Benjamin Swedlund

Fabienne Lescroart
email: fabienne.lescroart@univ-amu.fr

Cardiopharyngeal progenitor specification: multiple roads to the heart and head muscles

During embryonic development, the heart arise from various sources of undifferentiated mesodermal progenitors, with an additional contribution from ectodermal neural crest cells. Mesodermal cardiac progenitors are plastic and multipotent, but are nevertheless specified to a precise heart region and cell type very early during development. Recent findings have defined both this lineage plasticity and early commitment of cardiac progenitors, using a combination of single-cell and population analyses.

In this review, we discuss several aspects of cardiac progenitor specification. We discuss their markers, fate potential in vitro and in vivo, early segregation and commitment, and also intrinsic and extrinsic cues regulating lineage restriction from multipotency to a specific cell type of the heart. Finally, we also discuss the sub-divisions of the cardiopharyngeal field, and the shared origins of the heart with other mesodermal derivatives, including head and neck muscles.

Introduction

Throughout the animal kingdom, organogenesis takes place early during embryonic development. This developmental process consists in the formation of organs through the progressive commitment and specification of progenitor cells, their migration towards the final location of the organ and their signaling interactions with surrounding tissues. Organogenesis starts with the commitment of multipotent and plastic progenitors that will progressively acquire the molecular program of more restricted progenitors and finally differentiate into mature functional cells.

The heart is the first organ to form in mammals and arises from the specification of cardiac progenitor cells, which can be identified as early as during gastrulation [START_REF] Kinder | The orderly allocation of mesodermal cells to the extraembryonic structures and the anteroposterior axis during gastrulation of the mouse embryo[END_REF], and which represent the building blocks of cardiac development. Cardiac progenitors are included in a larger mesodermal population termed the cardiopharyngeal field (CPF) that contributes to both the heart and skeletal muscles of the head and neck [START_REF] Diogo | A new heart for a new head in vertebrate cardiopharyngeal evolution[END_REF]. In vertebrates, the first specified cardiac cells generally express Nkx2-5 [START_REF] Harvey | Patterning the vertebrate heart[END_REF], that notably also marks certain skeletal muscle progenitors of the head [START_REF] Diogo | A new heart for a new head in vertebrate cardiopharyngeal evolution[END_REF]. Nevertheless, cardiopharyngeal progenitor cells (CPCs) can be identified earlier during development, before the onset of Nkx2-5 expression, and there are now several lines of evidence showing that the fate of CPCs is specified during or prior to gastrulation.

In mammals, cardiac progenitors will build a four-chambered heart with two atria and two ventricles, consisting of different cell types including cardiomyocytes (CMs), endocardial cells (ECs) forming the inner layer, epicardial-derived cells (EPDCs) at the outer layer and smooth muscle cells (SMCs) [START_REF] Lescroart | Cell lineages, growth and repair of the mouse heart[END_REF]. It is now well established that vertebrate cardiogenesis occurs through progressive addition of several populations of progenitors to the forming linear heart tube. An early differentiating population of progenitors, termed the first heart field (FHF) gives rise to the left ventricle (LV) and some atrial cells, while a second wave of differentiation occurs from the second heart field (SHF) with a distinct contribution to the four-chambered heart, including the right ventricle (RV), the outflow tract (OFT) and the venous pole [START_REF] Buckingham | Building the mammalian heart from two sources of myocardial cells[END_REF]. Further subdivisions of the SHF distinguish the contribution of the anterior SHF (aSHF) to the OFT and RV from that of the posterior SHF (pSHF) to the OFT and venous region [START_REF] Meilhac | The deployment of cell lineages that form the mammalian heart[END_REF]. Subdivision of the FHF has not been clearly established but based on lineage tracing [START_REF] Bardot | Foxa2 identifies a cardiac progenitor population with ventricular differentiation potential[END_REF], we could wonder whether the FHF can also be patterned along the antero-posterior axis.

Throughout the evolution of vertebrates, the heart has acquired a complex multi-chambered structure, although conserved FHF and SHF populations can already be identified in tunicates, which are the closest living relatives of an early chordate precursor. Therefore, CPC specification has been studied in different models, ranging from the simple chordate Ciona robusta, that presents a simple beating heart tube, to the mammalian mouse model. Moreover, differentiation of mouse and human pluripotent stem cells (m-or h-PSCs) in vitro into different cardiac lineages has been shown to recapitulate the first steps of CPC specification, including the molecular pathways involved in this process, and are thus an attractive model to determine the minimal cues necessary for the formation of specific cardiac lineages.

In this review, we present currently known markers of early CPCs and their differentiation potential as studied by single-cell approaches. We also discuss insights into the regulation of CPC specification toward specific cardiac or pharyngeal lineages, which have recently been discovered through the multiplication of genetic techniques and reduction in costs of next-generation sequencing. Although cardiac neural crest cells also make important contributions to the OFT and valves [START_REF] Kirby | Neural crest cells contribute to normal aorticopulmonary septation[END_REF][START_REF] Kirby | Neural crest and cardiovascular patterning[END_REF], we choose here to focus only on the mesodermal contribution to the forming heart.

Markers of early CPCs

Several key studies have isolated and characterized CPCs and more specifically cardiac progenitors from mouse embryos and differentiating PSCs, which are capable of proliferating and giving rise to multiple cell types of the heart.

As the majority of cardiac and pharyngeal muscle cells derive from the mesoderm, genes that are expressed in the nascent mesoderm such as Brachyury, also called T, encoding a T-box transcription factor (TF) [START_REF] Kispert | The Brachyury gene encodes a novel DNA binding protein[END_REF][START_REF] Showell | T-box genes in early embryogenesis[END_REF], and Mesp1 (mesoderm posterior 1), encoding for a bHLH TF [START_REF] Saga | MesP1: a novel basic helixloop-helix protein expressed in the nascent mesodermal cells during mouse gastrulation[END_REF], are characteristic of early CPCs. Mesp1 is thereafter rapidly downregulated once the mesoderm is formed. During differentiation of both mouse and human PSCs, the expression of Mesp1 is also transient and precedes the expression of other cardiac markers [START_REF] Liu | Sox17 is essential for the specification of cardiac mesoderm in embryonic stem cells[END_REF][START_REF] Ueno | Biphasic role for Wnt/beta-catenin signaling in cardiac specification in zebrafish and embryonic stem cells[END_REF][START_REF] Bondue | Mesp1 acts as a master regulator of multipotent cardiovascular progenitor specification[END_REF][START_REF] Lindsley | Mesp1 coordinately regulates cardiovascular fate restriction and epithelialmesenchymal transition in differentiating ESCs[END_REF][START_REF] Hartogh | Dual reporter MESP1 mCherry/w-NKX2-5 eGFP/w hESCs enable studying early human cardiac differentiation[END_REF]. Mesp1 has spurred great interest in the last two decades as a potential marker of early cardiac progenitor specification from both heart fields [START_REF] Bondue | Mesp1: a key regulator of cardiovascular lineage commitment[END_REF]. Moreover, in the chordate Ciona, Mesp, the unique homolog of Mesp1, is expressed transiently in the B7.5 cells pair which will give rise to the Ciona heart, supporting a conserved role of Mesp genes in CPC specification throughout chordates [START_REF] Satou | The ascidian Mesp gene specifies heart precursor cells[END_REF]). Specific combinations of surface markers expressed in CPCs can now facilitate their isolation, purification and characterization. Flk1 (or Vegfr2) and Pdgfra are both expressed in nascent mesoderm [START_REF] Kataoka | Expressions of PDGF receptor alpha, c-Kit and Flk1 genes clustering in mouse chromosome 5 define distinct subsets of nascent mesodermal cells[END_REF][START_REF] Motoike | Evidence for novel fate of Flk1+ progenitor: contribution to muscle lineage[END_REF][START_REF] Ema | Deletion of the selection cassette, but not cis-acting elements, in targeted Flk1-lacZ allele reveals Flk1 expression in multipotent mesodermal progenitors[END_REF]. Interestingly, cells co-expressing Flk1 and Pdgfra during differentiation of PSCs are enriched for cardiac potential [START_REF] Hirata | Coexpression of platelet-derived growth factor receptor alpha and fetal liver kinase 1 enhances cardiogenic potential in embryonic stem cell differentiation in vitro[END_REF]). Cxcr4 has also been used in combination with Flk1 [START_REF] Nelson | CXCR4+/FLK-1+ biomarkers select a cardiopoietic lineage from embryonic stem cells[END_REF] or both Flk1 and Pdgfra [START_REF] Bondue | Defining the earliest step of cardiovascular progenitor specification during embryonic stem cell differentiation[END_REF] to isolate CPCs with cardiac potential. The expression of these three signaling receptors coincides with that of Mesp1, and overexpression of Mesp1 directly activates their transcription [START_REF] Bondue | Defining the earliest step of cardiovascular progenitor specification during embryonic stem cell differentiation[END_REF]. Flk1/Pdgfra/Cxcr4 also mark early Mesp1+ CPCs in vivo [START_REF] Lescroart | Early lineage restriction in temporally distinct populations of Mesp1 progenitors during mammalian heart development[END_REF]. Human PSCs undergoing differentiation into cardiac cell types also necessarily pass through an Flk1+ mesodermal stage [START_REF] Yang | Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population[END_REF]. Expression of Flk1 and Pdgfra together is generally predictive of CM potential in hPSCs [START_REF] Kattman | Stagespecific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines[END_REF]. Several other markers have recently been proposed for the isolation of hPSC-derived cardiac progenitors, such as Ror2 and Cd13 [START_REF] Drukker | Isolation of primitive endoderm, mesoderm, vascular endothelial and trophoblast progenitors from human pluripotent stem cells[END_REF][START_REF] Hartogh | Dual reporter MESP1 mCherry/w-NKX2-5 eGFP/w hESCs enable studying early human cardiac differentiation[END_REF][START_REF] Skelton | CD13 and ROR2 Permit Isolation of Highly Enriched Cardiac Mesoderm from Differentiating Human Embryonic Stem Cells[END_REF], or Fzd4 [START_REF] Yoon | FZD4 Marks Lateral Plate Mesoderm and Signals with NORRIN to Increase Cardiomyocyte Induction from Pluripotent Stem Cell-Derived Cardiac Progenitors[END_REF].

