MiR-195 enhances cardiomyogenic differentiation of the proepicardium/septum transversum by Smurf1 and Foxp1 modulation

Cardiovascular development is a complex developmental process in which multiple cell lineages are involved, namely the deployment of first and second heart fields. Beside the contribution of these cardiogenic fields, extracardiac inputs to the developing heart are provided by the migrating cardiac neural crest cells and the proepicardial derived cells. The proepicardium (PE) is a transitory cauliflower-like structure located between the cardiac and hepatic primordia. The PE is constituted by an internal mesenchymal component surrounded by an external epithelial lining. With development, cells derived from the proepicardium migrate to the neighboring embryonic heart and progressive cover the most external surface, leading to the formation of the embryonic epicardium. Experimental evidence in chicken have nicely demonstrated that epicardial derived cells can distinctly contribute to fibroblasts, endothelial and smooth muscle cells. Surprisingly, isolation of the developing PE anlage and ex vivo culturing spontaneously lead to differentiation into beating cardiomyocytes, a process that is enhanced by Bmp but halted by Fgf administration. In this study we provide a comprehensive characterization of the developmental expression profile of multiple microRNAs during epicardial development in chicken. Subsequently, we identified that miR-125, miR-146, miR-195 and miR-223 selectively enhance cardiomyogenesis both in the PE/ST explants as well as in the embryonic epicardium, a Smurf1- and Foxp1-driven process. In addition we identified three novel long non-coding RNAs with enhanced expression in the PE/ST, that are complementary regulated by Bmp and Fgf administration and well as by microRNAs that selectively promote cardiomyogenesis, supporting a pivotal role of these long non coding RNAs in microRNA-mediated cardiomyogenesis of the PE/ST cells.

Cardiovascular development is a complex developmental process in which multiple cell lineages are involved 1 . Soon after gastrulation, bilateral sets of procardiogenic cells align into the embryonic midline configuring a linear cardiac straight tube 2 . These cellular populations constitute the first heart field and will essentially contribute to the future left ventricle 3,4 . Additional cardiogenic progenitor cells emanate from the medial structures in the gastrulating embryo configuring the second heart field and contributing through both cardiac poles to the addition of the right ventricle and outflow at the arterial pole, and the atrioventricular canal and right and left atrial appendages at the venous poles [4][5][6] . Beside these cardiogenic fields, extracardiac contribution to the developing heart is provided by the proepicardial derived cells [7][8][9][10][11][12][13] .
The proepicardium (PE) is a transitory cauliflower-like structure located between the cardiac and hepatic primordia. With development, cells derived from the proepicardium migrate to the developing heart and progressive covers the most external surface 12,13 . Subsequently, the embryonic epicardium is trigger by the underlying myocardium to an epithelial-to-mesenchymal transition (EMT), migrate into the subepicardial space, generating

Materials and Methods
Tissue isolation and culture. Experimental protocols with chicken embryos were performed in agreement with the Spanish law in application of EU Guidelines for animal research. These protocols conformed to the Guide for Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH publication no. . Approved consent of the Ethic Committee of the University of Jaen was obtained prior to the initiation of the study. Fertilized eggs from white Leghorn chickens (Granja Santa Isabel, Cordoba, Spain) were incubated at 37.5 °C and 50% humidity for 2-7 days. Embryos were harvested and classified at different developmental stages (HH17, HH24 and HH32) according to Hamburger and Hamilton 26 . Embryos were removed from the egg by cutting the blastocyst margin with iredectomy scissors and placing them into Earle's balanced salt solution (EBSS) (Gibco). For qPCR analyses, HH17 embryonic hearts and PE/STs, respectively, were isolated, pooled and directly stored at −80 °C until used. HH24 and HH32 hearts were isolated, cultured as described by Ramesh et al. 27 , collected, pooled, and stored at −80 °C until used. Epicardial identity was validated by Wt1 and Tbx18 immunohistochemistry, resulting in >80% cells positive for these markers. For in vitro explants cultures, chicken HH17 were dissected in Earle's balanced salt solution (EBSS) (Gibco) and culture into collagen gels as previously described 28 or, alternatively, cultured in handing drops until collected, pooled and stored at −80 °C until used. microRNA and siRNA transfections. HH17 PE explants were cultured on collagen gels or hanging drops for 24 hrs at 37 °C in a cell culture incubator before pre-miRNAs (microRNA precursors) or siRNA transfection, respectively, as previously described 28 . HH24 and HH32 epicardial cells were cultured for 72hrs after the myocardial layer was removed and them transfected. Pre-miRNAs transfections were carried out with Lipofectamine 2000 (Invitrogen), following the manufacturer's guidelines. Briefly, 85 nM of pre-miRNA were applied to the explants (3-5 explants per well) for 24 hrs. siRNA transfections were also carried out using Lipofectamine 2000 (Invitrogen) as described above. After incubation, explants were either processed for qRT-PCR or immunohistochemical (IHC) analyses. Negative controls, i.e. HH17 explants treated only with Lipofectamine were run in parallel. To perform IHC analyses, the explants were fixed with 1% PFA for 2 hrs at 4 °C, rinsed for three times in PBS during 10 min, and stored in PBS at 4 °C. Each experimental condition was carried out in isolated tissues from at least 30 embryos. In all cases, 3-5 independent biological replicates were analyzed.
Immunofluorescence analysis by Confocal scanning laser microscopy. Immunofluorescence analyses were performed as previously reported 28 . Briefly, control and experimental HH17 PE explants and HH24, HH32 epicardial cell cultures were collected after the corresponding treatment, rinsed in PBS for 10 min at room temperature, and fixed with 1% PFA for 2 hrs at 4 °C. After fixation, the samples were rinsed three times (10 min each) in PBS at room temperature and then permeabilized with 1% Triton X-100 in PBS for 30 min at room temperature. To block nonspecific binding sites, PBS containing 5% goat serum and 1% bovine serum albumin (Sigma) was applied to the explants overnight at 4 °C. As primary antibody, a polyclonal goat anti-cardiac troponin I (Hytest) was used, diluted (1:200) in PBS, and applied to each culture overnight at 4 °C. Subsequently, the samples were rinsed three times (for 1 hr each) in PBS to remove excess primary antibody and incubated overnight at 4 °C with Alexa-Fluor 546 anti-goat (1:100; Invitrogen) as secondary antibody. After incubation with the secondary antibody, the explants were rinsed as described above. Finally, the explants and /or epicardial cell cultures, respectively, were incubated with DAPI (1:1,000; Sigma) for 7 min at room temperature and rinsed three times in PBS for 5 min each. Explants were stored in PBS in darkness at 4 °C until analyzed using a Leica TCS SP5 II confocal scanning laser microscope.

