Local retinoic acid signaling directs emergence of the extraocular muscle functional unit.

Coordinated development of muscles, tendons, and their attachment sites ensures emergence of functional musculoskeletal units that are adapted to diverse anatomical demands among different species. How these different tissues are patterned and functionally assembled during embryogenesis is poorly understood. Here, we investigated the morphogenesis of extraocular muscles (EOMs), an evolutionary conserved cranial muscle group that is crucial for the coordinated movement of the eyeballs and for visual acuity. By means of lineage analysis, we redefined the cellular origins of periocular connective tissues interacting with the EOMs, which do not arise exclusively from neural crest mesenchyme as previously thought. Using 3D imaging approaches, we established an integrative blueprint for the EOM functional unit. By doing so, we identified a developmental time window in which individual EOMs emerge from a unique muscle anlage and establish insertions in the sclera, which sets these muscles apart from classical muscle-to-bone type of insertions. Further, we demonstrate that the eyeballs are a source of diffusible all-trans retinoic acid (ATRA) that allow their targeting by the EOMs in a temporal and dose-dependent manner. Using genetically modified mice and inhibitor treatments, we find that endogenous local variations in the concentration of retinoids contribute to the establishment of tendon condensations and attachment sites that precede the initiation of muscle patterning. Collectively, our results highlight how global and site-specific programs are deployed for the assembly of muscle functional units with precise definition of muscle shapes and topographical wiring of their tendon attachments.

Retinoic acid controls early neurogenesis in the developing mouse cerebral cortex.

A tight regulation of neuron production is required to generate a functional cerebral cortex and is achieved by a proper balance between proliferation and differentiation of progenitor cells. Though the vitamin A (retinol) active derivative retinoic acid (RA) has been implicated as one of the signals acting during mammalian forebrain neurogenesis, its function at the onset of neurogenesis as well as during establishment of cortical layers and neuronal subtypes remains elusive. One limitation is that murine mutants for genes encoding key enzymes involved in RA synthesis die during early embryonic development. We analysed corticogenesis in Rdh10 null mutants, in which an RA deficiency is generated as the intracellular retinol to retinaldehyde conversion is abolished. When analysed at the latest stage before lethality occurs (embryonic day [E]13.5), the mutants show smaller telencephalic vesicles and the thickness of their cortical plate is strongly reduced. The first progenitors formed in the cortical plate are radial glial (RG) cells which generate neurons either directly, or through an indirect mechanism involving the production of intermediate neuronal progenitors (INPs) which then give rise to neurons. We show that in absence of RA, the RG progenitors proliferate less and prematurely produce neurons, leading to their depletion at E11.5. Furthermore, we could demonstrate that lack of RA impairs the generation of INPs at E13.5 and affects the cell cycle exit of progenitor cells during corticogenesis, altogether leading to a deficit in projection neurons and to microcephaly.

Mutations in the latent TGF-beta binding protein 3 (LTBP3) gene cause brachyolmia with amelogenesis imperfecta.

Inherited dental malformations constitute a clinically and genetically heterogeneous group of disorders. Here, we report on four families, three of them consanguineous, with an identical phenotype, characterized by significant short stature with brachyolmia and hypoplastic amelogenesis imperfecta (AI) with almost absent enamel. This phenotype was first described in 1996 by Verloes et al. as an autosomal recessive form of brachyolmia associated with AI. Whole-exome sequencing resulted in the identification of recessive hypomorphic mutations including deletion, nonsense and splice mutations, in the LTBP3 gene, which is involved in the TGF-beta signaling pathway. We further investigated gene expression during mouse development and tooth formation. Differentiated ameloblasts synthesizing enamel matrix proteins and odontoblasts expressed the gene. Study of an available knockout mouse model showed that the mutant mice displayed very thin to absent enamel in both incisors and molars, hereby recapitulating the AI phenotype in the human disorder.

Endogenous retinoic acid signaling is required for maintenance and regeneration of cornea.

