Ting Wang

Lauriane de Fabritus, ..., Zheng Wang

Susan M Kaech
email: skaech@salk.edu

Natalia B Ivanova
email: natalia.ivanova@uga.edu

Fabio R Santori
email: fabio.santori@yale.edu

Zheng Wang

Lauriane De Fabritus

Jinglian Tao

Essa M Saied

Ho-Joon Lee

Bulat R Ramazanov

Benjamin Jackson

Daniel Burkhardt

Mikhail Parker

Anne S Gleinich

Zhirui Wang

Dong Eun Seo

Ting Zhou

Shihao Xu

Irina Alecu

Parastoo Azadi

Christoph Arenz

Thorsten Hornemann

Smita Krishnaswamy

Serge A Van De Pavert

d Inhibition of sphingolipid synthesis impairs human cardiomyocyte differentiation d 1-deoxysphingosine supplementation promotes human cardiomyocyte maturation Authors

Identification of physiological modulators of nuclear hormone receptor (NHR) activity is paramount for understanding the link between metabolism and transcriptional networks that orchestrate development and cellular physiology. Using libraries of metabolic enzymes alongside their substrates and products, we identify 1-deoxysphingosines as modulators of the activity of NR2F1 and 2 (COUP-TFs), which are orphan NHRs that are critical for development of the nervous system, heart, veins, and lymphatic vessels. We show that these non-canonical alanine-based sphingolipids bind to the NR2F1/2 ligand-binding domains (LBDs) and modulate their transcriptional activity in cell-based assays at physiological concentrations. Furthermore, inhibition of sphingolipid biosynthesis phenocopies NR2F1/2 deficiency in endothelium and cardiomyocytes, and increases in 1-deoxysphingosine levels activate NR2F1/2-dependent differenti-ation programs. Our findings suggest that 1-deoxysphingosines are physiological regulators of NR2F1/2mediated transcription.

(legend on next page) Figure 2. Non-canonical alanine-based sphingolipids are NR2F1/2 ligands (A) Insect-cell-based system for metabolite screening. (B) Sphingolipids promote NR2F reporter activity in insect cells. Circles: active (green), inactive (gray), and not tested (white). Error bars are standard deviation. (C) 1-deoxySO-14Z promotes transcriptional activity of NR2F2 in cells transfected with native promoter-based NGF1A reporter. (D) 1-deoxySO-14Z binds recombinant NR2F2 LBD in FPA. (legend continued on next page) (E) 1-deoxySO-14Z binds recombinant NR2F2 LBD by SPR. (F) Summary of structure-activity relationships of 1-deoxysphingolipids in mammalian cells. Circles: active (green), inactive (gray), and white (not tested). (G) Metabolite pull-down experimental scheme. (H) Metabolites displaced from NR2F1 LBD by 1-deoxySO-14Z-d. (I) Summary of m/z ions (H) identified in the 25-33 min retention time interval. (J) Specificity and enrichment of 1-deoxySO-14Z-d in NR2F1 LBD compared with RAR LBD and NR2F1 LBD vehicle.

INTRODUCTION

Nuclear hormone receptors (NHRs) are transcription factors regulated by small molecules, such as endogenous metabolites, hormones, or diet-derived compounds including vitamins and fatty acids [START_REF] Tao | Nuclear hormone receptors and their ligands: metabolites in control of transcription[END_REF]. These ligands can either bind the ligand-binding pocket (orthosteric ligands) or to alternative sites in the ligand-binding domain (LBD) of the NHR (allosteric ligands) [START_REF] Van Westen | Chemical, target, and bioactive properties of allosteric modulation[END_REF]. Most natural NHR ligands are orthosteric, but 27-hydroxycholesterol was recently identified as a natural allosteric ligand for ERb [START_REF] Starkey | 27-Hydroxycholesterol is an estrogen receptor beta-selective negative allosteric modifier of 17beta-estradiol binding[END_REF]. There are 48 NHRs in the human genome, including 36 non-classic receptors and 12 classic NHRs for vitamins A, D, and steroid hormones. There are 19 orphan NHRs, where no natural ligand is known [START_REF] Tao | Nuclear hormone receptors and their ligands: metabolites in control of transcription[END_REF]. The identification of these ligands is central to understanding the relationship between metabolic activity and gene transcription.

The NR2F family is comprised of three members: NR2F1/ COUP-TFI, NR2F2/COUP-TFII, and NR2F6/EAR2 [START_REF] Jonk | Cloning and expression during development of three murine members of the COUP family of nuclear orphan receptors[END_REF]. NR2F1/2 have evolutionarily conserved roles in neurogenesis. NR2F1/2 homologs are expressed in the neural lineage of Hydra, a cnidarian [START_REF] Gauchat | The orphan COUP-TF nuclear receptors are markers for neurogenesis from cnidarians to vertebrates[END_REF], and in C. elegans the NR2F1/2 homolog unc-55 is required for the proper development of interneuron networks [START_REF] Petersen | A transcriptional program promotes remodeling of GABAergic synapses in Caenorhabditis elegans[END_REF][START_REF] Shan | Copulation in C. elegans males requires a nuclear hormone receptor[END_REF]. In Drosophila melanogaster the NR2F1/2 homolog Svp regulates the differentiation of photoreceptor cells [START_REF] Mlodzik | The Drosophila seven-up gene, a member of the steroid receptor gene superfamily, controls photoreceptor cell fates[END_REF] and neural lineages derived from type I and II neuroblasts [START_REF] Kanai | Seven-up controls switching of transcription factors that specify temporal identities of Drosophila neuroblasts[END_REF][START_REF] Maurange | Temporal transcription factors and their targets schedule the end of neural proliferation in Drosophila[END_REF][START_REF] Mettler | Timing of identity: spatiotemporal regulation of hunchback in neuroblast lineages of Drosophila by seven-up and Prospero[END_REF] as well as the development of valve-like ostiae in the fly heart [START_REF] Molina | Ostia, the inflow tracts of the Drosophila heart, develop from a genetically distinct subset of cardial cells[END_REF][START_REF] Ponzielli | Heart tube patterning in Drosophila requires integration of axial and segmental information provided by the bithorax Complex genes and hedgehog signaling[END_REF]. Vertebrates have two homologous NR2F receptors, NR2F1 and NR2F2, with partially redundant functions. Deletion of all four NR2F1/2 alleles in mice causes major eye defects such as coloboma and microphthalmia [START_REF] Tang | COUP-TFs regulate eye development by controlling factors essential for optic vesicle morphogenesis[END_REF]. NR2F1 and NR2F2 also possess non-redundant functions. NR2F1 regulates the development of oligodendrocytes [START_REF] Yamaguchi | The nuclear orphan receptor COUP-TFI is important for differentiation of oligodendrocytes[END_REF], formation of glossopharyngeal cranial nerves IX, and axon guidance and arborization in the peripheral nervous system [START_REF] Qiu | Null mutation of mCOUP-TFI results in defects in morphogenesis of the glossopharyngeal ganglion, axonal projection, and arborization[END_REF]. Haploinsufficiency of NR2F1 causes of Bosch-Boonstra-Schaaf optic atrophy syndrome in humans [START_REF] Bosch | NR2F1 mutations cause optic atrophy with intellectual disability[END_REF]. In contrast, NR2F2 is required for cardiac development in both mice [START_REF] Pereira | The orphan nuclear receptor COUP-TFII is required for angiogenesis and heart development[END_REF] and humans [START_REF] Al Turki | Rare variants in NR2F2 cause congenital heart defects in humans[END_REF], the formation of GABAergic interneurons in the mouse brain [START_REF] Kanatani | The COUP-TFII/Neuropilin-2 is a molecular switch steering diencephalon-derived GABAergic neurons in the developing mouse brain[END_REF], and the development of both venous [START_REF] You | Suppression of Notch signalling by the COUP-TFII transcription factor regulates vein identity[END_REF] and lymphatic endothelia in mice [START_REF] Lin | Direct transcriptional regulation of neuropilin-2 by COUP-TFII modulates multiple steps in murine lymphatic vessel development[END_REF][START_REF] Srinivasan | Lineage tracing demonstrates the venous origin of the mammalian lymphatic vasculature[END_REF]. The third member of the NR2F family, NR2F6, plays a role in the differentiation of pro-inflammatory CD4 + Th17 cells [START_REF] Hermann-Kleiter | The nuclear orphan receptor NR2F6 suppresses lymphocyte activation and T helper 17-dependent autoimmunity[END_REF]. Despite their ubiquity during development, the metabolic pathways that regulate the activity of NR2F NHRs are unknown.

Biochemical approaches and candidate metabolite screening methods have been used to identify the ligands of the orphan NHRs RXR [START_REF] Heyman | 9-cis retinoic acid is a high affinity ligand for the retinoid X receptor[END_REF][START_REF] Levin | 9-cis retinoic acid stereoisomer binds and activates the nuclear receptor RXR alpha[END_REF], LXR [START_REF] Janowski | An oxysterol signalling pathway mediated by the nuclear receptor LXR alpha[END_REF], and FXR [START_REF] Makishima | Identification of a nuclear receptor for bile acids[END_REF][START_REF] Parks | Bile acids: natural ligands for an orphan nuclear receptor[END_REF]. However, these approaches have not succeeded in all orphan NHRs. Here, we develop a strategy, which combines enzyme overexpression and metabolite screening, to identify 1-deoxysphingosines as modulators of NR2F1/2 activity. Our study suggests a physiological role for 1-deoxysphingosines as modulators of NR2F1/2-mediated transcription.

RESULTS

Metabolic enzyme overexpression screening identifies pathways that modulate NR2F1/2 transcriptional activity We reasoned that, if endogenous metabolites regulate NR2F1/2 proteins, then overexpression of enzymes within the metabolic pathways responsible for ligand synthesis would modulate NR2F1/2 transcriptional activity and such pathways could be identified using an appropriate reporter assay (Figure 1A). The LBDs of NR2F1 and NR2F2 are 95% identical [START_REF] Jonk | Cloning and expression during development of three murine members of the COUP family of nuclear orphan receptors[END_REF][START_REF] Qiu | Spatiotemporal expression patterns of chicken ovalbumin upstream promoter-transcription factors in the developing mouse central nervous system: evidence for a role in segmental patterning of the diencephalon[END_REF] (Figure S1A) and likely recognize the same ligand. Thus, modulation of reporter activity would be pro-moted by the same enzymes for both receptors. In contrast, the LBD of NR2F6 exhibits significantly lower amino acid identity to NR2F1/2 (~60%) (Figure S1A), suggesting that NR2F6 may bind a different ligand and serve as specificity control. We utilized wild-type full-length NR2F1/2/6 receptors to optimize the specificity and sensitivity of our luciferase reporter. We screened a set of DNA sequences composed of six conserved direct repeats (DRs) of the NR2F response element AGGTCA separated by spacers of zero (6xDR0A) to six (6xDR6A) adenines [START_REF] Montemayor | Genome-wide analysis of binding sites and direct target genes of the orphan nuclear receptor NR2F1/COUP-TFI[END_REF][START_REF] Pipao´ N | COUP-TF upregulates NGFI-A gene expression through an Sp1 binding site[END_REF]. A peak of NR2F1/2 reporter activity was observed with the 6xDR0A reporter when compared with the baseline empty vector control (Figure 1B). Previous studies suggested that NR2F1/2 could interact with other NHRs, such as RXRs, VDR, or PPARs, leading to activation or repression of their transcriptional activity [START_REF] Tsai | Chick ovalbumin upstream promoter-transcription factors (COUP-TFs): coming of age[END_REF]).

To evaluate the potential confounding effect from other NHRs in our reporter assays, we transfected HEK-293FT cells with native full-length constructs of VDR, RXRB, PPARG, and NR5A1 and found that, except for NR5A1, unrelated receptors did not increase the activity of the 6xDR0A reporter (Figure 1B). RXRB and VDR required reporters with different spacers (Figure 1B) plus addition of their respective ligands, 9-cis-retinoic acid and vitamin D, to induce their transcriptional activity (Figure S1B). These results confirmed that the activity of the 6xDR0A reporter in HEK-293FT cells was specific to NR2F1/2. Transfection of native full-length Nr2f1 or Nr2f2 cDNAs increased reporter activity in both serum-containing and serumfree media (Figure 1C), suggesting that HEK-293FT cells expressed the metabolic pathways required for production of NR2F1/2 ligand. NR2F1/2 activity was dependent on coactivator recruitment by the NR2F2 LBD. Indeed, the activity of the fulllength NR2F2 was inhibited by point mutations within the LBD as well as deletion of the coactivator recognition site (CRS) (Figure 1D). Furthermore, the activity of the NR2F1(LBD)-GAL4(DBD) fusion protein was inhibited by 4-methoxy-1-naphthol, a specific NR2F1/2 inhibitor that binds to the CRS [START_REF] Gue´ Vel | Inactivation of the nuclear orphan receptor COUP-TFII by small chemicals[END_REF] (Figure S1C). Taken together, these results indicate that reporter activity of NR2F1/2 in HEK-293FT cells is specific and coactivator dependent.

To identify metabolic pathways that regulate NR2F1/2 activity, we selected 516 cDNAs of enzymes that cover most reactions driving the metabolism of common biosynthetic molecules in mammalian cells, such as amino acids, fatty acids, retinoids, sterols, etc. These enzymes were tested individually in HEK-293FT cells by co-transfection with the native full-length NR2F1/2 receptor and 6xDR0A reporter (Figure 1A). We identified 14 metabolic enzymes that upregulated the transcriptional activity of both NR2F1 and NR2F2 by more than 2-fold compared with empty vector control, without increasing NR2F6 reporter activity. In addition, 9 enzymes downregulated transcriptional activity of NR2F1 and NR2F2 by more than 2-fold, without decreasing NR2F6 reporter activity (Figure 1E). The effect of each enzyme on NR2F2 reporter activity was confirmed using a luciferase reporter driven by the NGF1A promoter, a native NR2F2 target (Pi-pao´ n et al., 1999) (Figure S1F). Three enzymes in the NR2F1/2 set-AOC3, RNLS, and MMS19-had the opposite effect on NR2F6 reporter activity suggesting that ligands for NR2F1/2 and NR2F6 may be products of different branches of the same metabolic pathway. When re-tested against a panel of several unrelated full-length NHRs such as RXRB, VDR, PPARG, or NR5A1, most NR2F2 activators were specific and did not increase the reporter activity of these receptors (Figure 1F). Only three enzymes-CYP2J2, MMS19, and SLC27A2-increased reporter activity of NR5A1 on the 6xDR0A reporter (Figures 1B and1F). In contrast, none of the repressors identified in the screen were specific for NR2F1/2 (Figure 1F).