One subunit of a chromatin-remodelling complex, Smarcd3, has cardiac-specific expression. Smarcd3 is expressed in a subset of Mesp1+ CPCs, and a smaller 2.5 kb regulatory region of this gene can activate reporter gene expression in progenitors for both heart fields in mouse embryos [START_REF] Devine | Early patterning and specification of cardiac progenitors in gastrulating mesoderm[END_REF]. Interestingly, the same regulatory region marks zebrafish CPCs, even though this particular sequence was not conserved between both species [START_REF] Yuan | Heart enhancers with deeply conserved regulatory activity are established early in zebrafish development[END_REF]. This suggests that the same core transcriptional regulators are active in CPCs throughout vertebrates and can induce the expression of CPC-specific regulatory regions from other species. As regulatory regions of genes have readily been used to activate reporter gene expression in specific populations of CPCs for the whole heart [START_REF] Wu | Developmental origin of a bipotential myocardial and smooth muscle cell precursor in the mammalian heart[END_REF] or of the SHF [START_REF] Verzi | The right ventricle, outflow tract, and ventricular septum comprise a restricted expression domain within the secondary/anterior heart field[END_REF], the advent of whole-genome predictions of cardiac enhancers and validation of their activity in vivo [START_REF] He | Co-occupancy by multiple cardiac transcription factors identifies transcriptional enhancers active in heart[END_REF][START_REF] Yuan | Heart enhancers with deeply conserved regulatory activity are established early in zebrafish development[END_REF]) may accelerate the identification of cardiac-specific enhancers expressed as early as during gastrulation in the FHF, SHF, or whole CPF. It would then be interesting to investigate whether these enhancers are specifically active in multipotent pan-cardiac CPCs or rather independently activated in different lineages.

Lineage potential of CPCs

While in Ciona the cardiac lineage tree is highly ordered and reproducible, starting from only 2 Mesp+ progenitors at the time of gastrulation [START_REF] Satou | The ascidian Mesp gene specifies heart precursor cells[END_REF]), lineage diversification is more complex and plastic in vertebrates, with about 250 Mesp1+ progenitors contributing to the heart in the mouse model [START_REF] Chabab | Uncovering the Number and Clonal Dynamics of Mesp1 Progenitors during Heart Morphogenesis[END_REF].

The first experiments assessing the lineage potential of CPCs were performed in the chick and the mouse, using dye labeling or grafting experiments [START_REF] Stalsberg | The precardiac areas and formation of the tubular heart in the chick embryo[END_REF][START_REF] Tam | Mouse gastrulation: the formation of a mammalian body plan[END_REF][START_REF] Kinder | The orderly allocation of mesodermal cells to the extraembryonic structures and the anteroposterior axis during gastrulation of the mouse embryo[END_REF][START_REF] Lawson | Fate mapping the mouse embryo[END_REF][START_REF] Tzahor | Antagonists of Wnt and BMP signaling promote the formation of vertebrate head muscle[END_REF]. Grafting experiments have shown that cardiac progenitors are committed to a cardiac fate only after the formation of the cardiac crescent [START_REF] Tam | Mouse gastrulation: the formation of a mammalian body plan[END_REF][START_REF] Auda-Boucher | Staging of the commitment of murine cardiac cell progenitors[END_REF], suggesting a certain degree of plasticity of CPCs at gastrulation stages.

Lineage tracing experiments, clonal analyses and, more recently, single-cell RNA-sequencing (scRNA-seq) have allowed us to decipher the appearance of different cell lineages that arise from early CPCs.

Lineage tracing and in vitro differentiation demonstrates the multipotency of CPCs

Lineage tracing experiments with a Flk1-Cre knock-in mouse model showed that although originally involved in hematopoietic lineages, Flk1+ cells also contribute to the development of CMs and ECs [START_REF] Motoike | Evidence for novel fate of Flk1+ progenitor: contribution to muscle lineage[END_REF][START_REF] Ema | Deletion of the selection cassette, but not cis-acting elements, in targeted Flk1-lacZ allele reveals Flk1 expression in multipotent mesodermal progenitors[END_REF]. Furthermore, during differentiation of PSCs, T+Flk1+ cells present the ability to form colonies comprising CMs, SMCs and ECs, suggesting that this early mesodermal cell population is at least tri-potent [START_REF] Kattman | Multipotent flk-1+ cardiovascular progenitor cells give rise to the cardiomyocyte, endothelial, and vascular smooth muscle lineages[END_REF]. Tracking the progeny of Mesp1expressing cells revealed that they comprise the majority of cardiac cells including CM, ECs and EPDCs [START_REF] Saga | MesP1 is expressed in the heart precursor cells and required for the formation of a single heart tube[END_REF]). In addition, the Mesp1 lineage also contributes to non-cardiac derivatives, including skeletal muscles of the head and neck (see Table 1) [START_REF] Mcbratney-Owen | Development and tissue origins of the mammalian cranial base[END_REF][START_REF] Yoshida | Cell lineage in mammalian craniofacial mesenchyme[END_REF][START_REF] Asahina | Mesenchymal origin of hepatic stellate cells, submesothelial cells, and perivascular mesenchymal cells during mouse liver development[END_REF][START_REF] Harel | Distinct origins and genetic programs of head muscle satellite cells[END_REF]). Interestingly, this observation seems consistent with findings in Ciona, where Mesp+ cells also produce the anterior and tail muscles, indicating their multipotency (Figure 1) [START_REF] Stolfi | Early chordate origins of the vertebrate second heart field[END_REF]. During mouse and human PSC differentiation, isolated Mesp1-expressing cells have the potential to become ECs, SMCs, and CMs in a clonal assay [START_REF] Bondue | Defining the earliest step of cardiovascular progenitor specification during embryonic stem cell differentiation[END_REF][START_REF] Hartogh | Dual reporter MESP1 mCherry/w-NKX2-5 eGFP/w hESCs enable studying early human cardiac differentiation[END_REF]. Furthermore, an in vitro clonal assay using mPSCs demonstrated the bi-potential of Mesp1+ cells to generate skeletal and cardiac muscles, in agreement with their broad fate potential in vivo [START_REF] Chan | Heterogeneity of Mesp1+ mesoderm revealed by single-cell RNA-seq[END_REF].

Additional PSC studies have further shown that cardiac progenitors from slightly later stages, when Nkx2-5 is co-expressed with other markers such as the transcription factor Isl1, are able to generate colonies with potential to give rise to 2 or 3 cardiac cell types [START_REF] Moretti | Multipotent embryonic isl1+ progenitor cells lead to cardiac, smooth muscle, and endothelial cell diversification[END_REF][START_REF] Wu | Developmental origin of a bipotential myocardial and smooth muscle cell precursor in the mammalian heart[END_REF]. Therefore, early CPCs are multipotent and plastic. Nevertheless, we will discuss below that they are also specified very early towards a certain fate, and that when left in their endogenous environment, they are mostly uniproductive and contribute to specific cardiac regions and cell types.

Single cells analyses reveal heterogeneity among CPCs

Single-cell analyses have considerably changed our vision of CPC specification and have shown that the differential contribution of CPCs in the mouse model is complex, with many branching points and possible outcomes.

