Sonia Stefanovic
email: sonia.stefanovic@univ-amu.fr

Stéphane Zaffran
email: stephane.zaffran@univ-amu.fr

Mechanisms of retinoic acid signaling during cardiogenesis

Keywords:

Substantial experimental and epidemiological data have highlighted the interplay between nutritional and genetic factors in the development of congenital heart defects. Retinoic acid (RA), a derivative of vitamin A, plays a key role during vertebrate development including the formation of the heart. Retinoids bind to RA and retinoid X receptors (RARs and RXRs) which then regulate tissue-specific genes. Here, we will focus on the roles of RA signaling and receptors in gene regulation during cardiogenesis, and the consequence of deregulated retinoid signaling on heart formation and congenital heart defects.

Introduction

The heart is the first organ to function and is essential for the distribution of nutrients and oxygen in the growing mammalian embryo. Normal cardiac morphogenesis is thus vital for embryonic survival. Heart development is a complex process that requires the precise and coordinate interactions between multiple cardiac and extra-cardiac cell types. Any perturbation in the cells that contribute to heart formation leads to cardiac defects. Congenital heart defects affect 1-2% of live births, and are found in up to one-tenth of spontaneously aborted fetuses [START_REF] Bruneau | The developmental genetics of congenital heart disease[END_REF][START_REF] Fahed | Genetics of congenital heart disease: the glass half empty[END_REF]. Studies in the invertebrate Drosophila melanogaster have defined numerous regulators that determine cardiac cell specification and differentiation, revealing that the cardiac regulatory network is remarkably conserved during evolution.

More recently, genetic studies have identified mutations in genes encoding components of signaling pathways as well as proteins organizing chromatin structure that are responsible for congenital heart defects [START_REF] Miyake | KDM6A point mutations cause Kabuki syndrome[END_REF][START_REF] Vissers | Mutations in a new member of the chromodomain gene family cause CHARGE syndrome[END_REF][START_REF] Zaidi | De novo mutations in histone-modifying genes in congenital heart disease[END_REF].

The specification of multipotent heart progenitor cells and their differentiation into different cell lineages is under tight spatial and temporal transcriptional control. Defining the transcriptional networks underlying normal heart development is a prerequisite for understanding the molecular basis of congenital heart malformation. Vitamin A (or provitamin A carotenoid) deficiency is a major public health problem in underdeveloped countries [START_REF] Zile | Vitamin A-not for your eyes only: requirement for heart formation begins early in embryogenesis[END_REF]. Young children, pregnant and breast feeding women are the main groups affected because their requirements for Vitamin A are higher and the impact of deficiency more severe than the other population subgroups. Malformations following maternal vitamin A deficiency were first reported by [START_REF] Hale | The relation of vitamin A to anophthalmos in pigs[END_REF] (F., 1935). The mammalian embryo is strongly dependent on the maternal delivery of retinol (carotenoids and retinyl esters) through transplacental transfer. The fetus needs vitamin A throughout pregnancy [START_REF] Comptour | Nuclear retinoid receptors and pregnancy: placental transfer, functions, and pharmacological aspects[END_REF]. Consequently, both deficiency and excess of vitamin A cause severe damage during prenatal and postnatal development. Nutritional and clinical studies on animals and humans have shown that maternal vitamin A insufficiency can result in fetal death, or a broad range of abnormalities including cardiac malformations (D'Aniello and Waxman, 2015;[START_REF] Wilson | An analysis of the syndrome of malformations induced by maternal vitamin A deficiency. Effects of restoration of vitamin A at various times during gestation[END_REF]. Moreover, it is suggested that the elevated incidence of heart malformations in developing countries could be partly explained by a low availability of retinol due to vitamin A deficiency in the diet [START_REF] Sommer | Impact of vitamin A supplementation on childhood mortality. A randomised controlled community trial[END_REF][START_REF] Underwood | Vitamin A deficiency disorders: international efforts to control a preventable "pox[END_REF]. Conversely, a high level of retinol during pregnancy leads to toxicity of many organs including the heart. For example, maternal intake of isotretinoin has been shown to cause congenital cardiac defects in addition to other malformations [START_REF] Guillonneau | Teratogenic effects of vitamin A and its derivates[END_REF]. Importantly, genetic alterations reducing retinol uptake [START_REF] Golzio | Matthew-Wood syndrome is caused by truncating mutations in the retinol-binding protein receptor gene STRA6[END_REF][START_REF] Kawaguchi | A membrane receptor for retinol binding protein mediates cellular uptake of vitamin A[END_REF][START_REF] Pasutto | Mutations in STRA6 cause a broad spectrum of malformations including anophthalmia, congenital heart defects, diaphragmatic hernia, alveolar capillary dysplasia, lung hypoplasia, and mental retardation[END_REF] or retinoic acid (RA) production [START_REF] Pavan | ALDH1A2 (RALDH2) genetic variation in human congenital heart disease[END_REF][START_REF] Roberts | Cyp26 genes a1, b1 and c1 are down-regulated in Tbx1 null mice and inhibition of Cyp26 enzyme function produces a phenocopy of DiGeorge Syndrome in the chick[END_REF] have been implicated in human congenital heart disease. Altered RA signaling either genetically or nutritionally could be a predominant risk factor, increasing the frequency of congenital heart diseases in humans [START_REF] Huk | Increased dietary intake of vitamin A promotes aortic valve calcification in vivo[END_REF][START_REF] Jenkins | Noninherited risk factors and congenital cardiovascular defects: current knowledge: a scientific statement from the American Heart Association Council on Cardiovascular Disease in the Young: endorsed by the American Academy of Pediatrics[END_REF][START_REF] Underwood | Vitamin A deficiency disorders: international efforts to control a preventable "pox[END_REF]. In this review, we will discuss the role of retinoids in cardiac gene regulation and congenital heart defects.

Early heart development

The mammalian heart has four chambers and is composed of a variety of cell types. Distinct sets of cardiac progenitors differentiate to form the different parts of the heart. It develops from cardiac progenitors that can be traced back to the early gastrulating embryo (embryonic day (E) 6.5 in the mouse). The earliest progenitors originate from the primitive streak and migrate toward the anterior lateral region to form the cardiac crescent, defined as the first heart field (E7-7.5). By E7.5-8.0, during folding of the embryo and formation of the foregut, the two sides of the cardiac crescent are brought together to form the primary heart tube (Fig. 1). The embryonic myocardium of the tube is characterized by a primitive phenotype, i.e. lower proliferation, a poorly developed contractile apparatus and slow conduction [START_REF] Christoffels | Development of the pacemaker tissues of the heart[END_REF][START_REF] Moorman | Cardiac chamber formation: development, genes, and evolution[END_REF]. Growth of the heart tube depends on the addition of progenitor cells from adjacent pharyngeal mesoderm to the arterial and venous poles. This cell population, named the second heart field, was first identified in the mouse and the chick models [START_REF] Buckingham | Building the mammalian heart from two sources of myocardial cells[END_REF][START_REF] Zaffran | New developments in the second heart field[END_REF]. These progenitor cells, located in a dorsal/medial position relative to the linear heart tube, are kept in an undifferentiated and rapidly proliferating state. These cells ultimately contribute to the outflow tract, right ventricle and a major part of the atria, while the linear heart tube gives rises mainly to the left ventricle [START_REF] Buckingham | Building the mammalian heart from two sources of myocardial cells[END_REF][START_REF] Kelly | The arterial pole of the mouse heart forms from Fgf10-expressing cells in pharyngeal mesoderm[END_REF][START_REF] Zaffran | New developments in the second heart field[END_REF][START_REF] Zaffran | Right ventricular myocardium derives from the anterior heart field[END_REF]. Specific regions in the embryonic heart tube subsequently acquire a chamber-specific gene program, differentiate further and expand, or "balloon" by rapid proliferation to form the ventricular and atrial chamber myocardium (NE8.5) (Fig. 1). In contrast, the regions in between these differentiating chambers, the sinus venosus, the atrioventricular canal and the outflow tract, do not differentiate or expand, and consequently form constrictions. The inflow tract cells of the heart tube develop into atrial cells, pulmonary myocardial cells and myocardial cells of the superior caval veins.

