Développement`cardiaque`

Développement'cardiaque'
Damien'Bonnet''
M3C.Necker'
Morphogenèse'du'cœur''chez'la'drosophile'
FormaAon'du'mésoderme'pendant'l'’embryogenèse'de'la'drosophile'
Les'trois'principaux'dérivés'du'Assus'mésodermique'chez'la'drosophile'
Vue'dorsale'd'’un'embryon'de'drosophile.'
Marquage'spécifique'au'niveau'du'canal'dorsal'(ou'cœur)'
Le'cœur'ou'canal'dorsal'de'Drosophile'est'divisé'en'deux'régions'disAnctes'
Cell. péricard.!
rg!
Cell. Cardiaques!
gl!
Aorte!
Cœur !
Structure'du'canal'dorsal'en'fin'd'’embryogenèse'
Les'étapes'importantes'de'la'morphogenèse'
du'cœur'de'drosophile'
Stade 11
Spécification!
Stade 13
Différenciation!
Stade 15
Maturation!
Les''précurseurs'cardiaques'ne'sont'pas'tous'équivalents'
Rôle des gènes homéotiques le long
de l axe antéro-postérieur
-  Rôle fondamental et universel
dans l'édification des embryons
-  Informer les cellules de leur
position au cours de
l'embryogenèse
-  Préciser le positionnement
définitif des cellules dans
l'embryon au cours de la
formation des organes par
rapport aux axes
antéropostérieur et dorsoventral
-  Conservation entre les espèces
Svp/tin"
DiversificaAon'des'cellules'cardiaques'au'sein'du'tube'cardiaque'de'drosophile'
Lo'and'Frasch,'2001'
Stade 11
Spécification!
Stade 13
Différenciation!
Stade 15
Maturation!
La'perturbaAon'de'l'’acquisiAon'de'la'polarité'cellulaire'affecte'la'formaAon'du'cœur''
Fremion et al., 1999!
VisualisaAon'de'la'perte'de'polarité'cellulaire'en'absence'de'bkh'
Fremion et al., 1999!
Stade 11
Spécification!
Stade 13
Différenciation!
Stade 15
Maturation!
ReprésentaAon'de'la'migraAon'des'deux'rangées'de'cellules'cardiaques'
Chartier et al., 2002!
Le'contact'entre'l'’épithélium'cardiaque'et'l'’ectoderme'dorsal''
est'important'pour'l'’intégrité'de'la'structure'du'cœur'
Chartier et al., 2002!
Pericardin'est'essenAelle'au'mainAen'de'la'structure'du'tube'cardiaque'
Chartier et al., 2002!
La'souris'comme'modèle'd’étude'
de'la'cardiogenèse'des'vertébrés'
Poisson Zèbre
Xénope
Souris
Structure'du'cœur'chez'les'vertébrés'
Homme
Morphogenèse'précoce'du'coeur'de'souris'
Croissant cardiaque!
E7.5
Tube cardiaque !
E8
Morphogenèse'précoce'du'coeur'de'souris'
Croissant cardiaque
E7.5
Tube cardiaque !
E8
looping!
E8.5
pôle artériel
Nigel Brown!
pôle veineux!
Coeur'embryonnaire'(E10.5)'
OD!
LAOG!
OG!
OD!
CAV!
VE!
VD!
VG!
VD!
VG!
Christoffels et al., 2000 !
Coeur'foetal'(E14.5)'
OD!
OG!
VE!
O
G!
OD!
VG!
VD!
VG!
OD!
O
G!
VG!
VD!
VD!
Mésoderme)antérieur)
latéral)
Tinman'like'
factors'
MEF2c'
GATA4'
spécificaAon'
Myogenèse'
Morphogenèse'
Tube)cardiaque)
linéaire)
Nodal'
Le[y'
NKX2.5'
'
MEF2c'
Asymétrie'droite.gauche'
eHand'
'
dHand'
Looping)du)tube))
cardiaque)
'reAnoïc'
acid'
SpécificaAon'O.V'
ContribuAon'des'crêtes'
neurales'
Matura7on)des)chambres))
cardiaques)
Olson 1998!
Le'cœur'des'vertébrés'est'asymétrique'
La'noAon'de'latéralité'
J'23':'boucle'cardiaque'(loop)'
D.loop'
D.loop'
L.loop'
CFC1
L
ACVR2A
node
Primitive streak
ACVR1B
R
Expression spécifique de Nodal
du côté gauche
Lefty 1 restreint l expression de Nodal
du côté gauche de l embryon
L’asymétrie, un mécanisme physique?
Poisson zèbre:
-  tourbillon de fluide dans une petite
vésicule (Kuppfer)
-  Ce tourbillon de fluide est contrôlé
par des rotations de petits cils à
l’intérieur de cette vésicule
-  sens du tourbillon (vers le G ou la D)
détermine la future asymétrie interne
en concentrant certaines protéines
d’un côté de l’embryon
Autre hypothèse récente:
-  flux d’ions d’hydrogènes et potassium à l’intérieur de l’embryon
-  différence de potentiel entre la gauche et la droite
- ce flux d’ions crée un champ électrique dès les premières divisions cellulaires
Asymmetry in heart
development is
controlled by a
complex of interacting
and feedback pathways
Three molecular pathways to isomerism
•  Abnormal'iniAal'breaking'of'symmetry'
–  DefecAve/absent'node'monocillia'
–  Mix'of'solitus,'inversus,'le['and'right'isomerism'
•  Abnormal/loss'of'le['side'signal'pathway'
–  e.g.'pitx2c'
–  Right'isomerism'
•  Abnormal/loss'of'midline'barrier'
– 
– 
– 
– 
Spread'of'le['side'signals'to'right'
e.g.'le[y.1,'sonic'hedgehog'
Le['isomerism'
No'good'animal'model'(other'funcAons;'early'lethality)'
?
G
D
G
G
D D
D
G
Les'transgènes'et'l’idenAté'des'régions'cardiaques'
E9.5
E10.5
RA!
LA!
OFT!
RV!
LV!
RV!
Mlc1v-24
Mlc3f-9
Mlc3f-2
Mlc3f-2
Mlc1v-24
Mlc3f-9
Mlc3f-2 X iv/iv
E9.5!