The first clonal analyses performed in mouse, using retrospective clonal analysis, demonstrated the existence of two myocardial cell lineages, corresponding to the FHF and SHF. On the basis of the number of b-gal+ cells included in clones covering both lineages, lineage segregation between the first and second myocardial lineages was estimated to occur at the onset of gastrulation [START_REF] Meilhac | The clonal origin of myocardial cells in different regions of the embryonic mouse heart[END_REF]). More recent clonal tracing of Mesp1+ CPCs using two independent strategies (Mesp1-rtTA/tetO-Cre/Rosa-Confetti or a Mesp1-Cre/MADM system) established that lineage segregation of the FHF and SHF occurs before the onset of Mesp1 expression, as shown by the absence of clones in both FHF and SHF derivatives [START_REF] Devine | Early patterning and specification of cardiac progenitors in gastrulating mesoderm[END_REF][START_REF] Lescroart | Early lineage restriction in temporally distinct populations of Mesp1 progenitors during mammalian heart development[END_REF]. Similarly, clonal tracing of Smarcd3+ progenitors showed no evidence of a common progenitor for the FHF and SHF [START_REF] Devine | Early patterning and specification of cardiac progenitors in gastrulating mesoderm[END_REF].

Like in tunicates, multipotent progenitor cells giving rise to the SHF in the mouse also generate skeletal muscles of the head and neck, with at least three different populations of bi-potent progenitors found along the antero-posterior axis of the pharyngeal region, contributing to the masticatory muscles and RV, facial expression muscles and OFT or neck muscles and venous pole as well as OFT myocardium (Figure 1) [START_REF] Lescroart | Clonal analysis reveals common lineage relationships between head muscles and second heart field derivatives in the mouse embryo[END_REF][START_REF] Lescroart | Clonal analysis reveals a common origin between nonsomite-derived neck muscles and heart myocardium[END_REF]. Furthermore, Mesp1 lineage tracing also identified clones labeling both skeletal muscles of the head and the myocardium, suggesting that segregation between cranial and cardiac muscles occurs after the onset of Mesp1 expression [START_REF] Lescroart | Early lineage restriction in temporally distinct populations of Mesp1 progenitors during mammalian heart development[END_REF]). Nevertheless, most Mesp1+ CPCs are uniproductive and contribute in the heart to either the epicardium, CMs, SMCs or ECs, with only a few bipotent Mesp1-derived clones contributing to CMs and ECs, or SMCs and CMs, and only in SHF derivatives [START_REF] Devine | Early patterning and specification of cardiac progenitors in gastrulating mesoderm[END_REF][START_REF] Lescroart | Early lineage restriction in temporally distinct populations of Mesp1 progenitors during mammalian heart development[END_REF].

The heterogeneity of CPCs is exemplified by the different genetic lineage tracing experiments that have been performed and that label different compartments of the heart (Table 1) [START_REF] Meilhac | The deployment of cell lineages that form the mammalian heart[END_REF]. For example, Foxa2+ cells, found very early during gastrulation, preferentially contribute to the ventricles and epicardium. Interestingly, Foxa2+ cells seem to express lower levels of Mesp1 [START_REF] Bardot | Foxa2 identifies a cardiac progenitor population with ventricular differentiation potential[END_REF]. Tracing with a particular enhancer of Mef2c, activated during gastrulation, labels only a subpopulation of cardiac tissue and specifically the aSHF, comprising the OFT and RV, as well as some head muscles [START_REF] Verzi | The right ventricle, outflow tract, and ventricular septum comprise a restricted expression domain within the secondary/anterior heart field[END_REF][START_REF] Lescroart | Clonal analysis reveals common lineage relationships between head muscles and second heart field derivatives in the mouse embryo[END_REF][START_REF] Devine | Early patterning and specification of cardiac progenitors in gastrulating mesoderm[END_REF]. The anterior Hox genes (Hoxa1, Hoxa3, Hoxb1) distinguish pSHF progenitors with contributions to the inferior wall of the OFT and atria [START_REF] Bertrand | Hox genes define distinct progenitor sub-domains within the second heart field[END_REF]. Hand1+ cells only contribute to the CM of the LV [START_REF] Barnes | Analysis of the Hand1 cell lineage reveals novel contributions to cardiovascular, neural crest, extra-embryonic, and lateral mesoderm derivatives. Developmental dynamics : an official publication of the[END_REF]. Similarly, early Hcn4-expressing cells contribute specifically to the cardiac crescent and subsequently to LV CMs [START_REF] Liang | HCN4 Dynamically Marks the First Heart Field and Conduction System Precursors[END_REF][START_REF] Spater | A HCN4+ cardiomyogenic progenitor derived from the first heart field and human pluripotent stem cells[END_REF]). Interestingly, Sfrp5, which encodes a secreted receptor for Wnt signaling, is expressed in progenitors of all cardiac regions including CMs, ECs and EPDCs, with the exception of the RV [START_REF] Fujii | Sfrp5 identifies murine cardiac progenitors for all myocardial structures except for the right ventricle[END_REF]).

In addition, there is also clearly a dynamic temporal expression of markers during CPC specification such that CPCs expressing Mesp1 at different time points during gastrulation will have different contributions to the heart and head. Temporally controlled clonal analysis of Mesp1+ CPCs has thus shown that LV progenitors express Mesp1 early (induced with doxycycline at E6.25) while the OFT, RV and atria and head muscles derive preferentially from CPCs that express Mesp1 slightly later (induced at E7.25) [START_REF] Lescroart | Early lineage restriction in temporally distinct populations of Mesp1 progenitors during mammalian heart development[END_REF]. Similarly, clonal analysis using a Six2-Cre-ERT2 line revealed that Six2 is expressed in a subset of late CPCs and that Six2 is successively activated in different populations of CPCs that contribute to the RV (induction with tamoxifen at E6) and then to the OFT (induction at E7.5) together with head muscles [START_REF] Zhou | Temporally Distinct Six2-Positive Second Heart Field Progenitors Regulate Mammalian Heart Development and Disease[END_REF]. Interestingly, inducible lineage tracings of Hcn4+, Tbx5+ or Sma+ (smooth muscle actin, expressed in embryonic CMs) show a progressive contribution of cardiac progenitors to the heart with early differentiating cells contributing specifically to the left side of the interventricular septum [START_REF] Spater | A HCN4+ cardiomyogenic progenitor derived from the first heart field and human pluripotent stem cells[END_REF][START_REF] Devine | Early patterning and specification of cardiac progenitors in gastrulating mesoderm[END_REF][START_REF] Choquet | Segregation of Central Ventricular Conduction System Lineages in Early SMA+ Cardiomyocytes Occurs Prior to Heart Tube Formation[END_REF]).

Finally, with the emergence of new technologies such as high-throughput scRNA-seq, it is now possible to study the heterogeneity of CPCs in detail, as they arise and specify to a cardiac or muscular fate during gastrulation. A first study by Chan et al. applied scRNA-seq to Mesp1+ mPSCs during differentiation and revealed the existence of at least 6 subpopulations of Mesp1+ progenitors, with some primed for hematopoietic lineages while others were primed toward the cardiac lineages [START_REF] Chan | Heterogeneity of Mesp1+ mesoderm revealed by single-cell RNA-seq[END_REF]. Single cell expression profiling of nascent mesoderm at the late stage of mouse gastrulation demonstrated the existence of distinct populations of cardiac progenitors with different states of expression of Mesp1, Tbx5, Nkx2-5 and Isl1 [START_REF] Kokkinopoulos | Single-Cell Expression Profiling Reveals a Dynamic State of Cardiac Precursor Cells in the Early Mouse Embryo[END_REF]. scRNA-seq of Mesp1+ CPCs in vivo (from E6.75 to E7.25) has showed a continuum of cell states along distinct cardiopharyngealrelated trajectories towards 4 destination cell types. These distinct trajectories likely correspond to CMs, ECs, anterior CPF (including aSHF) or posterior CPF (including pSHF) lineages, with different markers enriched in these different populations. RNA-FISH using specific markers of these four types further demonstrated that these populations of pre-specified CPCs have distinct spatial localization in the gastrulating embryo (Figure 2). EC-primed Mesp1+ progenitors are found in the outer layer of mesodermal cells, in the vicinity of the visceral endoderm, while CM-primed Mesp1+ progenitors are found closer to the epiblast. aSHF and pSHF-Mesp1+ markers were largely non-overlapping, with Wnt2b (pSHF marker) being expressed close to the PS while FoxC2 (aSHF marker) was expressed more anterolaterally (Figure 2) [START_REF] Lescroart | Defining the earliest step of cardiovascular lineage segregation by single-cell RNA-seq[END_REF]. Tcf21, which labels cardiopharyngeal progenitors that contribute to head muscle formation, is expressed in the anterior CPF population.