Expression of the LIM homeobox 1 Islet1 (Isl1) in second heart field cells led to an appreciation of the full contribution of these progenitors to the venous, as well as the arterial pole of the heart [START_REF] Cai | Isl1 identifies a cardiac progenitor population that proliferates prior to differentiation and contributes a majority of cells to the heart[END_REF]. However, differences in gene expression between progenitors of the venous and arterial poles revealed that the second heart field is pre-patterned [START_REF] Galli | Atrial myocardium derives from the posterior region of the second heart field, which acquires left-right identity as Pitx2c is expressed[END_REF][START_REF] Snarr | Isl1 expression at the venous pole identifies a novel role for the second heart field in cardiac development[END_REF]. Recent genetic lineage analysis in the mouse has shown that anterior Homeobox (Hox) genes Hoxa1, Hoxa3 and Hoxb1 expression define distinct sub-domains within the posterior domain of the second heart field that contribute to a large part of the atrial and sub-pulmonary myocardium [START_REF] Bertrand | Hox genes define distinct progenitor sub-domains within the second heart field[END_REF][START_REF] Diman | A retinoic acid responsive Hoxa3 transgene expressed in embryonic pharyngeal endoderm, cardiac neural crest and a subdomain of the second heart field[END_REF]. This suggests that Hox-expressing progenitor cells in the posterior domain of the second heart field contribute to both poles of the heart tube. Indeed, fate mapping and clonal analysis experiments have confirmed that posterior second heart field cells contribute to outflow tract, and that sub-pulmonary and inflow tract myocardial cells are clonally related [START_REF] Dominguez | Asymmetric fate of the posterior part of the second heart field results in unexpected left/ right contributions to both poles of the heart[END_REF][START_REF] Laforest | Genetic lineage tracing analysis of anterior Hox expressing cells[END_REF][START_REF] Lescroart | Lineage tree for the venous pole of the heart: clonal analysis clarifies controversial genealogy based on genetic tracing[END_REF]. Genetic tracing of Hoxb1 lineages in deficient embryos for the transcription factor T-box1 (Tbx1) showed that the deployment of Hoxb1-positive cell during the formation of the heart is regulated by Tbx1 [START_REF] Rana | Tbx1 coordinates addition of posterior second heart field progenitor cells to the arterial and venous poles of the heart[END_REF].

RA signaling functions during heart development

Many studies have demonstrated that the formation of the heart depends on the vitamin A metabolite RA, which serves as a ligand for nuclear receptors (Fig. 2) [START_REF] Niederreither | Embryonic retinoic acid synthesis is essential for early mouse post-implantation development[END_REF][START_REF] Niederreither | Embryonic retinoic acid synthesis is essential for heart morphogenesis in the mouse[END_REF]. RA metabolic pathways have been the subject of some excellent recent reviews [START_REF] Niederreither | Retinoic acid in development: towards an integrated view[END_REF][START_REF] Rhinn | Retinoic acid signalling during development[END_REF]. Excess exposure in humans to vitamin A or its analogs, the retinoids, can cause embryonic defects and congenital heart disease, including conotruncal and aortic arch artery malformations such as transposition of the great vessels, double outlet right ventricle, and tetralogy of Fallot [START_REF] Lammer | Retinoic acid embryopathy[END_REF][START_REF] Mark | Function of retinoid nuclear receptors: lessons from genetic and pharmacological dissections of the retinoic acid signaling pathway during mouse embryogenesis[END_REF]. In rodents, treatment with RA was one of the earliest teratogenic models of heart defects [START_REF] Wilson | Congenital anomalies of heart and great vessels in offspring of vitamin A-deficient rats[END_REF]. Retinoic exposure produces transposition of the great arteries and a wide spectrum of great artery patterning defects [START_REF] Ratajska | Coronary artery embryogenesis in cardiac defects induced by retinoic acid in mice[END_REF][START_REF] Yasui | Morphological observations on the pathogenetic process of transposition of the great arteries induced by retinoic acid in mice[END_REF]. The variability, low penetrance, lack of molecular and electrophysiological data of any particular defect makes these RA-induced teratogenic defects difficult to identify in human patients. Indeed, early disturbances of RA signaling may lead to severe CHDs associated with embryonic death and thus be only rarely observed as a cause of congenital heart disease in humans.

The canonical RA synthetic pathway has been elucidated over the last two decades (Fig. 2), mainly via gene targeting studies of several enzymes in the mouse [START_REF] Niederreither | Retinoic acid in development: towards an integrated view[END_REF]. Two sequential reactions are required to transform retinol, the major source of retinoids, into retinaldehyde and RA. The first reversible oxidation is catalyzed by cytosolic alcohol dehydrogenases (ADHs) and microsomal retinol dehydrogenase (RDH), and retinaldehyde is then irreversibly oxidized to RA by retinaldehyde dehydrogenase (RALDHs also known as ALDHs). There are three members of the RALDH family, each with a unique developmental expression patterns [START_REF] Mic | Novel retinoic acid generating activities in the neural tube and heart identified by conditional rescue of Raldh2 null mutant mice[END_REF][START_REF] Mic | RALDH3, a retinaldehyde dehydrogenase that generates retinoic acid, is expressed in the ventral retina, otic vesicle and olfactory pit during mouse development[END_REF]Niederreither et al., 2002b) (Fig. 2). Analysis of knockout mice demonstrated that RALDH2 (ALDH1A2) is responsible for almost all RA production during early development. Studies in mouse and avian embryos have shown that RA deficiency is associated with anomalies of anteroposterior patterning of the primitive heart [START_REF] Hochgreb | A caudorostral wave of RALDH2 conveys anteroposterior information to the cardiac field[END_REF][START_REF] Osmond | The effects of retinoic acid on heart formation in the early chick embryo[END_REF][START_REF] Yutzey | Expression of the atrial-specific myosin heavy chain AMHC1 and the establishment of anteroposterior polarity in the developing chicken heart[END_REF]. Using in situ hybridization experiments, [START_REF] Hochgreb | A caudorostral wave of RALDH2 conveys anteroposterior information to the cardiac field[END_REF] have described two phases of Raldh2 expression [START_REF] Hochgreb | A caudorostral wave of RALDH2 conveys anteroposterior information to the cardiac field[END_REF][START_REF] Moss | Dynamic patterns of retinoic acid synthesis and response in the developing mammalian heart[END_REF]. The first phase is characterized by a large expression domain in lateral mesoderm in proximity with posterior cardiac precursors. The second phase is characterized by progressive encircling of cardiac precursors [START_REF] Hochgreb | A caudorostral wave of RALDH2 conveys anteroposterior information to the cardiac field[END_REF]. Furthermore, treatment of chick embryos with a pan-antagonist of RA signaling at stages HH4-7 causes changes in inflow architecture, indicating that a caudal to rostral wave of Raldh2 conveys anteroposterior information to the forming heart tube. The phenotype of Raldh2-null mice supports this notion (Niederreither et al., 2002a;[START_REF] Niederreither | Embryonic retinoic acid synthesis is essential for early mouse post-implantation development[END_REF][START_REF] Niederreither | Embryonic retinoic acid synthesis is essential for heart morphogenesis in the mouse[END_REF]. In the mouse, deletion of Raldh2 causes heart defects with poor development of the atria and sinus venosus (Niederreither et al., 2002a;[START_REF] Niederreither | Embryonic retinoic acid synthesis is essential for early mouse post-implantation development[END_REF][START_REF] Niederreither | Embryonic retinoic acid synthesis is essential for heart morphogenesis in the mouse[END_REF]. Interestingly, some of these abnormalities can be rescued by transient maternal RA supplementation from E7.5 to E8.5-9.5, suggesting that cardiac precursors commit to their fate early during cardiogenesis [START_REF] Mic | Novel retinoic acid generating activities in the neural tube and heart identified by conditional rescue of Raldh2 null mutant mice[END_REF][START_REF] Niederreither | The regional pattern of retinoic acid synthesis by RALDH2 is essential for the development of posterior pharyngeal arches and the enteric nervous system[END_REF]. Our investigation of the role of Raldh2 revealed that RA signaling plays a role in establishing the boundary of the second heart field in the embryo [START_REF] Ryckebusch | Retinoic acid deficiency alters second heart field formation[END_REF][START_REF] Sirbu | Retinoic acid controls heart anteroposterior patterning by down-regulating Isl1 through the Fgf8 pathway[END_REF]. Analysis of markers of the second heart field, including Isl1, Tbx1, Fgf8 and Fgf10 in Raldh2 mutant embryos has shown abnormal expansion of the expression domains of these genes in posterior lateral mesoderm, suggesting that RA signaling is required to define the posterior boundary of the second heart field [START_REF] Ryckebusch | Retinoic acid deficiency alters second heart field formation[END_REF][START_REF] Sirbu | Retinoic acid controls heart anteroposterior patterning by down-regulating Isl1 through the Fgf8 pathway[END_REF] (Fig. 3). Similarly, the zebrafish mutation neckless (nls), which disrupts function of raldh2, causes formation of large hearts [START_REF] Keegan | Retinoic acid signaling restricts the cardiac progenitor pool[END_REF]. This excess of cardiomyocytes results from an increase number of cardiac progenitor cells as revealed by increased number of nkx2.5-expressing cells [START_REF] Keegan | Retinoic acid signaling restricts the cardiac progenitor pool[END_REF]. The other RADLH enzymes do not play major roles during heart development since deletion of Raldh1 does not result in any observable phenotype [START_REF] Fan | Targeted disruption of Aldh1a1 (Raldh1) provides evidence for a complex mechanism of retinoic acid synthesis in the developing retina[END_REF], and Raldh3-null mice have only defects in ocular and nasal regions as well as neuronal differentiation in the brain [START_REF] Dupe | A newborn lethal defect due to inactivation of retinaldehyde dehydrogenase type 3 is prevented by maternal retinoic acid treatment[END_REF][START_REF] Molotkova | Role of retinoic acid during forebrain development begins late when Raldh3 generates retinoic acid in the ventral subventricular zone[END_REF].