Les'souris'iv'comme'modèle'd'’étude'de'la'latéralisaAon'
Mlc3f-2 X iv/iv
Situs inversus
E10.5!
Situs solitus
Situs ambigus
Un'marqueur'transgénique'pour'les'défauts'de'latéralisaAon'
Five'le['right'
asymmetric'
processes'in'
heart'
development'
Three types of
signalling
abnormality can lead
to isomerism
Pitx2'
Nodal'
Le[y2'
Pitx2)expression)in)the)le<)atrium)
Détermination très précoce de l identité
aortique et pulmonaire
Mlc1v-24 : VD et VE
Fgf 10
96-16 : Base du TP
T55 : Base de Ao
Sema3c
Hes1
Pitx2'mutants'develop'right'atrial'isomerism'
+/+'
normal'
δc/δc
Right'isomeric'
+/+
δc/δc
δc/δc
+/+
δc/δc
δc/δc
No'atrioventricular'septaAon'in'pitx2'c'nulls'
+/+'
+/+'
+/+'
δc/δc
δc/δc
δc/δc
-/-
Anterior'aorta'&'parallel'oujlows'in'pitx2'δc'
2.'Venous'sinus'&'tributaries'
 
Venous'vascular'corrosion'casts,'dorsal'views'
+/+
δc/δc
Bilateral'inferior'caval'veins'in'pitx2'δc'
DEXTROISOMERISME
Les grandes étapes du “late looping”
•  La convergence des segments d’admission et d’éjection
est nécessaire pour que la septation ventriculaire et
l’alignement puissent se produire de façon correcte
•  La petite courbure (inner curvature, primary fold) est le
pivot autour duquel s’organise le remodelage des
jonctions AV et VA
•  L’encastrement (wedging) de la partie gauche du
segment d’éjection entre mitrale et tricuspide :
indispensable à l’achèvement de la septation cardiaque
La'convergence'des'segments'd’admission'et'd’éjecAon'
parAcipe'à'la'concordance'normale'
Segment''
d'’admission'
Segment''
d’éjecAon'
La'convergence'des'segments'd’admission'et'd’éjecAon'
parAcipe'à'la'concordance'normale'
L’encastrement'(wedging)'de'la'parAe'gauche'du''
segment'd’éjecAon'entre'les'2'VAV'est'indispensable''
à'l’achèvement'de'la'septaAon'cardiaque'
SeptaAon'cardiaque':'J35'
SeptaAon':'achevée'par'la'réunion'de'5'septa,'dont'
l’alignement'est'nécessaire'pour'obtenir'un'cœur'normal'''
– 
– 
– 
– 
– 
'
septum'interauriculaire'primiAf''
septum'atrioventriculaire''
septum'interventriculaire'primiAf''''
septum'conal'''''
septum'aortopulmonaire'
La'septaAon'
les'bourgeons'endocardiques'
Bourgeons'du'conus'
Bourgeon'inférieur'
Bourgeon'supérieur'
Bourgeons'latéraux'
Repères'chronologiques'
Bourgeons'endocardiques'
Entonnoir'tricuspidien'
ElongaAon'voie'éjecAon'
Arcs'Ao'4et'6'
FormaAon'valve'semilunaires'
DélaminaAon'valve'tricuspide'
Wedging'
Convergence'
Croissant'
cardiaque'
SeptaAon'
cardiaque'
LOOP'
J18'
J23'
30'
Corne'D'du'sinus'veineux'
AppariAon'VP'1°'
Arcs'Ao'2'et'3'
40'
42' 44'
50'
Connexion'coronaires'.'aorte'
This'is'what'happens'but'not'how'it'
happens!!'
'
Human'heart'development,'from:'
Principles,of,Development'by'Lewis'Wolpert,'2nd'Edn'
'
An'outstanding'textbook'from'an'outstanding'scienAst.'
Tube'cardiaque'primiAf'
2'cavités':'antérieure'(ventricules),
postérieure'(oreilleres)'
Are'there'5'linearly'
arranged'heart'
segments,'and'is'a'
straight'tube?'–'No'&'
No'
There'are'never'specified'
“cylinders”'in'sequence.'
'
Gene'expression'domains'are'
varied'and'overlapping.'
Les champs cardiaques
•  Premier'champ'cardiaque'='tube'cardiaque'primiAf':'
ventricule'gauche'
•  Second'champ'cardiaque'
–  'ParAe'antérieure'='Aire'cardiaque'antérieure':'
ventricule'droit,'voie'd’éjecAon'
–  ParAe'postérieure'='Mésocarde'dorsal':'septaAon'
atrioventriculaire,'veines'pulmonaires,'sinus'venosus'
•  Cellules'de'la'crête'neurale'
•  Voie'd’éjecAon,'cellules'musculaires'lisses'des'gros'
vaisseaux'(arcs'aorAques)'
•  Cellules'de'l’épicarde'
–  Coronaires,'valves'AV,'voies'de'conducAon'
Cardiac precursor populations and lineages
Cardiac progenitor populations
Pro/epicardial3cells3
Cardiac NCC
Endocardial cells
Recruitment)of)cell)popula7ons)
Stolfi3et3al.320103
 'the'increase'in'complexity'and'size'of'the'heart'is'accompanied'by'an'increasing'recruitment'from'the'SHF'
 'the'cranial'mesoderm'and'occipital'lateral'mesoderm'contribuAon'may'have'been'redeployed'during'evoluAon'into'
the'SHF'
The'second'heart'field'is'further'subdivided'into'anterior'and'posterior'domains''
contribuAons'
Tbx13
Fgf83
aSHF:' Fgf103
Isl1'
pSHF:'
OFT'
RV'
RA'
LA'
Podoplanin3
Isl1,
Kelly3et3al.320013
GiCenberger3de3Groot3et3al.320073
Galli3et3al.320083
This'has'been'characterised'in'terms'of'gene'expression'
Myocardial)lineages)
 'the'second'myocardial'lineage'can'be'
anterior)second)myocardial)lineage)
masAcatory'muscles'
right)ventricle)
subdivided'into'an'anterior'and'a'posterior'
sub.lineage'contribuAng'to'the'
branchiomeric'and'non.somiAc'neck'muscles'
le['facial''
expression'muscles'
respecAvely'
right'facial''
expression'muscles'
 'further'sub.lineages'can'be'idenAfied'
pulmonary)trunk)
aorta)
posterior)second)myocardial)lineage)
le['neck'muscles'
pulmonary)trunk)
le<)atrium)
le<)superior)caval)vein)
pulmonary)vein)
right)atrium)
right'neck'muscles'
right)superior)caval)vein)
first)myocardial)lineage)
right)ventricle)
le<)ventricle)
right)atrium))
le<)atrium)
 'unexpected'relaAonship'between'the'
venous'and'arterial'pole'of'the'heart'
(A)
SM
NCC
FHF
SHF
AHF
PHF
OFT
Sinus
Venosus
PEO
DMP
Atrial
septum
Pulm.
