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\DOI{10.5802/crbiol.151}
\datereceived{2023-10-10}
\daterevised{2024-02-05}
\dateaccepted{2024-02-09}
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\dateposted{2024-03-15}
\begin{document}

\begin{noXML}

\CDRsetmeta{articletype}{review}

\title{On the involvement of the second heart field in congenital heart
defects}

\alttitle{Sur l'implication du second champ cardiaque dans les
malformations cardiaques cong\'{e}nitales}

\author{\firstname{Clara} \lastname{Guijarro}}
\address{Aix-Marseille Universit\'{e}, CNRS UMR 7288, IBDM, Marseille, France}

\author{\firstname{Robert G.} \lastname{Kelly}\CDRorcid{0000-0003-4357-0073}\IsCorresp}
\addressSameAs{1}{Aix-Marseille Universit\'{e}, CNRS UMR 7288, IBDM, Marseille, France}
%\curraddr[R. G. Kelly]{Aix-Marseille University, IBDM CNRS UMR 7288, Turing Centre for Living Systems, Luminy Campus Case 907, 73288 Marseille Cedex~9, France}
\email[R. G. Kelly]{Robert.Kelly@univ-amu.fr}

\keywords{\kwd{Heart development}\kwd{Embryology}\kwd{Cardiac
progenitor cells}\kwd{Congenital heart defects}}

\altkeywords{\kwd{D\'{e}veloppement
cardiaque}\kwd{Embryologie}\kwd{Cellules prog\'{e}nitrices
cardiaques}\kwd{Malformations cardiaques cong\'{e}nitales}}

\def\thanksname{Note}
\thanks{Robert G. Kelly is the recipient of the prix Lamonica de
Cardiologie - Fondation pour la Recherche Biom\'{e}dicale PCL 2022 from
the Acad\'{e}mie des sciences.}

%\def\thanksname{Funding}
\thanks{\textbf{Funding.} Agence National pour la Recherche (PP-heart
ANR-20-CE13-0029-01 and Heartbound ANR-22-CE13 projects), AFM-Telethon,
French National Research Agency (ANR-16-CONV-0001), Excellence
Initiative of Aix-Marseille University A$^{*}$MIDEX (Turing Centre for
Living Systems) and the Fondation pour la Recherche M\'{e}dicale}

\begin{abstract} 
Congenital heart defects (CHD) affect 1 in 100 live births and result
from defects in cardiac development. Growth of the early heart tube
occurs by the progressive addition of second heart field (SHF)
progenitor cells to the cardiac poles. The SHF gives rise to
ventricular septal, right ventricular and outflow tract myocardium at
the arterial pole, and atrial, including atrial septal myocardium, at
the venous pole. SHF deployment creates the template for subsequent
cardiac septation and has been implicated in cardiac looping and in
orchestrating outflow tract development with neural crest cells.
Genetic or environmental perturbation of SHF deployment thus underlies
a spectrum of common forms of CHD affecting conotruncal and septal
morphogenesis. Here we review the major properties of SHF cells as well
as recent insights into the developmental programs that drive normal
cardiac progenitor cell addition and the origins of CHD. 
\end{abstract}

