonic!stem!(ES)!cell!differentiation,!the!

different!cardiovascular!cell!lineages!that!compose!the!mature!heart!arise!from!different!

groups! of! cardiovascular! progenitors! (CPs),! which! contribute! to! the! formation! of!

different! heart! regions.! Mesp1! expression! is! the! earliest! sign! of! cardiovascular!

development.!Tracing!of!the!early!Mesp1/exressing!cells!reveals!that!all!cardiovascular!

cell! of! the! mature! heart! arise! from! Mesp1/derived! cells.! Various! functional! studies!

demonstrated! that! Mesp1! acts! as! a! central! regulator! of! cardiovascular! lineage!

commitment,! controlling! the! expression! of! early! cardiac! transcription! factors,! and!

promoting! the! differentiation! into! cardiomyocytes! (CMs),! endothelial! cells! (ECs)! and!

smooth!muscle!cells!(SMCs).!In!the!first!part!of!my!thesis!work,!we!generated!a!Mesp1/

GFP!reporter!ES!cell!line!allowing!tracking!and!isolating!the!earliest!Mesp1/expressing!

cells.!We!found!that!Mesp1!marks!an!early!population!of!CPs!that!presents!the!ability!to!

differentiate! into! CM,! EC! and! SMC! from! both! heart! fields.! Transcriptional! profiling! of!

Mesp1/CPs! uncovered! cell! surface! markers! allowing! their! isolation.! In! this! work,! we!

demonstrated!that!Mesp1!is!required!for!the!specification!of!these!early!CPs.!!

! !

To! ensure! harmonious! cardiovascular! development,! CP! specification! and!

migration!must!be!tightly!coordinated.!In!addition!to!the!induction!of!cardiovascular!cell!

fate,! Mesp1! seems! also! involved! in! the! migration! of! early! CPs,! as! suggested! by! the!

cardiac! malformation! associated! with! the! persistence! of! cardiovascular! specification!

and!differentiation!in!Mesp1!null!embryo.!The!upregulation!of!the!expression!of!Mesp2,!

its! closest! homologue,! in! the! cardiac! mesoderm,! suggest! that! Mesp2! could! partially!

compensate! for! Mesp1! function! during! early! cardiogenesis,! while! Mesp1! presents! a!

unique!and!non/redundant!function!during!CP!migration.!However,!Mesp2!KO!embryos!

do! not! present! cardiac! malformation! but! rather! major! skeletal! malformations.!

Inactivation! of! both! Mesp1! and! Mesp2! leads! to! the! complete! absence! of! mesoderm!

formation! and! consequently! heart! development,! precluding! the! assessment! of! the!

redundant! function! of! Mesp1! and! Mesp2! during! cardiovascular! specification! and!

differentiation.! The! second! aim! of! this! work! was! to! investigate! the! mechanisms! that!

compensate!for!Mesp1!functions!during!the!early!steps!of!cardiogenesis,!and!those!that!

control!the!unique!Mesp1!function!during!CP!migration.!Using!inducible!gain!of!function!

experiments! during! ES! cell! differentiation,! we! found! that! Mesp2! was! as! efficient! as!

Mesp1!at!promoting!CP!specification,!EMT!and!cardiovascular!differentiation.!However,!

only! Mesp1! stimulated! the! polarity! and! directional! cell! migration! through! a! cellular!

autonomous! mechanism.! Transcriptional! analysis! and! ChIP! experiments! revealed! that!

Mesp1!and!Mesp2!activate!a!common!set!of!target!genes!that!promote!CP!specification!

and!differentiation.!We!identified!two!direct!Mesp1!target!genes,!Prickle1!and!RasGRP3,!

that!are!strongly!induced!by!Mesp1!and!not!Mesp2!and!which!control!the!polarity!and!

the! speed! of! cell! migration.! Altogether! our! results! identify! the! molecular! interface!

controlled! by! Mesp1! that! links! CP! specification! and! migration! during! ES! cell!

differentiation.!

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Figure'1:'Evolution'of'the'heart'

(a)#Schematic#representation#of#the#heart#of#simple#chordates#composed#of#an#unique#cardiac#

chamber# that# pumps# for# some# bidirectionnaly# (arrows).# (b)# Fish# heart# possesses# a# single#

atrium# and# ventricle.# (c)# Reptiles,# birds# and# mammals# have# separated# circulations# for#

oxygenated# (red)# and# deoxygenated# (blue)# blood# @lows# allowed# by# the# aquisition# of# a# four#

chambered#heart.#A,#auricle;#V,#ventricle;#RA#and#LA,#right#and#left#atria;#RV#and#LV,#right#and#

left#ventricles.#Adapted#from#Olson#E.N.#et#al.#Science#2006.#

a b c

2.#Introduction!

2.1.!!At!the!heart!of!development!and!life!

The! heart! is! a! vital! organ! that! allows! circulation! of! blood! throughout! the! body!

and!thereby!the!distribution!of!oxygen!and!nutrients!to!the!cells!of!the!body.!Pumping!

organs! have! been! evolved! during! evolution! ¹ .! Pulsating! vessels,! which! lack! chambers!

and!valves!and!ensure!fluid!motion!by!peristaltic!contraction,!form!the!most!primitive!

heart!and!can!be!found!in!simple!organisms!such!as!annelids!(Figure!1a)! ² .!In!arthropods!

or!invertebrate!chordates,!the!pumping!organ!is!a!linear!tube!that!ends!with!valves! ^1,!3,!4 .!

Chambered! hearts! refer! to! multi/compartment! pumps! resulting! from! the! addition! of!

structural! units! dedicated! to! received! (atria)! or! propel! (ventricles)! the! blood! into! the!

circulatory! system.! Fish! present! a! two/chambered! heart! with! one! atrium! and! one!

ventricle!(Figure!1b).!Amphibians!exhibit!a!three!chambered/heart!with!two!atria!and!

one!single!ventricle.!Hearts!of!reptiles,!birds!and!mammals!include!two!separated!atria!

(left! and! right)! and! two! separated! ventricles! (left! and! right),! allowing! a! separated!

circulation!of!the!highly!(left!side)!and!the!poorly!(right!side)!oxygenated!blood!(Figure!

1c) ^1, ! ⁵ .!!

According!to!this!central!function!and!requirement!for!proper!development,!the!

heart! is! the! first! functional! organ! to! form! during! embryogenesis.! Perturbation! in! the!

morphogenesis! of! mammalian! hearts! leads! to! in! utero! cell! death,! or! congenital! heart!

diseases!(CHD).!In!human,!CHD!is!the!most!common!developmental!disorder,!occurring!

in!almost!3%!of!newborns! ⁶ ,!and!represent!the!leading!cause!of!death!during!the!first!

year!of!life! ⁷ .!!

The!functional!adaptation!of!the!heart!to!the!terrestrial!lifestyle!of!mammals!has!

required! an! increasing! complexity! of! the! heart,! in! terms! of! anatomy,! lineage!

contribution,!but!also!molecular!control.!