Retinoic acid (RA) is a biologically active metabolite of vitamin A (retinol) that serves as an important signaling molecule in orchestrating diverse developmental processes including multiple roles during ocular development. Loss-of-function studies using gene knockouts of RA-synthesizing enzymes encoded by Aldh1a1, Aldh1a2, and Aldh1a3 (also known as Raldh1, Raldh2, and Raldh3) have provided valuable insight into how RA controls eye morphogenesis including corneal development. However, it is unclear whether endogenous RA is required for maintenance and regeneration of adult cornea. Here, we investigated the role of Aldh1a genes in the adult cornea using a novel conditional Aldh1a1,2,3-flox/flox;Rosa26-CreERT2 loss-of-function mouse model to determine the biological function of RA. Our findings indicate that loss of RA synthesis results in corneal thinning characterized by reduced thickness of the stromal layer, impaired corneal epithelial cell proliferation, and increased apoptosis. Corneal thinning in Aldh1a-deficient mice was significantly rescued by RA administration, indicating an important role of endogenous RA signaling in adult corneal homeostasis and regeneration. Thus, Aldh1a1,2,3-flox/flox;Rosa26-CreERT2 mice provide a useful model for investigating the mechanistic role of RA signaling in adult corneal maintenance and could provide new insights into therapeutic approaches for controlling corneal repair to prevent vision loss.

Prokineticin receptor 1 is required for mesenchymal-epithelial transition in kidney development.

Identification of factors regulating renal development is important to understand the pathogenesis of congenital kidney diseases. Little is known about the molecular mechanism of renal development and functions triggered by the angiogenic hormone prokineticin-2 and its receptor, PKR1. Utilizing the Gata5 (G5)-Cre and Wilms tumor 1 (Wt1)(GFP)cre transgenic lines, we generated mutant mice with targeted PKR1 gene disruptions in nephron progenitors. These mutant mice exhibited partial embryonic and postnatal lethality. Kidney developmental defects in PKR(G5-/-) mice are manifested in the adult stage as renal atrophy with glomerular defects, nephropathy, and uremia. PKR1(Wt1-/-) embryos exhibit hypoplastic kidneys with premature glomeruli and necrotic nephrons as a result of impaired proliferation and increased apoptosis in Wt1(+) renal mesenchymal cells. PKR1 regulates renal mesenchymal-epithelial transition (MET) that is involved in formation of renal progenitors, regulating glomerulogenesis toward forming nephrons during kidney development. In the isolated embryonic Wt1(+) renal cells, overexpression or activation of PKR1 promotes MET defined by the transition from elongated cell to octagonal cell morphology, and alteration of the expression of MET markers via activating NFATc3 signaling. Together, these results establish PKR1 via NFATc3 as a crucial modifier of MET processing to the development of nephron. Our study should facilitate new therapeutic opportunities in human renal disorders.-Arora, H., Boulberdaa, M., Qureshi, R., Bitirim, V., Messadeq, N., Dolle, P., Nebigil, C. G. Prokineticin receptor 1 is required for mesenchymal-epithelial transition in kidney development.

Enamel and dental anomalies in latent-transforming growth factor beta-binding protein 3 mutant mice.

Latent-transforming growth factor beta-binding protein 3 (LTBP-3) is important for craniofacial morphogenesis and hard tissue mineralization, as it is essential for activation of transforming growth factor-ß (TGF-ß). To investigate the role of LTBP-3 in tooth formation we performed micro-computed tomography (micro-CT), histology, and scanning electron microscopy analyses of adult Ltbp3-/- mice. The Ltbp3-/- mutants presented with unique craniofacial malformations and reductions in enamel formation that began at the matrix formation stage. Organization of maturation-stage ameloblasts was severely disrupted. The lateral side of the incisor was affected most. Reduced enamel mineralization, modification of the enamel prism pattern, and enamel nodules were observed throughout the incisors, as revealed by scanning electron microscopy. Molar roots had internal irregular bulbous-like formations. The cementum thickness was reduced, and microscopic dentinal tubules showed minor nanostructural changes. Thus, LTBP-3 is required for ameloblast differentiation and for the formation of decussating enamel prisms, to prevent enamel nodule formation, and for proper root morphogenesis. Also, and consistent with the role of TGF-ß signaling during mineralization, almost all craniofacial bone components were affected in Ltbp3-/- mice, especially those involving the upper jaw and snout. This mouse model demonstrates phenotypic overlap with Verloes Bourguignon syndrome, also caused by mutation of LTBP3, which is hallmarked by craniofacial anomalies and amelogenesis imperfecta phenotypes.

Retinoic Acid Receptor ß Controls Development of Striatonigral Projection Neurons through FGF-Dependent and Meis1-Dependent Mechanisms.