veins
EPDC
AV
cushions
Card.
veins
Caval
veins
Dist.
cushions
Coronary
Vasc.
LV
RV
Aorta
IV
septum
AV
valves
Conduct.
system
(B)
Pharyng.
aches
Prox.
cushions
Atrium
OFT
septum
Semilun.
valves
Pulm.
trunk
ductus
arterios.
Auton.
Innerv.
Forming'the'chambers'from'the'tube'involves'
'the'addiAon'of'cells'at'both'ends,'bending,'and'
expanding'(or'ballooning)'
Cellules'de'la'crête'neurale'
Remodelage'du'conotruncus'
Remodelage'du'conotruncus'
Conotruncus'proximal'
.'Valves'et'sinus'
.'ConnecAons'ventriculo.arterielles'
'
Conotruncus'distal'
'
.'Arcs'aorAques'
.'Troncs'artériels'(AA'et'TP)'
'
'
Carnegie'14'
C'17'
C'20'
Composants'du'conotruncus'
VE'
VD'
VG'
Myocarde'
'
Endocarde'
coussins'endocardiques'
'
Cellules'de'la'crête'neurale'
Le'second'champ'cardiaque'
Second'heart'field':'three'lines'of'evidence'
. 'Gene'expression':'Fgf10'
'Kelly'et'al.,'Dev'Cell'(2001)'
. 'Fate'mapping':'Islet1'(Cre.lox)'
Cai'et'al.,'Dev'cell'(2003)'
'
'
. 'Clonal'analyses'
Meilhac'et'al.,'Dev'cell'(2004)'
Mlc1v/2433
(Fgf10)3
FGF10)reporter)construct)marks)
OFT)and)right)ventricle)
Kelly'et'al,'Dev'Cell'2001'
Clones'(cells'from'a'single'
progenitor)'do'not'populate'the'
right'and'le['ventricles.'
(Buckingham'lab,'Developmental'
Cell,'6:'1,'2004)'
Islet 1 (Cai et al, Dev Cell, 5: 877, 2003
Clones'(cells'from'a'single'
progenitor)'do'not'populate'the'
right'and'le['ventricles.'
(Buckingham'lab,'Developmental'
Cell,'6:'1,'2004)'
Embryologic origin of the coronary arteries
Gittenberger-de Groot AC, J Cell Mol Med 2010 ; 5 : 1056-60
Le'second'champ'
cardiaque''
Pitx2c'
Isl1'
Nkx2.5'
Tbx1'
Mef2c'
Fgf8.10'
Isl1'
Nkx2.5'
Wnt2'
•  Source'de'proliféraAon'
cellulaire'
–  Cellules'précurseurs'
cardiaques'
–  Cellules'myocardiques,'
endothéliales,'musc'lisses' SVC'
•  Plusieurs'«'sous.
domaines'»'
Tbx18'
AorAc'arch'
LA'
RA'
–  Antérieur'
•  Voie'd’éjecAon'
•  Ventricule'droit'
–  Postérieur'
•  Oreilleres''
•  SeptaAon'
auriculoventriculaire'
Pulm'Veins'
Pitx2c'depdt'
PT'myoc'
Sema3c+'
IVC'
RV'
LV'
ContribuAon'
from'a'subset'
Tbx1'or'Pitx2'
cells'
Vincent'D,'Buckingham'M,'2010'
1°CC'
2°CC'
Cell'endoth'vasc'
SCC'
Cell'myoc'
Crête'
neurale'
Sinus'venosus'
Organe'
proépic'
Vincent'D,'Buckingham'M,'2010'
Septation conotruncale
•  Crête'neurale':'
–  arcs'aorAques'
–  septaAon''
•  Aire'cardiaque'
antérieure:'
–  septum'conal'
–  convergence,wedging'
–  pénétraAon'des'
coronaires'dans'l’aorte'
Waldo et al, Dev Biol 2005 ; 281 : 78-90.
Comment'appliquer''
les'mécanismes'de'l’embryogenèse'cardiaque'
aux'cardiopathies'congénitales'
1'CHD'
1'CHD'
1'group'
of'CHDs'
1'CHD'
1'CHD'
Mechanism'1'
Many'
Genes'
1'Gene'
1'Mechanism'
Many'
Genes'
Mechanism'2'
Mechanism'3'
Group'of'
Genes'1'
1'Gene'
Group'of'
Genes'2'
Group'of'
Genes'3'
NoAon'd’hérédité'mulAfactorielle''
Concordant'congenital'heart'defects'
ToF
1'CHD'
1'Gene'
ToF
ToF
ToF
ToF
ToF : tetralogy of Fallot
ToF
ToF
Concordant'congenital'heart'defects'
'
ToF
ToF
ToF
ToF
ToF
ToF
ToF
Approche'mécanisAque'
Phylogeny'
•  Darwin'
–  Based'on'the'noAon'of'common'ancestor'
•  PhylogeneAc'groups'.phylum'
depicted schematically. S1, S2, S3, S4, somites 1–4.
Cardiac Development
Figure 11.5. Scanning electron micrograph of the pharyngeal
arches in a chick embryo. (A) Overview. The head is to the right.