2.2.!Cardiogenesis:!a!multiVlineage!contribution!

Structurally,! the! mammal! heart! is! a! multi/compartment! organ! with! four!

specialized!chambers!and!housing!two!separated!circulations:!the!systemic!circulation,!

that! allows! the! distribution! of! oxygenated! blood! throughout! the! body,! and! the!

pulmonary! circulation! that! carries! deoxygenated! blood! to! the! lungs,! and! returns!

Figure'2:'Scheme'of'the'adult'human'heart'

(a)#Schematic#representation#of#the#adult#heart:# #The#caval#veins#(inferior#and#superior)#drain#

into#the#right#atrium#(RA),#and#then#blood#enters#the#right#ventricle#(RV)#to#leave#through#the#

pulmonary# trunk.# In# the# left# side# of# the# heart,# the# pulmonary# veins# drain# into# the# left# atrium#

(LA).#Blood#then#enters#the#left#ventricle#(LV)#and#leaves#through#the#aorta.#The#left#and#right#

sides#of#the#heart#are#separated#by#septa#and#valves#which,#together#with#the#different#pressures#

in#the#cardiac#chambers,#allow#the#unidirectional#blood#@low.#The#heart#beat#is#controlled#by#the#

cardiac#conduction#system:#SAN,#sinoPatrial#node,#AVN,#atrioventricular#node,#the#His#bundles,#

the# bundle# branches# and# the# Purkinje# @ibers.# The# heart# is# itself# irrigated# by# the# coronary#

arteries.#Adapted#from#Harvey,#2002.#(b)#Schematic#section#through#an#adult#human#heart.#The#

heart#is#formed#by#three#layers#of#tissue:#an#inner#endocardium,#a#thick#middle#myocardium#and#

an# outer# epicardium.# Within# the# myocardium,# cardiac# @ibroblasts# and# coronary# arteries# are#

present.#Adapted#from#LiePVenema#et#al.#2007.#

#

a $$$ $$$

b

oxygenated!blood!back!to!the!heart!(Figure!1c).!The!blood!arrives!into!the!left!and!right!

atria!via!the!pulmonary!and!the!caval!veins,!goes!to!the!left!and!right!ventricles,!and!is!

propelled! respectively! into! the! aorta! and! the! pulmonary! trunk,! which! ensure! the!

systemic! and! the! pulmonary! circulation! (Figure! 2a).! Unidirectional! blood! flow! is!

ensured!by!the!different!pressures!in!each!chamber,!and!the!presence!of!valves!located!

at!the!bases!of!the!veins!and!the!arteries,!and!between!the!atria!and!the!ventricles! ⁸ .!

Histologically,! the! mammalian! heart! consists! of! three! layers:! an! inner!

endocardium,!a!thick!middle!myocardium!and!an!outer!epicardium.!The!myocardium!is!

the!main!constitutive!layer!of!the!heart!and!is!itself!irrigated!by!its!own!vasculature:!the!

coronary!vessels!(Figure!2b).!

At! the! cellular! level,! many! cell! types! contribute! to! the! heart.! Cardiomyocytes!

(CMs)! represent! the! contractile! unit,! but! also! give! rise! to! conductive! pacemaker! cells.!

Cardiac!fibroblasts!contribute!to!the!chamber!walls.!The!venous!and!arterial!vasculature!

is!made!of!smooth!muscle!cells!(SMCs)!and!endothelial!cells!(ECs),!which!also!contribute!

to! the! endocardium! and! valves! ⁵ .! CMs,! SMCs! and! ECs! arise! from! the! differentiation! of!

early! cardiovascular! progenitors! (CPs)! that! originate! from! the! primitive! streak! (PS)!

during!gastrulation.!

!

! 2.2.1.! Embryonic! origin! of! the! heart:! from! the! primitive! streak! to! the!

cardiovascular!cell!lineages!

Initial!studies!realized!in!chick!and!mouse!embryos!have!mapped!the!prospective!

cardiac!cells!in!the!anterior!region!of!the!PS!at! early!PS!stage!during!gastrulation! ^9,!10 .!

Transplantation!experiments!at!this!stage!demonstrated!that!their!commitment!toward!

a!cardiac!fate!remains!plastic! ¹¹ .!In!mice,!these!early!mesodermal!cells!leave!the!PS!at!

around! embryonic! day! 6.5! (E6.5)! and! migrate! bilaterally! in! an! anterior! direction!

forming! two! groups! of! cells! on! either! side! of! the! midline! (Figure! 3a)! ^11,!12 .! Once! these!

migrating! cells! reach! the! lateral! plate! mesoderm! (at! around! E7.25),! they! display!

restricted! cell! fate! potential,! and! are! called! the! “cardiogenic! mesoderm”,! ! ^{11,! 13} .! This!

cardiac! mesoderm! extends! then! through! the! midline! at! around! E7.5! to! form! the! first!

cardiac!structure!in!the!embryo,!the!“cardiac!crescent”!(Figure!3b)! ^10/12 .!The!first!beating!

unit! is! the! heart! tube,! which! appears! soon! after! at! around! E8.0! and! result! from! the!

midline! fusion! of! the! arms! of! the! cardiac! crescent! (Figure! 3c)! ^12,!13 .! This! cardiac! tube,!

Figure'3:'Morphogenesis'of'the'mouse'heart'

(a)#Cardiovascular#progenitor#originate#from#the#PS,#from#where#they#migrate#to#the#anterior#

region#of#the#embryo#at#about#E6.5.#(b)#These#cells#fuse#under#the#head#folds#(HF)#and#form#

the#cardiac#crescent,#where#differentiated#myocardial#cells#are#now#observed#(E7.5).#(c)#The#

early#cardiac#tube#appears#after#the#fusion#of#the#cardiac#crescent#arms#at#the#midline#(ML)#

(E8).#(d)#It#subsequently#undergoes#looping#(E8.5).#(e)#By#E10.5#the#heart#has#acquired#wellP de@ined# chambers,# but# they# are# not# yet# separated# (upper# panel,# ventral# view;# lower# panel,#

dorsal#view).#(f)#The#separated#chambers#appear#at#about#E14.5,#and#result#of#septation#of#

the# different# chambers.# They# are# connected# to# the# pulmonary# trunk# (PT)# and# aorta# (Ao),#

which# ensure# the# separate# pulmonary# and# systemic# circulation# of# the# blood,# respectively,#

after# birth.# Deoxygenated# blood# enters# the# heart# through# the# right# atrium# (RA)# and# is#

pumped#to#the#lungs#through#the#pulmonary#trunk#by#the#right#ventricle#(RV).#Oxygenated#

blood#returns#to#the#left#atrium#(LA)#and#is#pumped#by#the#left#ventricle#(LV),#through#the#

aorta,#to#the#systemic#circulation#that#serves#the#whole#body.#Anterior#(A)–posterior#(P)#and#

right#(R)–left#(L)#axes#are#indicated.#AA,#aortic#arch;#AVC,#atrioventricular#canal;#IFT,#in@low#

tract;#IVC,#inferior#vena#cava;#IVS,#interventricular#septum;#OFT,#out@low#tract;#PLA#primitive#

left# atrium;# PRA,# primitive# right# atrium;# PV,# pulmonary# vein;# SVC,# superior# vena# cava;# Tr,#

trabeculae.#Adapted#from#Buckingham#et#al.,#Nature+Reviews+Genetics+2005.#

which!consists!of!an!inner!layer!of!endocardium!and!an!outer!of!myocardium,!undergoes!

an!important!myocardial!expansion!and!a!complex!looping!which!lead!to!the!formation!

of!recognizable!cardiac!chambers! ^12,!13 !(Figure!3d/f).!