Notably, sphingosines bind and antagonize NR5A1, but this repression is dependent on ceramide degradation by ceramidases, which are interacting partners of NR5A1 [START_REF] Urs | Sphingosine regulates the transcription of CYP17 by binding to steroidogenic factor-1[END_REF], rather than the de novo synthesis of sphingo-sine. Several enzymes in the ceramide synthesis/degradation pathways including ACER1, ASAH2B, CERS2, and CERK were tested but had no significant effect on NR2F1/2 re-porter activity in our screen (data not shown). Likewise, overexpression of SPTLC2, the enzyme regulating sphingolipid biosynthesis, did not affect NR5A1 reporter activity . Thus, the activities of NR2F1/2 and NR5A1 may be modulated by different pools of sphingosines: NR2F1/2 by sphingosine biosynthesis while NR5A1 by sphingosine catabolism.

When assigned to known metabolic pathways [START_REF] Kanehisa | KEGG: new perspectives on genomes, pathways, diseases and drugs[END_REF][START_REF] Stehling | Human CIA2A-FAM96A and CIA2B-FAM96B integrate iron homeostasis and maturation of different subsets of cytosolic-nuclear iron-sulfur proteins[END_REF], the enzymes identified in our screen fell into four pathways: iron-sulfur cluster biosynthesis, amino acid metabolism, retinol and fatty acid metabolism, and sphingolipid biosynthesis (Figure 1G). Several enzymes that upregulated NR2F1/2 activity also upregulated the synthesis of D-erythro-sphingosine (Figure S1G). Previous reports suggested that at high concentrations, all-trans retinoic acid is a weak agonist of NR2F2 [START_REF] Kruse | Identification of COUP-TFII orphan nuclear receptor as a retinoic acid-activated receptor[END_REF]. Our screens tested 29 enzymes and retinoids in the retinoic acid pathway, but none affected NR2F1/2 transcriptional activity in our system (Figures S1H and S1I).

Taken together, our screen identified four metabolic pathways that modulated NR2F1/2 activity: iron-sulfur cluster biosynthesis, sphingolipid biosynthesis, and fatty acid/amino acid metabolism.

Non-canonical alanine-based sphingolipids are NR2F1/ 2 ligands Next, we tested 66 commercially available substrates and products of enzymes in the metabolic pathways that modulated NR2F1/2 activity (see STAR Methods). We utilized a GAL4-UAS-driven luciferase reporter to monitor the activity of the NR2F1(LBD)-GAL4 (DBD) and fly NR2F1/2 homolog Svp(LBD)-GAL4(DBD) fusion proteins in Drosophila melanogaster S2 cells in response to these metabolites (Figure 2A). This reporter system produces low background noise and is ideal for the detection of weak activity [START_REF] Santori | Identification of natural RORgamma ligands that regulate the development of lymphoid cells[END_REF]. While none of the metabolites in the iron-sulfur cluster biosynthesis, fatty acid, or amino acid metabolism pathways were active, transcrip-tional activity of the NR2F1-GAL4 fusion protein was promoted by several non-canonical sphingolipids, including 1-deoxysphingosine (1-deoxySO), 1-deoxysphinganine (1-deoxySA), and 1deoxymethylsphingosine (1-deoxymSO), all of which are products of SPTLC1/2 enzymes (Figure 2B).

To determine structure-activity relationship, we tested variants of active 1-deoxysphingolipids [START_REF] Saied | Stereoselective synthesis of novel sphingoid bases utilized for exploring the secrets of sphinx[END_REF][START_REF] Saied | Synthesis and characterization of some atypical sphingoid bases[END_REF]. 1-deoxySO with D14 double bond in the sphingoid base [START_REF] Steiner | Elucidating the chemical structure of native 1-deoxysphingosine[END_REF] promoted transcriptional activity of native full-length NR2F2 in HeLa cells with a Kd = 126 nM (Figure 2C). Under physiological conditions, sphingoli-pids are primarily derived from serine, with a minor fraction made using glycine or alanine. Strikingly, neither serine-based (Derythro-sphingosine) nor glycine-based (1-desoxymethylSO) sphingolipids had activity in our insect-cell-based metabolite screen (Figure 2B). The desaturation at positions D8 or D14 of the lipid tail of 1-deoxySO enhanced NR2F2 transcriptional activity (Figure S2A), suggesting that the absence of a C1 hydroxyl group and the presence of D4 and D14 unsaturation were key structural features of NR2F1/2 ligands.

Next, we tested the binding of sphingolipids to recombinant NR2F1 and 2-LBD in vitro. We found that 1-deoxySO-14Z bound to NR2F2-LBD with an affinity of 68 nM by fluorescence polarization assays (FPAs) (Figures 2D andS2B) and 19 nM by surface plasmon resonance (SPR) (Figure 2E). Thus, for 1-deoxySO-14Z, FPA and SPR measurements suggest an affinity in the range of 10-100 nM. In contrast, a canonical serine-based sphingosine (D-erythroSO) that had no detectable activity in our native reporter assay (Figure S2C) bound to NR2F1/2 LBD with an affinity of 59 nM determined by SPR (Figure S2D) and Kd > 300 nM by FPA (Figure S2B). The SPR measures direct interaction of metabolite and protein, whereas FPA measures the ability of a compound to promote coactivator peptide recruitment, which is a surrogate for agonist activity. Thus, our data suggest that both D-erythroSO and 1-deoxySO-14Z bind NR2F1/2 LBD at nanomolar concentrations, but of the two types of sphingosines 1-deoxysphingosines are better agonists. Other sphingolipids such as sphinga-4,14-dienine (sphinga-4,14) stabilized NR2F2 LBD in thermal shift assay (Figure S2E) but had low affinity for NR2F2 LBD (>300 nM by FP and >700 nM by SPR). We found that active sphingolipids bound both NR2F1 and NR2F2 LBDs, therefore supporting our hypothesis that these receptors are modulated by the same metabolites (Figure S2B). Our measurements suggest that the Kd of 1-deoxysphingosines for NR2F1/2 was well below their plasma concentrations of 300 nM [START_REF] Mwinyi | Plasma 1-deoxysphingolipids are early predictors of incident type 2 diabetes mellitus[END_REF][START_REF] Othman | Plasma 1-deoxysphingolipids are predictive biomarkers for type 2 diabetes mellitus[END_REF]. Thus, 1-deoxysphingosines could potentially modulate NR2F1/2 activity in physiological settings. The binding and biological activities of the different sphingolipids are summarized in Figures 2B, 2F, andS2A.

If 1-deoxysphingosines bind to NR2F1/2 produced in mammalian cells, then synthetic ligands would not only bind to NR2F1/2 LBD but would also displace endogenous metabolites bound to the receptor. Therefore, we tested whether deuterated 1-deoxySO-14Z (1-deoxySO-14Z-d) displaced endogenous metabolites bound to NR2F1 LBD in mammalian cells (Figure 2G). FLAG-tagged NR2F1 LBD was expressed and purified from HEK-293FT cells side by side with the LBD of retinoic acid receptor alpha (RARA), which was used as a control. The purified LBDs were incubated with either a vehicle or 1-deox-ySO-14Z-d, and displaced metabolites were isolated and identified by LC-MS. Several NR2F1-bound metabolites displaced by 1-deoxySO-14Z-d matched exact masses corresponding to those of sphingosines (Figures 2H and 2I). Furthermore, 1-deoxySO-14Z-d was enriched in the NR2F1 but not in the RARA samples (Figure 2J), suggesting that 1-deoxySO-14Z specifically bound to NR2F1 LBD.

Notably, while metabolites with exact masses of retinoic acid, a high-affinity ligand for RARs [START_REF] Giguere | Identification of a receptor for the morphogen retinoic acid[END_REF][START_REF] Petkovich | A human retinoic acid receptor which belongs to the family of nuclear receptors[END_REF], were found in the RARA preparations, no such metabolites were detected in the NR2F1 samples (see STAR Methods). Indeed, retinoic acid is a low-affinity ligand for NR2F1/2 with a Kd = 26 mM [START_REF] Kruse | Identification of COUP-TFII orphan nuclear receptor as a retinoic acid-activated receptor[END_REF]. This affinity is magnitudes below the 500 nM retinoic acid present in our medium and is too weak to be detected by our system.

1-deoxysphingosines could be either produced by cells themselves (autocrine route) or taken up from the medium (hormonal route). Sphingolipids, phospholipids, and glycerolipids are all synthesized from fatty acids (Figure S2F). Thus, if autocrine 1-deoxySO production is a significant source of NR2F1/2 ligand, NR2F1/2 transcriptional activity should be attenuated by the deletion of fatty acid synthase (FASN), the rate-limiting enzyme of fatty acid biosynthesis. In contrast, NR2F1/2 transcriptional activity should be increased by the deletion of enzymes, which decrease fatty acid availability such as PCYT1A and PCYT1B, which convert fatty acids to phosphatidylcholine [START_REF] Walker | A conserved SREBP-1/phosphatidylcholine feedback circuit regulates lipogenesis in metazoans[END_REF]. PCYT1A is normally expressed in HEK293FT cells, while PCYT1B is not and serves as a control. Loss of FASN significantly reduced the transcriptional activity of native full-length NR2F1/2 (Figure S2F), while deletion of PCYT1A increased NR2F2 and NR2F6 transcriptional activity (Figure S2F). The possible association of metabolic pathways identified in our study with sphingolipid biosynthesis are summarized in Figure S2G.

To estimate intracellular concentrations of canonical and noncanonical sphingolipids in NR2F2-expressing cells, we isolated cytosolic and nuclear fractions prepared from the telomeraseimmortalized human microvascular endothelium (TIME) cells (Figure S2H). TIME cells contained 0.05 pmole of 1-deoxysphin-gosine/10 6 cells, and more than half was allocated to the nuclear fraction (Figure S2I). These amounts correspond to 50 nM concentration, which is around the Kd measured by in vitro binding assays. 1-deoxysphingosine can also be imported from the media. To test this route, TIME cells were incubated with 1-deox-ySO fluorescently labeled with a nitrobenzoxadiazole group (NBD) and analyzed by confocal microscopy. Most NBD fluorescence was detected in the cytoplasm, but up to 20% of total fluorescence was observed in the nucleus (Figures S2J andS2K).

Phenotypes of Sptlc2 f/f Prox1 Cre-ERT2/+ and Sptlc2 f/f Cdh5 Cre-ERT2/+ embryos correlate to those of Nr2f2 deficient animals We hypothesized that if 1-deoxysphingosines are natural NR2F1/2 ligands, deletion of serine palmitoyltransferase (Sptlc2), which is the rate-limiting enzyme of sphingolipid biosynthesis, should correlate with Nr2f1/2 gene deficiency in vivo. We examined the venous and lymphatic endothelium, as well as the heart, where Nr2f2 gene function has previously been studied [START_REF] Lin | Direct transcriptional regulation of neuropilin-2 by COUP-TFII modulates multiple steps in murine lymphatic vessel development[END_REF][START_REF] Pereira | The orphan nuclear receptor COUP-TFII is required for angiogenesis and heart development[END_REF][START_REF] Srinivasan | Lineage tracing demonstrates the venous origin of the mammalian lymphatic vasculature[END_REF][START_REF] Srinivasan | The nuclear hormone receptor Coup-TFII is required for the initiation and early maintenance of Prox1 expression in lymphatic endothelial cells[END_REF][START_REF] You | Suppression of Notch signalling by the COUP-TFII transcription factor regulates vein identity[END_REF]. Importantly, sphingolipids and 1-deoxysphingolipids are present in HDL and LDL [START_REF] Hornemann | The SPTLC3 subunit of serine palmitoyltransferase generates short chain sphingoid bases[END_REF], and genetic deletion of Sptlc2 in both the liver and the heart has no effect on organ function in young animals (Lee et al., 2012a;[START_REF] Li | Liver-specific deficiency of serine palmitoyltransferase subunit 2 decreases plasma sphingomyelin and increases apolipoprotein E levels[END_REF]. Thus, we expected that conditional Sptlc2 deficiency could be partially rescued by HDL-and LDLderived sphingolipids present in the fetal circulation, resulting in a phenotype that is milder than that observed in Nr2f2-defi-cient animals.

We intercrossed mice with a conditional Sptlc f/f allele (Sptlc2 tm2.1Jia ) [START_REF] Li | Liver-specific deficiency of serine palmitoyltransferase subunit 2 decreases plasma sphingomyelin and increases apolipoprotein E levels[END_REF] and the BAC transgenic mice carrying the tamoxifen (TM)-inducible Cre recombinase integrated into the Prox1 locus (Prox1 Cre-ERT2 ) for deletion of Sptlc2 in lymphatic vessels, vascular endothelial precursors, heart, and liver [START_REF] Bazigou | Genes regulating lymphangiogenesis control venous valve formation and maintenance in mice[END_REF]. We also intercrossed Sptlc f/f mice with Cdh5 CreERT2 mice for deletion in endothelial cells [START_REF] Wang | Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis[END_REF]. Time-mated pregnant females were injected with TM at E10.5. Embryos were collected at E13.5 and E15.5 and stained and analyzed as whole-mount by light-sheet microscopy (Figure 3A). Deletion of floxed Sptlc2 allele was efficient, reaching 90% when measured in sorted PROX1+ cells and 60% when measured in whole heart, which is rich in PROX1+ cells (Figure 3B).

Deletion of Nr2f2 in endothelial cells affects angiogenesis and venous blood vessel development [START_REF] Pereira | The orphan nuclear receptor COUP-TFII is required for angiogenesis and heart development[END_REF][START_REF] You | Suppression of Notch signalling by the COUP-TFII transcription factor regulates vein identity[END_REF]. Accordingly, we found reduced vascularization when Sptlc2 was deleted in endothelial cells of Sptlc2 Cdh5CreERT2 embryos. We observed this to a lesser degree in Sptlc Prox1CreERT2 embryos (Figure 3C). However, we did not observe gross abnormalities in the heart myocardium of Prox1 Cre-ERT2 and Cdh5 Cre-ERT2 models such as those reported in full Nr2f2 knockouts at earlier stages of development [START_REF] Pereira | The orphan nuclear receptor COUP-TFII is required for angiogenesis and heart development[END_REF]. Sptlc2 deficiency in either the Prox1 Cre-ERT2 or Cdh5 Cre-ERT2 models failed to produce viable progeny for studies in adult animals.

We analyzed the lymphatic endothelium of TM-treated Sptlc2 f/f Prox-1 Cre-ERT2 (legend continued on next page) et al., 2007, 2010). Edema was consistently observed in Sptlc2 f/f Prox1 Cre-ERT2 (penetrance of 0.78), Sptlc2 f/f Cdh5 Cre-ERT2 (penetrance 0.87), and Nr2f2 f/f Prox1 Cre-ERT2 (penetrance 1) embryos at E15.5 (Figures 3D and S3A). Edema was visible by light-sheet microscopy (Figure 3E). We did not observe blood-filled lymphatics, even in the Nr2f2 conditional knockout model.