(B) Bulges are produced by cardiac neural crest in the pharyngeal
arches. 1md, mandibular part of pharyngeal arch 1; 1mx,
maxillary part of pharyngeal arch 1; 2–6, pharyngeal arches 2–6.
(Courtesy of K. K. Sulik, Embryo Images at
www.med.unc.edu/embryo_images/.)
neural crest cells before they migrate leads to abnormal patterning of the pharyngeal arches (Kirby et al., 1997).
The aortic arch arteries are a bilaterally paired series of
arteries that connect the aortic sac ventrally with the initially
paired dorsal aortas located dorsal to the foregut. These arteries
e 11.13. The morphological and functional
quences of cardiac neural crest ablation.
Kirby'2010'
development. One of the signaling factors expressed
by both
TransvecAon'
Ao PA
RV
Ao PA
Ao PA
LV
RV
LV
A
RV
B
C
Ao PA
Ao PA
LV
Ao PA
TA
LA
Conotruncal
RV
heart defects
RV
LV
RV
D
LV
LV
E
F
Ao PA
RV
Ao PA
Ao PA
LV
RV
LV
A
RV
B
C
Ao PA
Ao PA
LV
Ao PA
TA
LA
Conotruncal
RV
heart
defects
RV
LV
RV
D
LV
LV
E
F
MechanisAc'classificaAon'
  Neural'crest'cell'migraAon'defects''
Conotruncal3malformaKons3
  Flow'defects:''
HypoplasKc3leM3heart3
  Targeted'developmental'defects'
TAPVR3
  Extracellular'matrix'defects''
Ventricular3Septal3Defects3
  Endocardial'cushions'defects'3
Atrioventricular3septal3defects3
  3Looping'anomalies'
Heterotaxia3
1 group
of
CHDs
1 Mechanism
1 Gene
Mechanistic classification
•  Neural crest cell migration defects :
–  Conotruncal malformations
•  Flow defects:
–  Hypoplastic left heart
•  Targeted developmental defects
–  TAPVR
•  Extracellular matrix defects:
–  VSD
•  Endocardial cushions defects:
–  Atrioventricular septal defects
•  Looping anomalies:
–  heterotaxia
La'concordance'selon'la'classificaAon'
"mechanisAque"'
Tétralogie
de Fallot
Tétralogie
de Fallot
IAA
TAC
A
APSO
RVPA
Canal
atrioventriculaire
B
Coarctation
Hypothesis from the mechanistic
classification
One'geneAc'anomaly'(mutaAon,'deleAon)'
'
One'group'of'cardiac'malformaAons'
'
Homogeneous'in'the'mechanisAc'classificaAon'
chromosomes 22 (four green dots) but only one set has the 22q11
region (two red dots). (From Chen et al., 2005, with permission.)
1'CHD'
1'CHD'
1'group'
of'CHDs'
Many'
Genes'
1'Gene'
1'Mechanism'
Tbx1/Cre3
1'Gene'
Huynh et al. 2007 Genesis 45:470-75
Is'the'mechanisAc'classificaAon'
useful'in'human'geneAcs?'
Délétion 22q11
DistribuAon'of'conotruncal'defects''
in'newborns'with'22q11'deleAon'
'
'
'
'
'
''
•  Tetralogy'of'Fallot
'
•  PA.VSD '
'
'
•  Interrupted'aorAc'arch'
•  Truncus'arteriosus
'
•  Infundibular'VSD
'
•  AorAc'arch'anomalies '
Randomised'phenotype'
'%
'
'22.2
'10.8
'26
'11.4
'15.5
'13.9
''
''
''
''
''
''
A Tbx1-dependent population of outflow tract myocytes
-/-
Di'Felice'and'Zummo'2009'
Trends'Cardiovasc'Med'19:130.5'
Subpulmonary myocardium is Tbx1-dependent"
Tetralogy of Fallot – failure of normal expansile growth of"
sub-pulmonary myocardium"
TBX1 haploinsufficiency is the most frequent known genetic
of "
Tbx1-/-cause
tetralogy (10% of patients have DiGeorge syndrome)"
Pistes'pour'expliquer'
la'variabilité'du'phénotype'
MechanisAc'
Phenotypic'variability'for'a'given'mutaAon'
•  Modifiying'genes'and'allelic'heterogeneity'
–  VEGF'haplotypes'and'cardiac'phenotypes'in'22q11'deleAon'
Stalmans'I,'et'al.'Nat'Med.'2003;9:173.82'
•  Fetal'hemodynamics'
•  Surprises'of'complex'systems'
René'Thom'
Modèle'stochasAque'
Canal3atrio/ventriculaire3
Division '0'ou'1'
MigraAon'0'ou'1'
Adhésion'0'ou'1'
Modifiers and phenotypic variability
Stalmans'I,'et'al.''
VEGF:'a'modifier'of'the'del22q11'
(DiGeorge)'syndrome?'
Nat'Med.'2003;9:173.82.'
'
Phenotypic assessment
Phenotypic'conAnuum'
Reccurrence'within'the'conAnuum'
Cannes'2006'
A few years ago…
•  Everything has been
finally clearly understood
•  Get rid of old fashion
concepts
•  Become a member of
the new aristotelician
view of genetics of CHD
–  CHD are monogenic
diseases
–  One group of CHD-one
mechanism-one gene
Cannes'2006'
1'CHD'
1'CHD'
1'group'
of'CHDs'
Many'
Genes'
1'Gene'
1'Mechanism'
1'Gene'
1'CHD'
Many'
Genes'
Genes'for'syndromic'and'non'syndromic'defects'
are'different'
HOLT-ORAM
TBX 5
Basson.3PNAS319993
Atrial septal defect + AV-block
NKX 2.5
GeneAc'heterogeneity'
'
'
One'heart'defect.many'genes'
Human mutations in CHD
ASD:'TBX5,3NKX2.5,3GATA4,3MYH63
VSD:'NKX2.5,3TBX5,3GATA43
AVSD:'CRELD,3HEY2,3EVC,3EVC23
ToF:'TBX1,3NKX2.5,3FOG2,3JAGGED13
CAT:3NKX2.6,3TBX1,3Semaphorin3C3
DORV:'CFC1,3ZIC3,3TBX13
TGA:'CFC1,3PROSIT3240,3ZIC33
LVOTO:'NOTCH1,3GATA43
Pulmonary'stenosis:3PTPN11,3RAF/1,3BRAF,3SOS13
Gene'duplicaAon'
Nk2)
Mef2) Gata) Tbx) Hand)
DiploblasAc'
Cnideria'
Myoepithelium'
1'
1'
?'