\begin{altabstract}
Les malformations cardiaques cong\'{e}nitales touchent 1 naissance sur
100 et r\'{e}sultent d'anomalies du d\'{e}veloppement cardiaque. La
croissance du tube cardiaque pr\'{e}coce se produit par l'ajout
progressif de cellules prog\'{e}nitrices du second champ cardiaque
(SHF) aux p\^{o}les cardiaques. Le SHF contribue au myocarde septal
ventriculaire, au myocarde ventriculaire droit et au myocarde de la
voie de sortie au p\^{o}le art\'{e}riel, et au myocarde auriculaire, y
compris le myocarde septal auriculaire, au p\^{o}le veineux. Le
d\'{e}ploiement du SHF est essentiel pour la septation cardiaque et a
\'{e}t\'{e} impliqu\'{e} dans la formation du boucle cardiaque et, avec
les cellules de la cr\^{e}te neurale, dans l'orchestration du
d\'{e}veloppement de la voie eff\'{e}rente. Perturbation
g\'{e}n\'{e}tique ou environnementale du d\'{e}ploiement du SHF est
donc \`{a} l'origine d'un spectre de formes communes de maladies
cardiaques cong\'{e}nitales affectant la morphogen\`{e}se conotroncale
et septale. Ici, nous passons en revue les principales
propri\'{e}t\'{e}s des cellules du SHF ainsi que les d\'{e}couvertes
r\'{e}centes sur les programmes de d\'{e}veloppement qui contr\^{o}lent
l'ajout de cellules prog\'{e}nitrices cardiaques ainsi que les origines
des malformations cardiaques cong\'{e}nitales. 
\end{altabstract}

\maketitle

\vspace*{.5pc}

\twocolumngrid

\end{noXML}

\section{Introduction}\label{sec1}

Defects in the complex process of cardiac morphogenesis underlie
congenital heart defects (CHD) that affect 1 in 100 live
births~\cite{1}. The heart is derived from mesoderm, the central of the
three embryonic germ layers, and originates in the anterior region of
the early embryo as a tubular structure with venous (inflow) and
arterial (outflow) poles. Cardiac progenitor cells located in
splanchnic mesoderm, overlying future foregut endoderm, contribute
progressively to growth of the heart. These cells, known as the second
heart field (SHF), ultimately give rise to ventricular septal, right
ventricular and outflow tract myocardium at the arterial pole, and
atrial, including atrial septal myocardium, at the venous pole.
Deployment of SHF cells is coincident with looping morphogenesis, an
essential conserved process during which the heart tube rotates to the
right to correctly position the future regions of the definitive heart.
Subsequent steps of heart development \mbox{involve} cardiomyocyte
proliferation driving chamber morphogenesis, development of the
coronary arteries and cardiac conduction system coordinating the
heartbeat, and cardiac septation. During septation the formation of
ventricular and atrial septa generates the four chambered heart, and
the embryonic outflow tract is divided into the outlets of the left and
right ventricles, the ascending aorta and pulmonary trunk. The
initially tubular embryonic heart thus acquires independent right
atrioventricular and left ventriculoarterial junctions. Cardiac
septation is complete at birth when the pulmonary duct and the
interatrial foramen close, coincident with pulmonary perfusion,
isolating the pulmonary and systemic circulatory systems. 

Heart tube elongation by addition of SHF cells creates the template for
cardiac septation and therefore perturbation of SHF deployment in human
patients and animal models results in a spectrum of common forms of
CHD~\cite{2,3}. These include ventricular and atrial septal defects as
well as outflow or conotruncal defects, and lead to failure to
correctly separate the systemic and pulmonary circulation. Dissecting
the developmental programs driving normal cardiac progenitor cell
deployment is thus essential to understand how defects in these
processes result in CHD, as well as for directing stem cell derived
cardiomyocytes to different regional fates for drug testing and tissue
repair following cardiac damage. Here we review the contribution of the
SHF during heart development and its involvement in CHD, with focus on
the roles of two T-box transcription factors implicated in human CHD,
TBX1 and TBX5.

\section{Early heart development and the second heart field}
\label{sec2}

Nascent mesodermal cardiac progenitor cells emerge from the primitive
streak at gastrulation and migrate to the anterior lateral region of
the embryo (Figure~\ref{fig1}A). Two distinct waves of progenitor cells
have been identified that independently activate 
the gene encoding the early mesodermal
transcription factor MESP1~\cite{4,5,6}. The first wave generates first
heart field (FHF) progenitor cells that differentiate to form bilateral
cardiac primordia that converge in the ventral midline as the foregut
closes, giving rise to the cardiac crescent and ultimately to left
ventricular and atrial myocardium.\looseness=1 