The mammalian striatum controls sensorimotor and psychoaffective functions through coordinated activities of its two striatonigral and striatopallidal output pathways. Here we show that retinoic acid receptor ß (RARß) controls development of a subpopulation of GABAergic, Gad65-positive striatonigral projection neurons. In Rarb(-/-) knock-out mice, concomitant reduction of Gad65, dopamine receptor D1 (Drd1), and substance P expression at different phases of prenatal development was associated with reduced number of Drd1-positive cells at birth, in contrast to normal numbers of striatopallidal projection neurons expressing dopamine receptor D2. Fate mapping using BrdU pulse-chase experiments revealed that such deficits may originate from compromised proliferation of late-born striosomal neurons and lead to decreased number of Drd1-positive cells retaining BrdU in postnatal day (P) 0 Rarb(-/-) striatum. Reduced expression of Fgf3 in the subventricular zone of the lateral ganglionic eminence (LGE) at embryonic day 13.5 may underlie such deficits by inducing premature differentiation of neuronal progenitors, as illustrated by reduced expression of the proneural gene Ascl1 (Mash1) and increased expression of Meis1, a marker of postmitotic LGE neurons. In agreement with a critical role of FGF3 in this control, reduced number of Ascl1-expressing neural progenitors, and a concomitant increase of Meis1-expressing cells, were observed in primary cell cultures of Rarb(-/-) LGE. This defect was normalized by addition of fibroblast growth factor (FGF). Such data point to role of Meis1 in striatal development, also supported by reduced neuronal differentiation in the LGE of Meis1(-/-) embryos. Our data unveil a novel mechanism of development of striatonigral projection neurons involving retinoic acid and FGF, two signals required for positioning the boundaries of Meis1-expressing cells.

Correction: FGF Signalling Regulates Chromatin Organisation during Neural Differentiation via Mechanisms that Can Be Uncoupled from Transcription.

[This corrects the article DOI: 10.1371/journal.pgen.1003614.].

FGF signalling regulates chromatin organisation during neural differentiation via mechanisms that can be uncoupled from transcription.

Changes in higher order chromatin organisation have been linked to transcriptional regulation; however, little is known about how such organisation alters during embryonic development or how it is regulated by extrinsic signals. Here we analyse changes in chromatin organisation as neural differentiation progresses, exploiting the clear spatial separation of the temporal events of differentiation along the elongating body axis of the mouse embryo. Combining fluorescence in situ hybridisation with super-resolution structured illumination microscopy, we show that chromatin around key differentiation gene loci Pax6 and Irx3 undergoes both decompaction and displacement towards the nuclear centre coincident with transcriptional onset. Conversely, down-regulation of Fgf8 as neural differentiation commences correlates with a more peripheral nuclear position of this locus. During normal neural differentiation, fibroblast growth factor (FGF) signalling is repressed by retinoic acid, and this vitamin A derivative is further required for transcription of neural genes. We show here that exposure to retinoic acid or inhibition of FGF signalling promotes precocious decompaction and central nuclear positioning of differentiation gene loci. Using the Raldh2 mutant as a model for retinoid deficiency, we further find that such changes in higher order chromatin organisation are dependent on retinoid signalling. In this retinoid deficient condition, FGF signalling persists ectopically in the elongating body, and importantly, we find that inhibiting FGF receptor (FGFR) signalling in Raldh2-/- embryos does not rescue differentiation gene transcription, but does elicit both chromatin decompaction and nuclear position change. These findings demonstrate that regulation of higher order chromatin organisation during differentiation in the embryo can be uncoupled from the machinery that promotes transcription and, for the first time, identify FGF as an extrinsic signal that can direct chromatin compaction and nuclear organisation of gene loci.

Retinoic acid signalling during development.

Retinoic acid (RA) is a vitamin A-derived, non-peptidic, small lipophilic molecule that acts as ligand for nuclear RA receptors (RARs), converting them from transcriptional repressors to activators. The distribution and levels of RA in embryonic tissues are tightly controlled by regulated synthesis through the action of specific retinol and retinaldehyde dehydrogenases and by degradation via specific cytochrome P450s (CYP26s). Recent studies indicate that RA action involves an interplay between diffusion (morphogen-like) gradients and the establishment of signalling boundaries due to RA metabolism, thereby allowing RA to finely control the differentiation and patterning of various stem/progenitor cell populations. Here, we provide an overview of the RA biosynthesis, degradation and signalling pathways and review the main functions of this molecule during embryogenesis.