Next, we investigated the development of the jugular lymphatic sac (JLS). Formation of the JLS begins around E10 when progenitors of lymphatic endothelial cells (LECs) emerge from the primitive iliac and cardinal veins [START_REF] Srinivasan | Lineage tracing demonstrates the venous origin of the mammalian lymphatic vasculature[END_REF]. The JLS morphology of Sptlc2 f/f Prox1 Cre-ERT2 mice at E15.5 was relatively normal (Figure 3E) and similar to that reported for Nr2f2 -/-Prox1 CreERT2 mice [START_REF] Srinivasan | Lineage tracing demonstrates the venous origin of the mammalian lymphatic vasculature[END_REF][START_REF] Srinivasan | Prox1 dosage controls the number of lymphatic endothelial cell progenitors and the formation of the lymphovenous valves[END_REF]. In contrast, the Sptlc2 and Nr2f2 knockout phenotype was more severe in the Cdh5 Cre-ERT2 model, most likely due to the deletion of Sptlc2 in all endothelial cells [START_REF] Wang | Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis[END_REF]. Indeed, the JLS of the E13.5 Sptlc2 f/f Cdh5 Cre-ERT2 embryos were more disorganized and volumetrically larger (Figures 3E and3F) when compared with those from the Prox1 CreERT2 models and controls (Figure 3F). No E15.5 Nr2f2 f/f Cdh5 Cre-ERT2 embryos were obtained, suggesting embryonic lethality at an earlier stage. Furthermore, the dermal lymphatic networks of E15.5 embryos were altered (Figure 3G). In the WT embryos, the larger blood vessels branched to form a regular network of smaller vessels, while lymphatic vessels were narrow and well formed. In Nr2f2-and Sptlc2-deficient an-imals, the larger blood vessels branched less and instead formed a mesh-like capillary structure with bloated lymphatic vessels (Figure 3G), which is reminiscent of the Nr2f2 -/-phenotype [START_REF] Lin | Direct transcriptional regulation of neuropilin-2 by COUP-TFII modulates multiple steps in murine lymphatic vessel development[END_REF]. These results suggest a correlation between the cardiac, vascular, and lymphatic endothelial phenotypes of Sptlc2 and Nr2f2 deficient embryos.

To test whether 1-deoxysphingosines could rescue the Sptlc2 - /-phenotypes in mouse LECs, we used 4-hydroxy-tamoxifen (4OH-TMX) to delete floxed Nr2f2 f/f Prox1 CreERT2 or Sptlc2 f/f Prox1 CreERT2 alleles in primary cultures of mouse LECs (Figure S3B). The medium was supplemented with D-erythroSO to provide a precursor for the synthesis of canonical sphingolipids. We compared untreated cells with cells treated with 4OH-TMX combined with either a vehicle or 1-deoxySO-14Z. In the absence of 1-deoxySO-14Z, LECs in both Nr2f2 and Sptlc2 deleted cultures died between 24 and 48 h following 4OH-TMX administration. Addition of 1-deoxySO-14Z restored the number of viable cells in Sptlc2-deleted cultures, but not in Nr2f2-deleted cultures (Figure S3C). Within the 24-h interval after the 4OH-TMX administration, the presence of 1-deoxySO-14Z, but not the vehicle, partially restored PROX1 levels in LECs . Likely due to the short experimental time window, no effects were observed on other lymphatic endothe-lial markers, such as LYVE1 and NR2F2. Similarly, 1-deoxySO-14Z partially rescued cardiac development in E10.5 embryo cultures of Sptlc2 f/f Cdh5 CreERT2 mice (Figures S3F andS3G).

Taken together, these results suggest that 1-deoxysphingosine could partially rescue the Sptlc2 -/-phenotype in mouse LECs and embryonic hearts.

Sphingolipid biosynthesis modulates NR2F1/2regulated transcription in human ESC-derived cardiomyocytes

To understand the link between sphingolipid biosynthesis and NR2F1/2-regulated transcription, we used cardiomyocytes differentiated from human embryonic stem cell (hESC) (CM). NR2F2 plays a key role in CM maturation [START_REF] Churko | Defining human cardiac transcription factor hierarchies using integrated single-cell heterogeneity analysis[END_REF]. During CM differentiation, NR2F2 transcription was induced immediately after mesoderm specification concomitantly with the induction of cardiac progenitor factors NKX2-5 and TBX5 (Figures 4A and4B). NR2F1 was also induced with similar kinetics (Figure 4B) and was suggested to partially compensate for the loss of NR2F2 during CM differentiation [START_REF] Schwach | A COUP-TFII human embryonic stem cell reporter line to identify and select atrial cardiomyocytes[END_REF]. Since NR2F1 and NR2F2 share a common ligand, we used CRISPR-Cas9 gene editing to generate single NR2F2 knockout (KO) and NR2F1/2 double knockouts (DKO) hESCs (Figures S4A-S4C) to study the interplay between ligand biosynthesis and NR2F1/2-mediated transcription.

We characterized CM differentiation induced by modulation of the Wnt/b-catenin pathway [START_REF] Lian | Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/beta-catenin signaling under fully defined conditions[END_REF] in WT, KO, and DKO cells. The cellular composition of CM cultures was analyzed by single-cell RNA sequencing (scRNA-seq) at early (day 6) and late (day 19) stages of CM maturation. Cell populations, where identified using the PHATE single-cell analysis pipeline [START_REF] Moon | Visualizing structure and transitions in high-dimensional biological data[END_REF] and segregated according to genotype (Figure 4C). DKO cells contributed little to clusters in the KO group and negligibly to clusters in the WT group. Likewise, the KO cells had limited contribution to clusters of WT or DKO samples (Figures S4D andS4E). We then compared the CM maturation states in the three cultures using previously defined CM cluster classification methods [START_REF] Churko | Defining human cardiac transcription factor hierarchies using integrated single-cell heterogeneity analysis[END_REF]. At day 6, WT cultures contained cells of both intermediate and mature differentiation stages. In contrast, KO cultures contained cells of intermediate stages and DKO cultures predominantly contained cells of early proliferative stages with small numbers of mature-, mid-stage cells, and non-cardiac lineages (Figures 4C, 4D, S4D, andS4E). Mature CM cells were present in all cultures at day 19, albeit in reduced amounts in DKO cultures (Figure 4C).

The scRNA-seq data were validated by morphological examination of WT and DKO cultures. WT cultures consisted of dense sheets of cells with typical CM morphology and exhibited robust ''beating'' by day 9. DKO cultures were less dense, with few small ''beating'' clusters emerging by day 12 (Figure 4E; Videos S1, S2, S3, and S4). Cytometry profiling of the cultures showed a reduction in TNNT2 + CMs in DKO cultures (Figure 4F). qRT-PCR analyses of the stage-specific differentiation markers revealed that while both WT and DKO cells efficiently downregulated the pluripotency marker POU5F1 and induced the mesodermal marker MIXL1, cardiac progenitor specification and maturation was significantly delayed in DKO cultures (Figure 4G). Furthermore, a set of 175 curated NR2F2 ChIP targets [START_REF] Churko | Defining human cardiac transcription factor hierarchies using integrated single-cell heterogeneity analysis[END_REF] were significantly downregulated in DKO cells in comparison with WT cells (Figure 4H). Gene ontology (GO) analyses revealed that NR2F1/2 deletion impacted CM specification program and cellular metabolism (Figure 4I). Day 6 DKO CM cultures exhibited deregulated expression of genes involved in sterol, fatty acid, glycerolipid, phospholipid, and sphingolipid meta-bolism (Figures 4I,S4F,and S4G). Inspection of day 12 NR2F2-KO cells by transmission electron microscopy revealed increased size and numbers of white lipid droplets as well as autophagosomes and lysosomes filled with membranous debris (Figure S4H). Taken together, our findings show that NR2F1/2 modulate CM differentiation and cellular lipid metabolism.

To test the link between sphingolipid biosynthesis and NR2F1/ 2-regulated transcription, we treated WT CM cultures with myriocin, a specific inhibitor of canonical and non-canonical (legend continued on next page) sphingolipid biosynthesis [START_REF] Miyake | Serine palmitoyltransferase is the primary target of a sphingosine-like immunosuppressant, ISP-1/myriocin[END_REF]. Myriocin was administered from day 1 at 1 mM concentration, an amount sufficient to partially suppress NR2F2 reporter activity in HEK-293FT cells without loss of cell viability (Figures S5A andS5B). Bulk RNA-seq analysis of myriocin-and vehicle-treated cultures at day 6 of differentiation identified 1,121 upregulated and 850 downregulated genes. A large fraction of genes up or downregulated by myriocin in WT cells were similarly affected in DKO cells. Importantly, these co-regulated genes were not similarly affected in myriocin-treated DKO cells, suggesting that inhibition of sphingolipid biosynthesis in WT cells phenocopies NR2F2 deletion at the molecular level (Figures 5A and5B). Notably, out of 175 previously identified direct targets of NR2F2 [START_REF] Wu | Atrial identity is determined by a COUP-TFII regulatory network[END_REF], 69 were upregulated and 15 downregulated in day 6 WT cultures when compared with DKO cultures, suggest-ing that NR2F1/2 is an activator during early CM specification (Figure S5C). Key transcriptional regulators of CM lineage specification (GATA4 and NKX2-5), as well as structural proteins of mature CMs (MYH6, MYBPC3, TNNT2, and MYBPC3) were among the upregulated NR2F1/2 targets (Figure S5D). The median expression of activated NR2F2 genes was significantly downregulated in myriocin-treated WT cultures but not in myriocin-treated DKO cultures (Figures 5C, S5C, and S5D). We correlated bulk RNA-seq data from WT, DKO, and myriocintreated cultures with day 6 scRNA-seq data and found that the bulk transcriptome of myriocin-treated cultures exhibited the highest correlation with clusters of immature cells normally found in KO and DKO cultures (Figure 5D). This suggests that inhibition of sphingolipid biosynthesis mimics CM maturation defects arising from genetic deletion of NR2F1/2.

Exogenous or endogenous 1-deoxysphingosine supplementation activates NR2F1/2-regulated transcription in hESC-derived cardiomyocytes Next, we tested the effect of sphingosines on CM differentiation by adding vehicle, D-erythroSO, or 1-deoxySO-14Z to WT or DKO cultures after the completion of mesoderm induction but before the emergence of NKX2-5 + cardiac progenitors (Figure 6A). FACS analyses were performed at day 9, when NR2F1/2 expression peaked. Addition of 1-deoxysphingosine increased the fraction of TNNT2+ cells in WT cultures (Figures 6B and6C) and modulated the expression of 265/464 genes by >2-fold (increases/decreases) when compared with vehicletreated cultures (Figures 6D and6E). Upregulated gene list was enriched for gene ontologies associated with CM matura-tion, while downregulated genes were enriched for secreted and membrane-bound proteins (Figure 6D). Notably, 160 out of 265 genes (~60%) upregulated in 1-deoxysphingosine-treated WT cultures were also NR2F1/2-activated genes. Among the genes upregulated by 1-deoxysphingosine were 33 NR2F2 ChIP targets including the key CM-specific targets NKX2-5, MYBPC3, TNNT2, and MYH6 (Figures S6A andS6B). In contrast, genes downregulated in 1-deoxysphingosine-treated WT cul-tures overlapped poorly with NR2F1/2-repressed genes. Only 20 out of 464 downregulated genes (~4%) were NR2F1/2repressed genes. These numbers suggest that 1-deoxysphingosine activated CM differentiation and maturation programs by modulating NR2F1/2 activity but inhibited expression of secretory genes independently of NR2F1/2 (Figure S6A).

The canonical sphingosine, D-erythroSO, also influenced CM differentiation and maturation, but gene expression changes in WT cultures treated with this sphingosine were smaller in magnitude than those induced by 1-deoxysphingosine (Figures 6B, 6C, 6E, S6A, and S6B). This finding agrees with our binding and activity data, which showed that D-erythroSO bound to NR2F2 at nanomolar concentrations but was a less efficient agonist when compared with 1-deoxySO-14Z (Figure 2). Impor-tantly, treatment of DKO cultures with either 1-deoxySO-14Z or D-erythroSO did not increase the fraction of TNNT2+ cells and minimally affected gene expression (Figures 6B, 6C, 6E, S6A, and S6B). Thus, our data indicate that the uptake of exogenous 1-deoxysphingosine by differentiating CMs increased the numbers of mature CMs in culture at least in part by modulating NR2F1/2-dependent transcriptional networks.

Next, we investigated whether autocrine production of 1-deoxysphingosines could modulate NR2F1/2-dependent transcription during CM differentiation. Levels of 1-deoxysphingosines and D-erythro-sphingosine were regulated during differentiation (Figure 6H). In day 7 CM cultures, levels of 1-deoxysphingosines reached 0.68 pmol/10 6 cells, which corresponds to 680 nM concentration, above the Kd measured by in vitro binding assays. To further increase the levels of 1-deoxysphingosines in differentiating CMs, we took advantage of the mutant version of the SPTLC1 enzyme (SPTLC1 C133W ), which is associated with hereditary sensory and autonomic neuropathy type 1 (HSAN1) and increases production of 1-deoxysphingosines [START_REF] Garofalo | Oral L-serine supplementation reduces production of neurotoxic deoxysphingolipids in mice and humans with hereditary sensory autonomic neuropathy type 1[END_REF]. We used TALEN-mediated gene targeting to integrate Doxycycline (Dox)-inducible FLAG-SPTLC1 C133W (hereafter HSAN) and rtTA transgenes into a constitutively active AAVS1 locus of WT and DKO hESCs (Figures 6F andS6C). Transgene expression was induced by adding Dox to cultures at day 3, thus allowing a direct comparison of the same cell clone with or without induction of HSAN expression (Figure 6G). amino acids, fatty acids, ceramides, and sphingosine-1-phosphate (Figures 6H, S6D, andS6E). Induction of HSAN did decrease the levels of palmitic acid, a precursor of 1-deoxysphingosine, and other palmitic acid derivatives, such as palmitoyl-carnitine (Figure S6E). Thus, the introduction of the HSAN mutant specifically targeted 1-deoxysphingosine production and did not affect the general metabolism of cells.

Expression of HSAN increased the fraction of TNNT2+ cells in WT HSAN cultures, similar to what was observed with 1-deoxysphingosine supplemented medium (Figures 6I and6J). The bulk transcriptome of untreated day 7 WT HSAN cultures correlated with early immature and mature CM clusters in single-cell PHATE embedding. In contrast, Dox-treated WT HSAN cultures were depleted of immature CMs, suggesting that elevated 1-deoxysphingosine levels accelerate CM maturation (Figure 6K). Transcriptome analyses of DOX-treated cultures at day 7 and 9 identified 301/392 genes with more than 2-fold increases/decreases in expression levels. Dox-treated WT HSAN cultures and WT cultures supplemented with 1-deoxysphingosine exhibited similar profiles of up-and downregulated genes, including those of CM-specific NR2F2 targets (Figures 6L, S6B, andS6F). Reflective of these similarities, the enriched gene ontologies of Dox-treated WT HSAN cultures mirrored those of 1-deoxysphingosine treated WT samples. Specifically, genes associated with CM differentiation or maturation were overrepresented in the upregulated gene list, whereas secreted and membrane proteins were overrepresented in the downregulated gene list (Figure S6G). Importantly, HSAN expression in differentiating DKO CMs failed to increase the fraction of TNNT2+ cells and had a minimal effect on overall gene expression (Figures 6I, 6J, 6L, and S6F).