\begin{figure*}
{\vspace*{1pt}}
\includegraphics{fig01}
{\vspace*{1pt}}
\caption{\label{fig1}Cardiac development. Cartoon showing the
contribution of first (pink) and second (green) heart field progenitor
cells to the developing mouse heart. (A) Heart tube morphogenesis, 
(B)~heart tube looping and elongation and (C) chamber growth and cardiac
septation. FHF, first heart field; SHF, second heart field; L, lateral;
M, medial; A, anterior; P, posterior; D, dorsal; V, ventral; AP,
arterial pole; VP, venous pole; HT, heart tube; RA, right atrium; LA,
left atrium; RV, right ventricle; LV, left ventricle; NT, neural tube;
DM, dorsal mesocardium; OFT, outflow tract; Ao, aorta; PT, pulmonary
trunk; E: mouse embryonic day, D: human embryonic day.}
{\vspace*{2pt}}
\end{figure*}

The cardiac crescent is transformed into a transiently linear heart
tube that subsequently elongates and loops to the right, generating the
embryonic heart at midgestation in the mouse, equivalent to the 5th
week of human development (Figure~\ref{fig1}B). Heart tube elongation
is driven by the progressive addition of SHF cells, derived from the
second wave of \textit{Mesp1} expression, located in pharyngeal \mbox{mesoderm}
\mbox{dorsomedial} to the early heart tube, including the dorsal wall of the
pericardial cavity~\cite{5}. The SHF was first identified in 2001
through analysis of a \textit{Fgf10} enhancer trap transgene expressed
in murine pharyngeal mesoderm~\cite{7} and by cell labelling
experiments in avian embryos~\cite{8,9}. These findings built on
pioneering embryological evidence for the addition of extracardiac
cells to the growing cardiac poles~\cite{10,11}. Initially contributing
along the entire length of the cardiac primordium, SHF addition becomes
restricted to the arterial and venous poles after breakdown of the
dorsal mesocardium separates the heart tube from the dorsal pericardial
wall (Figure~\ref{fig1}B).  Genetic tracing and retrospective lineage
experiments have shown that the first and second heart fields
correspond to distinct genetic lineages defined by their exclusive
contributions to the outflow tract and left ventricle respectively, and
that SHF progenitor cells adding to the arterial and venous poles are
clonally related~\cite{12,13,14}. Subsequent labeling experiments and
genetic lineage analyses have revealed the progressive addition of SHF
progenitor cells to the elongating heart tube, giving rise successively
to the right ventricle, proximal and distal outflow tract, and atrial
myocardium, as well as smooth muscle cells of the pulmonary trunk
(Figure~\ref{fig1}C)~\cite{15,16,17,18}. Clonal relationships have been
established between these SHF subpopulations and branchiomeric skeletal
muscles of the head and neck, consistent with the existence of common
cardiac and skeletal muscle
progenitor cells in pharyngeal mesoderm~\cite{19,20,21,22}. This is
supported by the recent identification of multilineage primed
progenitor cells that contribute to skeletal muscle and myocardium,
using single-cell RNA-seq analysis~\cite{23}. Single-cell RNA
sequencing is a revolutionary approach that has yielded unprecedented
insights into heterogeneity and developmental trajectories of cardiac
progenitor cell populations~\cite{24}. These include the identification
of a population of FHF cells termed the juxtacardiac field, located at
the lateral boundary between the embryo and extraembryonic mesoderm,
that contributes to growth of the early cardiac primordium along its
ventrolateral margin, in contrast to SHF contributions along the
dorsomedial margin~\cite{25,26,27}. Single-cell transcriptomics has
also revealed differences in gene expression between left and right SHF
cells, consistent with the recent finding that the SHF is a driver of
looping morphogenesis~\cite{28,29}. Defects in cardiac looping, or in
the prior establishment of \mbox{embryonic} laterality, have been implicated
in a spectrum of CHD~\cite{29,30}. Dynamic imaging using
two-photon and laser scanning fluorescent microscopy have begun to
confirm the trajectories taken by cardiac progenitor cells during
growth of the early heart. These experiments have revealed 
a temporal delay between first and
second heart field cell deployment and distinct patterns of cell
behavior in the juxtacardiac and second heart fields during heart tube
assembly~\cite{6,31}. In addition, quantitative morphometric approaches
provide high-resolution understanding of the complex events associated
with early heart development~\cite{32}.