Statistical analyses.
For statistical analyses of datasets, unpaired Student's t-tests were used, as previously reported 28 . Significance levels or P values are stated in each corresponding figure legend. P < 0.05 was considered statistically significant.
microRNA mimics modulate cell lineage marker expression in the PE/ST. microRNAs play modulatory roles in multiple aspects of embryonic development and therefore also during cardiac formation. We have previously reported a pivotal role for miR-23b and miR-199 during epithelial-to-mesenchymal transition in cardiac valvulogenesis 28 . We herein tested whether microRNA administration can influence cell lineage determination of the PE/ST, focusing particularly on cardiomyogenic differentiation. Chicken HH17 PE/ST explants were isolated ( Fig. 2A-E) and cultured in collagen gels as previously described 28 . Treatment of the PE/ST explants was carried with nine distinct microRNAs (miR-21, miR-23b, miR-27b, miR-100, miR-125, miR-126, miR-146, miR-195 and miR-223), representing microRNAs that display distinct expression profiles during embryonic epicardial development (i.e. within the four distinct categories previously described), for 48 hrs and tissues were either processed for qPCR or immunohistochemistry and confocal image analyses, respectively. In all cases, at least 30 PE/ST explants were treated for each experimental conditions that were subsequently pooled. qPCR analyses was performed for markers of early (Mef2c, Nkx2.5 and Gata4) and terminal cardiogenic differentiation (Mhy15 and Tnnt2), epithelial to mesenchymal transition (Snail, Slug, Cdh1, Cdh2, Cdh5), and fibrogenesis (Col1a1). Analyses were always performed in 3-5 distinct biological replicates for each microRNA treatment. Overexpression of microRNA mimics were validated by qPCR as illustrated in Supplementary Fig. 1. Expression of early cardiomyogenic differentiation markers such as Mef2c, Nkx2.5 and/or Gata4 were selectively down-regulated (or not altered) by miR-21, miR-23b, miR-27b, miR-100, miR-195, miR-125 while miR-126 and miR-146, respectively, enhanced expression of Nkx2.5 and miR-223 administration increased expression of all three early cardiomyogenic markers. Cardiogenic terminal differentiation markers were significantly up-regulated in PE/ST treated with miR-223, miR-195, miR-125 and miR-146, respectively while they were selectively inhibited by miR-23b, miR-27b and miR-126 but not significantly altered by miR-100 and miR-21 administration (Fig. 2F). These data were further corroborated by immunohistochemical detection of cardiac troponin I as illustrated in Fig. 2G-R. Curiously, in most cases that terminal differentiation is enhanced, a selective down-regulation of most of the early cardiogenic markers is observed (miR-195, miR-125 and miR-146), except for miR-223 that were equally enhanced. PE/ST cells migrate into the nude myocardium and activate an epithelial-to-mesenchymal transition that is pivotal for it subsequent migration into the embryonic myocardium. However, it is unclear if EMT is required for PE/ST differentiation. Thus, we tested if EMT also display significant differences after microRNA administration, particularly those promoting cardiomyogenesis. miR-195 and miR-23 treatment decreased EMT markers such as Snail and/or Slug, whereas miR-27b, miR-21, miR-100, miR-146, miR-125, miR-126 and miR-223 increased those EMT transcriptional activators ( Supplementary Fig. 2). Our data support the notion that EMT and cardiomyogenic differentiation are not mutually exclusive biological processes, i.e. both can concur simultaneously. Curiously, expression of cell-cell junctional proteins, i.e. Cdh1, Chd2 and Chd5, was not always concomitantly up-regulated or down-regulated as the EMT transcriptional activators ( Supplementary Fig. 2), suggesting that different molecular mechanisms drive activation and cell-cell uncoupling, in line with previous reports 28 and/or that transcriptional overriding after microRNA administration is operative as suggested by Hill et al. 37 .
Fibrogenic deposition is a major hurdle for proper cardiomyocyte functional electrical coupling. We therefore tested if microRNAs promoting cardiomyogenesis would be concomitantly increasing fibrogenic differentiation. Our data demonstrate that fibrogenic differentiation, as assessed by Col1a1 expression, was significantly increased after miR-23b, miR-27b, miR-195 and miR-223, administration whereas miR-100 and miR-125 treatment decreased Col1a1 expression ( Supplementary Fig. 2). No significant changes were observed for miR-21, Figure 1. microRNA expression profile during PE and epicardium development. qPCR analyses of the differential expression of microRNAs during PE and epicardium development. Panel A illustrate microRNAs with increasing expression ranging from PE HH17 to embryonic epicardium at HH32. Panel B illustrate microRNAs with decreasing expression ranging from PE HH17 to embryonic epicardium at HH32. Panel C illustrate microRNAs with increased expression ranging from PE HH17 to embryonic epicardium at HH24 but decreasing at H32. HH17 PE were collected from >30 embryos and pooled to performed RNA isolation. Similarly, HH24 and HH32 epicardial cells were collected from >30 ventricular explants. In all cases, three distinct biological replicates of pooled PE/ST, HH24 and HH32 samples were subsequently tested by qPCR. (2020) 10:9334 | https://doi.org/10.1038/s41598-020-66325-x www.nature.com/scientificreports www.nature.com/scientificreports/ miR-146 and miR-126 administration, respectively ( Supplementary Fig. 2). Overall these data demonstrate that microRNA treatment can distinctly modulate cell differentiation behavior including cardiomyocyte, epithelial to mesenchymal transition and fibroblast differentiation.
In sum, these data demonstrate that single microRNA administration can exert different cell differentiation modulatory roles; e.g. miR-23 inhibits cardiogenesis and epithelial-mesenchymal transition while enhances fibrogenesis while miR-195 enhances terminal cardiomyocyte differentiation and fibrosis while inhibits epithelial-to-mesenchymal transition.