The negligible overlap of genes downregulated by HSAN/1deoxysphingosine and NR2F1/2-repression suggests that 1deoxysphingosine could have both NR2F1/2 dependent and independent effects on CM differentiation (Figure S6A). Genes downregulated by HSAN/1-deoxysphingosine were enriched in secreted proteins not typically expressed by CMs (Fig-ure S6G). Thus, 1-deoxysphingosines could be suppressing differentiation of mesoderm-derived non-CM cells. To test this hypothesis, we calculated median expression levels of genes up-or downregulated by HSAN and/or 1-deoxysphingo-sines in different cell types of the human heart using single-cell data from the Human Protein Atlas Project [START_REF] Uhle´ N | Proteomics. Tissue-based map of the human proteome[END_REF]. Indeed, expression of upregulated genes was higher in CM clusters, whereas downregulated genes were associated with fibroblasts, endothelial, smooth muscle, and immune cells (Figure 6M).

Our data collectively support the conclusion that 1-deoxysphingosines are developmentally regulated metabolites that activate NR2F1/2-dependent CM differentiation and maturation programs and inhibit differentiation of non-CM lineages independently of NR2F1/2.

DISCUSSION

We identified 1-deoxysphingolipids as modulators of NR2F1/2 using an approach consisting of two sequential screens. In the first screen, a comprehensive library of metabolic enzymes was overexpressed to identify metabolic pathways that modulate NR2F1/2 reporter activity in mammalian cells. For the second filter, substrates and products of these pathways were evaluated for their ability to activate NR2F1/2-dependent transcription in D. melanogaster cells, resulting in the identification of 1-deoxysphingolipids as NR2F1/2 modulators. Our approach is unbiased and makes no assumption of ligand structure and could be adapted to identify ligands of other orphan NHRs.

Data from the overexpression screen suggest that in addition to sphingolipid biosynthesis, which directly controls ligand production, three additional metabolic pathways, including amino acid, fatty acid, and iron-sulfur cluster metabolism, may indirectly regulate ligand synthesis. Indeed, iron depletion affects sphingolipid biosynthesis in yeast (Lee et al., 2012b;Sha-koury-Elizeh et al., 2010), and fatty acid metabolism provides precursors for sphingolipid biosynthesis [START_REF] Merrill | Kinetics of long-chain (sphingoid) base biosynthesis in intact LM cells: effects of varying the extracellular concentrations of serine and fatty acid precursors of this pathway[END_REF]. Amino acid metabolism controls the availability of serine, alanine, and glycine for sphingolipid biosynthesis [START_REF] Esaki | L-serine deficiency elicits intracellular accumulation of cytotoxic deoxysphingolipids and lipid body formation[END_REF]. Furthermore, the overexpression screen suggests that the components of the SPTLC complex play different roles in ligand synthesis. The SPTLC complex has three enzymatic subunits: SPTLC1, SPTLC2, or SPTLC3. SPTLC1 is the common component of the complex, while SPTLC2 and SPTLC3 modulate its specificity for fatty acids [START_REF] Lone | Subunit composition of the mammalian serine-palmitoyltransferase defines the spectrum of straight and methyl-branched long-chain bases[END_REF][START_REF] Russo | Myristatederived d16:0 sphingolipids constitute a cardiac sphingolipid pool with distinct synthetic routes and functional properties[END_REF]. Complexes with SPTLC1-SPTLC2 subunits generate sphingolipids with 18, 19, and 20 carbon bases [START_REF] Lone | Subunit composition of the mammalian serine-palmitoyltransferase defines the spectrum of straight and methyl-branched long-chain bases[END_REF], which agrees with our finding that SPTLC2 is the only component of sphingolipid biosynthesis that increased NR2F1/2 transcriptional activity.

The Kd range of 10-100 nM for in vitro binding and cell-based assays fits with the concentration ranges of 1-deoxysphingosines found in lymphatic endothelial cell lines and CM cultures and suggests that these ligands could be physiological. In contrast, canonical sphingolipids such as D-erythroSO bind to NR2F1/2-LBD with an affinity slightly lower than that of 1-deoxysphingosines but are either weak agonists of NR2F1/2 or inactive. Such a discrepancy between binding and activity could be due to the low availability of free canonical sphingosines, which are precursors for all other cellular sphingolipids [START_REF] Merrill | Sphingolipid and glycosphingolipid metabolic pathways in the era of sphingolipidomics[END_REF].

Unexpectedly, while mammalian cells exhibit NR2F1/2 transcriptional activity in the absence of added ligand, no basal Svp or NR2F1/2 transcriptional activity was observed in D. melanogaster S2 cells. This finding is surprising as mammalian and fly NR2F1/2 homologs share 92% identity [START_REF] Tsai | Chick ovalbumin upstream promoter-transcription factors (COUP-TFs): coming of age[END_REF], and most amino acid residues facing the ligand-binding pocket are conserved, suggesting that COUP-TFs of vertebrates and insects are under strong evolutionary pressure to bind the same ligand. Based on the lack of activity in fly tissues and cell lines, previous studies proposed that Svp is a repressor [START_REF] Palanker | Dynamic regulation of Drosophila nuclear receptor activity in vivo[END_REF]. Our findings, however, suggest that Svp is transcriptionally active in fly cells in the presence of 1-deoxysphingosines. The most plausible explanation for this discrepancy is that the ligand is not produced by the insect cell line utilized in our study but is generated by the few Svp-expressing cells in vivo. Most mammalian sphingolipids, including the NR2F1/2 ligands identified here, have sphingoid bases with 18 to 20 carbons, while flies preferentially make C14 and C16 sphingolipids [START_REF] Fyrst | Characterization of free endogenous C14 and C16 sphingoid bases from Drosophila melanogaster[END_REF][START_REF] Fyrst | Identification and characterization by electrospray mass spectrometry of endogenous Drosophila sphingadienes[END_REF]. Despite this preference, small amounts of mammalian-like C18 and C20 sphingolipids are made in a tissuespecific manner in D. melanogaster [START_REF] Fyrst | Characterization of free endogenous C14 and C16 sphingoid bases from Drosophila melanogaster[END_REF]) and C18 sphingolipids with 0, 1, or 2 double bonds are enriched in Sply mutant flies, which are deficient in sphingolipid catabolism [START_REF] Narayanaswamy | Lipidomic "deep profiling": an enhanced workflow to reveal new molecular species of signaling lipids[END_REF]. The identification of conditions for tissue-specific production of C18 and C20 deoxysphingosines in flies could shed light on the role of Svp in the development of D. melanogaster.

Our data support the conclusion that 1-deoxysphingolipids are activators of NR2F1/2-mediated transcription. However, others suggested that NR2F1/2 may also function as transcrip-tional repressors [START_REF] Churko | Defining human cardiac transcription factor hierarchies using integrated single-cell heterogeneity analysis[END_REF][START_REF] Wu | Atrial identity is determined by a COUP-TFII regulatory network[END_REF]. Given that 1deoxysphingosines can be further modified by CYP450 enzymes to generate new metabolites [START_REF] Alecu | Cytotoxic 1-deoxysphingolipids are metabolized by a cytochrome P450-dependent pathway[END_REF], it would be important to test whether these derivatives could func-tion as NR2F1/2 agonists or antagonists. Indeed, in the case of FXR, a receptor for bile acids, natural agonists [START_REF] Makishima | Identification of a nuclear receptor for bile acids[END_REF][START_REF] Parks | Bile acids: natural ligands for an orphan nuclear receptor[END_REF] and antagonists [START_REF] Sayin | Gut microbiota regulates bile acid metabolism by reducing the levels of tauro-beta-muricholic acid, a naturally occurring FXR antagonist[END_REF] have been found. These show a range of biological activ-ities from the regulation of intestinal stem cells to facilitating tu-mor development [START_REF] Fu | FXR regulates intestinal cancer stem cell proliferation[END_REF].

The modulation of CM differentiation and maturation by 1-deoxysphingosines is largely NR2F1/2 dependent, but the inhibition of non-CM differentiation programs appears to engage mechanisms that are independent of NR2F1/2. Indeed, similar effects have been reported for other NHR ligands. For example, estrogen, a ligand for ERa and ERb, also activates the G-proteincoupled receptor (GPCR) GPER [START_REF] Revankar | A transmembrane intracellular estrogen receptor mediates rapid cell signaling[END_REF]. Similarly, bile acids bind to both FXR [START_REF] Makishima | Identification of a nuclear receptor for bile acids[END_REF][START_REF] Parks | Bile acids: natural ligands for an orphan nuclear receptor[END_REF] and the GPCR TGR5 [START_REF] Kawamata | A G proteincoupled receptor responsive to bile acids[END_REF]. The mechanism behind NR2F1/2-independent functions of 1-deoxysphingosines remains to be elucidated.

Previous studies documented the cytotoxic nature of 1-deoxysphingosines accumulating in tissues and causing HSAN1 and macular degeneration [START_REF] Eichler | Overexpression of the wild-type SPT1 subunit lowers desoxysphingolipid levels and rescues the phenotype of HSAN1[END_REF][START_REF] Gantner | Serine and lipid metabolism in macular disease and peripheral neuropathy[END_REF][START_REF] Penno | Hereditary sensory neuropathy type 1 is caused by the accumulation of two neurotoxic sphingolipids[END_REF][START_REF] Rotthier | Mutations in the SPTLC2 subunit of serine palmitoyltransferase cause hereditary sensory and autonomic neuropathy type I[END_REF]. Our findings, however, suggest an alternative model where overproduction of 1-deoxysphingosines in disease engages normal gene regulatory networks induced by the autocrine or hormone-like function of 1-deoxysphingosine. An analogous situation is observed in autosomal aldosteronism, a disease that is characterized by hypertension and caused by mutations that induce the ectopic expression of aldosterone synthase, leading to increased levels of mineralocorticoids and chronic stimulation of the mineralocorticoid receptor [START_REF] Lifton | A chimaeric 11 beta-hydroxylase/aldosterone synthase gene causes glucocorticoid-remediable aldosteronism and human hypertension[END_REF]. So far, the link between the pathology induced by 1-deoxysphingosines and NR2F1/2 transcriptional activity has not been established. Our findings highlight the importance of identifying the natural ligands of NHRs to enhance our understanding of human physiology.

Limitations of the study

Sphingolipids elicit concentration dependent effects in aqueous solutions [START_REF] Sasaki | pH dependence of sphingosine aggregation[END_REF]. This limits the bioavailability and working concentrations of these lipids in cell-based assays and in vitro binding assays. Furthermore, ligand-receptor studies in vivo were limited by time constrains as primary LECs, and embryos ex vivo only survive for short periods after deletion of Sptlc2. The small amounts of tissues and cells in embryos limited our ability to measure the concentrations of 1-deoxy-sphingosines in embryonic tissues that express NR2F1/2. Thus, embryo phenotypes are mostly correlative. While the use of in vitro CM differentiation model provided a solution for some of these limitations, methods to study the receptor-ligand relationship in vivo should provide further support for our findings.

STAR+METHODS

Detailed methods are provided in the online version of this paper and include the following: Reporter assays in mammalian cells Mammalian reporter assays were performed as previously described [START_REF] Santori | Identification of natural RORgamma ligands that regulate the development of lymphoid cells[END_REF]. One day prior to transfection, HEK-293FT cells were plated at density of 5 x 10 4 cells per well in flat bottom 96 well plates in regular growth medium. Before transfection, medium was replaced with antibiotic-free DMEM supplemented with 1 % FBS. DNA mix consisted of 20-100 pg of Renilla normalization plasmid, 5-10 ng of reporter plasmid and 10-20 ng of NHR plasmid per well of 96 well plate. The DNA was diluted in 10 ml of OptiMEM media (Thermo Fisher) supplemented with 60 mg/mL polyethyleneimine (PEI) (Polyscience) and, after 10 min incubation at RT, added to the well. Transfected cells were maintained at 37 o C and 5% CO2 for 24 h, medium was replaced for regular growth medium and cells cultured for another 24 h. Luciferase activity was detected using the dual luciferase reporter kit (Promega).

d KEY RESOURCES

Reporter assays in insect cells

One day prior to transfection, S2 cells were plated at a density of 1.5 -2.5 x 10 5 cells per well in flat bottom 96 well plates in growth medium. DNA mixture consisted of 2-5 ng of Renilla normalization plasmid, 10-25 ng of reporter plasmid and 20-50 ng GAL4-NHR fusion plasmid per of 96 well plate. DNA was diluted in 75ml of growth media containing 0.5 ml of Cellfectin II (Invitrogen) and added to the cells. Cells were maintained for 48 h and luciferase activity was detected using the dual luciferase reporter kit (Promega).

Cell-based reporter assays for sphingolipids in mammalian cells Assays were performed in HEK-293FT or Hela cells. Cells were transfected with synthetic or native reporter, appropriate NHR plasmid and a Renilla normalization plasmid as described above. Sphingolipid-BSA complexes were diluted in media containing 1% FBS and added to the transfected cells 24 h after transfection. Vehicle-BSA complexes were used as controls. Cells were main-tained for another 24 h and luciferase activity was detected using the dual luciferase reporter kit (Promega).

Immunoprecipitation and lipidomics

Stable clones of HEK-293FT cells expressing 3xFLAG-NR2F1(LBD) or 3xFLAG-RARA(LBD) were established as follows. LBDs were cloned into pcDNA3.1 vector (Invitrogen). 300 ng of plasmid were diluted in 300 ml of serum-free DMEM medium containing 60 mg/mL PEI. DNA-Reagent mixture was incubated for 10 min at room temperature and added to the cells growing in a well of 6 well plate. After hygromycin (Invitrogen) selection the resistant cells were cloned and screened for expression of FLAG-LBD protein by FACS with Anti-FLAG-Alexa Fluor 647 or anti-FLAG FITC antibodies at 1:200-1:400 dilutions. The selected clones were grown as high-density suspension cultures in Expi293 medium (Thermo Fisher) supplemented with 500 nM all-trans-retinoic acid at 37 o C in 10% CO2.