\section{TBX1 and arterial pole development}\label{sec3}

Progenitor cells adding to the arterial pole of the heart, known as the
anterior SHF, give rise to right ventricular and outflow tract
myocardium. As these cells converge on the growing arterial pole, they
move through a transition zone in which progenitor cell gene expression
is downregulated and myocardial differentiation activated
(Figure~\ref{fig2}A)~\cite{33,34}. While lack of SHF deployment results
in severe early lethal phenotypes, milder defects in this process
generate an outflow tract that is too short to allow the normal
alignment between the left ventricle and ascending aorta during cardiac
septation. Consequently, anterior SHF defects contribute to conotruncal
CHD, that account for 30\% of CHD, ranging from partial
ventriculoarterial misalignment as in the case of overriding aorta and
tetralogy of Fallot, to double outlet right ventricle
(Figure~\ref{fig2}B).  Defective growth of the ascending aorta has also
been implicated in transposition of the great arteries~\cite{35}.

\begin{figure*}
\includegraphics{fig02}
\caption{\label{fig2}Arterial pole development and outflow tract
septation. (A) While SHF cells contribute to the myocardial wall of the
outflow tract at the transition zone, SHF addition is regulated by
cardiac neural crest cells that also mediate outflow tract septation.
Embryonic laterality impacts on rightward looping and
ventriculoarterial alignment. (B) An example of a conotruncal congenital
heart defect, tetralogy of Fallot. SHF, Second heart field; AP,
arterial pole; VP, venous pole; A, Anterior; P, Posterior; D, Dorsal;
V, ventral; RA, right atrium; LA, left atrium; RV, right ventricle; LV,
left ventricle; OFT, outflow tract; Ao, aorta; PT, pulmonary trunk.}
{\vspace*{2pt}}
\end{figure*}

Work in many groups worldwide has identified critical
transcriptional regulators, \mbox{intercellular} signaling pathways and
epithelial features that maintain SHF progenitor cell status and
regulate progressive differentiation at the cardiac
poles~\cite{36,37,38,39}. In addition to \textit{Fgf10} expression, SHF
cells are demarcated by expression of the transcription factors ISL1,
TBX1 and an enhancer from the \textit{Mef2c} locus~\cite{7,15,16,40}.
These transcription factors integrate the activity of signaling events
including autocrine signaling from pharyngeal mesoderm as well as
signal exchange with surrounding cell types, in particular adjacent
ventral pharyngeal endoderm and neural crest-derived mesenchyme. Heart
tube elongation is regulated by a balance between pro-proliferative FGF
signals and pro-differentiation BMP signaling; FGF10 expression in the
SHF, for example, acts together with FGF8 to control arterial pole
development~\cite{41}. As SHF cells differentiate in the transition
zone, \textit{Fgf10} expression is downregulated by NKX2-5, that blocks
ISL1-mediated activation of an intronic \textit{Fgf10}
enhancer~\cite{42}. SHF cells in the dorsal pericardial wall have been
shown to constitute an atypical epithelium, with apical tight and
adherens junctions and highly dynamic basal filopodia more typical of
mesenchymal cells~\cite{43}. The epithelial properties of SHF cells are
emerging as an important regulatory node controlling SHF deployment and
are regulated by cell adhesion molecules and components of the planar
cell polarity pathway, such as WNT5A and VANGL2, as well as TBX1,
required for focal adhesion and filopodial activity in the
SHF~\cite{33,38,43,44}. Moreover, analysis of apical junctions, cell
shape and actomyosin distribution in the SHF has suggested
biomechanical models driving SHF deployment, including planar cell
polarity driven push and pull mechanisms and an epithelial tension
driven circuit promoting proliferation in the posterior region of the
dorsal pericardial wall~\cite{45,46}.