In Ciona, the two Mesp+ progenitors divide once in the early gastrula to give rise to four equivalent multipotent progenitors, which then divide asymmetrically at the end of gastrulation to generate anterior tail muscle progenitors (ATMs) and trunk ventral cells (TVCs). Cardiac cells (CMs and pericardial cells) derive from TVCs [START_REF] Davidson | Uncoupling heart cell specification and migration in the simple chordate Ciona intestinalis[END_REF][START_REF] Davidson | FGF signaling delineates the cardiac progenitor field in the simple chordate, Ciona intestinalis[END_REF][START_REF] Beh | FoxF is essential for FGF-induced migration of heart progenitor cells in the ascidian Ciona intestinalis[END_REF][START_REF] Christiaen | The transcription/migration interface in heart precursors of Ciona intestinalis[END_REF]. TVCs are multipotent progenitors that divide asymmetrically a second time to form first heart precursors (FHPs) and second TVCs (STVCs) [START_REF] Stolfi | Early chordate origins of the vertebrate second heart field[END_REF]. STVCs divide asymmetrically again to form lateral atrial siphon muscle founder cells (ASMFs) and second heart precursors (SHPs) [START_REF] Stolfi | Early chordate origins of the vertebrate second heart field[END_REF][START_REF] Wang | One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering[END_REF]) (Figure 1). scRNA-seq of progenitor cells covering the early branches of the cardiopharyngeal lineage tree in Ciona intestinalis identified 3 distinct transcriptional clusters that correspond to the cardiac FHP and SHP populations as well as an Ebf+ pharyngeal muscle population. Interestingly, this analysis showed that specific transcriptional programs are activated in fate-committed progenitors. In addition, this single cell transcriptomic study identified new specific-lineage markers, including Mmp21 for the FHP and Dach as a SHP specific marker. Remarkably, in the mouse, Dach1 is first broadly expressed in pharyngeal mesoderm and then also restricted later to cardiac SHF progenitors [START_REF] Wang | Role of mesodermal FGF8 and FGF10 overlaps in the development of the arterial pole of the heart and pharyngeal arch arteries[END_REF].

While single-cell approaches have helped us investigate in more detail the heterogeneity of the CPC population at different stages of development mostly through gene expression, the combination of these techniques with rapidly advancing systems approaches to functional genomic analysis (single-cell CITE-seq, single-cell ATAC-seq) will further improve our understanding of the molecular mechanisms implicated in early cell fate decisions.

Regulation of CPC Specification

Early induction of cardiogenic mesoderm

The extrinsic signals given to cells during development in vivo can be used as guidance cues during PSC differentiation from pluripotency towards specific cell types. It is therefore important to understand the stage-specific requirements of signaling pathways and their downstream transcription factors during early differentiation [START_REF] Noseda | Previews. Unleashing cardiopoiesis: a novel role for G-CSF[END_REF][START_REF] Loh | Mapping the Pairwise Choices Leading from Pluripotency to Human Bone, Heart, and Other Mesoderm Cell Types[END_REF].

CPCs arise from epiblast cells that form the primitive streak (PS). Their specification therefore depends on correct positioning and formation of the PS: this process relies on several signaling pathways, including canonical Wnt/b-catenin and Tgfb /Nodal pathways [START_REF] Conlon | A primary requirement for nodal in the formation and maintenance of the primitive streak in the mouse[END_REF][START_REF] Haegel | Lack of beta-catenin affects mouse development at gastrulation[END_REF][START_REF] Noseda | Previews. Unleashing cardiopoiesis: a novel role for G-CSF[END_REF]. The same pathways are required to induce formation of PS-like cells and subsequent cardiogenic mesoderm from PSCs [START_REF] Xu | Abrogation of the Cripto gene in mouse leads to failure of postgastrulation morphogenesis and lack of differentiation of cardiomyocytes[END_REF][START_REF] Gadue | Wnt and TGF-beta signaling are required for the induction of an in vitro model of primitive streak formation using embryonic stem cells[END_REF]. Canonical Wnt signaling has a biphasic role during cardiogenesis, as it promotes early steps of cardiogenesis but then inhibits CM differentiation [START_REF] Marvin | Inhibition of Wnt activity induces heart formation from posterior mesoderm[END_REF][START_REF] Schneider | Wnt antagonism initiates cardiogenesis in Xenopus laevis[END_REF][START_REF] Tzahor | Wnt signals from the neural tube block ectopic cardiogenesis[END_REF][START_REF] Naito | Developmental stage-specific biphasic roles of Wnt/beta-catenin signaling in cardiomyogenesis and hematopoiesis[END_REF][START_REF] Liu | Sox17 is essential for the specification of cardiac mesoderm in embryonic stem cells[END_REF][START_REF] Qyang | The renewal and differentiation of Isl1+ cardiovascular progenitors are controlled by a Wnt/betacatenin pathway[END_REF][START_REF] Ueno | Biphasic role for Wnt/beta-catenin signaling in cardiac specification in zebrafish and embryonic stem cells[END_REF][START_REF] Paige | Endogenous Wnt/betacatenin signaling is required for cardiac differentiation in human embryonic stem cells[END_REF]. BMP signaling is also required for subsequent gastrulation and formation of the cardiac crescent [START_REF] Mishina | Bmpr encodes a type I bone morphogenetic protein receptor that is essential for gastrulation during mouse embryogenesis[END_REF][START_REF] Miura | BMP signaling in the epiblast is required for proper recruitment of the prospective paraxial mesoderm and development of the somites[END_REF]. The requirement of these signaling pathways for cardiac development is well conserved throughout chordates [START_REF] Noseda | Previews. Unleashing cardiopoiesis: a novel role for G-CSF[END_REF]. Modulation of these pathways and others has guided most protocols of differentiation of PSCs towards cardiac lineages [START_REF] Laflamme | Cardiomyocytes derived from human embryonic stem cells in prosurvival factors enhance function of infarcted rat hearts[END_REF][START_REF] Yang | Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population[END_REF][START_REF] Kattman | Stagespecific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines[END_REF]. Briefly, formation of cardiac mesoderm can be induced from PSCs by BMP4, Vegf, Activin A and Fgf2, and subsequent differentiation is enhanced by Wnt inhibition. These are the minimal requirements for the formation of cardiac lineages in vitro in serumfree conditions. These extrinsic signals induce an intrinsic cascade of molecular events, where the activation of core transcriptional regulators thereafter progressively promotes cardiac cell fates during differentiation. We will now discuss these intrinsic events.

In Drosophila, tinman, the ortholog of Nkx2-5, is considered to be the master regulator of cardiogenesis [START_REF] Azpiazu | tinman and bagpipe: two homeo box genes that determine cell fates in the dorsal mesoderm of Drosophila[END_REF][START_REF] Bodmer | The gene tinman is required for specification of the heart and visceral muscles in Drosophila[END_REF][START_REF] Frasch | Induction of visceral and cardiac mesoderm by ectodermal Dpp in the early Drosophila embryo[END_REF]. While mutations in Nkx2-5 are associated with congenital heart disease [START_REF] Schott | Congenital heart disease caused by mutations in the transcription factor NKX2-5[END_REF]), Nkx2-5 mouse mutants form an early heart tube [START_REF] Lyons | Myogenic and morphogenetic defects in the heart tubes of murine embryos lacking the homeo box gene Nkx2-5[END_REF][START_REF] Tanaka | The cardiac homeobox gene Csx/Nkx2.5 lies genetically upstream of multiple genes essential for heart development[END_REF] showing that during evolution, Nkx2-5 lost its central role in cardiac specification, likely due to expansion of the vertebrate Nkx gene family. A key role for Mesp1 has clearly been established for cardiac specification in chordates [START_REF] Saga | Mesp1 expression is the earliest sign of cardiovascular development[END_REF][START_REF] Bondue | Mesp1: a key regulator of cardiovascular lineage commitment[END_REF][START_REF] Liu | Earlier and broader roles of Mesp1 in cardiovascular development[END_REF]. Mesp1 mutant mouse embryos present cardia bifida, with differentiated CMs and ECs in separated heart fields [START_REF] Saga | MesP1 is expressed in the heart precursor cells and required for the formation of a single heart tube[END_REF]. Double mutants for Mesp1 and its closest homologue, Mesp2, on the other hand, have severe defects of gastrulation. Chimeras with Mesp1/2 null and wild type cells showed contributions of mutant cells to most of the embryo excluding the heart, indicating a cell autonomous requirement of Mesp1/2 for CPC specification [START_REF] Kitajima | MesP1 and MesP2 are essential for the development of cardiac mesoderm[END_REF].

Mesp1 Cre/Cre knock-out cells present a developmental block similar to that of double mutant embryos, with mesodermal precursors being stuck in the gene program of the epiblast, as shown by scRNA-seq. Transcripts of several regulators of pluripotency such as Nanog, Oct4 and Eras or markers of the epiblast including E-cadherin and Epcam, were thus upregulated in Mesp1-null cells [START_REF] Lescroart | Defining the earliest step of cardiovascular lineage segregation by single-cell RNA-seq[END_REF].