In!addition!to!this!initial!cardiac!tube,!cardiac!progenitor!cells!are!recruited!at!the!

poles! of! this! cardiac! tube! during! its! expansion! ^5,!14/19 .! In! accordance! to! the! timing! and!

their! anatomical! origin,! this! additional! progenitor! population! has! been! called! the!

secondary!heart!field!(SHF)!(Figure!4a!/!orange)! ¹⁴ !or!the!anterior!heart!field!(Figure!4a!/!

blue)! ¹⁵ .!

!

2.2.2.The!first!and!the!second!heart!fields!

! In!2001,!three!studies!realized!in!chick!and!in!mouse!embryos!demonstrated!that!

further! recruitment! of! heart! progenitor! cells! occurred! at! the! poles! of! the! cardiac! tube!

during! its! myocardial! expansion,! and! represented! the! source! of! outflow/tract!

myocardium!in!the!mature!heart! ^14/16 .!In!chick!embryos,!fate!mapping!studies,!explant!

experiments,! as! well! as! chick/quail! grafts! allowed! to! identify! the! origin! of! these!

additional! progenitor! cells! in! the! pharyngeal! mesoderm! (Figure! 4a)! ^{14,! 15} .! In! mouse!

embryo,!the!first!evidence!supporting!different!sources!of!cells!in!cardiac!development!

came!from!the!trapping!of!a!lacZ!reporter!under!the!control!of!regulatory!sequences!of!

the!Fgf10!gene!(fibroblast!growth!factor!10)! ¹⁶ .!ß/galactosidase!activity!was!observed!in!

the! embryonic! right! ventricle! and! the! outflow! tract,! as! well! as! in! part! of! pharyngeal!

mesoderm! ¹⁶ .!Di/I!labelling!analyses!showed!a!continuous!movement!of!the!cells!from!

the!pharyngeal!mesoderm!into!the!arterial!pole!of!the!heart!between!E8.25!and!E10.5,!

supporting!the!presence!of!an!“anterior”!heart!field!(Figure!4b)! ¹⁶ .!

! Further! evidences! for! the! existence! of! a! SHF! were! provided! by! studies! on! Isl1!

gene,! a! LIM! homeodomain! transcription! factor! ¹⁷ .! Isl1! knockout! (KO)! mice! exhibited!

several! defects! in! motor! neuron! and! pancreatic! development,! and! fail! to! develop! the!

cardiac! outflow! tract,! right! ventricle! and! much! of! the! atria! ¹⁷ .! In# situ! hybridization!

showed! that! Isl1! transcript! was! detected! in! cells! of! pharyngeal! mesoderm! during!

development,!but!not!in!the!myocardium!of!the!heart! ¹⁷ .!Lineage!tracing!on!these!Isl1+!

cells! demonstrated! that! these! cells! contributed! substantially! to! the! outflow! tract,! the!

right!ventricle,!and!the!atria!(Figure!4b)! ¹⁷ .!

! A! retrospective! clonal! analysis! realized! in! mouse! embryos! reinforced! the!

distinction! between! the! two! heart! fields! ²⁰ .! This! clonal! analysis! was! based! on! the!

Figure' 4:' Two' sources' of' myocardial' cells'in'the'developing'heart'

(a)# The# location# and# contribution# of# the#

‘second’# heart# @ield# (orange),# identi@ied#

by#Waldo#and#colleagues#(2001),#and#the#

‘anterior’#heart#@ield’#(blue),#identi@ied#by#

Mjaatvedt#and#colleagues#(2001)#in#chick#

embryo#are#shown#for#a#stageP16#embryo#

(lateral# view)# and# at# stage# 22# (frontal#

view).# (b)# The# location# and# contribution#

of# the# second# heart# @ield# in# mouse#

embryo# is# shown# in# green,# and# is#

compared# with# the# @irst# heart# @ield# (in#

red).# Frontal# views# are# shown# for# E7.5#

and# E10.5,# and# lateral# views# for# E8# and#

E8.5.# (c)# Retrospective# clonal# analysis#

performed# by# Meilhac# and# colleagues#

(2004).# Clones# of# βPgalactosidaseP positive#myocardial#cells#from#the#cardiac#

actin ^nlaacZ/+ # mouse# at# E8.5.# Frontal# views#

are# shown# and# clones# are# indicated# by#

the# numbered# arrows.# The# central# panel#

shows# a# large# clone# that# contribute# to#

both# heart# @ields.# The# left# and# right#

panels# show# @irst# and# second# lineage#

pro@iles,# respectively.# (d)# The# lineage#

contributions# to# the# heart# at# E8.5# with#

the# same# colour# coding# as# in# part# b.# LA,#

left# atria;# LV,# left# ventricle;# OFT,# out@low#

tract# (D,# distal;# P,# proximal);# PA,#

primitive# atria;# PhA,# pharyngeal# arches;#

RA,# right# atrium;# RV,# right# ventricle;# 1,#

out@low# region;# 2,# primitive# right#

ventricle;# 3,# primitive# left# ventricle;# 4/5,#

atrioventricular# region;# 6/7,# primitive#

left# and# right# atria.# Adapted# from#

Buckingham# M.# et# al.,# Nature+ Reviews+

Genetics+ 2005.# (e)# Early# lineage#

restriction# for# the# @irst# and# second# heart#

@ields# within# the# Mesp1# expressing# cells.#

Two#temporally#distinct#groups#of#Mesp1#

progenitors# contribute# to# the# heart#

development.# The# earlier# Mesp1#

progenitors#give#rise#to#the#FHF#(in#red)#

and# the# later# to# the# SHF# (in# green)#

derivatives,# with# an# overlapping#

expression# of# Mesp1# in# the# two#

populations# at# E6.75.# FHF# progenitors#

are#unipotent#and#give#rise#to#either#CMs#

or# ECs.# SHF# progenitors# are# either#

unipotent# or# bipotent.# Epicardium# and#

EPDCs# arise# as# an# independent# Mesp1P derived# lineage# at# the# early# time# points.#

PS:# primitive# streak.# Adapted# from#

Lescroart#et#al.,#Nature+Cell+Biology+2014#

e

spontaneous!mitotic!recombination!of!an!inactivated! nlaacZ#reporter,!targeted!into!the!

alfaGcardiac# actin# locus,! a! promoter! that! labels! all! early! cardiomyocytes.! Spontaneous!

recombination! of! this! cassette! give! a! functional! nlacZ! sequence! at! very! low! frequency!

allowing!to!label!the!cells!and!their!derivatives!at!the!clonal!level! ²¹ .!This!study!identified!

two!categories!of!clones!with!distinct!patterns!of!regionalization!in!the!cardiac!tube!at!