Nuclear extracts were prepared as follows. 1 x 10 8 cells were washed in PBS and resuspended in hypotonic buffer (20 nM Hepes pH 7.9, 1.5 mM MgCl, 10 mM KCl, 0.1 mM PMSF, 1 mM DTT, cOmplete protease inhibitor cocktail (Roche). The cells-to-buffer ratio was adjusted to 1 x 10 8 cells/mL and kept constant for the entire procedure. After 15-20 min incubation on ice, cells where homogenized 6-7 times with a Dounce homogenizer, transferred into Eppendorf tubes and centrifuged at 15,000 x g for 15 min. The pellet was resuspended in buffer containing 20 mM Hepes pH 7.9, 1.5 mM MgCl2, 0.2 mM EDTA, 25 % Glycerol, 420 mM KCl, 0.1 mM PMSF, 1 mM DTT and protease inhibitor cocktail. The pellet was homogenized 7-8 times with a Dounce homogenizer and placed on rotator shaker at 4

o C for 1-2 h. After incubation, the extract was centrifuged for 30 min at 15,000 xg. Supernatant was transferred into a new tube and incubated with 40 ml of anti-FLAG M2 magnetic beads (Sigma) on rotator shaker for 1 h at 4 o C. After incubation, beads were washed 4 times with buffer containing 50 mM Tris HCl pH 7.4, 100 mM NaCl and protease inhibitor cocktail. FLAG-LBDs were eluted by 30 min incubation with 150 ng/mL of 3xFLAG peptide (Sigma) at 4 o C. An aliquot corresponding to 1/20 of the IP was analyzed by Western blotting to confirm the presence of FLAG-LBD. The remaining IP material was processed following 2 treatment schemes -scheme 1 to isolate specifically hydrophobic ligands and scheme 2 to isolate ligands that are potentially hydrophilic. In the scheme 1, a lipid extraction was performed using 2:1 chloroformmethanol mixture following the Folch procedure [START_REF] Folch | A simple method for the isolation and purification of total lipides from animal tissues[END_REF]. The organic phase was collected and dried under N2 stream. Metabolite displacement experiments were performed with samples treated under scheme 1 by adding 5 mM of synthetic deuterated 1-deoxysphingosine-14Z-d [START_REF] Saied | Synthesis and characterization of novel atypical sphingoid bases[END_REF] to samples of both NR2F1-LBD and RARA-LBD during the immunoprecipitation procedure. The scheme 2 was used in general immunoprecipitation procedures to identify potential hydrophilic ligands bound to NR2F1-LBD but not RAR-LBD in C18+ and Hilic-modes, 3% MS grade acetic acid (Sigma) was added to the immunoprecipitates followed by addition of 40% MS grade methanol (Sigma) and 40% MS grade acetonitrile (Thermo Fisher) so that the ratio of of IP: MEOH:ACN is 20:40:40. This mixture was vortexed for 30 seconds and incubated overnight at -20 0 C. The next day samples were centrifuged at 15,000 x g for 15 min at 4 o C. The supernatant was collected and vacuum-dried. The protein pellet was frozen for further analysis. The tubes containing the dried metabolites and protein pellets were maintained at -80 o C until processing.

3-5 IP repeats for each set of experimental conditions were processed simultaneously by LC-MS. We first tested whether we could identify masses corresponding to derivatives of all-trans-retinoic acid in RARA samples. We found a 14.76 fold enrichment of a [M+H-H2O]+ adduct of exact mass m/z 283.2059 and mean retention time 27.5459 corresponding to retinoic acid in RARA but not NR2F1 samples (p value 0.01) in C18+ mode. To identify all possible metabolites bound to NR2F1 LBD we scanned all samples by C18+ and Hilic negative modes. Negative mode analysis was performed using a Phenomenex Luna-NH2 column (1.0x150 mm, 100A, 3 mm) at the flowrate of 50 ml/min, mobile phase A= 20mM NH4OAc/40 mM NH4OH and mobile phase B=5:95 H2O:ACN. The injection volume was 8 ml, gradient: T0: 0:100 T5:0:100 T45:100:0 T60:100:0, stop time= 60 min, 9 min post time to re-equilibrate the column. MS settings were: scanning m/z=70-1100, dry gas flowrate: 8L/min, nebulizer pressure 8 psi, dry gas temperature =325 0 C. Positive mode analysis used a C18 1.0x150mm column at a flowrate of 50 ml/min, mobile phase A= H2O/0.1% formic acid and mobile phase B=ACN/ 0.1% formic acid. The injection volume was 8 ml, gradient: T0: 95:5 T5:95:5 T50:5:95 T60:5:95 stop time= 61 min, 9 min post time to re-equilibrate the column. MS settings were: scanning m/z=70-1100, dry gas flowrate: 8L/min, nebulizer pressure 8 psi, dry gas temperature =325 0 C, acquisition in positive mode. Final data from the LC-MS run was analyzed using XCMS package [START_REF] Smith | XCMS: processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification[END_REF]. After XCMS analysis that included alignment, elimination of isotypic peaks we selected our data search parameters: Fold change > 3, maximum intensity > 30-50000, p value < 0.05. The files were inspected manually to confirm and check for accuracy or adjust for missing peaks.

Sphingolipidome

of TIME cells Cells were harvested, counted, and washed twice with PBS pH7.4. The nuclear extract was prepared by resuspending the cell pellet after the last wash with1 mL lysis buffer (10 mM Hepes pH7.6, 10 mM KCl, 1.5 mM MgCl2, 5% Glycerol, 0.2 mM PMSF, 1 mM DTT and cOmplete protease inhibitor cocktail (Roche)) and incubated on ice for 7-20 min. Cells were vortexed and spun at 21000 xg for 10 min at 4

o C. The supernatant is the cytosol. The pellet (nuclear fraction) was washed twice in lysis buffer at 21000 xg for 10 min at o C. final pellet was resuspended in 1 mL lysis buffer. Purity of each fraction was tested with anti-H3 antibodies by WB and protein quantified by Bradford assay. The cytosolic and nuclear fractions were stored at -80 0 C until processed. For sphingolipid analysis 100 ml cytosolic or nuclear fraction were further diluted with 500 ml of methanol and spiked with deuterated sphingosines as internal standards. Lipids were extracted in a shaker for 1 h at 37 o C. The mix was centrifuged at 15,000xg and supernatant transferred to a new tube. Lipids were hydrolyzed with 75 ml of methanolic HCL [1N HCL/10M water:methanol] and incubated for 16 h at 65 o C. The acid was neutralized with 100 ml of ammonium hydroxide (2N) plus 500 ml water. The sample was vortexed, and the lower phase collected and dried under N2 for analysis by MS.

Targeted metabolomics

Cell samples were collected and frozen at -80 0 C until further use. For targeted metabolomic analysis, 400 mL of 2:2:1 mix of MeO-H:ACN:H2O was added to the cell pellets. The cell suspension was vortexed, placed in liquid N2 for 1min, thawed, and sonicated for 15 min. This process was repeated twice. Samples were centrifuged to remove insoluble material and the supernatant was harvested and dried in Speedvac at 10 o C. 100 mL of MeOH was added, samples were sonicated for 5 min on ice, centrifuged, and transferred to autosampler vials. The insoluble protein material was used to determine protein concentration by Bradford assay for sample normalization.

Amino acids

Samples were run on an Agilent 6495 QqQ with jet stream source coupled to an Agilent 1290 liquid chromatography stack with an Agilent HILIC-Z column, 2.1 x 100mm. The mobile phase was composed of A= 20mM ammonium formate, pH=3, and B= 90:10 ACN/ H2O 20mM ammonium formate pH = 3. The gradient started at 100% B decreasing to 70% B in 12min and was held there for 3min (12 min -15 min) before a 7 min post-run for column re-equilibration. The flowrate was set to 400 mL/min and the sample injection volume was 5 mL. Operating in positive ion mode, the source conditions were as follows: drying gas temperature set to 290 o C with a flowrate of 11 L/min, the sheath gas temp was 400 o C with a sheath gas flowrate of 11 L/min, the neb pressure set to 45 psi, cap voltage set to 2500V and nozzle voltage set to 0V. Data were processed using Agilent MassHunter Quantitative analysis software with calibration ranges of 2 to 100 mM prepared using authentic synthetic standards.

Sphingolipids

Samples were run on Waters Xevo TQ-S micro triple quadrupole mass spectrometer. The mobile phase was composed of phase A= 1mM ammonium formate/0.1% formic acid, and phase B= 90:10 IPA/ACN 1 mM ammonium formate/0.1% formic acid. The column used was a Waters Acquity UPLC BEH-C18 2.1x100 mm. The gradient started at 80% A, holding for 1.5 min then ramping to 97% B at T=18 min holding for 4 min. There was a 4 min re-equilibration step. The flowrate was set to 200 mL/min and the sample injection volume was 2 mL. Operating in positive ion mode, the source conditions were as follows: capillary voltage =1500 V, desolvation flow = 600 L/hr, desolvation temp = 350 o C and cone flow = 50 L/hr. Data were processed using Waters TargetLynx software. For standard curves we used authentic synthetic standards and calibration levels ranges from 50 nM -10,000 nM with the exception of 1-deoxysphingosine-14Z, which ranged from 5 nM -350 nM.

Fatty acids

Samples were run on a Bruker Impact II Q-TOF coupled to an Agilent 1200 LC stack with an Agilent Zorbax 300SB-C18 column, 150 3 0.5 mm, mobile phase A = H2O/0.1% formic acid and mobile phase B = ACN/0.1% formic acid. The gradient started at 95% A, held there for 3 min, then ramped to 97% B at T= 21 holding for 4 min, followed by a 7 min re-equilibration step. The flowrate was set to 20 mL/min and the sample injection volume was 2 mL. Operating in negative ion mode, the source conditions were as follows: end plate offset = 500 V, capillary = 4,000 V, nebulizer pressure = 20 psi, drying gas flowrate = 7L/min at 180 o C. Sample information was taken from Bruker Data analysis and entered into Microsoft Excel where a calibration curve was generated, and sample concentrations calculated.

Purification of bacterial NR2F1 and NR2F2 LBDs

Mouse NR2F1 LBD (NP_005645.1, aa 184-423) and NR2F2 LBD (NP_033827.2, aa 176-414) were amplified by PCR and cloned into a modified pET45b(+) vector containing a N-terminal 6xHis-GST tag which improved protein solubility. Plasmids were transformed into E. coli T7 Express competent bacteria (NEB). Bacterial cultures were grown at 37 o C until the cultures reached an OD600 ~0.8. Protein expression was induced with 0.25-0.5 mM isopropyl-b-D-thiogalactopyranoside (IPTG)(American Bio) at 16 o C for 18 h. Cells were harvested by centrifugation at 15,000xg and lysed by passing through the French express cell disruptor (1-2 cycles of 500 psi followed by 3-5 cycles of 1000 psi) in lysis buffer (50 mM Tris•HCl pH 8.0, 500 mM NaCl, 5% Glycerol, 1 mM EDTA, 0.5 mM TCEP (Sigma) plus protease inhibitor cocktail (Roche)). When 6xHis tag was used for purification, EDTA was omitted from the lysis buffer.

Cell lysate was centrifuged at 20,000xg for 1 h and cleared supernatant was incubated with either Ni-NTA column (Qiagen) or Glutathione Sepharose-4B (GE Healthcare) pre-equilibrated with the lysis buffer. The 6xHis-tag purifications were extensively washed with wash buffer (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 20 mM imidazole, 5% glycerol with 0.5 mM TCEP) and eluted in same buffer with 200-500 mM imidazole. After washing with the lysis buffer, GST purifications were eluted with elution buffer (50 mM Tris•HCl pH 8.0, 250 mM NaCl, 10 mM reduced Glutathione (Sigma)). The eluate was pooled, concentrated, and further purified by SEC650 column on FPLC (Bio-Rad) in FPLC buffer (20 mM Tris$HCl pH 8.0, 250 mM NaCl) to produce the monomeric form of the receptor.

Purification of mammalian LBDs

We removed the first 22 amino acids and last 10 amino acids of NR2F2-LBD to increase protein stability. The truncated LBD was fused to a N-terminal His-tag and cloned into the pcDNA3.1 vector. A factor Xa site was introduced to allow for cleavage of the Histag. Protein was produced by transfecting suspension HEK293-FT cultures growing in FreeStlye F17 expression medium (Thermo Fisher). Transfections were performed with 200 mg of plasmid DNA complexed with PEI (0.5 mg DNA/1.5 mg PEI) prepared in FreeStlye F17 expression medium.

Fluorescence polarization assay (FPA) Fluorescein labelled co-activator and co-repressor peptides FL-SMRT-ID2, FL-SMRT-ID1, FL-PGC1A, FL-SRC2-1, FL-SRC2-2 and FL-SRC2-3 (Life Technologies) were first tested for specificity by incubating 5 nM of each peptide with 100 nM of the apo (empty) receptor LBDs of NR2F1/NR2F2/NR2F6 plus 5 mM vehicle to test whether peptide can bind to empty receptor. Next, we tested whether unrelated compounds could promote non-specific peptide binding by adding 5 mM vehicle, D-erythro-sphingosine, 7-dehydrocholesterol or 10-Nitrooleic acid. In these tests, SRC2-2 was determined to have the lowest background on Apo receptor or receptor incubated with unrelated compounds. Thus, the fluorescein conjugated SRC2-2 peptide was used in all FPA assays presented in the manuscript. FPAs were performed by incubating 5 nM of fluorescein-labeled SRC2-2 peptide with purified NR2F1 or NR2F2 LBDs in its apo-form or saturated by each individual compound for 1 h at room temperature. The proteins were assayed as serial dilutions in the binding buffer (20 mM Tris-HCl (pH 8.0), 200 mM NaCl, 10 mM DTT), the metabolites were used at 5 mM of concentration. The FPA measurements were carried out in triplicates using black 96-well plates or 384 well plates (Thermo Fisher). FPA signals were measured on the Spectramax M5 reader (Molecular Devices). The data processing and the Kd value calculations were performed using GraphPad Prism 5 (Graphpad).

Thermal shift assays using recombinant NR2F2-LBD Thermal shift assays were performed using SYPRO fluorescent dye [START_REF] Lo | Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery[END_REF]. 6xHis-tagged NR2F2-LBD was produced by overexpression in HEK293 cells and purified with a Nickel NTA chromatography column as described above. SYPRO Orange (Thermo Fisher) was diluted in H2O, from 5,000x commercial stock to 50x working stock. 12.5 mM working stock of NR2F2-LBD was prepared in elution buffer (50 mM Tris pH 8.5, 500 mM NaCl, 5% glycerol). The following metabolites were tested: 1-deox-ySO-14Z, D-erythro-sphingosine, Sphinga-4,14-diene, 1-desoxymSA and vehicle. The metabolites were prepared as a 125 mM working stock in ethanol. Reaction mixture was as follows: 10mL protein (5 mM final concentration), 1mL metabolite (5 mM final concentration), and 11.5 mL elution buffer. The reaction mixture without SYPRO was incubated at 4 o C for 30 min to allow for metabolite binding. After incubation, 2.5 mL of 50x SYPRO Orange stock was added to the reaction mixture to obtain final reaction concentration of 5X SYPRO Orange. Reaction mixtures were loaded, in sextuplicate, into Bio-Rad 96-well qPCR plates. Melting curves were generated using the Bio-Rad CFX Real-Time PCR system according the manufacturer's instructions.