Oral-facial-digital type I protein is required for primary cilia formation and left-right axis specification.

The oral-facial-digital type I (OFD1) syndrome (OMIM 311200) is a human developmental disorder; affected individuals have craniofacial and digital abnormalities and, in 15% of cases, polycystic kidney. The disease is inherited as an X-linked dominant male-lethal trait. Using a Cre-loxP system, we generated knockout animals lacking Ofd1 and reproduced the main features of the disease, albeit with increased severity, possibly owing to differences of X inactivation patterns between human and mouse. We found failure of left-right axis specification in mutant male embryos, and ultrastructural analysis showed a lack of cilia in the embryonic node. Formation of cilia was defective in cystic kidneys from heterozygous females, implicating ciliogenesis as a mechanism underlying cyst development. In addition, we found impaired patterning of the neural tube and altered expression of the 5' Hoxa and Hoxd genes in the limb buds of mice lacking Ofd1, suggesting that Ofd1 could have a role beyond primary cilium organization and assembly.

Retinoic acid deficiency impairs the vestibular function.

The retinaldehyde dehydrogenase 3 (Raldh3) gene encodes a major retinoic acid synthesizing enzyme and is highly expressed in the inner ear during embryogenesis. We found that mice deficient in Raldh3 bear severe impairment in vestibular functions. These mutant mice exhibited spontaneous circling/tilted behaviors and performed poorly in several vestibular-motor function tests. In addition, video-oculography revealed a complete loss of the maculo-ocular reflex and a significant reduction in the horizontal angular vestibulo-ocular reflex, indicating that detection of both linear acceleration and angular rotation were compromised in the mutants. Consistent with these behavioral and functional deficiencies, morphological anomalies, characterized by a smaller vestibular organ with thinner semicircular canals and a significant reduction in the number of otoconia in the saccule and the utricle, were consistently observed in the Raldh3 mutants. The loss of otoconia in the mutants may be attributed, at least in part, to significantly reduced expression of Otop1, which encodes a protein known to be involved in calcium regulation in the otolithic organs. Our data thus reveal a previously unrecognized role of Raldh3 in structural and functional development of the vestibular end organs.

Vax2 regulates retinoic acid distribution and cone opsin expression in the vertebrate eye.

Vax2 is an eye-specific homeobox gene, the inactivation of which in mouse leads to alterations in the establishment of a proper dorsoventral eye axis during embryonic development. To dissect the molecular pathways in which Vax2 is involved, we performed a transcriptome analysis of Vax2(-/-) mice throughout the main stages of eye development. We found that some of the enzymes involved in retinoic acid (RA) metabolism in the eye show significant variations of their expression levels in mutant mice. In particular, we detected an expansion of the expression domains of the RA-catabolizing enzymes Cyp26a1 and Cyp26c1, and a downregulation of the RA-synthesizing enzyme Raldh3. These changes determine a significant expansion of the RA-free zone towards the ventral part of the eye. At postnatal stages of eye development, Vax2 inactivation led to alterations of the regional expression of the cone photoreceptor genes Opn1sw (S-Opsin) and Opn1mw (M-Opsin), which were significantly rescued after RA administration. We confirmed the above described alterations of gene expression in the Oryzias latipes (medaka fish) model system using both Vax2 gain- and loss-of-function assays. Finally, a detailed morphological and functional analysis of the adult retina in mutant mice revealed that Vax2 is necessary for intraretinal pathfinding of retinal ganglion cells in mammals. These data demonstrate for the first time that Vax2 is both necessary and sufficient for the control of intraretinal RA metabolism, which in turn contributes to the appropriate expression of cone opsins in the vertebrate eye.

Retinoic acid in development: towards an integrated view.

Retinoic acid (RA) has complex and pleiotropic functions during vertebrate development. Recent work in several species has increased our understanding of the roles of RA as a signalling molecule. These functions rely on a tight control of RA distribution within embryonic tissues through the combined action of synthesizing and metabolizing enzymes, possibly leading to diffusion gradients. Also important is the switching of nuclear receptors from a transcriptionally repressing state to an activating state. In addition, cross-talk with other key embryonic signals, especially fibroblast growth factors (FGFs) and sonic hedgehog (SHH), is being uncovered. Some of these functions could be maintained throughout the life of an organism to regulate cell-lineage decisions and/or the differentiation of stem cell populations, highlighting possibilities for regenerative medicine.