1'
0'
'
'
TriploblasAc'
bilaterian'
Arthropods'
Tubular'heart'
1'
1'
2'
3'
1'
Cephalochordates' Vascular'system'
1'
1'
2'
?'
1'
Tunicates'
Centralized'pump'
1'
1'
2'
≥3'
1'
Fish'
2.chamber'
≥3'
4'
3'
≥4'
1'
Amphibians'
3.chamber'
≥3'
4'
3'
≥5'
2'
Amniotes'
3.'or'4.chamber'
≥2'
4'
3'
≥7'
2'
Modified from Olson EN. Science 2006;313:1922-7
Genetic heterogeneity of conotruncal defects
• 
• 
• 
• 
• 
Del'22q11'TBX1'
Alagille'JAGGED13
Wardenburg'PAX33'
Trisomie'21'
CHARGE''
•  NKX2.53
•  Fog2'mutaAons'
•  GATA,'etc…..'
Hétérogénéité'généAque'des'CAV'
Ellis'van'Creveld'syndrome'
Looping'anomalies'
Smith.Lemli.Opitz'
8p23'del'
Down'syndrome'
Noonan'syndrome'
Phenotype.genotype'correlaAons'
Down'syndrome'
Normal'
chromosomes'
Normal'
Chromosomes'
isomerism'
Non del 22q11
del 22q11
Principe'd’incerAtude'd’Heisenberg'
• Pour une cardiopathie
donnée, on ne peut connaître
l’anomalie génique
• Pour une anomalie génique
donnée, on ne peut connaître
la cardiopathie
Cannes'2006'
To be…
Or not to be…
…a
conotruncal
defect
CFC1 mutations and TGA/DORV
Goldmuntz et al. 2002
Lignée'cardiosensor':'96.16'
Expression'de'96/16'dans'un'contexte'Splotch///'
VDDI'
Défauts'de'rotaAon'?'
96-16 expression in Splotch heart with PTA
- Persistent truncus arteriosus with a rotation defect
-  In addition to a septation defect
-  Abnormal neural crest cell migration
Expression'de'96.16'dans'un'contexte'Pitx2c///''.'latéralité'
WT'
Pitx2c///'
TP'
TGV'
TP'
WT3
Pitx2c///3
Défauts'd’asymétrie'?'
96-16 expression in Pitx2δc heart with TGA
ao
pt
ao
pt
-  Transposition of the great arteries with a rotation defect
-  Normal septation and normal neural crest cell migration
-  Defect of left-right signalling
A quel groupe appartient la TGV ?
•  Ce'n’est'pas'une'cardiopathie'conotroncale'
•  C’est'un'défaut'segmentaire'de'la'latéralité'(rotaAon)'
•  Certaines'cardiopathies'(TAC,'VDDI,…)'peuvent'être'
considérés'comme'tels'
•  Hétérogénéité'généAque'sous.tendue'par'
hétérogénéité'des'mécanismes/gènes'
Familles'TGV'
Familles'TGV'+'DD'
VDDI
AR
D
G
AV
Ao
AP
Ao
AP
Ao
Ao
AP
VD
VD
Ao
AP
VD
AP
VD
VD
S,D,L
Ao
AP
Ao
AP
Ao AP
AP
Ao
AP
Ao
•  TransposiAon'of'the'great'arteries'is'a'segmental'
looping'anomaly'limited'to'the'oujlow'tract'
'
•  Could'other'segmental'discordances'be'allelic'to'situs'
anomalies?'
–  Corrected'transposiAon'of'the'great'arteries'
–  Isolated'atrio.ventricular'discordance'
–  Peristent'le['superior'vena'cava'
Défauts segmentaires latéralité
La'persistance'de'la'VCSG'
Cardiopathies'associées':'CoarctaAon'
CoarctaAon'='spectre'vers'hypoVG'
L’anomalie'iniAale'est'la'VCSG':'le'diagnosAc'
est'coarctaAon'
•  7'familles'idenAfiées':'hypoVG'avec'VCSG'+'
isomérisme'
• 
• 
• 
• 
Intrasegmental diversification of myocardial cells
Prc'
Tin'
α.Spec'
Mef2'
ndae1'
Zaffran'et'al.,'CSH'Symposium'2002'
to about somites 2–3, and mediolaterally from the PARAXIAL
almost to the edge of the embryonic disc (Abu-Issa
and Kirby, unpublished). In addition, because different parts of
MESODERM
22
tinuous across the midline, cranial to the prechordal plate
(DeHaan, 1963, 1963; Rosenquist, 1970). However, when tritiated thymidine-labeled mesoderm/endoderm from the crescent
Cardiac Development
posteriorly 0.4 mm caudal to the node (Rawles, 1943).
Myocardial potency diminishes toward the periphery of these
regions, and there are no asymmetric differences in the shape
Figure 3.3. Location of Nkx2.5 and Bmp4 expression in
extent
twofields
areas.
One of the problems with the
relationand
to the various
maps of
of thethe
cardiogenic
in
the mesoderm in chick at approximately stage 5.
early potency mapping has to do with the method of tissue
explantation. Many of these studies used all the layers of the
Figure 3.4. Expression of Isl1 (green) and MLC2a (red) messages showing subdivision of the
blastodisccardiogenic
and thus
myocardial
potential
of(2cells
in the
epifield in the
mouse. (A) ED7; (B)
ED7.5; (C) ED8.0
somite pairs);
(D) ED8.25
somite pairs); (E) ED8.5 (8 somite pairs). (From Cai et al., 2003, with permission.)
blast was(5not
distinguished from myocardial potential of cells
located in the mesoderm.