Another important enzyme for RA synthesis and for early embryogenesis is RDH10. Rdh10 expression is localized in the lateral plate mesoderm of the cardiac crescent and later in the venous pole of the heart tube [START_REF] Sandell | RDH10 is essential for synthesis of embryonic retinoic acid and is required for limb, craniofacial, and organ development[END_REF]. Using the RARE-hsp68-lacZ reporter transgene, it has been shown that RA activity in Rdh10 null embryos is almost completely eliminated at the critical E8.0-E8.5 stage of development [START_REF] Rhinn | Involvement of retinol dehydrogenase 10 in embryonic patterning and rescue of its loss of function by maternal retinaldehyde treatment[END_REF][START_REF] Sandell | RDH10 oxidation of Vitamin A is a critical control step in synthesis of retinoic acid during mouse embryogenesis[END_REF][START_REF] Sandell | RDH10 is essential for synthesis of embryonic retinoic acid and is required for limb, craniofacial, and organ development[END_REF]. RDH10 loss-of-function is lethal between E10.5 and E14.5 [START_REF] Cammas | Expression of the murine retinol dehydrogenase 10 (Rdh10) gene correlates with many sites of retinoid signalling during embryogenesis and organ differentiation[END_REF][START_REF] Romand | Dynamic expression of the retinoic acid-synthesizing enzyme retinol dehydrogenase 10 (rdh10) in the developing mouse brain and sensory organs[END_REF][START_REF] Sandell | RDH10 is essential for synthesis of embryonic retinoic acid and is required for limb, craniofacial, and organ development[END_REF]. Rdh10 mutant embryos exhibit abnormalities characteristic of RA deficiency. Some severally affected mutant (b 10%) fail to undergo normal looping and chamber formation, remaining, instead, simple tubes, which can be partly rescued by maternal RA supplementation [START_REF] Rhinn | Involvement of retinol dehydrogenase 10 in embryonic patterning and rescue of its loss of function by maternal retinaldehyde treatment[END_REF][START_REF] Sandell | RDH10 oxidation of Vitamin A is a critical control step in synthesis of retinoic acid during mouse embryogenesis[END_REF]. Rdh10 mutants obtained at E12.5-E14.5 have poor myocardial trabeculation. Zebrafish Rdh10a deficient embryos have enlarged hearts with increased cardiomyocyte number (D'Aniello et al., 2015).

The retinaldehyde reductase DHRS3 regulates retinoic acid biosynthesis through a feedback inhibition mechanism and the interaction between RDH10 and DHRS3. Dhrs3 mutant embryos die late in gestation and display defects in cardiac outflow tract formation, atrial and ventricular septation [START_REF] Adams | The retinaldehyde reductase activity of DHRS3 is reciprocally activated by retinol dehydrogenase 10 to control retinoid homeostasis[END_REF][START_REF] Billings | The retinaldehyde reductase DHRS3 is essential for preventing the formation of excess retinoic acid during embryonic development[END_REF][START_REF] Feng | Dhrs3a regulates retinoic acid biosynthesis through a feedback inhibition mechanism[END_REF].

The transport of vitamin A appears to be mediated by STRA6, a membrane bound protein that can interact with cellular retinol binding proteins (CRABPs and CRBP), which bind retinol in the serum (Fig. 2). Human mutations in STRA6 underlie Matthew-Wood syndrome, associated with multiple developmental defects including, occasionally, outflow tract, atrial and ventricular septal defects [START_REF] Golzio | Matthew-Wood syndrome is caused by truncating mutations in the retinol-binding protein receptor gene STRA6[END_REF][START_REF] Pasutto | Mutations in STRA6 cause a broad spectrum of malformations including anophthalmia, congenital heart defects, diaphragmatic hernia, alveolar capillary dysplasia, lung hypoplasia, and mental retardation[END_REF]. Surprisingly, deletion of Stra6 in the mouse has only a modest effect on the levels of RA signaling in most tissues, with the exception of the eye [START_REF] Amengual | STRA6 is critical for cellular vitamin A uptake and homeostasis[END_REF].

Cyp26A1 is a RA degrading enzyme that belongs to the p450 family (Fig. 2). Interestingly, Cyp26A1 expression is spatially restricted in the cardiac crescent and later at the poles of the E8.0 heart tube [START_REF] Maclean | Cloning of a novel retinoic-acid metabolizing cytochrome P450, Cyp26B1, and comparative expression analysis with Cyp26A1 during early murine development[END_REF][START_REF] Rydeen | Cyp26 enzymes are required to balance the cardiac and vascular lineages within the anterior lateral plate mesoderm[END_REF]. Loss of Cyp26 enzymes in zebrafish and mice results in severe phenotypes with embryonic lethality which include smaller atria, looping defects and outflow tract defects [START_REF] Abu-Abed | The retinoic acid-metabolizing enzyme, CYP26A1, is essential for normal hindbrain patterning, vertebral identity, and development of posterior structures[END_REF][START_REF] Emoto | Retinoic acid-metabolizing enzyme Cyp26a1 is essential for determining territories of hindbrain and spinal cord in zebrafish[END_REF][START_REF] Hernandez | Cyp26 enzymes generate the retinoic acid response pattern necessary for hindbrain development[END_REF]Niederreither et al., 2002a;[START_REF] Sakai | The retinoic acid-inactivating enzyme CYP26 is essential for establishing an uneven distribution of retinoic acid along the anterio-posterior axis within the mouse embryo[END_REF]. Although Cyp26c1 knock-out mice do not have significant defects, double Cyp26A1 and Cyp26C1 mutants have more severe looping defects [START_REF] Uehara | CYP26A1 and CYP26C1 cooperatively regulate anterior-posterior patterning of the developing brain and the production of migratory cranial neural crest cells in the mouse[END_REF]. Loss of CYP26 enzymes in humans is associated with numerous developmental syndromes [START_REF] Rydeen | Cyp26 enzymes are required to balance the cardiac and vascular lineages within the anterior lateral plate mesoderm[END_REF]. Inhibition of CYP26A1 is associated with DiGeorge syndrome-like phenotypes that causes heart defects such as conotruncal malformations (interrupted aortic arch, persistent truncus arteriosus, tetralogy of Fallot, and ventricular septal defects [START_REF] Roberts | Cyp26 genes a1, b1 and c1 are down-regulated in Tbx1 null mice and inhibition of Cyp26 enzyme function produces a phenocopy of DiGeorge Syndrome in the chick[END_REF]). The role for CRABPs and CRBP is less clear. However, studies using knock-out mouse suggest that these proteins appear not to be essential for heart development [START_REF] Lampron | Mice deficient in cellular retinoic acid binding protein II (CRABPII) or in both CRABPI and CRABPII are essentially normal[END_REF].

RA regulates development by acting as a diffusible signaling molecule that controls the activity of retinoic acid receptors (RARs). A total of six receptors (RARα, -β, -γ, RXRα, -β, and -γ) transduce the activities of RA [START_REF] Metzger | Contribution of targeted conditional somatic mutagenesis to deciphering retinoid X receptor functions and to generating mouse models of human diseases[END_REF]. Unlike the specific and restricted pattern of Raldh2 or the spatial and temporal availability of RA during development, RARα, RXRα, and RXRβ are ubiquitously expressed in embryonic and adult tissues, whereas RARβ, RARγ, and RXRγ expression is more restricted [START_REF] Dolle | Developmental expression of murine retinoid X receptor (RXR) genes[END_REF][START_REF] Dolle | Retinoic acid receptors and cellular retinoid binding proteins. I. A systematic study of their differential pattern of transcription during mouse organogenesis[END_REF][START_REF] Ruberte | Retinoic acid receptors and cellular retinoid binding proteins. II. Their differential pattern of transcription during early morphogenesis in mouse embryos[END_REF][START_REF] Ruberte | Specific spatial and temporal distribution of retinoic acid receptor gamma transcripts during mouse embryogenesis[END_REF]. Studies with knockout strategies for RARs and RXRs in mutant mice, have demonstrated their crucial role in many developmental processes [START_REF] Kastner | Genetic analysis of RXR alpha developmental function: convergence of RXR and RAR signaling pathways in heart and eye morphogenesis[END_REF][START_REF] Li | Normal development and growth of mice carrying a targeted disruption of the alpha 1 retinoic acid receptor gene[END_REF][START_REF] Lohnes | Function of retinoic acid receptor gamma in the mouse[END_REF][START_REF] Lufkin | High postnatal lethality and testis degeneration in retinoic acid receptor alpha mutant mice[END_REF][START_REF] Mendelsohn | Function of the retinoic acid receptors (RARs) during development (II). Multiple abnormalities at various stages of organogenesis in RAR double mutants[END_REF][START_REF] Sucov | RXR alpha mutant mice establish a genetic basis for vitamin A signaling in heart morphogenesis[END_REF]. As in vitamin A deficient syndrome, fetal or postnatal damages were found in RAR or RXR single-mutant mice, but the defects are less severe, suggesting functional redundancy among these receptors. Whereas RXRα-null mice exhibit embryonic lethality, functional redundancy between the RAR and other RXR isotypes has been demonstrated [START_REF] Mendelsohn | Function of the retinoic acid receptors (RARs) during development (II). Multiple abnormalities at various stages of organogenesis in RAR double mutants[END_REF]. Mutation of either Raldh2 or RXRα results in similar phenotypes characterized by profound embryonic lethality with prominent myocardial defects suggesting a role of RXRα in myocardial growth [START_REF] Dyson | Atrial-like phenotype is associated with embryonic ventricular failure in retinoid X receptor alpha-/-mice[END_REF][START_REF] Gruber | RXR alpha deficiency confers genetic susceptibility for aortic sac, conotruncal, atrioventricular cushion, and ventricular muscle defects in mice[END_REF][START_REF] Kastner | Genetic analysis of RXR alpha developmental function: convergence of RXR and RAR signaling pathways in heart and eye morphogenesis[END_REF][START_REF] Li | Normal development and growth of mice carrying a targeted disruption of the alpha 1 retinoic acid receptor gene[END_REF][START_REF] Sucov | RXR alpha mutant mice establish a genetic basis for vitamin A signaling in heart morphogenesis[END_REF]. On the other hand, other defects in double RXR-RAR mutants are not observed in Raldh2 mutants. RA signaling through RARα1/RXRα regulates differentiation of second heart field cells and outflow tract formation [START_REF] Li | Retinoic acid regulates differentiation of the secondary heart field and TGFbeta-mediated outflow tract septation[END_REF].