Involvement of retinol dehydrogenase 10 in embryonic patterning and rescue of its loss of function by maternal retinaldehyde treatment.

Retinoic acid (RA), an active vitamin A metabolite, is a key signaling molecule in vertebrate embryos. Morphogenetic RA gradients are thought to be set up by tissue-specific actions of retinaldehyde dehydrogenases (RALDHs) and catabolizing enzymes. According to the species, two enzymatic pathways (ß-carotene cleavage and retinol oxidation) generate retinaldehyde, the substrate of RALDHs. Placental species depend on maternal retinol transferred to the embryo. The retinol-to-retinaldehyde conversion was thought to be achieved by several redundant enzymes; however, a random mutagenesis screen identified retinol dehydrogenase 10 [Rdh10(Trex) allele; Sandell LL, et al. (2007) Genes Dev 21:1113-1124] as responsible for a homozygous lethal phenotype with features of RA deficiency. We report here the production and characterization of unique murine Rdh10 loss-of-function alleles generated by gene targeting. We show that although Rdh10(-/-) mutants die at an earlier stage than Rdh10(Trex) mutants, their molecular patterning defects do not reflect a complete state of RA deficiency. Furthermore, we were able to correct most developmental abnormalities by administering retinaldehyde to pregnant mothers, thereby obtaining viable Rdh10(-/-) mutants. This demonstrates the rescue of an embryonic lethal phenotype by simple maternal administration of the missing retinoid compound. These results underscore the importance of maternal retinoids in preventing congenital birth defects, and lead to a revised model of the importance of RDH10 and RALDHs in controlling embryonic RA distribution.

Non-cell-autonomous retinoid signaling is crucial for renal development.

In humans and mice, mutations in the Ret gene result in Hirschsprung's disease and renal defects. In the embryonic kidney, binding of Ret to its ligand, Gdnf, induces a program of epithelial cell remodeling that controls primary branch formation and branching morphogenesis within the kidney. Our previous studies showed that transcription factors belonging to the retinoic acid (RA) receptor family are crucial for controlling Ret expression in the ureteric bud; however, the mechanism by which retinoid-signaling acts has remained unclear. In the current study, we show that expression of a dominant-negative RA receptor in mouse ureteric bud cells abolishes Ret expression and Ret-dependent functions including ureteric bud formation and branching morphogenesis, indicating that RA-receptor signaling in ureteric bud cells is crucial for renal development. Conversely, we find that RA-receptor signaling in ureteric bud cells depends mainly on RA generated in nearby stromal cells by retinaldehyde dehydrogenase 2, an enzyme required for most fetal RA synthesis. Together, these studies suggest that renal development depends on paracrine RA signaling between stromal mesenchyme and ureteric bud cells that regulates Ret expression both during ureteric bud formation and within the developing collecting duct system.

Endogenous retinoic acid regulates cardiac progenitor differentiation.

Retinoic acid (RA) has several established functions during cardiac development, including actions in the fetal epicardium required for myocardial growth. An open question is if retinoid effects are limited to growth factor stimulation pathway(s) or if additional actions on uncommitted progenitor/stem populations might drive cardiac differentiation. Here we report the dual effects of RA deficiency on cardiac growth factor signaling and progenitor/stem biology using the mouse retinaldehyde dehydrogenase 2 (Raldh2) knockout model. Although early heart defects in Raldh2(-/-) embryos result from second-heart-field abnormalities, it is unclear whether this role is transient or whether RA has sustained effects on cardiac progenitors. To address this, we used transient maternal RA supplementation to overcome early Raldh2(-/-) lethality. By embryonic day 11.5-14.5, Raldh2(-/-) hearts exhibited reduced venticular compact layer outgrowth and altered coronary vessel development. Although reductions in Fgf2 and target pERK levels occurred, no alterations in Wnt/beta-catenin expression were observed. Cell proliferation is increased in compact zone myocardium, whereas cardiomyocyte differentiation is reduced, alterations that suggest progenitor defects. We report that the fetal heart contains a reservoir of stem/progenitor cells, which can be isolated by their ability to efflux a fluorescent dye and that retinoid signaling acts on this fetal cardiac side population (SP). Raldh2(-/-) hearts display increased SP cell numbers, with selective increases in expression of cardiac progenitor cell markers and reduced differentiation marker levels. Hence, although lack of RA signaling increases cardiac SP numbers, simultaneous reductions in Fgf signaling reduce cardiomyocyte differentiation, possibly accounting for long-term defects in myocardial growth.