The early fate mapping studies, by contrast, were able to
mark EPIBLAST or MESODERM. However, many of these early
maps were generated before precise embryo staging was established and before a procedure for whole embryo culture was
available (Hamburger and Hamilton, 1951; New, 1955). Thus,
the earlier studies are less precise and repeatable than studies
done since the late 1950s. Also, because whole embryos in
culture do not develop well after 36–48 h and myocardial cells
contributed to the heart during the looping period are added
very late, their existence was unappreciated until very recently.
Fate mapping has been done using several methods: labeling
with vital dyes, iron particles, autoradiography, quail-chick
chimeras, and most recently fluorescent dye labeling. Vital dye
labeling first showed that tissues with cardiogenic potential
were present in the early blastoderm, an imprecisely defined
cell origin (Graper, 1907; Pasteels, 1936).
Figure 3.1. Location of the myocardial progenitors in the chick
primitive streak, cardiogenic fields and heart tube. (A–C) Position in
the primitive streak and heart tube but it was not understood until
recently how the organization in the cardiogenic fields related to that
Á
Epiblast Origin of Cardiogenic Cells
The fate maps of all the vertebrates appear to follow a gener-
Laterality'defects'in'human'
Asymmetry'disease'
•  Heterotaxy'
–  Right'isomerism'
• 
• 
• 
• 
Abnormal'systemic'and/or'pulmonary'vein'connecAon'
«'AVSD'»'
«'Single'ventricle'»'
«'DORV'»'
–  Le['isomerism'
•  Asymmetry'disease'limited'to'an'«'antero.posterior'»'segment'
– 
– 
– 
– 
– 
TGA'
Double'discordance'
Some'types'of'AVSD'?'
Atrioventricular'discordance'
Abnormal'vein'connecAons'?'
TGA'is'a'LR'asymmetry'defect'?'
Fuzzy Logic
•  Le truncus est une cardiopathie
conotroncale ou une anomalie de
rotation
• Le VDDI est une cardiopathie
conotroncale et une anomalie de rotation
• La TGV est une cardiopathie de la
région conotroncale non liée à la crête
neurale
Cardiopathies conotroncales
+
RVPA
• 
• 
• 
• 
Groupes'embryologiques'différents'
23'paAents'idenAfiés'–'4'formes'familiales'
Cardiopathies'Second'heart'field?'
Truncus'arteriosus'
–  Conotroncal'crêtes'neurales'
–  RotaAon'defect'ou'second'heart'field'defect'
Coarctation de l’aorte
•  Maladie'de'l’isthme'aorAque'
•  Cardiopathie'de'débit':'spectre'hypovg'
•  Cardiopathie'conotroncale'
–  InterrupAon'de'crosse''
–  Syndrome'de'coarctaAon'
•  ParAe'émergée'de'l’iceberg':'VCSG'au'sinus'coronaire'
Une'cardiopathie':'plusieurs'mécanismes'donc'
hétérogène'généAquement'
1'CHD'
1'CHD'
1'group'
of'CHDs'
1'CHD'
1'CHD'
Mechanism'1'
Many'
Genes'
1'Gene'
1'Mechanism'
Many'
Genes'
Mechanism'2'
Mechanism'3'
Group'of'
Genes'1'
1'Gene'
Group'of'
Genes'2'
Group'of'
Genes'3'
Le['superior'caval'vein'
CoarctaAon'of'the'aorta'
1'CHD'various'mechanisms'
1'CHD'
•  Disease'of'the'aorAc'isthmus'
•  Flow'defect':'spectrum'of'HLHS'
•  Contruncal'defect'
Mechanism'1'
Mechanism'2'
Mechanism'3'
–  Interrupted'aorAc'arch'
•  Laterality'defect'with'persisAng'LSCV'
Group'of'
Genes'1'
Group'of'
Genes'2'
Group'of'
Genes'3'
Société'de'Biologie'2008'
Coronary patterning
•  La'connexion'des'coronaires'définit'l’idenAté'aorAque'
•  La'posiAon'des'coronaires'est'un'marqueur'de'la'rotaAon'
•  Dans'le'TAC'
–  Coronaires'normales':'il's’agit'd’une'aorte'='atrésie'pulmonaire,'défaut'
de'septaAon'
–  Coronaires'anormales':'défaut'de'rotaAon'
Gittenberger-De Groot et al. The embryology of the common arterial trunk.
Progress Ped Cardiol 2002 ; 15 : 1-8
Coronaires':'le'pôle'veineux'
Pôle'veineux'
Origine'des'vaisseaux'
coronaires''
•  Les'coronaires'se'forment'à'
parAr'de'l’épicarde'
OPE'
(péricarde'viscéral)'
•  Organe'proépicardique'
•  Rentre'en'contact'avec'le'
myocarde'près'du'pôle'
veineux'(sinoatrial)'du'cœur'
OPE'
Pôle'
artériel'
Embryologie':'transformaAon'épithélio.
mésenchymateuse'
•  Les'Ç'épithéliales'du'pro.
épicarde'se'transforment'en'Ç'
mésenchymateuses'(FOG2,'
GATA4)'
•  Migrent'dans'le'Assu'conjoncAf'
de'l’espace'sous.épicardique'
•  Puis'dans'des'espaces'
nouvellement'créés'dans'le'
OPE'
myocarde'en'développement'
•  Ces'deux'espaces'
communiquent'entre'eux'mais'
pas'avec'la'lumière'ventriculaire'
(endocarde'intact)':'Pas,de, Mesenchyme'
sinusoïdes,
OPE'
Epithelium'
Endocarde'
Espaces'
intramyoc'
Embryologie : les sinusoïdes
•  Existent'de'façon'transitoire….'Chez'les'oiseaux'!'