Mechanisms of transcriptional regulation

RA has been characterized as a diffusible morphogen that acts directly on cells in a concentration-dependent manner to assign positional identities [START_REF] Briscoe | Morphogen rules: design principles of gradient-mediated embryo patterning[END_REF]. RA has a non-cell-autonomous (paracrine) effect on neighboring cells but there is also evidence for it acts in an intracrine manner in cells that synthesize it [START_REF] Azambuja | Retinoic acid and VEGF delay smooth muscle relative to endothelial differentiation to coordinate inner and outer coronary vessel wall morphogenesis[END_REF]. RA signaling is dependent on cells that have the ability to metabolize retinol to RA. RA can form gradients capable of inducing sharp boundaries of target gene expression. The underlying mechanisms include activities of RA-degrading enzymes [START_REF] White | How degrading: Cyp26s in hindbrain development[END_REF]. Several enzymatic activities such as RDHs and CYP26s are required in addition to RALDHs to control RA distribution within the embryo. In zebrafish, it has been demonstrated that RA degradation by CYP26 enzymes progressively determines the limits of RA-dependent gene expression [START_REF] Hernandez | Cyp26 enzymes generate the retinoic acid response pattern necessary for hindbrain development[END_REF]. CYP26s enzymes would thus function to establish boundaries in RA responsiveness. RA gradients induce sharply defined domains of gene expression also through tight feedback regulation of RA synthesis and interactions with other localized morphogens [START_REF] Schilling | Dynamics and precision in retinoic acid morphogen gradients[END_REF][START_REF] Shimozono | Visualization of an endogenous retinoic acid gradient across embryonic development[END_REF]. This has been explored mainly in the context of brain development.

The basic mechanism for transcriptional regulation by RARs relies on DNA binding to specific sequence elements, the RA response elements (RAREs). RARs and RXRs are highly conserved among mammals. Unlike RARs, RXRs are not specific to the retinoic pathway, and can be involved in other signaling by binding vitamin D receptors, liver X receptors, thyroid receptors, and peroxisome proliferator-activated receptors. RXR can act as either a homodimer or heterodimer with RARs. In the latter case, regulation of gene transcription is achieved by the binding of the heterodimer RAR/RXR to a specific sequence composed classically of two direct repeats of a hexameric motif. In the classical view, functional RAREs near genes that require RA for normal expression during development typically consist of hexameric direct repeats (DRs) (A/G)G(T/ G) TCA with interspacing of 5 bp (DR5 elements) or 2 bp (DR2 elements), unlike vitamin D and thyroid hormone response elements, which typically exhibit DR3 and DR4 configurations, respectively. Even if spacing is required, the specificity of RAR and RXR binding seems also regulated by interactions of other transcription factors and the epigenetic landscape around the RARE. The cell specificity of the response to RA signaling is probably due to interactions with different regulatory proteins. Recently a two-hybrid assay in yeast demonstrated that RXRα interacts with the cardiac transcription factor Nkx2.5 [START_REF] Waardenberg | Prediction and validation of protein-protein interactors from genome-wide DNA-binding data using a knowledge-based machine-learning approach[END_REF]. In humans, mutations of NKX2-5 result in congenital heart defects such as atrial septal defects and conduction block [START_REF] Prendiville | Insights into the genetic structure of congenital heart disease from human and murine studies on monogenic disorders[END_REF]. Mutations of Nkx2.5 alters this interaction suggesting that defects seen in patients carrying Nkx2.5 mutations may in part due to disrupted protein partner interaction between Nkx2.5 and RXRα. Local chromatin environment, nearest neighboring factor binding motifs are likely important parameters underlying the RARE recognition code. Identifying RARs and RXRs co-factors has the potential to shed light on the complex gene regulatory processes underlying normal development and is likely critical for better differentiation protocols used to drive stem cells into specific cardiac cell types.

The use of chromatin immunoprecipitation (ChIP), with antibodies against RARs has demonstrated a greater diversity of RAREs than previously appreciated [START_REF] Boergesen | Genome-wide profiling of liver X receptor, retinoid X receptor, and peroxisome proliferator-activated receptor alpha in mouse liver reveals extensive sharing of binding sites[END_REF][START_REF] Chatagnon | RAR/RXR binding dynamics distinguish pluripotency from differentiation associated cis-regulatory elements[END_REF][START_REF] He | The role of retinoic acid in hepatic lipid homeostasis defined by genomic binding and transcriptome profiling[END_REF][START_REF] Lalevee | Genome-wide in silico identification of new conserved and functional retinoic acid receptor response elements (direct repeats separated by 5 bp)[END_REF][START_REF] Mendoza-Parra | Dissecting the retinoid-induced differentiation of F9 embryonal stem cells by integrative genomics[END_REF][START_REF] Moutier | Retinoic acid receptors recognize the mouse genome through binding elements with diverse spacing and topology[END_REF]. Other hexameric repeat configurations have been found to bind to RARs in cell line studies involving ChIP-seq, but there in vivo importance is unknown. A recent ChIP study coupled to sequencing and performed in ES cells suggested that the presence of RA might also induce de novo RAR/RXR binding to numerous RAREs that are not bound by unliganded receptors [START_REF] Mahony | Ligand-dependent dynamics of retinoic acid receptor binding during early neurogenesis[END_REF]. There is also evidence that inverted repeats with no spacer can also be targets for RARs. RAR ChIP studies and in silico analyses have discovered 13,000-15,000 potential RAREs. Many of these RAREs have not been attributed to endogenous RA signaling and seem to be off-targets due to treatment with high amounts of RA or RAR antagonists. RXR ChIP-seq analyses also revealed that a large fraction of genomic regions occupied by RXR are not associated with a recognizable DR binding site, indicating indirect binding via DNA looping and interaction with co-factors [START_REF] Delacroix | Cell-specific interaction of retinoic acid receptors with target genes in mouse embryonic fibroblasts and embryonic stem cells[END_REF]. Consistent with this, in vitro reporter assays suggest that the transcriptional activities of RARs and RXRs do not necessarily require direct DNA binding [START_REF] Clabby | Retinoid X receptor alpha represses GATA-4-mediated transcription via a retinoid-dependent interaction with the cardiac-enriched repressor FOG-2[END_REF][START_REF] Molkentin | Transcription factor GATA-4 regulates cardiac muscle-specific expression of the alpha-myosin heavy-chain gene[END_REF]. Since ChIP assesses protein-DNA proximity by cross linking, and not direct binding, it will be necessary to verify RAR and RXR binding using in vivo foot printing. Importantly, the presence of an RAR or RXR does not conclusively show that RA will bind to the receptor and regulate gene expression in an RA-dependent manner.