Surface plasmon resonance (SPR) SPR was measured using the T100 system (Biacore). Recombinant 6xHis-tagged NR2F2-LBD was produced in mammalian cells as described above. The protein was non-covalently bound to the nickel NTA sensor chip (Series S) (GE Healthcare Life Sciences) with an RU of 1,000 using the running buffer HBS-P+ (10 mM Hepes pH 7.4, 150 mM NaCl, 0.05% surfactant P20) (Fisher Scientific). For metabolite capture and regeneration, we used HBS-P+ as running buffer and the following program: surface activation 0.5 mM NiCl2 with a flow rate of 10 mL/min, contact time 90 sec and washing time 60 sec. Protein was captured from 20 mg/mL solution at a flow rate of 10 m L/min for 500 sec. After metabolite binding, the chip was regenerated twice, once with 350 mM EDTA with a flow rate of 30 mL/ min, contact time 60 sec and washing time 60 sec, and a second regeneration with 10 mM Glycine pH2.5 in HBS-P+ buffer with a flow rate of 30 mL/min, contact time 60 sec and washing time 60 sec. For binding assessment, measurements were run using the HBS-P+ buffer with a flow rate of 30 mL/min, association time 180 sec, dissociation time 600 sec and stabilization time of 10 sec. Metabolites were prepared in HBS-P+ buffer as serial dilutions of 800 nM, 600 nM, 400 nM, 200 nM, 100 nM, 50 nM and 0 nM. The 0 nM concentration was used to define the blank for background subtraction of the sensorgrams. Each dilution was run and then the two regeneration cycles performed before the next dilution was tested. Three metabolites were tested: sphinga-4,14-dienine, 1-deoxySO and D-erythroSO.

Conditional deletion of Sptlc2 and Nr2f2 in mice Sptlc2 f/f and Nr2f2 f/f mice were crossed with Cdh5 CerERT2 or Prox1 CreERT2 mice to obtain Sptlc2 f/+ Cdh5 CerERT2/+ , Nr2f2 f/+ Cdh5 CerERT2/+ , Sptlc2 f/+ Prox1 CerERT2/+ and Nr2f2 f/+ Prox1 CerERT2/+ animals. For genotyping of young animals, genomic DNA was extracted from the ears or tails. Embryo genotyping was performed using extraembryonic tissues (placenta, amnion) or embryonic tails. DNA extractions were performed using the HotStart mouse genotyping kit (Kapa Biosystems) and PCR were performed using the GoTaq green master mix (Promega) using the primers listed in

Deletion efficiency after tamoxifen injection

The efficiency of deletion after tamoxifen injection was estimated using hearts and tails of E15.5 embryos or sorted Prox1+ cells. To obtain Prox1+ cells, embryos were homogenized and digested in tissue digestion buffer (1%FCS in DMEM, 0.5mg/mL Collagenase D (Sigma), 40mg/mL DNase I(Sigma)) for 1 h in a shaker at 90 rpm at 37 o C. The digested cells were filtered through a 100 mm cell strainer (Fisher Schientific) and centrifuged at 700xg for 5 min. The cells were fixed using fixation/permeabilization solution (Thermo Fisher) according to the manufacturer's instructions. Fixed and permeabilized cells were incubated with anti-mouse CD16/CD32 (Bio Cell) at 4 o C for 15 min in the dark, then incubated for 1 h with AF405 labelled anti-mouse Prox1 antibody (Novus Biologicals) at 4 o C for 1 hour in the dark. Cells were washed, resuspended and Prox1 positive cells were sorted using the BD Aria II sorter. DNA was extracted as described above. Quantitation was performed by PCR using two sets of primers listed in Table S1, Primers-PCR deletion efficiency. Set 1 amplifies a fragment of 199 bp in the floxed region of Sptlc2 deleted by Cre recombinase and Set 2 amplifies a 200 bp region in the non-deleted exon2 of the Sptlc2 gene. Control primer set was used to normalize DNA quantities across the samples. PCR products were resolved in 1% agarose gels and imaged. Band intensities were obtained with ImageJ and normalized to the intensity of the control PCR products.

Embryo processing, staining, and imaging For time mated pregnancies, mating of 6-12 weeks old Sptlc2 f/f Prox1 CreERT2 or Sptlc2 f/f Cdh5-CreERT2 males and Sptlc2 f/f or Sptlc2 f/+ females was performed as follows. Embryonic day 0 was defined as the day males and females were placed together. At day 10, pregnant females were injected with 100 mL of 10 mg/mL of Tamoxifen (Sigma) dissolved in Sunflower seed oil (Sigma). Embryos were collected at days E13.5 and E15.5, washed twice in ice cold PBS pH7.5 and photographed before further processing. The embryos were placed in fixative containing 0.4% paraformaldehyde (PFA) (Electron Microscopy Sciences) in Ca ++ MgCl2 ++ containing DPBS (Gibco). The embryos were fixed overnight at 4 o C on a rocking platform. Next day the embryos were transferred to Ca ++ MgCl2 ++ containing DPBS with 0.1% NaN3 and stored at 4 o C until use. For light sheet microscopy, the embryos were stained using the iDisco protocol [START_REF] Renier | iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging[END_REF]. Embryos were dehydrated in methanol and incubated overnight in 5% H2O2 in methanol at 4 o C, rehydrated and permeabilized at 37 o C in DPBS, 0.3 M Glycine, 0.2% TX-100 and 20% DMSO for maximum 2 days. After blocking in 5% donkey serum, the embryos were incubated with primary antibodies for 4 days (E13.5) or 5 days (E15.5) at 37 o C. After washing, embryos were incubated with secondary antibodies following primary antibody incubation protocol. Finally, the embryos were dehydrated and cleared in methanol and benzyl benzoate. The Ultramicroscope II (Lavision Biotec) was used for the image acquisition. Images were processed using the Imaris v9 software (Bitplane Technologies) for data analysis and 3D rendering. The lymphatic surface area was measured in Imaris by applying the surface area tool. To stain the lymphatic endothelial cells, we used anti-Lyve1 (Reliatech) and anti-Prox1 (R&D technologies). Blood vessels and lymphatic vessels were visualized with anti-CD31 (Thermo Fisher). Secondary antibodies were donkey anti-rabbit Alexa 555 (Thermo Fisher), donkey anti-rat Alexa 790 (Jackson Immunoresearch), and donkey anti-goat Alexa 647 (Thermo Fisher).

Rescue experiments using mouse lymphatic endothelium Lymphatic endothelial cells (LECs) were isolated from mouse lymph nodes digested with Collagenase D (Sigma) and Dispase (Stem Cell Technologies). LECs were cultured as previously described (Jordan-Williams and Ruddell, 2014). For LECs grown in semi-synthetic medium, LECs were plated on Lab-TEK II chamber slides (Nunc) and cultured overnight in semi-synthetic medium (SSM) containing DMEM, 10 mM Hepes, 1%BSA, and 1 mM D-erythroSO-BSA complexes, 1x Insulin-Transferrin-Selenium (ITS-A) (Thermo Fisher), 10 mM b-mercaptoethanol, 0.2 mM Glutamine and 1% FBS. 0.5-1 mM of 4-hydroxytamoxifen (4-OH-TMX) (Sigma) were added to induce deletion of Nr2f2 or Sptlc2. The D-erythroSO was added to all samples to replenish the precursor required for synthesis of membrane sphingolipids and is essential for cell survival [START_REF] Hanada | Sphingolipids are essential for the growth of Chinese hamster ovary cells. Restoration of the growth of a mutant defective in sphingoid base biosynthesis by exogenous sphingolipids[END_REF]. The 4-OH-TMX-treated cells were divided into three groups: plus or minus desired concentration of 1-deoxySO (14Z)-BSA complexes and vehicle. In LECs grown in SSM the concentrations of 1-deoxySO-14Z used was 50-100 nM. Cells that did not receive 4-OH-TMX were used as controls. All groups of cells received fresh media and appropriate metabolites daily for continued culture. Cells were processed for immunostaining 24 h after the 4-OH-TMX administration as follows. Cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences), washed 3 times with PBS supplemented with 0.3% Triton X-100 (PBST), blocked for 2 h with PBST containing 5% goat serum and stained overnight with anti-NR2F2 antibody (Abcam). Cells were washed 3 times with PBST, stained for 1 h with goat anti-mouse-Alexa 488 antibody (Thermo Fisher) and washed 3 times with PBST. Cells were then blocked for 30 min with PBST containing 5% goat serum and stained for 1 h with anti-mouse Prox1antibody conjugated to Alexa 405 or Alexa 647 as indicated (Novus Biologicals) and antimouse MECA-79 Alexa 488 (Thermo Fisher). Cells were washed 3 times with PBST, mounted and imaged using the Leica S5 confocal microscope. Images were analyzed and processed using Leica LAS X software. For flow cytometry, LECs were grown in 12-well tissue culture plates using vascular cell basal medium (ATCC) containing 2.5% FBS supplemented with endothelial cell growth kit-VEGF (ATCC) and 1 mM D-erythroSO-BSA complexes. Control samples received 1.25 mM vehicle (ethanol)-BSA conjugate, experimental group 1 samples received 1.25 mM vehicle-BSA conjugate plus 5 mM 4-OH-TMX while experimental group 2 received 1.25 mM 1-deoxySO-14Z-BSA conjugate plus 5 mM 4-hydroxytamoxifen 4-OH-TMX. After 48 h of culture, single cell suspensions were obtained using TrypLE (Thermo Fisher), cells were washed once in PBS, fixed and analyzed on Cytoflex flow cytometer (Beckman Coulter). Data analyses were performed using FlowJo version 10 software.

Rescue experiments using embryo culture E10.5 Sptlc2 f/f Cdh5 CreERT2/+ embryos were harvested, washed once with pre-warmed PBS, placed in DMEM-F12 media (Thermo Fisher) supplemented with 5% FBS and equilibrated in 37 o C incubator with 5% CO2. The extra-embryonic tissues were removed using a dissection microscope. Embryos were divided in two groups. The experimental group was cultured in embryo culture medium containing DMEM-F12 (Thermo Fisher), 15% KSR (Thermo Fisher), 1x N2 supplement (Thermo Fisher), 1x Glutamax (Thermo Fisher), 2.5 mL/mL of 100x Pen-Strep solution (Thermo Fisher), 1 mM 4-OH-TMX (Sigma), 5 mM D-erythroSO-BSA complexes and 25 nM 1-deoxySO-BSA complexes. Control group embryos were cultured similarly but received vehicle-BSA complexes in place of 1-deoxySO-BSA complexes. Cultures were carried out in 50 mL conical tubes in high O2 atmosphere which was created by attaching a Steriflip filter unit (Millipore Sigma) connected to an O2 tank for 20-30 sec. The tubes were sealed with parafilm and placed in roller bottle in a 37 o C incubator with 5% CO2 for 24 h. The embryos were then washed in PBS with Ca +2 and Mg +2 (Thermo Fisher) and fixed overnight in Ca +2 Mg +2 PBS plus 0.4% PFA (Electron Microscopy Sciences 15710). The fixed embryos were washed with PBS and stored in PBS plus 0.1% Sodium Azide. Embryos were genotyped and processed for light sheet microscopy as described above.

Generation of NR2F2-KO and NR2F1/2-dKO hESC lines CRISPR/Cas9 editing of NR2F2 gene was performed with two gRNAs targeting exon 2 that are predicted to delete ~300 bp region of the gene (Table S1 -gRNAs). gRNAs were purchased as custom-made oligonucleotides (Eurofins Genomics), annealed and cloned into the LentiCRISPR V2 vector [START_REF] Sanjana | Improved vectors and genome-wide libraries for CRISPR screening[END_REF][START_REF] Shalem | Genome-scale CRISPR-Cas9 knockout screening in human cells[END_REF]. H9 hESCs were treated with Accutase (Stem Cell Technologies), washed and resuspended in 800 mL cold PBS, and mixed with 40 mg of each gRNA. Cells were electroporated using Gene Pulser XceII (Bio-Rad) at 250 V, 500 mF in a 0.4 cm Gene Pulser cuvette (Bio-Rad). Cells were plated onto a Matrigel (Corning)-coated tissue culture dish in mTeSR1 media (Stem Cell Technologies) supplemented with 5 mM ROCK inhibitor Y27632 (Tocris) for the first 24 h. Two days after electroporation, Puromycin (Sigma) was added at 1 mg/mL concentration for 48 h. Individual colonies were picked and expanded in 6-well plates. Genomic DNA was purified using the KAPA Express Extract Kit (Kapa Biosystem). Deletions were validated by PCR using primers listed in Table S1-PCR primers for deletion screen. The PCR products were sequenced to confirm sequences of each NR2F2 allele using the Table S1-sequencing primers. The absence of protein in the KO clones was confirmed by Western blotting of day 7 cardiomyocyte lysates. To generate NR2F1/2-DKO hESCs, two NR2F2-KO clones were electroporated with a mixture of NR2F1 gRNAs. Clone selection and validation was as described above for a single NR2F2 knockout. Two independent DKO clones showed similar behavior in the differentiation protocol.

Generation of Dox-inducible SPTLC1 C133W (HSAN) hESC lines

The targeted knockin of the SPTLC1 C133W and rtTA transgenes into the AAVS1 locus was performed as previously described [START_REF] Wang | A non-canonical BCOR-PRC1.1 complex represses differentiation programs in human ESCs[END_REF][START_REF] Wang | Combining CRISPR/ Cas9-mediated knockout with genetic complementation for in-depth mechanistic studies in human ES cells[END_REF]. WT or DKO hESCs were treated with Accutase, washed and 2-3 x 10 6 cells resuspended in 100 mL cold R buffer from the Neon transfection system kit (Thermo Fisher) and mixed with plasmid mix consisting of 2.5 mg AAVS1-TALEN-L, 2.5mg AAVS1-TALEN-R, 10 mg Puro-FLAG-SPTLC1 C133W donor and 10 mg Neo-M2rtTA donor. Cells were electroporated using the Neon Transfection System (Invitrogen) at 1050 V 2x30 millisecond pulses and plated on Matrigel-coated tissue culture plates in Stem-Flex™ medium (Thermo Fisher), supplemented with 5 mM ROCK inhibitor for the first 24 h. Two days after electroporation, cells were selected with 50 mg/mL Geneticin (Life Technologies) for 4 days, followed by treatment with 0.5 mg/mL Puromycin (Life Technologies) for another 2 days. Individual colonies were picked and expanded in StemFlex™ medium. Integration into the AAVS1 locus was confirmed by PCR. Dox dependent transgene expression was validated by Western blotting with anti-FLAG antibody.

Cardiomyocyte differentiation of hESCs

Cardiomyocyte differentiation was performed as described in [START_REF] Burridge | Chemically defined generation of human cardiomyocytes[END_REF][START_REF] Lian | Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/beta-catenin signaling under fully defined conditions[END_REF] with minor modifications. Prior to differentiation, hESCs were cultured in E8 medium. Single cells were prepared using Accutase and plated onto Matrigel-coated 12-well tissue culture plates in E8 medium supplemented with 5 mM ROCK inhibitor for the first 24 h. Plating densities were adjusted to obtain 75-85% confluent wells at day 4 after plating. This time point was considered a day 0 of differentiation protocol. To initiate differentiation, cells were cultured in RPMI-1640 medium (Gibco) supplemented with Insulin-free B-27 (Thermo Fisher) and 6 mM CHIR99021 (Tocris) for 48 h followed by a 24-hour culture in Insulin-free RPMI/B27 medium without inhibitor. 72 h after addition of CHIR99021, cells were switched into the Insulin-free RPMI/B27 supplemented with 5 mM IWP2 (Tocris). On day 5 of differentiation, the media was replaced for the Insulin-free RPMI/B27 medium without inhibitor. On day 7 of differentiation and every two days there-after, media was replaced for fresh Insulin-containing RPMI/B27 medium.