•  Disparaissent'avec'la'condensaAon'du'myocarde'
•  Et'persistent'de'façon'pathologique'
–  Si'hyperpression'ventriculaire'(APSI)'
–  Si'défaut'de'maturaAon'du'myocarde'V'
–  Si'dévpt'anormal'(fistules'coronaro.cardiaques)'
•  Mais3:3pas3de3sinusoïdes3au3cours3du3
développement3des3coronaires3chez3l’humain3
Origine des
vaisseaux
coronaires
Pôle'veineux'
•  Les'coronaires'se'forment'à'
parAr'de'l’épicarde'
OPE'
(péricarde'viscéral)'
•  Organe'proépicardique'
•  Rentre'en'contact'avec'le'
myocarde'près'du'pôle'
veineux'(sinoatrial)'du'cœur'
Pôle'
artériel'
Origine des vaisseaux coronaires
•  Cellules'endothéliales'du'sinus'veineux'(pôle'
veineux)'+'endocarde'
•  DédifférenciaAon'des'cellules'veineuses'
•  Invasion'du'myocarde':'formaAon'des'artères'
•  Vaisseaux'superficiels':'veines''
K'Red.Horse'et3al.'Nature'464,'549.553'(2010)'
Origine des vaisseaux coronaires : organe
proépithelial ou pôle veineux ??
•  Pas'd’épicarde'='pas'd’artères'coronaires'
–  Poulet'(physical'inhibiAon)
'
–  Souris'(α4'integrin,'VCAM.1) '
'Girenberger.de'Groot'2000'
'Yang'1995,'Kwee'1995'
•  Poulet,'souris'et'homme:'les'vaisseaux'sanguins'
apparaissent'uniquement'quand'l’épicarde'est'formé'
Embryologic'origin'of'the'coronary'arteries'
Girenberger.de'Groot'AC,'J'Cell'Mol'Med'2010';'5':'1056.60'
Coronary sinuses are formed
via ingrowth of the peritruncal capillary plexus
Ao'
Ao'
Peritruncal''
Capillary'plexus'
D'
R'
PenetraAng''
plexus'
L'
LCA'
RCA'
D'
R'
L'
SMC'
LCA'
Hypoxia'and'apoptosis'are'correlated'with'the'invasion'of'the'Aorta'
Tomanek'RJ,'Angiogenesis,'2005'
Coronary'artery'parerning'in'Tbx1///'hearts'
LCA!
Ao!
RCA!
PT!
Tbx1+/-
Connexin403
eGFP3
Tbx1+/+3
Tbx1///3
Embryologie : pénétration des coronaires
dans l’aorte
•  Les'coronaires'sont'
«'aárées'»'par'l’aorte'
(domaine'sous.aorAque)'
•  Elles'pénètrent'dans'l’aorte'
au'point'le'plus'près'de'leur'
trajet'épicardique'
•  Mais'en'«'fuyant'»'l’artère'
pulmonaire'(domaine'sous.
pulmonaire)'
Ao
RCA
LCA
PT
Coronary artery patterning in Tbx1-/- embryos"
*"
*"
*"
RCA"
Tbx1-/- common outflow"
Control aorta"
Coronary stem"
Right"
LCA"
Ostium position (valve cusp)"
Left" Dorsal"
Right"
Left"
Dorsal"
Left"
0"
13"
1"
17"
5"
2"
Right"
14"
0"
0"
20"
0"
4"
Common"
0"
0"
0"
9"
0"
1"
Additional"
2"
0"
0"
8"
2"
2"
n=14"
n=34"
Théveniau-Ruissy et al 2008
Circulation Research 103:142-8
Coronary'artery'parerning'in'Tbx1///'hearts'
PECAM'
LCA!
Ao!
RCA!
PT!
Tbx1+/-
RCA!
LCA!
Tbx1+/-
Tbx1-/-
Tbx1-/-
PECAM
Embryonic''
truncus''''''''''''''''' '
'Control'''
'CAT''''''''''''''''DORV''''''''''''''' 'TOF'''''''''''
'TOF&PA'''''''
Ao'''''''''PA'
.'
Degree'of'rotaAon'
Anomalies'of'OFT'rotaAon'responsible'for'CT'defects'significantly'affect'the'posiAon'of'
coronary'osAa'
+'
Anomalies'of'OFT'rotaAon'responsible'for'CT'defects'significantly'affect'the'posiAon'of'
coronary'osAa'
1'CHD'
1'
CHD'
1'group'
of'CHDs'
1'CHD'
1'
CHD'
1'CHD'
Mechanism'1'
Many'
Genes'
1'
Gene'
1'
Mechanism'
Many'
Genes'
Mechanism'2'
1'
Gene'
Mechanism'3'
Group'of'
Genes'1'
1'
Gene'
Variety'of'
phenotypes'
Group'of'
Genes'2'
Group'of'
Genes'3'
Bmp4)and)Morphological)Varia7on)of)Beaks)in)Darwin's)Finches)
Arhat'Abzhanov1,'Meredith'Protas1,'B.'Rosemary'Grant2,'Peter'R.'Grant2'and'Clifford'J.'Tabin1,*'
Testing the refractory potential of
subpulmonary myocardium in explant assays
E12.5'
In'vitro'explants'
Subpulmonary'
myocardium'
Tbx1///3
Tbx1+/+3
Pecam'
Ventricular'
myocardium'
Tbx1///3
AcAn.GFP'
Experiments'are'aimed'at'generaAng'fluorescent'microvessels'in'culture'
E12 explants cultured for 48h in collagen gel
Tbx13+/+3
Tbx13+/+3,33
AcKn3GFP/+3
GFP'
Pecam'
merged'
No'GFP.posiAve'vessels'are'observed'at'subpulmonary'explant'level'
E12'may'be'to'late'for'invesAgaAons'
Semaphorin3c is expressed in subpulmonary myocardium"
"
Semaphorins - a family of secreted or membrane associated glycoproteins"
that regulate cell motility and attachment in axon guidance and vascular growth"
SMA-FITC
+/+
Sema3c
-/-
Sema3c
SMA-FITC
+/+
Sema3c
-/-
Sema3c
SMA-FITC
-/-
Sema3cSMA-FITC
Kruger'et'al'2005'
Nat'Rev'Cell'Biol'6:789.800'
-/-
Sema3c
Gitler'et'al'2004'
Dev'Cell'7:107.16'
Théveniau-Ruissy et al 2008
Circulation Research 103:142-8
Coronary patterning defects in mice lacking the DiGeorge"
Syndrome candidate gene Tbx1"
Outflow tract hypoplasia in Tbx1 mutant embryos is associated "
with coronary artery patterning anomalies"
Sema3c is a Tbx1-dependent gene expressed in subpulmonary"
myocardium but is not the critical downstream mediator of the"
Tbx1 coronary artery phenotype"
What are the molecular and cellular mechanisms underlying the Tbx1 "
coronary artery phenotype? "
"
Magali Théveniau-Ruissy, Karim Mesbah, Pauline Parisot"
Identification of a new regulator of the second heart field"
T55
X
T55
16'B2.B4'
B" B"
lacZ3
B"B"
B"
B"
BamHI"
5538"
B"
3766"
inserAon'site'
WT" Tg"
Hes13is'a'T55'candidate'gene'expressed'in'the'SHF'
bHLH'transcripAonal'repressor,homologue'of'Drosophila'Hairy/Enhancer'of'Split"
Hes1'acts'as'a'negaAve'regulator'of'myogenesis'and'neurogenesis'
The'role'of'Hes1'is'to'ensure'maintainance'of'undifferenAated'progenitor'cells:'
"
Congenital'heart'defects'in3Hes1'null'embryos'
Hes1+//3
Hes1+//3
Hes1///3
Hes1///3
Hes1+/+3
Hes1///3
p27kip1'Hoechst'
D’Arcy Thompson
Of'growth'and'forms'
transvecAon'
IdenAficaAon'of'novel'subpulmonary'enriched'genes'
Identification of other differentially expressed genes – Affymetrix "
microarray using microdissected superior and inferior E10.5 OFT walls"
superior'
inferior'
45000'probes'screened'in'triplicate;'20'genes'enriched'>2.fold'in'inferior'wall'
'
'
'
'
ValidaAon'of'candidate'genes'
Tbx1-dependence of future subpulmonary myocardial genes?"