E8.5.! One! first! lineage! contributed! to! both! ventricles! and! both! atria! but! never! to! the!

outflow!tract,!and!the!other!contributed!to!the!outflow!tract!and!all!other!heart!regions,!

except! the! left! ventricle! (Figure! 4c)! ²⁰ .! This! second! lineage! corresponds! to! the!

contribution!of!the!SHF.!With!the!assumption!that!cell!proliferation!is!homogenous!and!

synchronous!before!E8.5,!the!size!of!clones!(in!term!of!number!of!labelled!cells!within!a!

clone)! indicated! the! time! of! the! mitotic! recombination.! Beside! these! two! main!

populations! contributing! to! either! first! or! second! heart! field,! the! identification! of! rare!

and!large!clones!that!have!labelled!cells!in!both!lineages!suggested!that!the!first!and!the!

second! lineages! could! segregate! from! a! common! progenitor,! possibly! at! the! onset! of!

gastrulation!(Figure!4c)! ^12,!20 .!!

Mesoderm! posterior! 1! (Mesp1)! gene! is! a! transcription! factor! whose! earlier!

expression! is! detected! at! the! onset! of! gastrulation! at! about! E6.5! in! the! PS! ²² .! Using! a!

temporal! clonal! labelling! of! the! Mesp1! expressing! cells! in! mouse! embryos,! a! recent!

study! demonstrated! that! as! soon! as! Mesp1! expression! during! gastrulation,! two!

temporally! distinct! groups! of! Mesp1! expressing! cells! can! already! be! distinguished! by!

their! restricted! contribution! to! either! the! first! heart! field! (FHF)! (earlier! Mesp1!

expressing!cells)!or!the!SHF!(later!Mesp1!expressing!cells)!(Figure!4e)! ²³ .!The!absence!of!

common! FHF! and! SHF! progenitors! among! Mesp1! expressing! cells! suggests! that! the!

common! progenitor! identified! in! the! retrospective! clonal! analysis! of! Meilhac! and!

colleagues!(2004)!exists!before!gastrulation,!within!the!epiblast.!

!

2.2.3.!The!neural!crest!contribution!

! Beside!the!first!and!second!heart!field,!cells!originating!from!the!neural!crest!also!

contribute! to! the! heart.! Neural! crest! cells! originate! from! the! region! of! the!

neuroepithelium!that!borders!the!surface!ectoderm! ²⁴ .!A!subregion!of!the!cranial!neural!

crest,! called! “cardiac! neural! crest”! play! a! role! in! heart! development.! Ablation! of! these!

cardiac!neural!crest!cells!in!chick!leads!to!a!number!of!cardiovascular!defects!including!

defective! development! of! the! cardiac! outflow! tract,! abnormal! patterning! of! the! great!

arteries! and! abnormal! myocardial! function! ²⁴ .! In! mice,! neural! crest/ablated! embryos!

showed! a! defect! in! second! heart! field! development,! observed! by! an! abnormal! cardiac!

tube! looping! due! to! a! failure! of! addition! of! second! heart! field/derived! myocardium! ²⁴ .!

Lineage! tracing! experiments! showed! that! the! neural! crest! derivatives! invaded! the!

cardiac!outflow!tract,!contributing!to!the!septation!of!the!arterial!trunk!in!the!aorta!and!

the!pulmonary!artery,!but!also!plays!a!major!role!in!the!development!of!the!valves!and!

the!cardiac!conduction!system! ^24/26 .!!

!

2.2.4.!The!epicardial!lineage!

! The! epicardium! is! the! outermost! epithelial! layer! of! the! mature! heart! that!

surrounds! the! myocardium.! Initial! studies! using! chicken/quail! chimera,! retroviral!

lineage!tracing,!and! in#vitro!data,!and!more!recent!mouse!lineage!tracings!using! Tbx18!

and! Wt1! expression! as! drivers,! showed! that! the! epicardium/derived! cells! (EPDCs)!

contribute! to! the! development! of! coronary! smooth! muscle! cells,! endothelial! cells,!

cardiac! fibroblasts,! and! myocardial! cells! in! the! cardiac! chambers! and! the!

interventricular!septum! ^27/29 .!Epicardial!outgrowth!inhibition!induces!several!defects!in!

cardiac!morphogenesis!including!defective!ventricular!inlet!and!outflow!tract!septation!

27,! 30 .! So! epicardium! not! only! provides! for! the! diverse! cell! types! that! contribute! to!

different! structures! of! the! mature! heart,! but! it! additionally! plays! modulatory! roles! in!

myocardial!development.!

! While! in! the! adult! zebrafish,! the! epicardium! contributes! also! to! myocardial!

homeostasis! throughout! life! by! maintaining! its! potential! and! providing! continual! CM!

turnover!in!the!adult!zebrafish!heart! ³¹ ,!in!postnatal!mice,!the!epicardium!loss!its!ability!

to! provide! precursor! cells! for! the! various! cardiac! cell! types,! and! becomes! relatively!

dormant!by!P4,!and!consequently!unable!to!respond!adequately!to!a!myocardial!injury!

occurring! in! adult! heart! ^32,!33 .! After! heart! injury,! the! “quiescence”! of! the! epicardium! is!

rapidly! lost! and! epicardial! cells! revert! to! an! embryonic/like! phenotype,! re/expressing!

Tbx18,!Wt1!and!Raldh2!(normally!expressed!during!heart!development),!proliferating!at!

the!site!of!injury!and!secreting!factors!to!modulate!wound!leading! ^34,!35 .!In!adult!mice,!

this! response! is! characterised! by! mass! fibrosis! and! scar! formation,! which,! whilst!

required!as!an!immediate!and!early!response!to!prevent!organ!rupture,!ultimately!leads!

to!pathological!remodelling!and!heart!failure! ³⁶ .!!

! During! heart! development,! epicardial! cells! arise! from! a! transient! embryonic!

structure! composed! by! embryonic! progenitor! cells! and! called! “proepicardial! organ”!