RA acts as a ligand for RAR and RXR nuclear receptors, switching them from potential repressors to transcriptional activators. Whether a change of RA gradient concentrations guide this function is unknown. Since the spatial organization of the nucleus may impact on activation or repression of gene expression and interaction with co-factors, assessment of the localization of RA receptors within the nucleus might be relevant. When RA is absent, RAR/RXR heterodimers bind RAREs where they recruit repressive complexes that inhibit transcription. In the presence of RA, however, the repressive complex bound to the receptor is exchanged for an activating complex and transcription at the target site is activated. As the receptors are already present on many target genes, this makes RA the limiting factor in deciding whether or not target genes are activated. The main determinant that drives RA signaling is RA availability rather than nuclear-receptor abundance, which is likely to be secondary. Mechanisms underlying the function of governing the decision of whether RARs and RXRs function as activators or active repressors of a targets gene have been studied in depth using in vitro systems and in the context of the several developmental processes [START_REF] Gillespie | Retinoid regulated association of transcriptional co-regulators and the polycomb group protein SUZ12 with the retinoic acid response elements of Hoxa1, RARbeta(2), and Cyp26A1 in F9 embryonal carcinoma cells[END_REF][START_REF] Janesick | Active repression by RARgamma signaling is required for vertebrate axial elongation[END_REF][START_REF] Kashyap | Epigenetic regulatory mechanisms distinguish retinoic acidmediated transcriptional responses in stem cells and fibroblasts[END_REF][START_REF] Kumar | Retinoic acid controls body axis extension by directly repressing Fgf8 transcription[END_REF][START_REF] Nagy | Nuclear receptor repression mediated by a complex containing SMRT, mSin3A, and histone deacetylase[END_REF]. HDAC inhibitors increase RA sensitivity by promoting dissociation of repressive complexes from RAR [START_REF] Lee | High histone acetylation and decreased polycomb repressive complex 2 member levels regulate gene specific transcriptional changes during early embryonic stem cell differentiation induced by retinoic acid[END_REF]. Indeed, RARs associate with histones acetylases (HATs) and histone deacetylases (HDACs) to modulate gene activity and dictate cell fate [START_REF] Weston | Active repression by unliganded retinoid receptors in development: less is sometimes more[END_REF]. In the repressive unliganded state, the RAR-RXR heterodimer recruits co-repressors such as histone deacetylase (HDAC) protein complexes and Polycomb repressive complex 2 (PRC2). This results in histone H3 lysine 27 trimethylation, chromatin condensation and gene silencing. RA binding to RAR-RXR induces a conformational change in the heterodimer, which promotes the replacement of repressive factors by co-activators such as histone acetylase (HAT) complexes and Trithorax proteins, which mediate H3K4me3, chromatin relaxation and gene activation. These epigenetic factors thus act as mediators or partners in the action of cardiac RARs and RXRs on chromatin structure.

In summary, in the presence of RA, RARs bind RA response elements (RAREs) and recruit HATs. In the absence of RA, RARs can actively repress gene transcription by recruiting HDACs that promote chromatin compaction and gene repression. Surprisingly, there are exceptions to this classical model: during neurogenesis RARE sequences upstream of Fgf8 and Hoxb1 mediate gene repression, rather than activation, because RA binding to RAR leads to the recruitment of PRC2 and HDACs, and triggers H3K27me3 [START_REF] Boudadi | The histone deacetylase inhibitor sodium valproate causes limited transcriptional change in mouse embryonic stem cells but selectively overrides Polycomb-mediated Hoxb silencing[END_REF][START_REF] Kumar | Retinoic acid controls body axis extension by directly repressing Fgf8 transcription[END_REF][START_REF] Studer | Role of a conserved retinoic acid response element in rhombomere restriction of Hoxb-1[END_REF].

Transcriptional activities of retinoic receptors in mammalian heart development

Activities during early cardiogenesis

Several members of the Hox gene family, including Hoxa1 and Hoxb1, are regulated by RAREs, which has been demonstrated in vitro and in vivo [START_REF] Dupe | In vivo functional analysis of the Hoxa-1 3′ retinoic acid response element (3′RARE)[END_REF][START_REF] Huang | A conserved retinoic acid responsive element in the murine Hoxb-1 gene is required for expression in the developing gut[END_REF][START_REF] Langston | Retinoic acid-responsive enhancers located 3′ of the Hox A and Hox B homeobox gene clusters. Functional analysis[END_REF][START_REF] Marshall | A conserved retinoic acid response element required for early expression of the homeobox gene Hoxb-1[END_REF][START_REF] Oosterveen | The direct context of a hox retinoic acid response element is crucial for its activity[END_REF]. The transcription factor homeobox gene Hoxa1 (LaRosa and [START_REF] Larosa | Early retinoic acid-induced F9 teratocarcinoma stem cell gene ERA-1: alternate splicing creates transcripts for a homeobox-containing protein and one lacking the homeobox[END_REF], is a direct target of RA and possesses an enhancer containing a RARE. Consistent with such regulation, reduction or increase of RA signaling causes defects in the contribution of Hoxa1-; Hoxa3-and Hoxb1-expressing progenitor cells to the heart [START_REF] Bertrand | Hox genes define distinct progenitor sub-domains within the second heart field[END_REF]. Our study has demonstrated that RA is required to activate Hoxa1 expression in the posterior second heart field, a subpopulation of cardiac progenitor cells that will later give rise to atrial and sub-pulmonary myocardium [START_REF] Bertrand | Hox genes define distinct progenitor sub-domains within the second heart field[END_REF][START_REF] Ryckebusch | Retinoic acid deficiency alters second heart field formation[END_REF]. Reduction or excess of RA signaling causes abnormalities in the cardiac contribution of Hoxa1 and Hoxb1 expressing progenitors [START_REF] Bertrand | Hox genes define distinct progenitor sub-domains within the second heart field[END_REF]. Enhancers for Hoxa3 and Hoxb1 genes driving expression in cardiac progenitors have been identified [START_REF] Diman | A retinoic acid responsive Hoxa3 transgene expressed in embryonic pharyngeal endoderm, cardiac neural crest and a subdomain of the second heart field[END_REF][START_REF] Nolte | Shadow enhancers flanking the HoxB cluster direct dynamic Hox expression in early heart and endoderm development[END_REF]. It has been reported that enhancers for Hoxb1 gene mediate reporter expression in the second heart field and the proepicardium. These cardiac enhancers have RAREs and may be the direct targets of RA signaling. RA signaling is maintained by an autoregulatory mechanism via Hox genes. Indeed, in the context of brain development, Raldh2 expression is under the direct transcriptional control of HOX, PBX and MEIS complex [START_REF] Vitobello | Hox and Pbx factors control retinoic acid synthesis during hindbrain segmentation[END_REF]. HOXA1-PBX1/2-MEIS2 binds a regulatory element required to maintain normal Raldh2 expression [START_REF] Vitobello | Hox and Pbx factors control retinoic acid synthesis during hindbrain segmentation[END_REF]. The expression profile of Pbx and Meis factors overlaps Hox genes in the second heart field [START_REF] Chang | Pbx1 functions in distinct regulatory networks to pattern the great arteries and cardiac outflow tract[END_REF][START_REF] Stankunas | Pbx/Meis deficiencies demonstrate multigenetic origins of congenital heart disease[END_REF][START_REF] Wamstad | Dynamic and coordinated epigenetic regulation of developmental transitions in the cardiac lineage[END_REF]. Mice deficient for Pbx1, Meis1 and Hox genes have similar cardiac phenotypes [START_REF] Makki | Cardiovascular defects in a mouse model of HOXA1 syndrome[END_REF][START_REF] Paige | A temporal chromatin signature in human embryonic stem cells identifies regulators of cardiac development[END_REF]Stankunas et al., 2008). Together it suggests that PBX/MEIS and HOX proteins may cooperatively regulate Raldh2 gene expression in cardiac progenitors. RA signaling activates another marker of cardiac progenitor cells, the Tbox transcription factor Tbx5 [START_REF] Liberatore | Ventricular expression of tbx5 inhibits normal heart chamber development[END_REF][START_REF] Niederreither | Embryonic retinoic acid synthesis is essential for heart morphogenesis in the mouse[END_REF][START_REF] Sirbu | Retinoic acid controls heart anteroposterior patterning by down-regulating Isl1 through the Fgf8 pathway[END_REF]. Tbx5 is expressed in the posterior domain of the second heart field as well as the first heart field and is required to activate chamber-specific genes, such as atrial natriuretic factor (Nppa) and atrial natriuretic factor (Nppb) [START_REF] Bruneau | A murine model of Holt-Oram syndrome defines roles of the T-box transcription factor Tbx5 in cardiogenesis and disease[END_REF][START_REF] Mori | Tbx5dependent rheostatic control of cardiac gene expression and morphogenesis[END_REF]. RA signaling specifies Tbx5 expressing cells, progenitors of the first heart field to a venous and atrial cell fate [START_REF] Xavier-Neto | A retinoic acid-inducible transgenic marker of sino-atrial development in the mouse heart[END_REF].