Cardiac neural crest cells, like the SHF, move through the pharyngeal
region into the developing arterial pole of the heart, playing an
important role in dividing the embryonic outflow tract into the
pulmonary trunk and ascending aorta~\cite{47}. Ablation of neural crest
cells thus leads to persistent common arterial trunk due to a failure
of outflow tract septation; cardiac neural crest cell ablation also
leads to a shortened outflow tract through reduced SHF deployment
(Figure~\ref{fig2}A)~\cite{48,49}. This observation in avian embryos
was among the first to show the significance of SHF addition for
cardiac looping and outflow tract alignment. These phenotypes reflect
signal exchange between SHF and neural crest cells as they converge on the
arterial pole of the heart, orchestrating normal outflow tract
elongation. The T-box transcription factor TBX1 has been identified as
a critical regulator of SHF development, promoting progenitor cell
proliferation and delaying differentiation in the SHF~\cite{40}.
\textit{TBX1} is the major gene implicated in 22q11.2 deletion or
DiGeorge syndrome, characterized by craniofacial and cardiovascular
\mbox{defects,} including a range of conotruncal CHD~\cite{50,51}. Single-cell
RNA-seq experiments have shown that Tbx1 controls myogenic trajectories
from the multilineage primed progenitor cell population to cardiac and
skeletal muscle fates~\cite{23}. \textit{Tbx1} null embryos display
common arterial trunk associated with defective cardiac neural crest
cell development; in addition, cells giving rise to myocardium at the
base of the pulmonary trunk, the outlet of the right ventricle, fail to
contribute to the arterial pole of \textit{Tbx1} mutant hearts~\cite{40,52,53}.
Underdevelopment of subpulmonary myocardium has been proposed to be a
primary upstream mechanism leading to tetralogy of Fallot~\cite{54}.
Indeed, tetralogy of Fallot is genetically heterogenous but the major
known genetic cause is haploinsufficiency for \textit{TBX1}, 22q11.2 DS
accounting for over 15\% of patients~\cite{50}. Moreover, SHF ablation
in avian embryos leads to a tetralogy of Fallot-like
phenotype~\cite{55}. Distinct genetic programs have been defined in
subpulmonary and subaortic myocardium, reflecting earlier differences
in the distribution of progenitor cells in the SHF; recently
\textit{Pparg} and \textit{Dlk1} have been shown to demarcate
subpulmonary and subaortic myocardium respectively~\cite{53,56,57}.
Future subpulmonary myocardium, initially located in the inferior wall
of the midgestation outflow tract, becomes positioned ventrally during
outflow tract septation through counterclockwise rotation of the
myocardial wall driven by ongoing SHF addition 
(Figure~\ref{fig2}A)~\cite{58,59}.

Genetic studies have identified mutations associated with sporadic and
familial CHD, including mutations in genes encoding T-box transcription
factors and chromatin regulators~\cite{60}. Examples include
\textit{TBX1} and \textit{CHD7}, the latter implicated in cardiovascular defects
associated with CHARGE syndrome. CHD7 is an ATP-dependent chromatin
remodeler required for normal contributions of both neural crest and
SHF lineages during heart development, including regulation of
\textit{Fgf10} expression in the SHF through the ISL1-bound intronic
enhancer~\cite{61}. Integration of genetic studies with single cell
RNA-seq datasets can provide further mechanistic insights and has, for
example, highlighted the importance of outflow tract progenitor cells
in tetralogy of Fallot~\cite{62}. However, genetic defects only account
for one third of CHD cases to date, suggesting an important, yet poorly
understood, effect of \textit{in utero} exposure to environmental
factors. Indeed, recent studies in the mouse have identified the SHF as
the target of short-term gestational hypoxia, maternal iron deficiency
and prenatal alcohol exposure, in each case leading to reduced
deployment of SHF progenitor cells and a shortened OFT, resulting in a
range of conotruncal CHD~\cite{63,64,65}. 