(PEO).!The!PEO!is!observed!between!E8.5!and!E9.5!in!mouse!embryo!at!the!venous!pole!

of! the! heart! ³⁷ .! At! around! E8.5,! the! left! and! right! PE0! primordium! appear!

simultaneously,! moved! towards! the! embryonic! midline! where! they! merged! to! form! a!

single! structure! ³⁷ .! At! around! E9.5,! epicardial! sheet! formation! begins! on! contact! of! a!

“proepicardium! cysts”! with! the! myocardium! ³⁰ .! Cells! of! each! cyst! flatten! out,! forming!

island!like!structures!of!epithelial!sheet!on!the!surface!of!the!myocardium,!which!fuse!

together!to!give!rise!the!entire!epicardium!at!around!E11! ³⁰ .!Different!Cre/mediated!cell!

fate!in!mouse!embryos!showed!that!PEO!cells!derive!from!mesodermal!cells!which!has!

transiently!expressed!Mesp1,#Isl1!or!Nkx2.5#genes! ^29,!30,!38 .!!

!

2.2.5.!Conclusion!

Heart! formation! is! a! sequential! process! that! requires! the! coordinated!

contributions!of!distinct!groups!of!cardiovascular!progenitors!(CPs),!which!seems!to!be!

specified! at! different! times,! and! migrate! to! different! heart! regions.! To! ensure! a!

harmonious!cardiac!development,!the!specification!and!migration!of!these!distinct!CPs!

must!be!tightly!linked.!!

! !

Figure'5:'The'cellular'aspects'of'EMT''

(i)#Epithelial#cells#expressing#adherent#junctions#proteins#such#as#EPcadherin,#catenins#and#actin.#

Tight# junctions# are# associated# with# apical# polarity# complexes,# while# integrins# interact# with#

components# of# the# basal# membrane.# (ii)# Loss# of# cellPcell# adhesion.# EMTPrelated# transcription#

factors#repress#the#transcription#of#the#adherent#and#tight#junctions#genes,#inducing#the#loss#of#

EPcadherin.# (iii)# Breakdown# of# the# basal# membrane# and# apical# constriction.# Cytoskeletal#

remodelling#is#required#for#cell#delamination#inducing#apical#constriction#and#reorganization#of#

the# basal# membrane.# (iv)# Cell# delamination# and# invasion.# Expression# of# integrin# and#

metalloproteases#and#continued#cytoskeletal#reorganization#will#favour#the#migration#of#the#cell.#

Adapted#from#Acloque#et#al.,#Journal+of+Clinical+Investigation+–+Review+Series+2009.#

Figure'6:'Recrutement'and'internalization'of'epiblast'cells'in'the'PS'and'generation'of'the'3' germ'layers'

In$gastrula.ng$embryo,$epiblast$cells$are$recruted$to$the$PS,$undergo$EMT$and$are$internalied$(arrows)$

to$ generate$ mesoderm$ and$ endoderm,$ while$ the$ cells$ remaining$ in$ the$ epiblast$ become$ ectoderm.$

Adapted#from#Dias#and#McLone,#Pediatric#Neurosurgery#2001.#

2.3.!Cardiac!specification!and!morphogenetic!cell!movements!occur!concomitantly!

during!early!embryonic!development!

According!to!the!rapid!requirement!for!a!circulatory!system!that!ensure!further!

development!of!the!embryo,!CPs!are!specified!very!early!during!embryogenesis,!during!

the!early!step!of!gastrulation.!Gastrulation!is!characterized!by!a!dynamic!morphogenetic!

cell!movement!that!allows!the!induction!and!establishment!of!the!three!germ!layers,!the!

endoderm,! mesoderm! and! ectoderm.! During! gastrulation,! epithelial! to! mesenchymal!

transition! (EMT)! promotes! the! exit! from! the! PS! and! the! migration! of! the! early! CPs.!

Extrinsic! signals! such! as! FGF,! Wnt! and! BMP! signalling! pathways! have! been! shown! to!

regulate! the! migration! of! the! prospective! cardiac! mesoderm,! especially! through! the!

regulation!of!EMT!and/or!actin!cytoskeletal!remodelling.!!

!

2.3.1.!Epithelial!to!mesenchymal!transition!!

! The!cellular!process!of!EMT!has!been!initially!described!by!Elizabeth!Hay,!using!

the!chick!PS!as!model! ³⁹ .!Elizabeth!Hay!showed!that!epithelial!cells!in!the!PS!cells!can!

turn!into!mesenchymal!cells!by!undergoing!several!dramatic!phenotypic!changes!such!

as!the!loss!of!E/cadherin!and!claudins!adhesion!proteins,!combined!with!the!loss!of!the!

apico/basal! polarity,! and! a! profound! cytoskeletal! remodelling,! which! will! favour! cell!

delamination! and! invasion! ^{39,! 40} ! (Figure! 5).! Since! this! process! is! reversible! and!

mesenchymal! cells! can! revert! to! epithelial! cells! via! a! mesenchymal! to! epithelial!

transition!(MET),!the!term!“transition”!has!been!preferred!to!“transformation”! ³⁹ .!

!

2.3.1.1.%EMT%allows%to%the%prospective%cardiac%mesoderm%to%leave%the%PS%

In!the!developing!embryo,!the!first!EMT!event!occurs!during!gastrulation,!where!

a! subset! of! the! epiblast! epithelial! cells! are! recruited! to! the! PS,! undergo! EMT! and! are!

internalized!to!generate!mesoderm!and!endoderm,!while!those!remaining!in!the!epiblast!

become! ectoderm! (Figure! 6)! ^40/42 .! The! first! mobilised! mesodermal! cells! migrate! from!

proximal/posterior!region!of!the!PS!into!extraembryonic!regions,!forming!the!yolk!sac!

endothelium!and!blood!islands! ⁴³ .!The!prospective!cardiac!mesoderm!is!derived!from!a!

more!anterior!region!of!the!streak!and!undergoes!EMT!at!a!slightly!later!time!point! ⁴⁴ .!

Mutant! embryos! carrying! defects! in! EMT! during! gastrulation! often! exhibit! a! cell!

accumulation!in!the!PS,!du!to!an!apparent!failure!of!cell!movement!within!the!PS! ⁴¹ .!The!

result! of! such! defect! can! be! associated! with! several! development! abnormalities,!

including!critical!heart!defects! ⁴¹ .!

!

2.3.1.2.%Transcriptional%regulation%of%EMT%

Multiple! studies! demonstrated! that! EMT! is! controlled! by! Wnt,! TGF/ß! and! FGF!

signalling!pathways.!These!signals!activate!the!expression!of!transcription!factors!that!in!

turn! induce! EMT! ^{40,! 42} .! During! gastrulation,! the! Snail! family! genes! encoding! for! DNA!

binding! zinc! finger! transcription! factors! Snail1! and! Snail2! (also! known! as! Slug)! are!

expressed!in!the!PS,!and!Snail1!protein!has!been!demonstrated!to!be!a!key!regulator!of!