Myriocin treatments were initiated on day1 and included a vehicle (DMSO) control and 1 mM Myriocin (Sigma). D-erythroSO-BSA and 1-deoxySO-BSA complexes were added at 1.5 mM concentration on day 3 of differentiation and every two days thereafter. Expression of SPTLC1 C133W (HSAN) mutant in WT HSAN and DKO HSAN hESC lines was induced by adding 2 mg/mL Doxycycline (Sigma) on day 3 and every two days thereafter.

TNNT2 staining of cardiomyocytes

Cardiomyocyte cultures were treated with Accutase (Stem Cell Technologies) to obtain single cell suspension. Cells were pelleted, washed once in PBS and then fixed and permeabilized using the BD Cytofix/Cytoperm fixation/permeabilization kit (BD Biosciences). The cells were stained with anti-cardiac troponin T Alexa 647 antibody (BD biosciences) for 30 min, washed once in PBS and resuspended in PBS a density of 1x10 6 cells/mL. Data were collected on the CytoFlex instrument (Beckman Coulter) and analyzed in FlowJo v.10.

Electron

microscopy of hESC-derived cardiomyocytes Day 12 cardiomyocyte cultures were fixed in 0.1 M sodium cacodylate buffer (pH 7.4) containing 2.5% glutaraldehyde for 1 h at room temperature. After cells were rinsed in the same buffer twice, they were post-fixed in 1% OsO4 at room temperature for 1 h. Specimens were stained en bloc with 2% aqueous uranyl acetate for 30 min, dehydrated in a graded series of ethanol to 100% and embedded in EMbed 812 resin. Blocks were polymerized at 60 o C overnight. Thin 60 nm sections were cut using a Leica ultramicrotome and post-stained with 2% uranyl acetate and lead citrate. Cell sections were examined with a FEI Tecnai transmission electron microscope at 80 kV accelerating voltage; digital images were recorded with an Olympus Morada CCD camera and iTEM imaging software.

RNA isolation

Total RNA was isolated using Trizol Reagent (Thermo Fisher), treated with DNAse I (Ambion) to remove genomic DNA, precipitated, washed with 70% ethanol and dissolved in water.

Reverse Transcription and quantitative PCR (RT-qPCR) 1 mg of total RNA was reverse transcribed using random hexamers and SuperScript II Reverse Transcriptase (Invitrogen). Resultant cDNA was diluted in water and 20 ng was used in each RT-qPCR reaction. Reactions were run on a CFX96 instrument using iTaq Universal SYBR Green Supermix (Bio-Rad). Standard curves were generated for each primer set and were used to calculate Ct values with the expression threshold set to 100 RFU. In order to compare expression levels between different samples, all expression values were scaled to GAPDH. The gene-specific primers used in PCR-based analyses, included those published previously [START_REF] Osafune | Marked differences in differentiation propensity among human embryonic stem cell lines[END_REF][START_REF] Wang | Distinct lineage specification roles for NANOG, OCT4, and SOX2 in human embryonic stem cells[END_REF]are listed in Table S1-qPCR Primers.

Immunoblotting

Cells were washed twice in ice-cold PBS and incubated in 1X RIPA buffer (Thermo Fisher) with 1X PhosSTOP protease inhibitor(Roche) and 1X cOmplete Protease Inhibitor Cocktail (Roche) for 5 min on ice. Lysates were pre-cleared by maximum speed centrifugation in a benchtop centrifuge for 30 min and protein was quantified using the Bio-Rad Protein Assay Kit II (Bio-rad). 20 mg of protein lysate was resolved on SDS-PAGE gel and transferred onto an Immobilon membrane (Millipore). Blots were blocked in 5% non-fat dry milk, incubated with primary antibody overnight at 4 o C, washed, incubated with secondary antibody for 1 h at room temperature, washed, and developed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher). TUBULIN, GAPDH or histone H3 were used as loading controls. A complete list of antibodies used in this study is listed in Table S1-Antibodies.

Basic statistical analyses

Statistical analyses were performed using GraphPad Prism 5 and Excel software. Unless otherwise stated, data are shown as averages with standard deviations. Exclusion of samples with at least ±2 sd from the mean was only performed in statistical procedures that require two independent samples of equal size. Whenever possible, experiments were designed so that samples under comparison are of equal sizes. Basic comparisons were performed using non-randomized data analyzed by two-tailed Student's t test either paired or unpaired using equal or unequal variance as indicated.

Bulk RNA-seq analyses RNA samples were processed by the Yale Center for Genome Analyses according to standard Illumina protocols. Libraries were sequenced on the NovaSeq flow cell with 100 bp paired-end reads. Signal intensities were converted to individual base calls during the run using the system's Real Time Analysis (RTA) software. Base calls were transferred from the machine's dedicated personal computer to the Yale High Performance Computing cluster via a 1 Gigabit network mount for downstream analysis. Sample de-multiplexing and alignment to the human genome -was performed using Illumina's CASAVA 1.8.2 software suite. Untrimmed FASTA files were mapped with Salmon (version 0.8.2) using GENECODE version 38 (GRCh38.p12) reference transcriptome. DeSeq2 package was used to determine differential expression between samples. Data were processed on the Galaxy server (https://usegalaxy.org/).

Genes with expression fold changes greater than 2 and p values less than 0.05 were considered differentially expressed. Additional filtering was applied to remove genes with expression levels below 5 TPM in all samples. Gene ontologies were defined using Panther (www.pantherdb.org/) and NIH David (https://david.ncifcrf.gov/) packages. List of NR2F2 target genes identified by ChIP was obtained from [START_REF] Churko | Defining human cardiac transcription factor hierarchies using integrated single-cell heterogeneity analysis[END_REF][START_REF] Wu | Atrial identity is determined by a COUP-TFII regulatory network[END_REF].

Single-cell (sc)RNAseq of cardiomyocyte differentiation and data analyses Live cells from WT, NR2F2-KO and NR2F1/2-DKO cultures were sorted and analyzed in duplicates on day 6 and 19 of cardiomyocyte differentiation. Single cell separation, library preparation, and sequencing were performed by the Yale Center for Genome Analyses according to standard 10x Genomics protocols. Minimum sequencing depth was 20,000 read pairs per cell. Cell Ranger™ analysis pipeline was used for initial analysis and visualization.

Data from all samples for each time point (Day 6 and Day 19) were concatenated into a single matrix for analysis. The number of cells in each sample were as follows: WT-R1-11,506 cells, WT-R2 -9,836 cells, KO-R1-9,272 cells, KO-R2-19,454 cells, DKO-R1-12,658 cells, DKO-R2 -21277 cells for day 6, and WT-R1-8,821 cells, WT-R2 -13,424 cells, KO-R1-16,214 cells, KO-R2-19,422 cells, DKO-R1-37,637 cells, DKO-R2-33,183 cells for day 19. For data pre-processing, we followed the current best practices in single-cell RNA sequencing analysis based on the SCANPY toolkit [START_REF] Luecken | Current best practices in single-cell RNA-seq analysis: a tutorial[END_REF][START_REF] Wolf | SCANPY: large-scale single-cell gene expression data analysis[END_REF]. Genes detected in less than 50 cells were removed. Cells with mitochondrial read percentages larger than 20% were removed. Cells with the total UMI count larger than 50,000 for Day 6 and 25,000 for Day 19 were removed. All filtering decisions were made by manual inspection of the data distributions using SCANPY. The filtered data contained 73,202 cells/19,008 genes at day 6 and 119,758 cells/ 19,134 genes at day 19. The filtered data were then library-size normalized and square-root transformed using scprep (www. github.com/KrishnaswamyLab/scprep), as previously described [START_REF] Burkhardt | Quantifying the effect of experimental perturbations in single-cell RNAsequencing data using graph signal processing[END_REF][START_REF] Moon | Visualizing structure and transitions in high-dimensional biological data[END_REF]. To build a cell similarity graph, 100 PCA dimensions were calculated and edge weights between cells were calculated using an alpha-decay kernel as implemented in the graphtools library (www.github.com/KrishnaswamyLab/graphtools) using knn=9 and decay=10. This graph was then used for the MELD analysis [START_REF] Burkhardt | Quantifying the effect of experimental perturbations in single-cell RNAsequencing data using graph signal processing[END_REF] to calculate the enhanced experimental signal (EES) for each sample with default parameters. The graph was also used for PHATE embedding [START_REF] Moon | Visualizing structure and transitions in high-dimensional biological data[END_REF] visualization using default parameters. For clustering, we used k-means clustering with k = 30 and the PHATE diffusion potential [START_REF] Moon | Visualizing structure and transitions in high-dimensional biological data[END_REF]. Analysis of groups of genes was done by the scoring method, ''scanpy.tl.score_genes'', in SCANPY. Identification of marker genes for each cluster were done by the ranking method, ''scanpy.tl.rank_genes_groups'', with logistic regression in SCANPY.

Figure 1 .

 1 Figure 1. Enzyme overexpression screening identifies modulators of NR2F1/2 transcriptional activity (A) Reporter system used in the overexpression screen. (B) Reporter constructs with DR0A spacing are specific for NR2F2. Cells were grown in medium with 10% FCS with no added ligand. Values: relative light units (RLU). Error bars are standard deviation. (C) Reporter activity is induced by native full-length NR2F1, NR2F2, and NR2F6 receptors in both FCS-containing and serum-free medium. Error bars are standard deviation and p-values were defined using two tailed Student's t test. (D) NR2F2 activity is abolished by mutations in the coactivator recognition site (CRS). Error bars are standard deviation. (E) Metabolic enzymes modulate the activity of native full-length NR2F1/2 receptors. Activators (left) upregulated reporter activity by >2-fold. Repressors (right) downregulated reporter activity by >2-fold. Error bars are standard deviation. (F) Activating enzymes are specific for native full-length NR2F1/2. Error bars are standard deviation. (G) Summary of pathways modulating NR2F1/2 activity.

Figure 3 .

 3 Figure 3. Phenotypes of Sptlc2 f/f Prox1 Cre-ERT2/+ and Sptlc2 f/f Cdh5 Cre-ERT2/+ embryos correlate to those of Nr2f2 deficient animals (A) Genetic manipulation of Sptlc2. (B) Deletion efficiency of Sptlc2 f/f in Prox1 Cre-ERT2/+ mice by genomic qPCR. Error bars are standard deviation. (C) Surface rendering of blood vessels in E13.5 embryos. (D) Summary of edema phenotype in Sptlc2 -/-embryos. (E) Edema in Nr2f2 -/-and Sptlc2 -/-embryos by light-sheet microscopy. Ortho slices (5 mm) of representative embryos (right panels). (F) Comparison of JLS volume across the strains used in (E). Error bars are standard deviation. (G) Blood vessel branching and dilated dermal lymphatics in Nr2f2 -/-and Sptlc2 -/-E15.5 embryos.

Figure 4 .

 4 Figure 4. Simultaneous deletion of NR2F1 and NR2F2 negatively impacts cardiomyocyte differentiation (A) CM differentiation protocol. (B) Expression of NR2F1 and NR2F2 and key differentiation markers during CM differentiation. Error bars are standard error. (C) Combined scRNA-seq data of CMs derived from WT, KO, and DKO hESCs.

  (D) Distribution of mature-, mid-, and early-stage CM and non-CM populations in cells derived from WT, KO, and DKO hESCs. (E) Morphology of CM cultures (days 9 and 12). Arrows: ''beating'' cell clusters in DKO cells. (F) TNNT2 expression in CMs derived from WT and DKO hESCs. (G) Expression of CM markers in WT and DKO cells defined by qPCR. Error bars are standard error. (H) Expression of NR2F2 targets in WT and DKO CMs. Error bars are standard deviation. p-Value was determined using a two tailed Student's t test. (I) GO analyses of genes up/downregulated in DKO cells (day 6).

Figure 5 .

 5 Figure 5. Sphingolipid biosynthesis modulates NR2F1/2-regulated gene network in hESC-derived cardiomyocytes (A) Myriocin modulates NR2F1/2-dependent gene expression in day 6 CMs. p-Values were calculated using the hypergeometric distribution. (B) Heatmap data from (A). (C) Expression profiles of genes modulated by NR2F1/2 in day 6 CMs. Boxes denote second and third distribution quartiles, whiskers show the first and the fourth quartiles, the median is marked by horizontal line. p-values are from the Student's t test.****p values < 0.0001. (D) Projection of bulk RNA-seq data from day 6 myriocin-treated cultures onto PHATE embedding. Red = cells with R > 0.6 correlation with corresponding bulk RNA-seq transcriptome.

Figure 6 .

 6 Figure 6. Exogenous and endogenous 1-deoxysphingosines modulate NR2F1/2-regulated transcription in human ESC-derived cardiomyocytes (A) Schedule of ligand supplementation during CM differentiation. (B) 1-deoxysphingosine supplementation increases the fraction of TNNT2+ cells. Error bars are standard deviation. p-Values were calculated using two tailed Student's t test. (C) Representative FACS profiles of TNNT2 expression in WT and DKO CMs treated with vehicle, D-erythroSO and 1-deoxySO-14Z.