Genetic evidence that oxidative derivatives of retinoic acid are not involved in retinoid signaling during mouse development.

Retinoic acid, the active derivative of vitamin A (retinol), is a hormonal signaling molecule that acts in developing and adult tissues. The Cyp26a1 (cytochrome p450, 26) protein metabolizes retinoic acid into more polar hydroxylated and oxidized derivatives. Whether some of these derivatives are biologically active metabolites has been debated. Cyp26a1(-/-) mouse fetuses have lethal morphogenetic phenotypes mimicking those generated by excess retinoic acid administration, indicating that human CYP26A1 may be essential in controlling retinoic acid levels during development. This hypothesis suggests that the Cyp26a1(-/-) phenotype could be rescued under conditions in which embryonic retinoic acid levels are decreased. We show that Cyp26a1(-/-) mice are phenotypically rescued by heterozygous disruption of Aldh1a2 (also known as Raldh2), which encodes a retinaldehyde dehydrogenase responsible for the synthesis of retinoic acid during early embryonic development. Aldh1a2 haploinsufficiency prevents the appearance of spina bifida and rescues the development of posterior structures (sacral/caudal vertebrae, hindgut, urogenital tract), while partly preventing cervical vertebral transformations and hindbrain pattern alterations in Cyp26a1(-/-) mice. Thus, some of these double-mutant mice can reach adulthood. This study is the first report of a mutation acting as a dominant suppressor of a lethal morphogenetic mutation in mammals. We provide genetic evidence that ALDH1A2 and CYP26A1 activities concurrently establish local embryonic retinoic acid levels that must be finely tuned to allow posterior organ development and to prevent spina bifida.

Identification and characterization of rod-derived cone viability factor.

Retinitis pigmentosa is an untreatable, inherited retinal disease that leads to blindness. The disease initiates with the loss of night vision due to rod photoreceptor degeneration, followed by irreversible, progressive loss of cone photoreceptor. Cone loss is responsible for the main visual handicap, as cones are essential for day and high-acuity vision. Their loss is indirect, as most genes associated with retinitis pigmentosa are not expressed by these cells. We previously showed that factors secreted from rods are essential for cone viability. Here we identified one such trophic factor by expression cloning and named it rod-derived cone viability factor (RdCVF). RdCVF is a truncated thioredoxin-like protein specifically expressed by photoreceptors. The identification of this protein offers new treatment possibilities for retinitis pigmentosa.

Hox genes define distinct progenitor sub-domains within the second heart field.

Much of the heart, including the atria, right ventricle and outflow tract (OFT) is derived from a progenitor cell population termed the second heart field (SHF) that contributes progressively to the embryonic heart during cardiac looping. Several studies have revealed anterior-posterior patterning of the SHF, since the anterior region (anterior heart field) contributes to right ventricular and OFT myocardium whereas the posterior region gives rise to the atria. We have previously shown that Retinoic Acid (RA) signal participates to this patterning. We now show that Hoxb1, Hoxa1, and Hoxa3, as downstream RA targets, are expressed in distinct sub-domains within the SHF. Our genetic lineage tracing analysis revealed that Hoxb1, Hoxa1 and Hoxa3-expressing cardiac progenitor cells contribute to both atria and the inferior wall of the OFT, which subsequently gives rise to myocardium at the base of pulmonary trunk. By contrast to Hoxb1(Cre), the contribution of Hoxa1-enhIII-Cre and Hoxa3(Cre)-labeled cells is restricted to the distal regions of the OFT suggesting that proximo-distal patterning of the OFT is related to SHF sub-domains characterized by combinatorial Hox genes expression. Manipulation of RA signaling pathways showed that RA is required for the correct deployment of Hox-expressing SHF cells. This report provides new insights into the regulatory gene network in SHF cells contributing to the atria and sub-pulmonary myocardium.