EMT!during!mouse!gastrulation! ^45,!46 .!Mouse!embryos!invalidated!for!Snail1!gene!exhibit!

profound!defects!of!the!mesoderm!germ!layer,!which!displays!an!epithelial!morphology!

and!still!express!E/Cadherin! ^46,!47 .!By!contrast,!Snail2!(or!Slug)!deletion!in!mice!shows!no!

EMT!failure!during!gastrulation! ⁴⁸ .!Snail1!acts!as!a!transcriptional!repressor!by!binding!

to! the! conserved! E/box! cis/regulatory! sequences! within! the! promoters! of! its! target!

genes,!such!as! EGCadherin,!as!well!as!the!genes!encoding!claudin/3,!claudin/4,!claudin/7!

and!occluding,!and!repressing!their!expression! ^49,!50 .!

Several!other!transcription!factors!acting!as!EMT!inducers!have!been!identified,!

including! the! helix/loop/helix! (HLH)! factors! Twist1! and! Twist2,! and! the! zinc/finger!

factors! ZEB1! and! ZEB2! ^51/53 .! During! embryonic! development,! the! ZEB! proteins! are!

involved!in!migration!of!the!neural!crest!cells!and!formation!of!the!derivative!structures!

during! subsequent! embryonic! development! ⁵⁴ .! ZEB! proteins! display! a! strong! binding!

affinity!for!E/box!regions!that!flank!the!EGcadherin!gene!and!are!able!to!transcriptionally!

repress! E/cadherin! expression! ⁵⁴ .! Conversely,! the! Zeb! factors! can! also! activate! the!

expression! of! genes! promoting! cell! invasion! such! as! the! pro/invasion! MMP! (Matrix!

Metalloproteinase)!family!members,!especially!MMP2! ^42,!53 .!ZEB1!and!ZEB2!bind!also!to!

the! E/box! promoters! and! repress! miR/200! micro/RNAs! (miRNAs),! a! group! of! five!

miRNAs! that! share! similar! targeting! sequences,! modulating! the! abundance! of! ZEB!

proteins,!creating!a!reciprocal!feedback!loop!controlling!EMT! ^42,!55 .!

The! repression! of! E/cadherin! is! often! considered! to! be! one! of! the! most! critical!

events!during!EMT.!While!required!for!the!exit!from!the!PS!of!the!early!CPs,!the!loss!of!E/

cadherin! alone! is! not! necessarily! indicative! of! the! migratory! phenotype! ⁴² .! In!the! next!

sections,!I!will!describe!other!key!cellular!processes!that!control!cell!migration.!!

!

Figure'7:'Cell'migration'is'dependent'on'different'actin'Hilament'structures'

(a)# Cell# movement# begins# with# an# actinPdependent# protrusion# of# the# leading# edge,# inducing#

formation# of# lamellipodia# and/or# @ilopodia.# (b)# Adhesion# molecules# stabilizes# the# membrane#

protrusion#and#serves#as#traction#point#during#cell#migration.#Adhesion#molecules#are#linked#with#

actin#@ilament.#(c)#The$actomyosin@based$contrac.on$forces$mediated$by$these$trac.on$sites$allow$to$

the#nucleus#and#the#cell#body#to#move#forward.#(d)#Then,#retraction#@ibres#pull#the#rear#of#the#cell#

forward,#adhesions#at#the#rear#of#the#cell#disassemble#and#the#trailing#edge#retracts.#Adapted#from#

Mattila#P.#et#al.,#Nature+Reviews+Molecular+Cell+Biology+2009.#

2.3.2.!Actin!cytoskeletal!remodelling!

! Cell!movement!can!be!conceptualized!as!a!cyclic!process!requiring!dynamic!actin!

cytoskeletal! remodelling.! The! initial! response! of! a! cell! to! a! pro/migratory! signal! is! to!

polarize! and! extend! membrane! protrusions! in! the! direction! of! migration! (Figure! 7a).!

These!membrane!protrusions,!which!can!be!spike/like!filopodia!or!broad!lamellipodia,!

are!driven!by!rapid!actin!polymerization.!The!cell!protrusions!are!stabilized!by!adhering!

to! the! extracellular! matrix! through! adhesion! molecules! such! as! integrins,! which! are!

linked!via!adapters!with!actin!filaments!(=!polarized!actin)!(Figure!7b).!These!adhesion!

molecules,! acting! as! the! “feet”! of! the! migrating! cell,! serve! as! traction! sites! for! cell!

migration,!and!are!disassembled!at!the!cell!rear!(Figure!7b/d).!The!force!transmitted!to!

these!adhesion!sites!derives!from!the!interaction!of!myosin!II!with!actin!filaments!that!

attach!to!these!sites,!which!“pull”!the!cells!to!the!leading!edge! ^56,!57 .!

!

2.3.3.!Extrinsic!controls!of!early!mesoderm!migration!

! Various!extrinsic!signals!such!as!FGF,!Wnt,!BMP!and!ERK!have!been!shown!to!be!

important! mediators! of! cell! migration! during! early! embryonic! development,! through!

regulation!of!different!cellular!mechanisms.!The!mechanisms!described!below!concern!

essentially! the! ones! that! will! be! further! analysed! and! described! in! this! work.! Among!

these! extracellular! signals,! some! are! also! known! to! be! important! for! the! induction! of!

cardiac!specification.!However,!it!this!section!I!will!focus!on!their!migratory/role!during!

the!early!steps!of!cardiogenesis.!

!

2.3.3.1.%FGF%signalling%and%cardiac%mesoderm%migration%

i.#Fgfr1#mediated#EMT#in#early#mesodermal#cells#

! The!involvement!of!FGF!signalling!in!mice!gastrulation!and!mesoderm!formation!

has!been!initially!characterized!in!embryos!invalidated!for!the!FGF!receptor!1!(Fgfr1)! ⁵⁸ .!

In!the!absence!of! fgfrG1,!embryos!displayed!gastrulation!defects,!aberrant!mesodermal!

patterning!and!die!early!during!development! ⁵⁸ .!Chimeric!analyses!have!been!performed!

to! further! study! the! function! of! Fgfr1! in! morphogenesis! and! patterning! of! the!

mesodermal! germ! layer! ⁵⁹ .! At! E9.5,! fgfrG1! deficient! cells! failed! to! contribute! to! all!

mesodermal!cell!lineages,!including!the!heart,!and!accumulate!within!the!PS!of!chimeric!

embryos! ⁵⁹ .!This!cell!accumulation!is!attributed!to!deficient!EMT!within!the! fgfrG1!null!

cells,!which!displayed!high!levels!of!E/cadherin!and!downregulated!expression!of! Snail,!

providing! the! first! evidence! that! FGF! signalling! is! required! during! gastrulation,!

mediating!EMT!and!morphogenesis!of!early!mesoderm! ^59,!60 .!!