  The induction of HSAN by Dox increased 1-deoxysphingosine concentration in differentiating CM cultures by nearly 2-fold but minimally affected the metabolism of canonical sphingosines, (D) Summary of GO analysis of genes up/downregulated by 1-deoxysphingosine in day 9 WT CM cultures. (E) Expression profiles of up/downregulated genes in day 9 CMs after treatment with vehicle, D-erythroSO or 1-deoxysphingosine. (F) The reverse tetracycline-controlled transactivator (rtTA) and SPTLC1 C133W (HSAN1) transgenes were integrated into the AAVS1 locus, protein expression induced by Dox analyzed by WB. (G) Schedule of Dox supplementation during CM differentiation. (H) Quantitation of canonical and non-canonical sphingosines in hESCs and day 7 CMs. (I) Effect of HSAN expression on % of TNNT2+ cells in WT HSAN and DKO HSAN CMs during the 15-day differentiation time-course. (J) Representative FACS profiles of TNNT2 expression in WT HSAN and DKO HSAN CM cultures. (K) Projection of bulk RNA-seq data from day 7 WT HSAN cultures onto the day 6 PHATE embedding. (L) Genes up/downregulated after addition of 1-deoxysphingosine or Dox. ****p values < 0.0001. (M) Genes up/downregulated after addition of 1-deoxysphingosine or Dox in different cell types of the human heart.

  o C for 30-90 min and imaged. For tissue culture experiments the complexes were added directly to culture medium cells every time medium was changed.

  and Sptlc2 f/f Cdh5 CreERT2 embryos. Deletion of Nr2f2 in lymphatic endothelium results in fetal edema, bloodfilled lymphatics, defects in the development of lymphatic sacs, and dilated skin lymphatic vessels[START_REF] Lin | Direct transcriptional regulation of neuropilin-2 by COUP-TFII modulates multiple steps in murine lymphatic vessel development[END_REF] Srinivasan

TABLE

	B Deletion efficiency after tamoxifen injection
	B Embryo processing, staining, and imaging
	B Rescue experiments using mouse lymphatic endo-
	thelium
	B Rescue experiments using ex-vivo embryo culture
	B Generation of NR2F2-KO and NR2F1/2-dKO
	hESC lines
	B Generation of Dox-inducible SPTLC1 C133W (HSAN)
	hESC lines
	B Cardiomyocyte differentiation of hESCs
	B TNNT2 staining of cardiomyocytes
	B Electron microscopy of hESC-derived cardiomyocytes

d RESOURCE AVAILABILITY B Lead contact B Material availability B Data and code availability d EXPERIMENTAL MODEL AND SUBJECT DETAILS d METHOD DETAILS B Insect and mammalian reporter systems B Enzyme overexpression screening B Metabolite screening B Preparation of sphingolipid-BSA complexes B Reporter assays in mammalian cells B Reporter assays in insect cells B Cell-based reporter assays for sphingolipids in mammalian cells B Immunoprecipitation and lipidomics B Sphingolipidome of TIME cells B Targeted metabolomics B Amino acids B Sphingolipids B Fatty acids B Purification of bacterial NR2F1 and NR2F2 LBDs B Purification of mammalian NR2F2 LBDs B Fluorescence polarization assay (FPA) B Thermal shift assays using recombinant NR2F2-LBD B Surface plasmon resonance (SPR) B Conditional deletion of Sptlc2 and Nr2f2 in mice B RNA isolation B Reverse Transcription and quantitative PCR (RT-qPCR) B Immunoblotting B Basic statistical analyses B Bulk RNA-seq analyses B Single-cell (sc)RNAseq of cardiomyocyte differentiation and data analyses

Table S2

 S2

	Continued
	Reagent or resource	Source	Identifier
	Critical commercial assays
	Deposited data
	Bulk RNA-seq	This paper	GEO: GSE184913
	Single-cell RNA-seq	This paper	GEO: GSE185456
	Experimental models: Cell lines
		This paper	N/A

related to key resources table. Oligos.

Table S1 ,

 S1 Primers-Mouse Genotyping. The PCR conditions were as follows.

	95 o C 3 min, 35 cycles of 95 o C for 15 sec, 60 o C for 15 sec, 72 o C for 15 sec followed by 72 o C for 5 min. Cdh5-CreERT2 transgene
	produced a 548 bp product. PCR products were resolved in 1% agarose gel.
	For Sptlc2-Flox: o C for 5 min. Wild type allele produced a 420 bp product, Sptlc2-loxP allele produced a 600 bp product. For Nr2f2-Flox we used primers designed by (Takamoto et al., 2005): 95 94 o C 5 min, 30 cycles of 94 o C for 1 min, 59 o C for 1 min and 72 o C for 1 min, followed by 72 o C 8 min, 35 cycles of 95 o C for 15 sec, 56 o C for 30 sec, 72 o C for 1 min followed by 72 o C for 5 min. Wild type allele produced a 785 bp product, Nr2f2-loxP allele produced a 394 bp product. For Prox1-CreERT2: 95 o C for 2 min, 35 cycles 95 o C for 15 sec, 60 o C for 30 sec, 72 o C for 1 min followed by 72 o C for 2 min. The Prox1-CreERT2 transgene produced a 730 bp product. For Cdh5-CreERT2:

ACKNOWLEDGMENTS

We thank Nancy Ruddle (Yale University), Benjamin S. Lowry, and Harrison B. Chong (University of Georgia) for critical reading of the manuscript, Vishwa Deep Dixit (Yale University) for Sptlc2 mice, Robert G. Kelly (IBDM, Marseille) for analysis of heart phenotype in Sptlc2 deficient animals, Christopher Castaldi and Guilin Wang (Yale Center for Genome Analysis) for RNA-seq and scRNA-seq, Bill Webb (the Scripps Center for Mass Spectrometry and Metabolomics) for LC-MS, Narinder Ghumman (University of Georgia) for bioinformatics, Timothy Nottoli (Yale University) for mouse rederivation, and Nikhil Joshi (Yale University) for help with mouse protocols. Mice were provided by MMRRC (Nr2f2 f/f ) and Cancer Research UK (Prox1-CreERT2 and Cdh5-CreERT2). The work was supported by NIH grants R21-AI131015 (F.R.S./ S.M.K.), R01 GM107092 (N.B.I.), and R24GM137782 (P.A.), the Yale University institutional grant YD000234 (F.R.S.), CT RMRF grant 16-RMB-Yale-07 (S.K./ N.B.I.), French National Research Agency grants ANR-16-ACHN-0011 and ANR-17-CE13-0029 (S.A.v.d.P.), FRM Amorcage de jeunes equipes AJE20150633331 (S.A.v.d.P.), the Swiss National Foundation grant project 31003A-179371 (T.H.), and the National Natural Science Foundation of China project 81600093 (T.W.). N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Cat#MMM1013-202739977 Cat#MMM1013-202769921 Cat#MMM1013-202762257 N/A N/A N/A N/A 20 N/A N/A N/A Cat#71327 N/A N/A Imaris version 9 software Oxford Instruments N/A

This paper This paper This paper This paper This paper This paper This paper paper This paper This paper This paper This paper Open Biosystems Open Biosystems Open Biosystems This paper This paper This paper This paper Invitrogen This paper This paper This paper Millipore Sigma This paper This paper N/A

SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j. devcel.2021.10.018.

AUTHOR CONTRIBUTIONS

Conceptualization, methodology, writing, review, and editing, F.R.S. and N.B.I. Supervision and project administration, F.R.S., N.B.I., and S.M.K. Investigation and validation, T.W., L.d.F., Z.W., J.T., B.R.R., B.J., T.Z., A.G., Zhirui Wang, P.A., S.X., M.P., D.E.S., T.H., C.A., E.M.S., I.A., S.A.v.d.P., N.B.I., and F.R.S. Resources, Zheng Wang, J.T., T.H., C.A., E.M.S., and I.A. Formal data analysis and curation, N.B.I., B.R.R., H.J.-L., D.B., and S.K.

DECLARATION OF INTERESTS

The authors declare no competing interests.

STAR+METHODS KEY RESOURCES TABLE

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Fabio Santori (fabio.santori@yale.edu).

Material availability

All plasmids, reagents, and cell lines are available upon request. The Prox1 and Cdh5 CreERT2 mouse models require written permission from Cancer Research UK/Ximbio.

Data and code availability

Bulk-RNA-seq and single-cell RNA-seq data are available through NCBI GEO datasets: access numbers GSE184913 (bulk RNA-seq) and GSE185456 (single-cell RNA-seq).

This manuscript did not generate new code.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

HEK-293FT and HeLa cells were grown in DMEM (Gibco) supplemented with 2 mM Glutamine, 10,000 units/mL Penicillin/Streptomycin, and 10 % FBS at 37 o C and 5% CO2. Cultures were passaged every 5-6 days by adding TrypLE (Gibco) and re-plating at 1:10 ratio. Drosophila melanogaster S2 cells were maintained in Express Five SFM media (Gibco) at room temperature in the dark and passaged once a week. Sptlc2 tm2.1Jia [START_REF] Li | Liver-specific deficiency of serine palmitoyltransferase subunit 2 decreases plasma sphingomyelin and increases apolipoprotein E levels[END_REF], Prox1 CreERT2 [START_REF] Bazigou | Genes regulating lymphangiogenesis control venous valve formation and maintenance in mice[END_REF] and Cdh5 CreERT2 [START_REF] Wang | Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis[END_REF] mice were obtained from the Cancer Research Consortium (UK) under an MTA. Nr2f2 f/f mice were re-derived from frozen sperm (B6;129S7-Nr2f2 tm2Tsa/Mmmh ) received from the Mutant Mouse Regional Resource Center (MMRRC) at the University of Missouri. Mice were maintained in the Yale Animal Resources TAC facility and used under the approved IACUC protocols.

H9 human embryonic stem cells (WA09, NIH approval number NIHhESC-10-0062) were grown in feeder-free conditions in plates coated with Matrigel (BD Biosciences) in E8 media (Thermo Fisher) or mTeSR1 media (Stem Cell Technologies) at 37 o C and 5% CO2. Cultures were passaged every 4 days by adding 1mg/mL Dispase (Stem Cell Technologies) for 5 min at 37 o C and re-plating at a 1:10 ratio.

METHOD DETAILS

Insect and mammalian reporter systems pGL4.31[luc2p/GAL4UAS/Hygro] vector (Promega) containing firefly luciferase under GAL4(UAS) transcriptional control was modified to replace the late SV40 promoter with the Drosophila melanogaster hsp70 promoter [START_REF] Santori | Identification of natural RORgamma ligands that regulate the development of lymphoid cells[END_REF]. This reporter (referred to as pHM1) was used in experiments with GAL4(DBD)-NHR(LBD) fusion proteins in insect cells. GAL4(DBD)-NR2F1(LBD) and GAL4(DBD)-Svp(LBD) proteins were cloned in insect expression vector pAc driven by the Drosophila melanogaster actin promoter [START_REF] Huh | Digoxin and its derivatives suppress TH17 cell differentiation by antagonizing RORgt activity[END_REF].

Mammalian synthetic reporter plasmids for NR2F1 and NR2F2 receptors were constructed by replacing the GAL4 (UAS) sequence of pHM1 with six tandem repeats of the conserved AGGTCA NR2F binding site (AGGTCAAGGTCAAGGTCAAGGTCAAGGTC AAGGTCAAGGTCAAGGTCA) (pHM1 6xDR0A plasmid) [START_REF] Montemayor | Genome-wide analysis of binding sites and direct target genes of the orphan nuclear receptor NR2F1/COUP-TFI[END_REF][START_REF] Pipao´ N | COUP-TF upregulates NGFI-A gene expression through an Sp1 binding site[END_REF][START_REF] Zelhof | Seven-up inhibits ultraspiracle-based signaling pathways in vitro and in vivo[END_REF].

Native mammalian NR2F2 reporter used in Figures S1F,2C, and S2C was constructed by replacing the GAL4 (UAS) sequence of pHM1 with a single NR2F2 binding site found in the NGF1A promoter (TCACGGCGGAGGCGGGCCCGGGTATATAA) (pHM1-NGF1A) [START_REF] Pipao´ N | COUP-TF upregulates NGFI-A gene expression through an Sp1 binding site[END_REF]. Native NR2F2 mammalian reporter used in Figures 2C and S2C utilized a fluorescent-bioluminescent fusion of Renilla luciferase and Venus fluorescent protein nanolantern (pHM1-NGF1A-nano) [START_REF] Saito | Luminescent proteins for high-speed single-cell and whole-body imaging[END_REF] in place of the firefly luciferase. The NR2F6 reporter was constructed using the NR2F6 bindings site found in the IL-17 promoter (AATGGAAAGTTTTCTGACCC ACTTTAAATC) (pHM1-IL17) [START_REF] Hermann-Kleiter | Nuclear orphan receptor NR2F6 directly antagonizes NFAT and RORgt binding to the Il17a promoter[END_REF].

6x(DR0A-DR6A) reporters were constructed by replacing the GAL4 (UAS) sequence of pHM1 with synthetic oligos described in Table S1.

CMV-driven NHR expression plasmids were obtained from commercial sources (Table S1-Plasmids).

The Renilla luciferase (RL) plasmid pRL-CMV (Promega) was used for normalization in mammalian cells, RL under a Pol2 promoter [START_REF] Huh | Digoxin and its derivatives suppress TH17 cell differentiation by antagonizing RORgt activity[END_REF] was used for normalization in insect cells. In experiments utilizing the nanolantern bioluminescent reporter (Figures 2C andS2C) normalization was performed with the filrefly luciferase expression plasmid pluc2p-ffluc-CMV (Promega).

Enzyme overexpression screening

We identified all known metabolic genes in the mouse and human genomes using data from the Mouse Genomics Informatics (MGI) and the Human Protein Atlas databases [START_REF] Eppig | Mouse genome informatics (MGI) resource: genetic, genomic, and biological KnowledgeBase for the laboratory mouse[END_REF][START_REF] Uhlen | Towards a knowledge-based Human Protein Atlas[END_REF]. The resulting list contained 1336 enzymes and excluded protein kinases, phosphatases, DNA and RNA polymerases as well as enzymes involved in protein translation and modification. Enzymes with no known human homologues were removed from the list. The list was further filtered to select a set of 516 enzymes that represent rate limiting metabolic enzymes and enzymes involved in secondary metabolism (Key Resources Table S1-Metabolic Enzyme cDNA Clones). The cDNA clones were purchased from commercial suppliers and validated by sequencing.

For the screening, transfections were performed as described below (Reporter assays in mammalian cells) using the following DNA amounts: 20-100 pg of Renilla normalization plasmid, 5-10 ng of reporter plasmid, 10-20 ng of NHR plasmid and 10 ng of enzyme/ empty vector per well. Expression of transfected proteins was tested for a subset of clones by WB with anti-FLAG antibody (Sigma).

Metabolite screening

Synthetic metabolites were purchased from suppliers indicated in Table S1-Reagents. Mammalian or insect cells were trans-fected with the appropriate NHR plasmids and reporters as described below (Reporter assays in mammalian cells and Reporter assays in insect cells). 24 h after transfection, synthetic metabolite diluted in the appropriate solvent at concentration of 5-10 mM was added. Reporter activity was measured 16 h after the addition of metabolite as relative luciferase unit (RLU) ratio to vehicle (solvent alone).

Preparation of sphingolipid-BSA complexes

In mammalian cells, the uptake of sphingolipids is greatly improved by conjugation to BSA [START_REF] Penno | Hereditary sensory neuropathy type 1 is caused by the accumulation of two neurotoxic sphingolipids[END_REF]. For staining and delivery of sphingolipids to live and fixed cells sphingolipid-BSA complexes were prepared as follows. Sphingolipids were dissolved in ethanol at 200 mM concentration. Fatty acid free BSA (Sigma) was dissolved in PBS pH7.4 supplemented with 10 mM Hepes pH7.4 at 20 mM concentration. A tube with 10 mL of BSA solution was placed on a vortex mixer and 200 mL sphingolipid stock solution was added while it was vortexing. This produces a complex of 20 mM BSA: 20 mM sphingolipid that can be stored at -20 0 C.

For measurements of the sphingolipid uptake, cells were washed with serum-free medium and BSA-sphingolipid complexes were added at the desired concentration. Cells were incubated at 4 o C for 30 min, washed several times with ice-cold medium, incubated at