Integration of these genes in the Tbx1 genetic network controlling "
OFT morphogenesis"
Patterning of the second heart field
•  Before'addiAon'to'the'heart,'SHF'cells'express'Islet13(Cai'et'al.,'Dev'cell'2003)'
•  The'SHF'has'two'components:'
.'“anterior”'which'expresses'FGF10'and'makes'the'right'ventricle'
and'the'OFT'(Kelly'et'al.,'Dev'Cell'2001,'Zaffran'et'al.,'Circ'Res'2004)'
.'An'atrial,'or'inflow,'component'(pSHF)'(Galli'et'al.,'Dev'2008)'
AHF'
SHF'
Isl13
Fgf10/lacZ3
What are the signals that control the patterning of the heart fields ?
Courtesy'Stéphane'Zaffran'
TAPVC to the coronary sinus
PAPVC to the superior caval vein
PAPVC to the inferior caval vein
PAPVC to the inferior caval vein (pulmonary
atresia)
PAPVC to the innominate vein (absent
pulmonary valve)
Discordant ventriculoarterial connections IVS
TAPVC to the coronary sinus
TAPVC to the inferior caval vein
PAPVC to the right atrium
TAPVC to the coronary sinus (+ coarctation)
Interrupted aortic arch
TAPVC to the coronary sinus
TAPVC to the innominate vein
Subarterial ventricular septal defect
TAPVC to the innominate vein
TAPVC to the portal vein (+ coarctation)
Double outlet right ventricle
TAPVC to the superior caval vein
PAPVC to the innominate vein
Right pulmonary artery from ascending aorta
PAPVC to the right atrium
subarterial ventricular septal defect was diagnosed (and
subsequently repaired) in the final patient after repair of
an abnormal venous connection.
Of the 23 patients, 17 had both the conotruncal defect
and the APVC repaired. No treatment was proposed for three
patients (in the early 1980s). Eight of nine TAPVC were
n=4
n=2
n=2
n=2
n=1
IAA: interrupted aortic arch; IVS: intact ventricular septum;
PAPVC: partially anomalous pulmonary venous connection;
TAPVC: totally anomalous pulmonary venous connection.
Ao, interrupted aortic arch, and aortopulmonary window.
Nomenclature was based on the European Paediatric Cardiac
Code [13]. We reviewed patient files for clinical presentation, family history, diagnostic and surgical procedures and
outcome.
Results
We identified 23 patients with conotruncal defects and APVC
in our database. The types of defects are shown in Table 1,
and two examples of the association are shown in Fig. 1.
Diagnosis of the conotruncal defect was made during
fetal life in five patients (CAT, n = 2; tetralogy of Fallot,
n = 1; discordant ventriculoarterial connections, n = 1; interrupted aortic arch, n = 1). APVC was detected during fetal
life in four patients. The presenting symptoms were as
follows:
• cyanosis, n = 20;
Figure 1. Schemes of conotruncal defects with associated anomalous pulmonary venous connections. A. Tetralogy of Fallot and
totally anomalous pulmonary venous connection to the coronary
sinus. B. Common arterial trunk and partially anomalous pulmonary
venous connection of both right pulmonary veins to the inferior
caval vein.
Schémas de malformations complexes associant une cardiopathie
conotroncale et un retour veineux pulmonaire anormal (RVPA). A.
Bajolle'F.'et'al.ACVD'2009'
Contribution of Hox-expressing cardiac progenitors
  Expression of Hoxa1, Hoxa3 and Hoxb1 define distinct sub-domains within the SHF
  Cardiac progenitor cells expressing anterior Hox genes contribute to atrial and subpulmonary myocardium
E14.5
Courtesy'Stéphane'Zaffran'
Different'domains'of'expression'at'the'venous'pole'of'the'heart'
GiCenberger3de3Groot3et3al.320073
Christoffels3et3al.320063
DeRuiter3et3al.319953
Christoffels3et3al.320063
What'is'the'origin'of'the'venous'pole'of'the'heart?'
1st)model))
caval'vein'and'pulmonary'vein'myocardium''
both'derive'from'the'posterior'SHF''
2nd)model)
caval'vein'and'pulmonary'vein'myocardium''
have'a'disAnct'origin'
Clonal analysis of the venous pole
relationships between atrial and venous myocardium
LA and LSCV are clonally related
LA and PV are clonally related
RA and RSCV are clonally related
morphology''
β.gal'
2044
LA+LSCV
1804
LA+PV
1638
RA+RSCV
atrial and venous myocardium are clonally related
Lescroart'et'al.'2011'