\section{TBX5 and cardiac septation}\label{sec4}

Dye labelling and genetic lineage tracing experiments have shown that
subpulmonary myocardium originates in \textit{Hoxb1} expressing cells
in the posterior lateral region of the dorsal pericardial wall,
corresponding to the location of proliferative multilineage primed
progenitor cells~\cite{23,53,66}. Downregulation of \textit{Hoxb1}
expression is required for normal arterial pole and right ventricular
development~\cite{67}. Cardiac progenitor cells in the \textit{Isl1},
\textit{Hoxb1}, \textit{Tbx1}, \textit{Wnt5a} and \textit{Mef2c}
enhancer genetic lineages contribute not only to the arterial pole but
also to the venous pole of the heart, making critical contributions to
atrial septal structures including the primary atrial septum and dorsal
mesenchymal protrusion that bridges the atrial septum with the
atrioventricular cushions (Figure~\ref{fig3}A)~\cite{53,67,68,69,70}.
Defects in dorsal mesenchymal protrusion development result in primum
type atrial septal defects, a class of atrioventricular septal
defect~\cite{71}.

\begin{figure*}
\includegraphics{fig03}
{\vspace*{3pt}}
\caption{\label{fig3}Venous pole development and atrial septation. (A)
Scheme of the region boxed in Figure~\ref{fig2}A, showing progenitor
cell segregation into anterior SHF (aSHF) and posterior SHF (pSHF)
domains contributing to the arterial and venous poles. Note the
contribution of first heart field (FHF) cells from the juxtacardiac
field (JCF) to the venous pole. (B) Diagram showing that Tbx1 expression
is maintained in the aSHF, while Tbx5 is activated in the pSHF. (C)
Left: the SHF genetic lineage (blue) labelled by a conditional
${\upbeta}$-galactosidase reporter gene activated by Cre recombinase in
the SHF, showing labeling in the right ventricle (RV), interventricular
septum (VS) and atrial septal structures: the primary atrial septum
(PAS) and dorsal mesenchymal protrusion (DMP). Right: the DMP fails to
form in \textit{Tbx1} mutant hearts resulting in a primum type atrial
septal defect (asterisk). AP, arterial pole; VP, venous pole; A, atrium; V,
ventricle; RV, right ventricle; LV, left ventricle; HT, heart tube.
Scale bar in C: 100~${\upmu}$m} 
{\vspace*{3pt}}
\end{figure*}

In contrast to the anterior SHF, which retains the early SHF genetic
program, posterior SHF cells activate \textit{Tbx5}, a key regulator of
the FHF and venous pole differentiation program, under the influence of
retinoic acid signaling~\cite{72,73}. \textit{Tbx5} is upregulated in
TBX1 positive pharyngeal mesoderm as the dorsal mesocardium breaks down
and progenitor cells segregate to alternate poles (Figure~\ref{fig3}B).
Transient co-expression of TBX1 and TBX5 is followed by downregulation
of the anterior SHF program and the establishment of a transcriptional
boundary within the dorsal pericardial wall, separating TBX1 positive
arterial pole progenitors in the anterior SHF from TBX5 positive venous
pole progenitors in the medial posterior SHF~\cite{73}. Mutations in
\textit{TBX5} result in Holt-Oram syndrome, characterized by atrial
septal CHD and forelimb defects~\cite{74}. TBX5 has recently been shown to
activate an enhancer of \textit{Aldh1a2}, encoding a retinoic acid
biosynthetic enzyme, pointing to a feed-forward mechanism between TBX5
and retinoic acid signaling as the posterior SHF domain is established
in the dorsal pericardial wall~\cite{75}. The \mbox{boundary} between anterior
and posterior domains is perturbed in the absence of \textit{Tbx1},
resulting in failure of dorsal mesenchymal protrusion development and
atrial septal defects in 30\% of \textit{Tbx1} null embryos,
potentially accounting for a low frequency of atrial septal defects in
22q11.2 DS patients (Figure~\ref{fig3}C)~\cite{53}. Similar atrial
septal defects are observed in conditional mutant mouse embryos lacking
\textit{Tbx5} in the SHF~\cite{76}. The posterior SHF shares lineage
contributions with pulmonary mesenchyme and signal exchange between the
SHF and adjacent pulmonary endoderm plays an important role in venous
pole development~\cite{75,77}. TBX5 activates Wnt ligand expression in
the posterior SHF that signals to pulmonary endoderm, in turn
controlling the timing of differentiation in the posterior SHF through
the Hedgehog signaling pathway~\cite{78,79}.