DNA elements conferring tissue-type specific gene expression are ideal to analyse the molecular mechanisms that underlie the localized cardiac gene expression. For example, in the context of somite development a site-directed mutagenesis study demonstrated that a RARE upstream of Fgf8 is required for RA repression of Fgf8 in transgenic mouse embryos, thereby showing that RA directly represses Fgf8 transcription in vivo [START_REF] Kumar | Retinoic acid controls body axis extension by directly repressing Fgf8 transcription[END_REF]. Studies of Raldh2 null embryos showed that RA restricts the size of the second heart field by repressing Fgf8 expression in the second heart field [START_REF] Ryckebusch | Retinoic acid deficiency alters second heart field formation[END_REF][START_REF] Sirbu | Retinoic acid controls heart anteroposterior patterning by down-regulating Isl1 through the Fgf8 pathway[END_REF], and zebrafish heart development also requires FGF8 repression by RA [START_REF] Sorrell | Restraint of Fgf8 signaling by retinoic acid signaling is required for proper heart and forelimb formation[END_REF]; however, the of use CRISPR/Cas9-mediated genomic deletion of the Fgf8 RARE showed no defect in heart development or cardiac Fgf8 expression [START_REF] Kumar | Nuclear receptor corepressors Ncor1 and Ncor2 (Smrt) are required for retinoic acid-dependent repression of Fgf8 during somitogenesis[END_REF].

Several RXR/RAR target genes have been identified, including genes within the retinoid pathway, such as the cardiac expressed genes Rarα [START_REF] Dolle | Retinoic acid receptors and cellular retinoid binding proteins. I. A systematic study of their differential pattern of transcription during mouse organogenesis[END_REF][START_REF] Leroy | Mouse retinoic acid receptor alpha 2 isoform is transcribed from a promoter that contains a retinoic acid response element[END_REF][START_REF] Ruberte | Retinoic acid receptors and cellular retinoid binding proteins. II. Their differential pattern of transcription during early morphogenesis in mouse embryos[END_REF]), Rarβ2 (de The et al., 1990), Cyp26a1 [START_REF] Loudig | Transcriptional cooperativity between distant retinoic acid response elements in regulation of Cyp26A1 inducibility[END_REF][START_REF] Maclean | Cloning of a novel retinoic-acid metabolizing cytochrome P450, Cyp26B1, and comparative expression analysis with Cyp26A1 during early murine development[END_REF] Crbp1 [START_REF] Smith | A retinoic acid response element is present in the mouse cellular retinol binding protein I (mCRBPI) promoter[END_REF] and Crabp2 [START_REF] Durand | All-trans and 9-cis retinoic acid induction of CRABPII transcription is mediated by RAR-RXR heterodimers bound to DR1 and DR2 repeated motifs[END_REF]. RA represses the expression of the major embryonic production enzymes RDH10 and RALDH2 [START_REF] D'aniello | Depletion of retinoic acid receptors initiates a novel positive feedback mechanism that promotes teratogenic increases in retinoic acid[END_REF][START_REF] Niederreither | Restricted expression and retinoic acid-induced downregulation of the retinaldehyde dehydrogenase type 2 (RALDH-2) gene during mouse development[END_REF][START_REF] Strate | Retinol dehydrogenase 10 is a feedback regulator of retinoic acid signalling during axis formation and patterning of the central nervous system[END_REF]. Whether this feedback mechanism implies a direct transcriptional mechanism is currently unknown. Ectopic RA signaling affects outflow tract cushion development through the direct repression of a functional RARE in the promoter region of the myocardial Tbx2 gene [START_REF] Sakabe | Ectopic retinoic acid signaling affects outflow tract cushion development through suppression of the myocardial Tbx2-Tgfbeta2 pathway[END_REF]. The chicken slow MyHC3 promoter (slow myosin heavy chain 3) directs transgene expression in the cardiac venous pole at E8.5 and in the atrium at E9.5 with a persistent expression at later stages. This 168 bp regulatory element contains a RARE, suggesting that atrial specific gene expression is controlled directly by the localized synthesis of RA. An inhibitory protein complex composed of RXRα and IRX4 that binds this RARE to inhibit slow MyHC3 expression in primary cultures of embryonic atrial and ventricular quail cardiomyocytes [START_REF] Wang | Irx4 forms an inhibitory complex with the vitamin D and retinoic X receptors to regulate cardiac chamber-specific slow MyHC3 expression[END_REF]. GATA factors also bind the slow MyHC3 regulatory element in vitro, suggesting a cooperative effect between RAR/RXR and GATA factors [START_REF] Wang | A positive GATA element and a negative vitamin D receptor-like element control atrial chamber-specific expression of a slow myosin heavy-chain gene during cardiac morphogenesis[END_REF]. Similarly, RA receptors regulate the chamber-specific genes Nppa (Anf, atrial natriuretic factor) and Nppb (Bnf, brain natriuretic factor) via direct interaction with Gata4 and its co-repressor, Fog2 [START_REF] Clabby | Retinoid X receptor alpha represses GATA-4-mediated transcription via a retinoid-dependent interaction with the cardiac-enriched repressor FOG-2[END_REF][START_REF] Wu | 1,25(OH)2 vitamin D3, and retinoic acid antagonize endothelin-stimulated hypertrophy of neonatal rat cardiac myocytes[END_REF]. Fog factors facilitate the chromatin occupancy of Gata factors and interact with the repressive nucleosome remodeling and deacetylase (NuRD) complex [START_REF] Chlon | Combinatorial regulation of tissue specification by GATA and FOG factors[END_REF][START_REF] Stefanovic | GATA-dependent transcriptional and epigenetic control of cardiac lineage specification and differentiation[END_REF][START_REF] Vakoc | Proximity among distant regulatory elements at the beta-globin locus requires GATA-1 and FOG-1[END_REF]. Thus, RARE elements could act as a platform to recruit these cardiac co-factors and drive chamber specific gene programs [START_REF] Prendiville | Insights into the genetic structure of congenital heart disease from human and murine studies on monogenic disorders[END_REF].

RA activities and cardiac stem cells biology

Unlike many other adult tissues, the myocardium of mammals has a limited ability to compensate for the loss of cells after cardiac damage. The ability of RA to stimulate cellular differentiation has been exploited in regenerative medicine differentiation of atrial and ventricular myocytes from human embryonic stem cells [START_REF] Devalla | Atrial-like cardiomyocytes from human pluripotent stem cells are a robust preclinical model for assessing atrial-selective pharmacology[END_REF][START_REF] Gassanov | Retinoid acid-induced effects on atrial and pacemaker cell differentiation and expression of cardiac ion channels[END_REF][START_REF] Wobus | Retinoic acid accelerates embryonic stem cellderived cardiac differentiation and enhances development of ventricular cardiomyocytes[END_REF][START_REF] Zhang | Direct differentiation of atrial and ventricular myocytes from human embryonic stem cells by alternating retinoid signals[END_REF]. Treatment of differentiating embryonic stem cells with RA promotes atrial specification [START_REF] Devalla | Atrial-like cardiomyocytes from human pluripotent stem cells are a robust preclinical model for assessing atrial-selective pharmacology[END_REF]. Several studies have indicated the involvement of COUP-TFs in RA signaling [START_REF] Jonk | Isolation and developmental expression of retinoic-acid-induced genes[END_REF][START_REF] Van Der Wees | Developmental expression and differential regulation by retinoic acid of Xenopus COUP-TF-A and COUP-TF-B[END_REF]. Atrial identity is determined by a COUP-TFII regulatory network [START_REF] Pereira | The orphan nuclear receptor COUP-TFII is required for angiogenesis and heart development[END_REF][START_REF] Wu | Atrial identity is determined by a COUP-TfiI regulatory network[END_REF]. Furthermore COUP-TFI and COUP-TFII are upregulated in differentiated cardiomyocytes in response to RA [START_REF] Devalla | Atrial-like cardiomyocytes from human pluripotent stem cells are a robust preclinical model for assessing atrial-selective pharmacology[END_REF], indicating that a RA-COUP-TF network module participates in the normal control of atrial specification. COUP-TFII is also present in a complex with the HAT p300 and RXR/RAR at the RAREs and enhances RA's actions on target genes during development [START_REF] Vilhais-Neto | Rere controls retinoic acid signalling and somite bilateral symmetry[END_REF]. COUP-TFI and COUP-TFII receptors bind DR elements used by RARs [START_REF] Kliewer | Retinoid X receptor-COUP-TF interactions modulate retinoic acid signaling[END_REF]. Further studies will reveal whether COUP-TfiI is also a RXR/RAR co-factor or a direct target of RA signaling during cardiogenesis. In cell culture, RA can also promote epicardial lineage specification. The combined action of RA, BMP and WNT signaling is required in specifying an epicardium-like lineage from human embryonic stem cells under chemically defined conditions [START_REF] Iyer | Robust derivation of epicardium and its differentiated smooth muscle cell progeny from human pluripotent stem cells[END_REF]. Varying concentration of RA may be responsible for generating in vitro these different cardiac subtypes of cells. Cardiac specific differentiation is also certainly influenced by the use of temporal and combined morphogens.