!

ii.#FGF8#and#FGF4##are#essential#for#early#mesoderm#migration#

! In!gastrulating!mouse!embryo,!the!FGF!ligands!Fgf4!and!Fgf8!are!both!expressed!

within! the! PS.! Embryos! invalidated! for! Fgf8! gene! fail! to! express! Fgf4,! resulting! to! a!

double!invalidation!at!about!E7.25! ⁶¹ .!In!absence!of!both!FGF8!and!FGF4,!epiblast!cells!

undergo! EMT! during! gastrulation,! but! most! of! them! then! fail! to! exit! from! the! PS! and!

accumulate! in! the! posterior! side! of! the! embryo! ⁶¹ .! Consequently,! no! embryonic!

mesoderm/derived!tissues!develop,!although!extraembryonic!tissues!form! ⁶¹ .!

! Further!studies!realized!in!Drosophila!and!in!Caenorhabditis#elegans!demonstrate!

that! FGF! signalling! is! necessary! for! cell! migration! and! required! for! normal! embryonic!

development! ^62,!63 .!In!both!organisms,!ectopic!expression!induced!a!migratory!activity!of!

the!cells,!suggesting!that!FGF!signalling!is!a!driver!for!cell!migration!in!early!embryos,!

and!possibly!function!as!attractants!for!cell!migration! ^62,!63 .!To!test!if!FGF!ligands!display!

the! same! properties! during! murine! embryonic! development,! chimeric! embryos! were!

obtained!by!injection!of!WT!embryonic!stem!(ES)!cells!into!FGF8!KO!blastocyst! ⁶¹ .!These!

chimeric!embryos!indicated!that!FGF!signalling!is!required!in!the!PS!itself,!and!not!in!the!

more! distant! visceral! endoderm,! presumably! to! regulate! the! production! of! proteins!

necessary!for!cell!migration! ⁶¹ .!

!

2.3.3.2.%Wnt%signalling%roles%during%early%cardiac%morphogenesis%

! Wnt!are!a!family!of!secreted!proteins,!which!initiate!several!signal!transduction!

pathways.! The! classic! canonical! Wnt! (or! Wnt/ß/Catenin)! signalling! pathway! involves!

the!signalling!mediator!ß/Catenin.!In!absence!of!Wnt,!cytosolic!ß/Catenin!is!recruited!by!

the! destruction! complex! composed! of! axin,! APC! and! GSK3ß,! and! thereby! is!

phosphorylated! and! degraded! by! the! proteasome.! In! the! presence! of! Wnt! ligand,! the!

destruction! complex! is! disassembled,! ß/catenin! is! stabilized,! enters! the! nucleus! and!

regulates! target! gene! transcription! through! interaction! with! TCF/LEF! transcription!

factors.! The! noncanonical! Wnt! signalling! pathways! are! independent! of! ß/catenin,! and!

Figure' 8:' Convergent' extension' movements' elongate' the' anteriorKposterior' axis' of' the' vertebrate'body'plan'

Example# of# convergent# extension# movement# in# the# frog# embryo.# During# gastrula# and# neurula#

stage,# the# notochordal# (red)# and# somitic# (pink)# tissues# turn# inside# and# converge# (narrow)# and#

extend#(lengthens)#during#embryogenesis.#The#overlying#presumptive#hindbrain#and#spinal#cord#

(blue)#tissues#converge#and#extend#coordinately.#These#movements#push#the#head#away#from#the#

tail# and# elongate# the# body# axis.# Similar# movements# elongate# the# body# axis# of# mammals# (on# the#

right).#Adapted#from#Keller#R.,#Science+Review+2002.#

involved! diverse! intracellular! mediators! ⁶⁴ .! The! Wnt! signalling! pathway! regulates! in!

different! manner! and! at! different! times! early! cardiac! development,! impacting! cardiac!

mesoderm! specification! and/or! migration.! In! the! following! sections! I! will! mainly!

summarize! the! effects! of! Wnt! signalling! pathways,! especially! the! Wnt/PCP! pathway,!

during!early!cardiac!mesodermal!cell!movements.!

!

i.# Canonical# and# noncanonical# Wnt# signalling# during# early# cardiac# mesoderm#

development#

! Several! studies! have! shown! that! cardiac! mesoderm! formation! is! dependent! on!

the! Wnt/ß/Catenin! signalling! pathway.! Mice! invalidated! for! ßGCatenin! fail! to! form!

mesodermal! germ! layer! ⁶⁵ .! Also,! the! loss! of! Wnt3a,! which! activates! the! ß/Catenin!

dependent! Wnt! pathway,! leads! to! an! absence! of! mesoderm/specific! marker! genes! ⁶⁶ .!

Experiments! in! zebrafish! and! in! mouse! ES! cells! showed! that! canonical! Wnt! signalling!

displays! a! biphasic! role! in! cardiac! specification! ^67/70 .! The! canonical! Wnt! signalling!

exhibits! a! pro/cardiogenic! effect! only! at! early! stages,! before! the! expression! of! early!

cardiac!genes,!promoting!the!expression!of!many!of!them,!while!it!inhibits!cardiac!cell!

differentiation!at!a!slightly!later!time!point! ^67/70 .!While!these!results!show!that!in!mice!

the! Wnt/ß/Catenin! signalling! plays! a! major! role! in! the! induction! of! the! mesodermal!

identity,!which!is!a!prerequisite!for!the!formation!of!the!CPs,!the!Wnt/PCP!pathway,!is!

involved!in!early!cardiac!morphogenesis!through!the!control!of!the!cardiac!progenitor!

cell!movement! ⁶⁴ .!

!

ii.#Convergent#and#extension#movements#and#Wnt/PCP#signalling#

During!embryonic!development,!the!head/to/tail!axis!of!vertebrates!is!elongated!

by! “convergent! and! extension”! (CE)! tissue! movements,! which! narrow! (converge)! the!

mediolateral!axis!and!elongate!(extend)!the!anterior/posterior!axis!of!the!embryo,!and!

thereby!establish!its!morphological!and!functional!polarity,!with!a!head!on!one!end!and!

a!tail!on!the!other!(Figure!8)! ⁷¹ .!The!forces!for!CE!movement!are!generated!within!the!

tissues! by! polarized! cell! motility.! The! polarity! of! the! cells! within! the! plane! of! the!

epithelium!is!regulated!by!the!Wnt/PCP!pathway! ⁷¹ .!

Wnt/PCP! pathway! has! been! initially! described! in! Drosophila,! and! the! principal!

genes! components! include! frizzled! (fz),! which! encodes! a! seven/pass! transmembrane!

receptor,! dishevelled! (dsh),! which! encodes! a! cytoplasmic! signalling! protein,!Strabismus!