Cells expressing both \textit{Tbx5} and the SHF program have also been
identified in the interventricular septal region using an
intersectional lineage reporter approach~\cite{4}. Conditional
inactivation using Cre recombinase driven by the \textit{Mef2c}
enhancer has shown that \textit{Tbx5} is required in the SHF lineage
for formation of the muscular ventricular septum as well as for atrial
septation~\cite{76,80}; moreover, the transcription factors MEF2C and
TBX5 coregulate target genes required for ventricular septal
development~\cite{81}. Thus both atrial and ventricular septal
structures form at sites where \textit{Tbx5} expression and the SHF
program transiently overlap, suggesting that this defines a boundary
program in the developing heart required to initiate septal
morphogenesis. Atrial and ventricular CHD together account for over
50\% of CHD~\cite{82}. Further dissection of this regulatory program,
operating at the interface between the heart fields, will be essential
to increase our understanding of the origins of septal CHD.

\section{Conclusions and perspectives}\label{sec5}

Since the discovery of the SHF extensive evidence has revealed the
importance of this spatiotemporally heterogenous progenitor cell
population in the etiology of CHD affecting development of the cardiac
poles and septa. While severe heart tube elongation defects result in
early embryonic lethality, milder perturbation of cell addition at the
cardiac poles results in alignment defects and failure to separate the
systemic and pulmonary circulation at birth, contributing to a spectrum
of common forms of CHD encountered in human patients. Technological
advances, such as single-cell transcriptomics and quantitative
morphometric approaches, as well as dynamic imaging and establishment
of a variety of stem cell derived cardiac organoid models, are
providing new insights into the regulation of SHF
development~\cite{31,83}. Current objectives include elucidation of the
regulatory networks controlling distinct SHF subpopulations implicated
in CHD, the identification of downstream pathways effecting regional
heart morphogenesis and cardiac septation, and dissection of the
disease mechanisms by which genetic and environmental factors interact
in the SHF to cause CHD.

\section*{Declaration of interests}

The authors do not work for, advise, own shares in, or receive funds
from any organization that could benefit from this article, and have
declared no affiliations other than their research organizations.

\section*{Funding}

Work in RK's group is supported by the Agence National pour la
Recherche (PP-heart ANR-20-CE13-0029-01 and Heartbound ANR-22-CE13
projects) and the AFM-Telethon. CG acknowledges the support of the
French Government program managed by the French National Research
Agency (ANR-16-CONV-0001) and the Excellence Initiative of
Aix-Marseille University A$^{*}$MIDEX (Turing Centre for Living
Systems) and the Fondation pour la Recherche M\'{e}dicale. RK is an
Inserm research fellow.

\CDRGrant[ANR]{ANR-20-CE13-0029-01}
\CDRGrant[ANR]{ANR-22-CE13}
\CDRGrant[ANR]{ANR-16-CONV-0001}

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