Interestingly, during mPSC differentiation, overexpression of human or murine Mesp1 accelerates and increases cardiac differentiation, as shown by the appearance of beating areas and enhanced troponin T expression [START_REF] Bondue | Mesp1 acts as a master regulator of multipotent cardiovascular progenitor specification[END_REF][START_REF] David | MesP1 drives vertebrate cardiovascular differentiation through Dkk-1-mediated blockade of Wnt-signalling[END_REF][START_REF] Lindsley | Mesp1 coordinately regulates cardiovascular fate restriction and epithelialmesenchymal transition in differentiating ESCs[END_REF]). However, continuous expression of Mesp1 inhibits cardiac differentiation [START_REF] Bondue | Mesp1 acts as a master regulator of multipotent cardiovascular progenitor specification[END_REF]. Mesp1 has been shown to directly bind to the promoter of key cardiac TFs, such as Mef2c, Tbx20, Gata4 and Nkx2-5, thereby upregulating their expression and promoting further upregulation of downstream cardiac genes (Figure 3). Nevertheless, in the context of reprogramming, forced Mesp1 expression in cardiac fibroblasts is not sufficient to induce cardiac specification. In contrast, Smarcd3, Tbx5 and Gata4 have been shown to be sufficient for cardiac reprogramming [START_REF] Takeuchi | Directed transdifferentiation of mouse mesoderm to heart tissue by defined factors[END_REF][START_REF] Ieda | Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors[END_REF].

The key role of Mesp genes during cardiac progenitor specification appears to be highly conserved in chordates, as shown by the induction of ectopic cardiac differentiation upon expression of a constitutive form of Mesp ortholog in Ciona and human Mesp1 in Xenopus [START_REF] Davidson | Uncoupling heart cell specification and migration in the simple chordate Ciona intestinalis[END_REF][START_REF] David | MesP1 drives vertebrate cardiovascular differentiation through Dkk-1-mediated blockade of Wnt-signalling[END_REF]. Knock-down experiments using morpholinos also demonstrated the importance of Mesp for cardiac specification in ascidians [START_REF] Satou | The ascidian Mesp gene specifies heart precursor cells[END_REF]). In fish, the master regulator role of Mesp is not clear. While there are four mesp genes, only mespaa has a pro-cardiogenic role after animal cap transplants with mespaa over-expressing cells. Surprisingly, mespaa or even quadruple mespaa/ab/ba/bb CRISPR/Cas9 mutants did not show any cardiac defects [START_REF] Deshwar | Mespaa can potently induce cardiac fates in zebrafish[END_REF].

Mesp1 is activated in nascent mesoderm by key regulators of endoderm and mesoderm development Eomesodermin (Tbr2) and Brachyury (T) [START_REF] Costello | The T-box transcription factor Eomesodermin acts upstream of Mesp1 to specify cardiac mesoderm during mouse gastrulation[END_REF][START_REF] David | Induction of MesP1 by Brachyury(T) generates the common multipotent cardiovascular stem cell[END_REF][START_REF] Van Den Ameele | Eomesodermin induces Mesp1 expression and cardiac differentiation from embryonic stem cells in the absence of Activin[END_REF]. In Ciona embryos, Mesp is activated by Tbx6 and Lhx3, only in B7.5 cells co-expressing these factors [START_REF] Davidson | Uncoupling heart cell specification and migration in the simple chordate Ciona intestinalis[END_REF][START_REF] Christiaen | Spatio-temporal intersection of Lhx3 and Tbx6 defines the cardiac field through synergistic activation of Mesp[END_REF]). Consistent with these findings, a recent report searching for mesoderm-inducing factors in mouse embryonic fibroblasts found that Tbx6 was sufficient to activate Mesp1 and subsequent CPC gene expression. During PSC differentiation, Tbx6 promoted mesoderm and endoderm induction in the absence of serum and growth factors in both a cellautonomous and non-cell-autonomous manner [START_REF] Sadahiro | Tbx6 Induces Nascent Mesoderm from Pluripotent Stem Cells and Temporally Controls Cardiac versus Somite Lineage Diversification[END_REF]. Further studies in mouse will be required to determine how the precise domain of Mesp1 expression is defined within the nascent mesoderm, although other factors are also implicated in early specification of cardiac progenitors, such as long non-coding RNAs [START_REF] Klattenhoff | Braveheart, a long noncoding RNA required for cardiovascular lineage commitment[END_REF][START_REF] Ounzain | CARMEN, a human super enhancer-associated long noncoding RNA controlling cardiac specification, differentiation and homeostasis[END_REF][START_REF] Guo | A Linc1405/Eomes Complex Promotes Cardiac Mesoderm Specification and Cardiogenesis[END_REF], which have even been used to promote trans-differentiation to cardiac cell types [START_REF] Hou | Long noncoding RNA Braveheart promotes cardiogenic differentiation of mesenchymal stem cells in vitro[END_REF]) (Figure 3). Finally, the dynamic gene expression changes involved in transitions from pluripotency to cardiogenic mesoderm are accompanied by modifications of the epigenetic landscape or chromatin accessibility, with regulatory regions of genes being first primed then activated, and other regions involved in alternative fates being de-commissioned [START_REF] Paige | A temporal chromatin signature in human embryonic stem cells identifies regulators of cardiac development[END_REF][START_REF] Wamstad | Dynamic and coordinated epigenetic regulation of developmental transitions in the cardiac lineage[END_REF][START_REF] Racioppi | Combinatorial chromatin dynamics foster accurate cardiopharyngeal fate choices[END_REF]. Transcription factors cooperate to induce these changes, through interactions with each other and specific chromatin modifiers [START_REF] Chang | Epigenetics and cardiovascular development[END_REF][START_REF] Luna-Zurita | Complex Interdependence Regulates Heterotypic Transcription Factor Distribution and Coordinates Cardiogenesis[END_REF]. Combining data from single-cell expression and broad epigenetic studies, such as repressive histone mark ChIP-seq data from many tissues, may help us to identify new genetic drivers of cell fate specification [START_REF] Shim | Comparative analysis of diverse cell states establishes an epigenetic basis for inferring regulatory genes governing cell identity[END_REF].

CPC specification to the different cardiac lineages

In vitro, mPSCs cultured in serum conditions can form three populations with differential expression of T and Flk1 [START_REF] Kouskoff | Sequential development of hematopoietic and cardiac mesoderm during embryonic stem cell differentiation[END_REF][START_REF] Kattman | Multipotent flk-1+ cardiovascular progenitor cells give rise to the cardiomyocyte, endothelial, and vascular smooth muscle lineages[END_REF]. Hemangioblast specification usually precedes cardiac specification, as the former derives from a first wave of Flk1-expresssing Brachyury+ cells while the cardiogenic mesoderm arises from a Brachyury+ population, with later expression of Flk1 [START_REF] Kattman | Multipotent flk-1+ cardiovascular progenitor cells give rise to the cardiomyocyte, endothelial, and vascular smooth muscle lineages[END_REF]). Interestingly, if Mesp1 is induced earlier during mPSC differentiation, there is an increase in the hematopoietic progenitor population through direct activation of Tal1, encoding a vascular TF, at the expense of cardiac fates [START_REF] Chan | Mesp1 patterns mesoderm into cardiac, hematopoietic, or skeletal myogenic progenitors in a context-dependent manner[END_REF]. Conversely, in the absence of Tal1, extra-embryonic tissue (also expressing Mesp1) can differentiate into CM [START_REF] Van Handel | Scl represses cardiomyogenesis in prospective hemogenic endothelium and endocardium[END_REF].

CM and EC lineages of the FHF segregate early during gastrulation [START_REF] Mikawa | The polyclonal origin of myocyte lineages[END_REF][START_REF] Wei | Fate diversity of primitive streak cells during heart field formation in ovo[END_REF][START_REF] Lescroart | Early lineage restriction in temporally distinct populations of Mesp1 progenitors during mammalian heart development[END_REF]. scRNA-seq of Mesp1+ CPCs showed that several Notch signaling actors, including Notch1, are specifically downregulated in CM-fated cells and conversely upregulated in EC-fated cells [START_REF] Lescroart | Defining the earliest step of cardiovascular lineage segregation by single-cell RNA-seq[END_REF]). The Notch pathway has been implicated in several key processes of cardiac development, including trabeculation and valve formation [START_REF] Macgrogan | Notch signaling in cardiac development and disease[END_REF][START_REF] Macgrogan | Notch and interacting signalling pathways in cardiac development, disease, and regeneration[END_REF]. In vitro activation of Notch reduces CM differentiation [START_REF] Nemir | Induction of cardiogenesis in embryonic stem cells via downregulation of Notch1 signaling[END_REF]) and repression of Notch increases CM differentiation [START_REF] Schroeder | Recombination signal sequence-binding protein Jkappa alters mesodermal cell fate decisions by suppressing cardiomyogenesis[END_REF]. Lineage tracing of early Notch1+ cells preferentially marks the endocardium, suggesting a potential role for the Notch pathway in early CPC fate decisions between CMs and ECs [START_REF] Lescroart | Defining the earliest step of cardiovascular lineage segregation by single-cell RNA-seq[END_REF]. Moreover, further crosstalk between these two lineages happens in vitro and in vivo, suggesting paracrine mechanisms governing CPC specification and differentiation [START_REF] Saba | Sox17 expression in endocardium precursor cells regulates heart development in mice[END_REF][START_REF] Saint-Jean | Myocardial differentiation is dependent upon endocardial signaling during early cardiogenesis in vitro[END_REF]. Interestingly, endocardial cells with "valve-like" properties can be derived from hPSCs through the activation of other signaling pathways. VEGF, together with FGF2 and FGF8, can induce EC differentiation at the expense of CMs, thus favoring the specification of a pre-valvular cell type [START_REF] Neri | Human pre-valvular endocardial cells derived from pluripotent stem cells recapitulate cardiac pathophysiological valvulogenesis[END_REF]).