During embryonic stem cell differentiation RA treatment does not affect Hcn4 expression [START_REF] Wobus | Retinoic acid accelerates embryonic stem cellderived cardiac differentiation and enhances development of ventricular cardiomyocytes[END_REF], a major gene in the cardiac conduction tissue [START_REF] Liang | Insights into cardiac conduction system formation provided by HCN4 expression[END_REF], indicating that RA signaling may not be implicated in the differentiation of pacemaker-like cells. In line with this, there is currently interest in reprogramming cells to pacemaker cells by transducing transcription factor genes [START_REF] Boink | The past, present, and future of pacemaker therapies[END_REF]. Through such a reprogramming attempt, it has been shown that the use of RARγ or RXRα together with the cardiac transcription factors Gata6 and Tbx3 does not generate cells with spontaneous beating activity, a key feature of pacemaker cells [START_REF] Nam | Induction of diverse cardiac cell types by reprogramming fibroblasts with cardiac transcription factors[END_REF]. It also remains to be determined whether manipulating the RARs-RXRs-HAT/RARs-RXRs-HDAC complexes could target many silent cardiac-specific sites, open the chromatin for active transcription and enhance reprogramming toward human atrial cells.

Activities during late stages of cardiogenesis

RA is also involved in processes taking place during late cardiac development (reviewed in Xavier-Neto et al., (2015)). RA signaling acts on neural crest cells orientation and positioning, myocardial specification, and the endothelial-to-mesenchymal transition of endocardial cells process to allow proper endocardial cushion fusion and complete outflow tract septation [START_REF] El Robrini | Cardiac outflow morphogenesis depends on effects of retinoic acid signaling on multiple cell lineages[END_REF][START_REF] Niederreither | Embryonic retinoic acid synthesis is essential for heart morphogenesis in the mouse[END_REF]. RA signaling is involved in the formation of the epicardium [START_REF] Braitsch | Pod1/Tcf21 is regulated by retinoic acid signaling and inhibits differentiation of epicardium-derived cells into smooth muscle in the developing heart[END_REF][START_REF] Moss | Dynamic patterns of retinoic acid synthesis and response in the developing mammalian heart[END_REF][START_REF] Von Gise | WT1 regulates epicardial epithelial to mesenchymal transition through beta-catenin and retinoic acid signaling pathways[END_REF]. Indeed both Raldh2 and RARE-hsp68-lacZ transgene are expressed in the epicardium from stage E11.5 [START_REF] Moss | Dynamic patterns of retinoic acid synthesis and response in the developing mammalian heart[END_REF][START_REF] Xavier-Neto | Sequential programs of retinoic acid synthesis in the myocardial and epicardial layers of the developing avian heart[END_REF]. The heart phenotypes of Raldh2 and RXRα deficient embryos are very similar and are characterized by a severe hypoplasia of the ventricular myocardium, a phenotype mimicking other mutants with defective epicardial function [START_REF] Brade | Retinoic acid stimulates myocardial expansion by induction of hepatic erythropoietin which activates epicardial Igf2[END_REF][START_REF] Merki | Epicardial retinoid X receptor alpha is required for myocardial growth and coronary artery formation[END_REF]. Raldh2 is a direct target of Wt1 in epicardial cells [START_REF] Guadix | Wt1 controls retinoic acid signalling in embryonic epicardium through transcriptional activation of Raldh2[END_REF]. Wt1 regulates epicardial epithelial to mesenchymal transition through β-catenin and RA signaling pathways [START_REF] Von Gise | WT1 regulates epicardial epithelial to mesenchymal transition through beta-catenin and retinoic acid signaling pathways[END_REF]. The transcription factor Tcf21 is regulated by RA signaling and inhibits differentiation of epicardium-derived cells into smooth muscle in the developing heart [START_REF] Braitsch | Pod1/Tcf21 is regulated by retinoic acid signaling and inhibits differentiation of epicardium-derived cells into smooth muscle in the developing heart[END_REF]. It was found that epicardium-derived cells that maintain the expression of Wt1 and Raldh2 initially populate the subepicardial space and subsequently invade the ventricular myocardium. As epicardiumderived cells differentiate into the smooth muscle and endothelial cell lineage of the coronary vessels, the expression of Wt1 and Raldh2 becomes downregulated [START_REF] Perez-Pomares | Experimental studies on the spatiotemporal expression of WT1 and RALDH2 in the embryonic avian heart: a model for the regulation of myocardial and valvuloseptal development by epicardially derived cells (EPDCs)[END_REF]. RA stimulates myocardial expansion by induction of hepatic erythropoietin which activates epicardial Igf2 [START_REF] Brade | Retinoic acid stimulates myocardial expansion by induction of hepatic erythropoietin which activates epicardial Igf2[END_REF]. Erythropoietin and RA, secreted from the epicardium, are required for cardiac myocyte proliferation [START_REF] Stuckmann | Erythropoietin and retinoic acid, secreted from the epicardium, are required for cardiac myocyte proliferation[END_REF]. The RA pathway regulates myocardial growth signals such as Pi3k/ERK, Fgf2-9 [START_REF] Kang | Convergent proliferative response and divergent morphogenic pathways induced by epicardial and endocardial signaling in fetal heart development[END_REF][START_REF] Lin | Endogenous retinoic acid regulates cardiac progenitor differentiation[END_REF][START_REF] Merki | Epicardial retinoid X receptor alpha is required for myocardial growth and coronary artery formation[END_REF] and Wnts [START_REF] Merki | Epicardial retinoid X receptor alpha is required for myocardial growth and coronary artery formation[END_REF]. RA and VEGF delay smooth muscle relative to endothelial differentiation to coordinate inner and outer coronary vessel wall morphogenesis [START_REF] Azambuja | Retinoic acid and VEGF delay smooth muscle relative to endothelial differentiation to coordinate inner and outer coronary vessel wall morphogenesis[END_REF]. RA deficiency reduces expression of Sonic Hedgehog targets and the factors required in the coronary vasculature [START_REF] Lavine | Endocardial and epicardial derived FGF signals regulate myocardial proliferation and differentiation in vivo[END_REF]. Wt1 and RA signaling in the subcoelomic mesenchyme control the development of the pleuropericardial membranes and the sinus horns [START_REF] Norden | Wt1 and retinoic acid signaling in the subcoelomic mesenchyme control the development of the pleuropericardial membranes and the sinus horns[END_REF]. RA signaling is activated in the postischemic heart suggesting that it may play a role in regulation of damage and repair during remodeling. [START_REF] Bilbija | Retinoic acid signalling is activated in the postischemic heart and may influence remodelling[END_REF], Tables 1 and2).

Future directions

Although there has been progress in characterizing the function of RA signaling, many gaps remain with respect to the underlying mechanisms of RA-mediated gene regulation. Interpretation of experimental data are complicated by the fact that exposure to RA (in cultured cells, whole embryos or explants) may have different, sometimes opposite, effects depending on the concentration, stage or duration of exposure. Strategies that interfere with endogenous retinoid signaling through genetic loss-of-function appear more reliable than approaches using exogenous retinoids, including RAR/RXR antagonists that may lead to the forced repression of target gene loci. Given the ability of RA to signal across cells, understanding the site of action of RA receptors remains difficult. Recent studies have led to novel insights into the interplay between retinoid and other transcription factors in several developing systems. Our knowledge of the relationship between RA signaling and other signaling pathways also remains rudimentary. In the context of heart development, our understanding of the transcriptional targets of RA signaling is also limited. As previously mentioned several cardiac genes have been identified as regulatory targets of RA. In a few cases this regulation is direct, driven by a heterodimer of retinoid receptors bound to a DNA response element; in others, it has either not been investigated in depth or it is indirect, reflecting the actions of intermediate factors. However, our understanding of the role of retinoids will be enhanced if such a distinction can be made for each regulated target gene. Treated epicardium-derived cells Activation [START_REF] Braitsch | Pod1/Tcf21 is regulated by retinoic acid signaling and inhibits differentiation of epicardium-derived cells into smooth muscle in the developing heart[END_REF] Table 2 Some known co-factors associated proteins that regulates RAR/RXR function.

Coactivator proteins Function References

NcoA-1 Histone acetylation [START_REF] Kashyap | Epigenetic regulatory mechanisms distinguish retinoic acidmediated transcriptional responses in stem cells and fibroblasts[END_REF][START_REF] Kumar | Nuclear receptor corepressors Ncor1 and Ncor2 (Smrt) are required for retinoic acid-dependent repression of Fgf8 during somitogenesis[END_REF] HAT p300 Histone acetylation [START_REF] Gillespie | Retinoid regulated association of transcriptional co-regulators and the polycomb group protein SUZ12 with the retinoic acid response elements of Hoxa1, RARbeta(2), and Cyp26A1 in F9 embryonal carcinoma cells[END_REF][START_REF] Kashyap | Epigenetic regulatory mechanisms distinguish retinoic acidmediated transcriptional responses in stem cells and fibroblasts[END_REF][START_REF] Vilhais-Neto | Rere controls retinoic acid signalling and somite bilateral symmetry[END_REF]