(stbm)!or!van!Gogh!(Vang),!which!encodes!a!probable!membrane!protein,!flamingo!(fmi),!

a!serpentine!membrane!protein,!and!prickle!(pk)! ⁷¹ .!Various!studies!realized!in!zebrafish,!

and! xenopus! embryos! demonstrate! that! both! loss! and! gain! of! function! of! Wnt/PCP!

pathway! components,! such! as! Wnt11,! Wnt5a,! or! Pk1! impair! CE! movements! during!

gastrulation! ^72,!73 .!Similar!study!realized!in!chicken!embryos!demonstrated!that!Wnt11b!

and!Wnt5a,!which!are!both!expressed!in!the!avian!PS,!are!also!essential!for!normal!cell!

migration! during! gastrulation,! and! disruption! of! their! expression! leads! to! a! cell!

accumulation! within! the! PS! ⁷⁴ .! In! mice,! several! Wnt/PCP! components! have! been!

reported!to!be!required!for!cardiac!development! ⁷⁵ .!Studies!on!the!core!PCP!component!

Pk1! realized! in! mice! have! shown! that! this! gene! plays! an! essential! role! in! early!

embryonic!development! ^76,!77 .!Deletion!of!Pk1!gene!results!to!early!critical!morphological!

defect!and!tissue!disorganization!associated!with!abnormal!cell!polarity,!leading!to!early!

embryonic! lethality! at! about! E5.5! ^{76,! 77} .! To! bypass! this! early! embryonic! lethality! ⁷⁶ ,!

different! other! Prickle1! mutant! alleles,! predicted! to! generate! varied! gene/dosage! in!

different!tissues,!have!been!engineered!in!mice! ⁷⁷ .!This!further!study!demonstrated!the!

requirement! of! Pk1! in! cell! polarity! and! actin! filament! organization! during! cardiac!

development,! and! shown! also! that! Pk1! mediates! Wnt5a! signalling! to! modulate!

Dishevelled!activation! ⁷⁷ .!

!

2.3.3.3.% Bone% morphogenetic% proteins% as% negative% regulators% of% CE%

movement%in%zebrafish%

! In! vertebrates,! the! Bone! morphogenetic! proteins! (Bmps)! are! expressed! during!

gastrulation!and!form!a!ventral/to/dorsal!gradient!in!the!embryo! ⁷³ .!Studies!realized!in!

zebrafishs!have!shown!that!in!addition!to!a!role!in!cell!fate!specification,!Bmps!serves!

also!as!negative!regulators!of!CE!movement! ⁷⁸ .!Bmps!inhibit!CE!movement!by!negatively!

regulating!the!expression!of!noncanonical!Wnt/PCP!pathway!component! ⁷⁸ .!It!has!been!

suggested! that! Bmps! mediate! their! negative! regulation! through! inhibition! of! adhesion!

point! stability! of! the! lamellipodia! within! the! migrating! mesodermal! cells! ⁷⁹ .! It! still!

remains! unclear! whether! Bmp! signalling! has! a! similar! function! in! regulation! the! CE!

movements!in!other!vertebrate!species! ⁷³ .!

!

2.3.4.!Conclusion!

! EMT! and! cytoskeletal! remodelling! are! crucial! for! the! exit! from! the! PS! and! the!

migration!of!early!cardiac!mesoderm.!While!extrinsic!signals!such!as!FGF,!Wnt,!and!BMP!

signalling! pathways! have! been! shown! to! regulate! these! key! cellular! events! and! the!

migration! of! the! early! CPs,! the! intrinsic! mechanisms! driving! their! migration,! and!

potentially!linking!CP!specification!and!migration!during!development!remain!currently!

unknown.!

!

! !

2.4.!Molecular!control!of!early!cardiovascular!development!

! In! the! last! 10! years,! many! advances! have! been! done! in! understanding! the!

molecular! control! of! early! cardiovascular! development.! This! is! partly! related! to! the!

important! conservation! of! the! embryological! events! across! species,! which! allowed! to!

study! “simple”! organisms! to! decipher! the! complexity! of! the! gene! network! in! more!

complex! species! ^80,!81 .! Studies! of! the! fruit! fly! Drosophila#melanogaster! first! allowed! to!

identify! the! core! set! of! evolutionarily! conserved! transcription! factors! namely:! NK2,!

MEF2,! GATA,! Tbx! and! Hand,! which! form! the! cardiac! transcriptional! network,! and!

governs!heart!development! ⁵ .!This!ancestral!cardiac!network!controls!cardiac!cell!fates,!

the!expression!of!genes!encoding!contractile!proteins!and!cardiac!morphogenesis! ^80,!81 .!

With!evolution,!the!heart!evolved!to!a!more!complex!structure.!The!mammalian!

heart!is!a!four/chambered!organ!composed!of!three!layers!of!cardiac!tissues!and!several!

cell! types.! According! to! the! progressive! complexity! of! the! anatomy! of! the! heart,! the!

molecular! inputs! driving! the! expression! of! the! core! transcriptional! network! became!

progressively!more!complex:!although!in!fruit!fly!every!single!transcription!factor!of!the!

ancestral!network!is!required!for!cardiac!development,!in!vertebrates,!the!duplication!of!

genes! of! this! cardiac! network! occurred! during! evolution,! leaded! to! a! functional!

redundancy!within!the!transcription!factors!of!the!same!family! ⁸⁰ .!

Nevertheless,! while! this! elaborated! cardiac! structure! is! very! different! to! the!

“primitive! heart”! of! the! fruit! fly! Drosophila# melanogaster,! which! displays! a! simple!

beating!dorsal!vessel,!the!core!transcriptional!network!for!cardiac!specification!has!been!

conserved! across! evolution,! although! the! molecular! control! of! its! expression! became!

more!and!more!complex! ^80,!81 .!

!

2.4.1.!An!ancestral!network!of!regulatory!genes!

Studies! in! Drosophila# have! shown! that! the! dorsal! vessel! (or! heart)! of! the!

Drosophila#arises!from!the!mesoderm,!and!that!expression!of!the!NK2!homeobox!gene!

tinman!(tin)!within!mesodermal!cells!is!a!central!event!in!heart!development! ⁸⁰ .!The!tin!

gene! encodes! the! homeodomain! transcription! factor! Tinman.! In! absence! of! Tin!

function,! there! is! a! complete! loss! of! cardiac! and! visceral! muscle! cells,! and! mutant!

embryos!die!at!the!end!of!embryogenesis,!likely!from!a!lack!of!circulation! ⁸² .!!

Tin! promotes! cardiac! specification! and! differentiation! through! binding! the!

a$

b$

Figure'10:'Cardiac'regulatory'genes'across'evolution'

Complexi@ication# and# multiplication# of# cardiac# structures# is# associated# with# an# increase# in# the#

number# of# cardiac# regulatory# genes# during# evolution.# Adapted# from# Olson# E.N.# et# al.# Science#

Review#2006.#

Figure'9:'Cardiac'transcriptional'network$

Organization# of# the# core# transcriptional# network# of# Drosophila# (a)# and# vertebrate# (b)# heart#

development.#Adapted#from#Davidson#E.H.#at#al.,#Science+Review+2006.#