The PSC model has also been used to specify PSCs into other cardiac cell lineages (Figure 4). Epicardial cells, for example, can be produced from hPSCs. Indeed, stage-specific activation of BMP, retinoic acid (RA) and Wnt/b-catenin signaling pathways after induction of CPCs induces the specification of epicardial-like progenitors expressing TBX18 and WT1, two markers of the proepicardial organ [START_REF] Iyer | Robust derivation of epicardium and its differentiated smooth muscle cell progeny from human pluripotent stem cells[END_REF][START_REF] Bao | Long-term self-renewing human epicardial cells generated from pluripotent stem cells under defined xeno-free conditions[END_REF][START_REF] Guadix | Human Pluripotent Stem Cell Differentiation into Functional Epicardial Progenitor Cells[END_REF]. In line with these findings, mouse embryos lacking the Wnt inhibitors Dkk1 and Dkk2 have a thicker epicardium and thinner myocardium [START_REF] Phillips | Dkk1 and Dkk2 regulate epicardial specification during mouse heart development[END_REF], showing again the necessity and usefulness of translating our current in vivo knowledge for in vitro generation of specific cell types. Epicardial cells can differentiate into cardiac fibroblasts or vascular SMCs through epithelial-to-mesenchymal transition, which can be induced in vitro by addition of Tgf-b1 to the cells. Nevertheless, as the epicardium gives rise to multiple cell lineages, the resulting cell population is heterogeneous. In order to obtain a pure population of cardiac fibroblasts without going through an epicardial-like stage, another protocol has recently been developed where activation of the FGF pathway in hPSC-derived CPCs induces formation of Periostinand Vimentin-expressing cardiac fibroblasts, presenting many functional similarities with their in vivo counterparts [START_REF] Zhang | Functional cardiac fibroblasts derived from human pluripotent stem cells via second heart field progenitors[END_REF].

Pacemaker CMs can also be generated from mPSC by induction of Tbx3 [START_REF] Jung | Programming and isolation of highly pure physiologically and pharmacologically functional sinus-nodal bodies from pluripotent stem cells[END_REF] or Shox2 [START_REF] Ionta | SHOX2 overexpression favors differentiation of embryonic stem cells into cardiac pacemaker cells, improving biological pacing ability[END_REF]. Sino-atrial node (SAN)-like pacemaker cells arise from Nkx2.5-, Tbx18+ CPCs [START_REF] Mommersteeg | The sinus venosus progenitors separate and diversify from the first and second heart fields early in development[END_REF]. As a first demonstration of generating SAN-like pacemaker cells from hPSCs without recurring to overexpression, inhibition of the FGF pathway with activation of BMP and RA signaling generated a NKX2-5-but SIRPA+ cardiac cell type, presenting pacemaker properties similar to SAN cells. These cells were able to display pacemaker activity when grafted into rat hearts [START_REF] Protze | Sinoatrial node cardiomyocytes derived from human pluripotent cells function as a biological pacemaker[END_REF].

Regional specification

Multiplication of the cardiac chambers has significantly increased the efficiency but also complexity of the heart during evolution. The specification of early CPCs toward a particular cardiac region is not yet fully understood and there may be a lot to glean from in vitro PSC models and in vivo studies.

One of the main regional distinctions is between atrial and ventricular cardiac tissue. As discussed previously, lineage-tracing experiments in the mouse have shown the existence of distinct CPCs that contribute to different cardiac regions. For example, Foxa2+ CPCs specifically label ventricular derivatives. Formation of chimeric embryos from wild type and Foxa2 -/-mutant mPSCs have shown that Foxa2 is cell-autonomously required for ventricular specification such that there is no contribution of mutant cells to the ventricles. Similarly, Foxa2 -/-mPSCs have a decreased ability to form ventricular cardiomyocytes [START_REF] Bardot | Foxa2 identifies a cardiac progenitor population with ventricular differentiation potential[END_REF]. Retinoic acid (RA) signaling is also a known player for regional specification, such that exogenous RA treatment patterns the CPCs along the antero-posterior axis and promotes atrial identity [START_REF] Xavier-Neto | A retinoic acid-inducible transgenic marker of sino-atrial development in the mouse heart[END_REF][START_REF] Hochgreb | A caudorostral wave of RALDH2 conveys anteroposterior information to the cardiac field[END_REF][START_REF] Huang | Retinoic acid signaling sequentially controls visceral and heart laterality in zebrafish[END_REF][START_REF] Stefanovic | Mechanisms of retinoic acid signaling during cardiogenesis[END_REF][START_REF] Bono | T-box genes and retinoic acid signaling regulate the segregation of arterial and venous pole progenitor cells in the murine second heart field[END_REF]. The specific addition of RA at the cardiac mesoderm-CPC stage during hPSC differentiation favors specification toward an atrial-like phenotype (KCNJ3+) at the expense of ventricular fate (MYL2+) [START_REF] Zhang | Direct differentiation of atrial and ventricular myocytes from human embryonic stem cells by alternating retinoid signals[END_REF][START_REF] Lee | Human Pluripotent Stem Cell-Derived Atrial and Ventricular Cardiomyocytes Develop from Distinct Mesoderm Populations[END_REF]. A study has shown that ventricular hPSC-derived CPCs are marked by the expression of CYP26A1, which encodes an enzyme responsible for RA degradation. In contrast, atrial hPSC-derived CPCs express RALDH2 (an enzyme required for the synthesis of RA). RALDH2+ CPCs will preferentially generate atrial CMs [START_REF] Lee | Human Pluripotent Stem Cell-Derived Atrial and Ventricular Cardiomyocytes Develop from Distinct Mesoderm Populations[END_REF]. Consistently, in the mouse model, early Mesp1-expressing CPCs, which contribute preferentially to the LV, express Cyp26a1, while atrial Mesp1+ CPCs that express Mesp1 at later stages preferentially express Raldh2 [START_REF] Lescroart | Early lineage restriction in temporally distinct populations of Mesp1 progenitors during mammalian heart development[END_REF]. Interestingly, ISL1 knock-out hPSCs resemble PSC cultures exposed to RA signaling and lead to the preferential specification of atrial CMs. Further investigation showed that ISL1 suppresses the atrial specifier NR2F1. Specification of hPSCs into atrial CMs through RA signaling appears to be mediated by activation of the TALE transcription factor MEIS2, which antagonizes ISL1 expression [START_REF] Quaranta | Revised roles of ISL1 in a hES cell-based model of human heart chamber specification[END_REF].

In fish, RA signaling also indirectly counteracts the activity of Isl1 through Ajuba, a LIM domain protein [START_REF] Witzel | The LIM protein Ajuba restricts the second heart field progenitor pool by regulating Isl1 activity[END_REF]). In addition, in vitro studies have demonstrated that the proportion of atrial versus ventricular progenitors can be modulated by different concentrations of Activin A and Bmp4 early during hPSC differentiation [START_REF] Lee | Human Pluripotent Stem Cell-Derived Atrial and Ventricular Cardiomyocytes Develop from Distinct Mesoderm Populations[END_REF]) (Figure 4). It would be very informative to evaluate the levels of BMP4 and Activin A in vivo to see whether this reflects a distinct environmental context for atrial and ventricular progenitors. Furthermore, differentiation protocols established so far do not yet allow us to differentiate between LV versus RV or between atrial cardiac cell types, such as right versus left atrial CMs or pacemaker cells. These might be achieved by understanding the specification of the FHF and anterior and posterior SHF as they contribute to different regions of the heart.

The establishment of spheroids from HCN4-GFP/Tbx1-Cre; Rosa-tdTomato mPSCs has allowed study of FHF versus SHF specification, despite the fact that Tbx1 is only expressed in a subpopulation of the SHF. Transcriptomic analysis demonstrated that Bmp signaling is enriched in HCN4+ FHF progenitors while Wnt signaling appears to be specific to Tbx1-derived SHF cells. Modulating levels of Bmp4 or Wnt3A demonstrated that Bmp4 can promote FHF or Tbx1+ SHF specification depending on its concentration, while Wnt3a specifically promotes the appearance of the Tbx1-derived SHF population. Interestingly, the same study showed that Cxcr4, which initially marks early CPCs, is not expressed later in the FHF and specifically labels the Tbx1+ SHF population [START_REF] Andersen | Precardiac organoids form two heart fields via Bmp/Wnt signaling[END_REF].