FOG2

Transcription factor [START_REF] Clabby | Retinoid X receptor alpha represses GATA-4-mediated transcription via a retinoid-dependent interaction with the cardiac-enriched repressor FOG-2[END_REF] Baf60a/c Recruit SWI/SNF complex [START_REF] Chiba | Two human homologues of Saccharomyces cerevisiae SWI2/SNF2 and Drosophila brahma are transcriptional coactivators cooperating with the estrogen receptor and the retinoic acid receptor[END_REF][START_REF] Flajollet | The core component of the mammalian SWI/SNF complex SMARCD3/BAF60c is a coactivator for the nuclear retinoic acid receptor[END_REF] GATA4 Transcription factor [START_REF] Clabby | Retinoid X receptor alpha represses GATA-4-mediated transcription via a retinoid-dependent interaction with the cardiac-enriched repressor FOG-2[END_REF] Nkx2.5 Transcription factor [START_REF] Waardenberg | Prediction and validation of protein-protein interactors from genome-wide DNA-binding data using a knowledge-based machine-learning approach[END_REF] COUP-TFI-II Transcription factor [START_REF] Vilhais-Neto | Rere controls retinoic acid signalling and somite bilateral symmetry[END_REF] HDACs Histone deacetylation [START_REF] Kashyap | Epigenetic regulatory mechanisms distinguish retinoic acidmediated transcriptional responses in stem cells and fibroblasts[END_REF] There is no doubt that emerging molecular technologies will help in understanding the function of retinoic receptors in cardiac lineage specification. Studies using ChIP-chip and ChIP-seq against RARs and RXRs are available for cultured cell lines and adult tissues [START_REF] Boergesen | Genome-wide profiling of liver X receptor, retinoid X receptor, and peroxisome proliferator-activated receptor alpha in mouse liver reveals extensive sharing of binding sites[END_REF][START_REF] Chatagnon | RAR/RXR binding dynamics distinguish pluripotency from differentiation associated cis-regulatory elements[END_REF][START_REF] He | The role of retinoic acid in hepatic lipid homeostasis defined by genomic binding and transcriptome profiling[END_REF][START_REF] Lalevee | Genome-wide in silico identification of new conserved and functional retinoic acid receptor response elements (direct repeats separated by 5 bp)[END_REF][START_REF] Mendoza-Parra | Dissecting the retinoid-induced differentiation of F9 embryonal stem cells by integrative genomics[END_REF][START_REF] Moutier | Retinoic acid receptors recognize the mouse genome through binding elements with diverse spacing and topology[END_REF]. The exploitation of ChIP-seq technologies from embryonic tissue will enhance the ability to distinguish direct and indirect regulation of cardiac gene expression. For most targets, RA receptors will be present on cardiac regulatory regions regardless of whether the associated gene is transcriptionally active or not and thus interpreting the information will require additional epigenetic data at those sites to determine the likely transcriptional status associated with specific RAREs. The ENCODE project has provided access to valuable data on genome-wide chromatin occupancy of transcription factors, chromatin modifying and remodeling enzymes and histone modifications in heart tissues [START_REF] Bernstein | An integrated encyclopedia of DNA elements in the human genome[END_REF]. Retinoic receptors can mediate looping of distant DNA sequences, enabling transcriptional regulation by far-upstream enhancers [START_REF] Yasmin | DNAlooping by RXR tetramers permits transcriptional regulation "at a distance[END_REF]. Whether this is the case in the context of heart development is unknown. Chromosome conformation capture technologies (e.g. 3-5C, Hi-C) were developed to identify long-range chromatin interactions [START_REF] De Laat | Topology of mammalian developmental enhancers and their regulatory landscapes[END_REF]. Merging these data sets can further facilitate identification of RA regulatory elements. The functional importance of RAREs can be assessed in vivo using recent genome editing technologies [START_REF] Harrison | A CRISPR view of development[END_REF]. Another technical issue is obtaining the starting material from small, localized populations of cardiac progenitor cells. This represents a significant challenge but will be necessary to determine the specificity of the cardiac gene programs. Overcoming these technical challenges will provide important new data in our understanding of RA signaling and its role in cardiac development. Addressing this question is critical for understanding the origin of congenital heart defects. Finally, defining how RA signaling and its interacting factors act to enable epigenetic regulatory events will provide insight into the biology of cardiac progenitor cells leading to methods for increasing the efficiency of directed differentiation of pluripotent cells and cellular reprogramming into cardiac subtypes.

Fig. 1 .

 1 Fig. 1. Heart fields and their contributions to the developing heart. (A) The second heart field (light grey) is located dorsally from the forming heart derived from the first heart field (dark grey). The second heart field is added at the venous and arterial poles of the definitive heart. Ballooning model of cardiac chamber formation (B). The early heart tube has an embryonic phenotype (dark grey). Chamber myocardium (light grey) expands from the outer curvature, whereas non-chamber myocardium (grey) of the inflow tract, atrioventricular canal, outflow tract and inner curvature does not expand. a indicates atrium; ift, inflow tract; la, left atrium; Iv, left ventricle; ra, right atrium; rv, right ventricle; sv, sinus venosus.

Fig. 2 .

 2 Fig. 2. Metabolism of vitamin A. Retinyl esters, retinol, and β-carotene are taken into the body from the diet. Both retinol and β-carotene may be converted into the transcriptionally active vitamin A forms after first being converted to retinaldehyde. RA then regulates transcription of vitamin A-responsive genes. When RA is no longer needed, it is catabolized by cytochrome enzymes (CYP26 enzymes).

Fig. 3 .

 3 Fig. 3. Retonic acid is required to define the posterior limits of the second heart field. Fgf10 is a molecular marker of the murine SHF. The use of the Mlc1v-nlacZ-24 reporter line, in which a lacZ transgene has been integrated upstream of Fgf10 gene, shows the SHF. Ventral views of wild-type (A) or Raldh2-/-(B) embryos at embryonic day 8.5 showing posterior expansion of Fgf10-lacZ transgene expression (arrowhead). The heart has been removed to allow observation of the X-gal staining, compare Raldh2-/-(B) with WT (A) embryos.

Table 1

 1 Retinoid-responsive cardiac genes.

	Gene	Source	Response to RA Mode	References
	MHCα	Neonatal rat cardiomyocytes	Activation		(Rohrer et al., 1991)
	Cardiac α actin	Chicken cardiac mesoderm	Repression		(Wiens et al., 1992)
	MHC1a	Treated chicken embryo	Activation		(Yutzey et al., 1994)
	MLC2a	Treated mouse embryos	Repression		(Dyson et al., 1995)
	a-Actinin	Treated chicken embryos	Repression		(Dickman and Smith, 1996)
	SERCA	Neonatal rat cardiomyocytes	Activation		(Rohrer et al., 1991)
	Na/K/ATP1A3 subunit	Neonatal rat cardiomyocytes	Repression		(He et al., 1996)
	Large chloride conducting channel	Sheep ventricular cells	Activation		(Rousseau et al., 1996)
	G protein-coupled endothelin signaling Neonatal rat cardiomyocytes	Repression		(Zhou et al., 1995)
	G protein-coupled a-adrenergic signals	Neonatal rat cardiomyocytes	Repression		(Zhou et al., 1995)
	ANF	Neonatal rat cardiomyocytes	Activation	Direct (Clabby et al., 2003; Wu et al., 1996; Zhou et al., 1995)
	GLUT4	Adult mouse heart	Activation		(Castello et al., 1994)
	GATA4	Cell culture	Activation		(Arceci et al., 1993; Clabby et al., 2003)
	FGF8	Raldh2-/-embryos	Repression		(Ryckebusch et al., 2008; Sirbu et al., 2008)
	ISL1	Raldh2-/-embryos	Repression		(Ryckebusch et al., 2008; Sirbu et al., 2008)
	TBX2	Treated mouse embryos, C2C12	Repression	Direct (Sakabe et al., 2012)
	TBX5	Raldh2-/-, chicken embryos	Activation		(Liberatore et al., 2000; Niederreither et al., 2001; Sirbu et al., 2008)
	RARα	P19 cells	Activation	Direct (Ruberte et al., 1991)
	RARβ2	Treated chicken embryos	Activation		(Kostetskii et al., 1998)
	Cyp26α1	P19 cells	Activation		(MacLean et al., 2001)
	Raldh2	Treated mouse embryos	Activation		(Niederreither et al., 1997)
	Beta-integrin	Treated mouse embryos	Activation		(Hierck et al., 1996)
	Flectin	Treated chicken embryos	Activation		(Tsuda et al., 1996)
	Heart lectin-associated matrix protein	Treated chicken embryos	Activation		(Smith et al., 1997)
	JB4/fibrillin-related protein	Treated chicken embryos	Repression		(Smith et al., 1997)
	EPO3-IGF2	Raldh2-/-embryos	Activation	Direct (Brade et al., 2011)
	TGF2	Treated mouse embryos	Activation		(Mahmood et al., 1992)
	Tcf21

Acknowledgments

We thank Dr. R. Kelly who critically read this manuscript and offered valuable suggestions. S. Stefanovic is supported by post-doctoral awards from l'Institut de France Lefoulon-Delalande and H2020-MSCA-IF-2014. S. Zaffran is an INSERM research fellow. Work in S. Zaffran's laboratory is supported by the INSERM, the Agence Nationale pour la Recherche (ANR-13-BSV2-0003-01) and the Association Française contre les Myopathies (AFM-Telethon).