Finally, scRNA-seq has enabled prediction of lineage specifiers for specific cardiac regions. For example, Hopx, Chd3, Ncor1 and Shox2, Sox4 and Hoxb4 were among the major genes predicted to play a role in the specification of the atrial or sinus venosus lineages, respectively. Similarly, Nr1d1 could be a cell fate determinant for the RV while Hand2, Msx1 may specify the OFT (de Soysa et al. 2018). Indeed, scRNA-seq of Hand2-null cells shows disregulation of the RV and OFT gene program with abnormal activation of posterior genes, but these data suggest a failure of RV cells to migrate to their final location rather than a defect in their specification [START_REF] De Soysa | Single-cell transcriptome analysis during cardiogenesis reveals basis for organ level developmental anomalies[END_REF]. Similarly, ectopic expression of Hoxb1 in aSHF CPCs causes deregulation of the aSHF gene program, suggesting that homeodomain genes play a key role in the antero-posterior patterning and cell identity of CPCs [START_REF] Stefanovic | Hox-Dependent Coordination of Cardiac Cell Patterning and Differentiation[END_REF]. Relatedly, in tunicates, knock-out of the SHP-specific gene Dach in multipotent CPCs led to ectopic expression of the FHP marker Mmp21, suggesting a change in cell fate identity [START_REF] Wang | Role of mesodermal FGF8 and FGF10 overlaps in the development of the arterial pole of the heart and pharyngeal arch arteries[END_REF].

Cardiomyocyte versus skeletal muscle fate

As discussed above, the CPF is able to differentiate into both cardiac tissue and skeletal muscles of the head and neck. Several studies in mice, chick and fish have shown that there is a conserved regulatory network (including Tbx1, Pitx2, Nkx2-5 and Isl1) that governs cardiac and skeletal muscle progenitor specification [START_REF] Tirosh-Finkel | Mesoderm progenitor cells of common origin contribute to the head musculature and the cardiac outflow tract[END_REF][START_REF] Nathan | The contribution of Islet1-expressing splanchnic mesoderm cells to distinct branchiomeric muscles reveals significant heterogeneity in head muscle development[END_REF][START_REF] Sambasivan | Distinct regulatory cascades govern extraocular and pharyngeal arch muscle progenitor cell fates[END_REF][START_REF] Nevis | Tbx1 is required for second heart field proliferation in zebrafish[END_REF][START_REF] Paffett-Lugassy | Unique developmental trajectories and genetic regulation of ventricular and outflow tract progenitors in the zebrafish second heart field[END_REF][START_REF] Guner-Ataman | Failed Progenitor Specification Underlies the Cardiopharyngeal Phenotypes in a Zebrafish Model of 22q11.2 Deletion Syndrome[END_REF]. Interestingly, fundamental insights on how the CPF is able to differentiate into cardiac or skeletal muscle lineages arise from studies in Ciona robusta. It is now clear that in tunicates, there is an antagonism between Nk4 (homolog of Nkx2-5) and Tbx1/10 to regulate cardiac versus skeletal muscle specification. Thus, in committed cardiac progenitors, NK4 inhibits Tbx1/10-dependent activation of Ebf, while in ASM progenitors, NK4 is repressed, thereby allowing Tbx1/10 to activate the skeletal muscle program [START_REF] Wang | One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering[END_REF]. Ebf is then required for differentiation of ASMs by promoting the pharyngeal myogenic program [START_REF] Razy-Krajka | Collier/OLF/EBFdependent transcriptional dynamics control pharyngeal muscle specification from primed cardiopharyngeal progenitors[END_REF].

Fgf/MAPK signaling also plays a critical role in Ciona in skeletal vs cardiac specification. Activated in multipotent progenitors, Fgf signaling is then maintained only in the lateral-most daughter cells during cell division within the CPF lineage. Fgf signaling is thereafter restricted to ASM progenitors, thus promoting skeletal muscle identity [START_REF] Razy-Krajka | An FGF-driven feedforward circuit patterns the cardiopharyngeal mesoderm in space and time[END_REF]. Even if a role for Fgf signaling in cardiac versus skeletal muscle specification has not yet been shown in vertebrates, it is required for development of the CPF [START_REF] Abu-Issa | Fgf8 is required for pharyngeal arch and cardiovascular development in the mouse[END_REF][START_REF] Vitelli | A genetic link between Tbx1 and fibroblast growth factor signaling[END_REF][START_REF] Brown | Cre-mediated excision of Fgf8 in the Tbx1 expression domain reveals a critical role for Fgf8 in cardiovascular development in the mouse[END_REF][START_REF] Hu | Tbx1 regulates fibroblast growth factors in the anterior heart field through a reinforcing autoregulatory loop involving forkhead transcription factors[END_REF][START_REF] Aggarwal | Dissection of Tbx1 and Fgf interactions in mouse models of 22q11DS suggests functional redundancy[END_REF][START_REF] Ilagan | Fgf8 is required for anterior heart field development[END_REF][START_REF] Park | Required, tissue-specific roles for Fgf8 in outflow tract formation and remodeling[END_REF][START_REF] Kelly | Visualization of outflow tract development in the absence of Tbx1 using an FgF10 enhancer trap transgene[END_REF][START_REF] Park | An FGF autocrine loop initiated in second heart field mesoderm regulates morphogenesis at the arterial pole of the heart[END_REF][START_REF] De Pater | Distinct phases of cardiomyocyte differentiation regulate growth of the zebrafish heart[END_REF][START_REF] Wang | Role of mesodermal FGF8 and FGF10 overlaps in the development of the arterial pole of the heart and pharyngeal arch arteries[END_REF][START_REF] Lazic | Mef2cb regulates late myocardial cell addition from a second heart field-like population of progenitors in zebrafish[END_REF][START_REF] Watanabe | Fibroblast growth factor 10 gene regulation in the second heart field by Tbx1, Nkx2-5, and Islet1 reveals a genetic switch for down-regulation in the myocardium[END_REF]. sc-RNAseq in Ciona has further enabled precise investigation of temporal CPC specification. It led to the characterization of ten discrete regulatory states along specification pseudotime, with identification of "primed" and "de novo" markers that are respectively expressed either throughout these states or only in more committed progenitors. About 50% of cardiac or skeletal muscle genes are "primed" and therefore already expressed in multipotent progenitors. Cross-comparison with other published sc-RNAseq of developing mouse hearts showed high conservation of the transcriptional network governing cardiac versus skeletal muscle specification [START_REF] Wang | Role of mesodermal FGF8 and FGF10 overlaps in the development of the arterial pole of the heart and pharyngeal arch arteries[END_REF].

Tbx1 mutant embryos show defects in both craniofacial skeletal muscles and heart [START_REF] Lindsay | Tbx1 haploinsufficieny in the DiGeorge syndrome region causes aortic arch defects in mice[END_REF][START_REF] Kelly | The del22q11.2 candidate gene Tbx1 regulates branchiomeric myogenesis[END_REF][START_REF] Lescroart | Clonal analysis reveals a common origin between nonsomite-derived neck muscles and heart myocardium[END_REF]. Through RNA-sequencing of wild-type versus tbx1 mutant zebrafish embryos, gdf3-ALK4, a TGFµ superfamily ligand has been identified as a downstream effector of tbx1 involved in CPC specification [START_REF] Guner-Ataman | Failed Progenitor Specification Underlies the Cardiopharyngeal Phenotypes in a Zebrafish Model of 22q11.2 Deletion Syndrome[END_REF]. In fish, tbx1 is also required to specify the nkx2.5+ CPF. Moreover, recent studies have also shown a role for Wnt signaling in the balance between cardiac and skeletal muscle lineages, as Wnt activation during or prior to gastrulation inhibits pharyngeal muscle specification at the expense of cardiac lineages [START_REF] Mandal | Wnt signaling balances specification of the cardiac and pharyngeal muscle fields[END_REF]. Such a role for Wnt has not been found in tunicates [START_REF] Kaplan | Initial characterization of Wnt-Tcf functions during Ciona heart development[END_REF]). Other factors have been implicated in pharyngeal muscle specification. NR2F or COUP-TF proteins, downstream targets of RA signaling, have been shown, in fish, to promote posterior pharyngeal muscle specification (muscles analogous to the vertebrate neck muscles) while restricting ventricular CM progenitors [START_REF] Dohn | Nr2f-dependent allocation of ventricular cardiomyocyte and pharyngeal muscle progenitors[END_REF].

BMP signaling, when applied to differentiating mPSCs, leads to the downregulation of skeletal muscle markers while promoting cardiac genes. RNA-seq showed that Podxl and Cdh4 are surface markers that could be used to distinguish between committed skeletal and cardiac progenitors respectively [START_REF] Chan | Heterogeneity of Mesp1+ mesoderm revealed by single-cell RNA-seq[END_REF]. After mesoderm induction, dual inhibition of Wnt and Nodal promotes CPC specification, and cells thus express Tbx1, Isl1 and Nkx2-5. With specific culture conditions, it is then possible to specify the CPF into skeletal muscle fibers with a cocktail of signaling molecules including a Bmp inhibitor, Hgf, Fgf2 and Igf1 or into beating cardiomyocytes when supplemented with ascorbic acid, Vegf, Fgf2 and Fgf10 [START_REF] Nandkishore | Divergent early mesoderm specification underlies distinct head and trunk muscle programmes in vertebrates[END_REF].

Concluding remarks

The specification of CPCs is a complex developmental process that occurs concomitantly with their migration. Mesp1, a key transcription master regulator of CPC specification, has also a critical role in CPC migration by controlling the epithelial-to-mesenchymal transition, cell polarity and migration speed [START_REF] Bondue | Mesp1 acts as a master regulator of multipotent cardiovascular progenitor specification[END_REF][START_REF] Lindsley | Mesp1 coordinately regulates cardiovascular fate restriction and epithelialmesenchymal transition in differentiating ESCs[END_REF][START_REF] Chiapparo | Mesp1 controls the speed, polarity, and directionality of cardiovascular progenitor migration[END_REF]. Therefore, it would be important to integrate both CPC migratory trajectories and lineage behavior to investigate the influence of each process on the other. In addition, it is well established that left-right asymmetry is critical for proper heart development [START_REF] Desgrange | Left-right asymmetry in heart development and disease: forming the right loop[END_REF]) but it is not clear how this affects CPC specification.

With the emergence of single-cell approaches, the model of CPC specification has changed, and it is now clear that CPCs represent a very heterogeneous and dynamic population of progenitors, reflecting distinct physical positions in space and time but also some stochastic factors. In addition, recent research has shown that early CPCs are "primed" toward a particular lineage very early during embryonic development. The determinants of this early "primed" transcriptional heterogeneity are not yet understood. Comprehending the regulation of CPC specification will help to decipher the developmental mechanisms leading to congenital heart disease, which affect about 1% of newborns world-wide [START_REF] Srivastava | Genetic regulation of cardiogenesis and congenital heart disease[END_REF]).

In addition, investigating the embryonic regulation of CPC specification has already significantly improved differentiation protocols of PSCs toward a specific cell type. This is particularly relevant in the context of cell therapy after cardiac injury. The identification of multipotent CPCs has brought significant hope to the field of heart repair and further research into the early steps of CPC specification will undoubtedly lead to the design of strategies to specify multipotent CPCs into a specific mature cardiac cell type, that could then be used for cell therapy. As a recent example, hPSC-derived CMs have recently been grafted into infarcted pig hearts and showed significant engraftment, although accompanied by ventricular tachyarrhythmia [START_REF] Romagnuolo | Human Embryonic Stem Cell-Derived Cardiomyocytes Regenerate the Infarcted Pig Heart but Induce Ventricular Tachyarrhythmias[END_REF]. Further improvements are thus required to successfully repair infarcted hearts in patients.

Figure 2 :

 2 Figure2: Heterogeneity of CPC specification as revealed by single-cell analysis A. In scRNA-seq experiments, four distinct Mesp1+ CPC subpopulations have been identified that emerge from epiblast cells and that correspond to the endothelial (EC -black), cardiomyocyte (CMred), anterior cardiopharyngeal field (aCPF, light green) and posterior CPF (pCPF, dark green) progenitors. These populations are enriched for some transcripts (in brackets) and are found in particular locations in the embryo (B). B. Drawing of the gastrulating embryo, where the different CPC subpopulations are found. The embryo is bilateral: on one side is depicted the localization of the Mesp1+ EC versus Mesp1+ CM populations found either in the outer layer or close to the epiblast, while on the other side the localization of aCPF and pCPF progenitors is represented along the anteromedial axis of the embryo. These distinct CPC subpopulations make different regional contributions to the definitive heart as illustrated in C. AA, aortic arch, PT, pulmonary trunk, Ao, aorta, PV, pulmonary vein, SCV, superior caval vein, ICV, inferior caval vein, RA, right atrium, LA, left atrium, RV, right ventricle, LV, left ventricle.

Figure 3 :

 3 Figure 3: Early intrinsic regulation of cardiac specificationRepresentation of the molecular hallmarks of cardiac progenitor cell commitment. When cardiac progenitors express Mesp1 in the nascent mesoderm, they turn off the genetic program of pluripotency and turn on the gene regulatory network of cardiac specification and migration. Green arrows represent direct activation, red arrows represent repression. Genes grouped together are regulated in a similar manner by Mesp1. Although Linc1405 was shown to interact with Eomes, it was not the case for Braveheart, another cardiogenic long non-coding RNA.

Figure 4 :

 4 Figure 4: Extrinsic regulation of cardiac specificationBy differentiating mouse and human pluripotent stem cells (PSCs) under serum-free conditions, the minimal cues necessary for their specification and differentiation to a particular cardiopharyngeal cell lineage were defined. Arrows indicate different steps of differentiation. All protocols pass through a stage where cells present features of multipotent cardiopharyngeal progenitors (CPCs), then diverge in terms of given signaling modulators and cell markers. AA, ascorbic acid; BMP, bone morphogenetic protein; CFs, cardiac fibroblasts; CMs, cardiomyocytes; FGF, fibroblast growth factor; IGF-1, insulin-

Table 1 : Lineage tracing in mouse has revealed different early CPCs subpopulations CPCs subpopulation Genetic construct used Labeled cardiac regions

 1

	Labeled	Non-cardiac	References
	cardiac cell	derivatives
	types

Steps from cardiopharyngeal progenitor cells to cardiac and skeletal muscle cell lineages: comparison between tunicates and mouse A.

  The Ciona intestinalis CPC lineage tree is found on top. Two Mesp+ CPCs (green) are found at around 4 hours post fertilization (hpf). After two cell divisions, these CPCs form four anterior tail muscle cells (ATMs) and four multipotent trunk ventral cells (TVCs). These TVCs divide asymmetrically to form a second TVC (STVC) and a first heart precursor (FHP -red). The STVC (dark grey) divides to form an atrial siphon muscle progenitor cell (ASM -blue), analogous to the vertebrate head muscles and a second heart precursor (SHP -orange). A subset of the lineage branches is depicted from a single Mesp CPC, the other branches being identical in sequence and outcome. Below, layout of the tunicate larva. Black lines indicate sister cells. Time after fertilization is indicated, in hours postfertilization (hpf). At around 28 hpf, ASM progenitor derivatives form an ASM ring while FHP-and SHP-derived cells form the developing heart, including pericardial cells and cardiomyocytes. B. The mouse CPC lineage. On top, early CPCs (in green) are found in the gastrulating embryo and are marked by the expression of Mesp1, Flk1, Pdgfra and Cxcr4 as soon as they leave the primitive streak (PS). At embryonic day (E) 8.5, the primary heart tube is mainly formed from first heart field (FHF, red) progenitors, with additional contribution to the arterial and venous pole respectively from anterior and posterior second heart field (aSHF, blue and pSHF, purple) progenitors that lie behind the heart tube, within the cardiopharyngeal mesoderm. On the right, the contribution of FHF and anterior and posterior pharyngeal mesoderm to the head and neck muscles and to the heart; below, the lineage tree deduced from retrospective clonal analysis. Interestingly, there are at least three reiteration of the Ciona ontogenetic motif along the antero-posterior axis of the embryo. The expression window of Mesp1 is indicated by green dotted lines. Mesp1 is first expressed in the FHF progenitors (red) while expressed later in the anterior (blue) and posterior (purple) progenitors of the cardiopharyngeal field. EOM, extraocular muscles, ao, aorta, pt, pulmonary trunk, RA, right atrium, LA, left atrium, RV, right ventricle, LV, left ventricle.

Skeletal myofibers of head and neck muscles MyoD+ Myogenin+ Pre-valvular endocardial cells

	Figure 4
						-BMP
						+HGF
						+FGF2
						+IGF1
			-Wnt	Bipotent progenitors Tbx1+ Tcf21+	+VEGF +FGF +AA	c-TnT+ SHF-derived CMs
			-Nodal	Flk1-/Pgfra+
						Atrial CMs
					+RA	SIRPA+ Nkx2.5+
					+BMP/	Raldh2+
					Activin
					-RA	Ventricular CMs
	PSCs	+Wnt/β-catenin	CPCs	+BMP -FGF		SIRPA+ Nkx2.5+ CD235a+
		+ BMP		+RA
	Oct4+ Sox2+ Nanog+	+ Activin/Nodal +FGF	Brachyury+ Mesp1+ Flk1+/Pgfra+	+VEGF +Fgf2 +Fgf8	SAN-like pacemaker cells Tbx3+ SIRPA+ Nkx2.5-
			Ror2+/Cd13+
			Cxcr4+
			Fzd4+
			+BMP	Nfatc+
			+RA	Tbx20+
			+Wnt	CD31+
						+Tgf-β1
					Epicardial cells
			+Wnt +FGF		Tbx18+ WT1+	Epicardial-derived SMCs and CFs
					Cardiac fibroblasts
					POSTN+
					Vimentin+

Acknowledgments

We regret that many relevant references were not cited due to space constraints. We thank L. Christiaen, R. Kelly and the members of the Zaffran lab for helpful comments. B.S. is supported by the FNRS. FL is an INSERM fellow.