Modulatory effects of miR-195 and miR-233 is partially promoted in the embryonic epicardium.
In order to dissect if the modulatory roles exerted by distinct microRNAs in the HH17 PE/ ST explants is also applicable to the embryonic epicardium, HH24 epicardial explants were analyzed after microRNA over-expression of a selected number of microRNAs, i.e. those reporting enhanced cardiomyogenesis miR-125, miR-126, miR-146, miR-195 and miR-223, and miR-100 as a negative control. Regulation of early myogenic markers is partially discordant as compared to PE/ST explants. For example, miR-100, miR-125 and Arrows demarcates the PE. Panel C illustrates PE culturing just right after dissection (panel C) and 24 h after culturing (panel D). Panel E represents an schematic overview of the experimental design. Panel F shows qPCR results of cardiomyogenic (Nkx2.5, Mef2c, Gata4, Mhy15, Tnnt2) markers expression after microRNA mimic administration in HH17 PE/ST explants. Observe that miR-23 and miR-27 over-expression leads to down-regulation of all cardiomyogenic markers, miR-100 does not modify most of them and miR-223, miR-195, miR-125 and miR-146 increased terminally differentiation markers such as cardiac troponin T (Tnnt2). Confocal image analyses of cTnI expression in controls (panels G-J), miR-27 (panels K-N) and miR-195 (panels O-R) treated HH17 PE/ST explants. Observe that miR-195 administration selectively increases the overall cTnI immunohistochemical signal (panel P and R). HH17 PE were dissected from >30 embryos, treated with the corresponding microRNA mimics and subsequently pooled to perform RNA isolation. On each case, three-tofive distinct biological replicates were subsequently tested by qPCR. (2020) 10:9334 | https://doi.org/10.1038/s41598-020-66325-x www.nature.com/scientificreports www.nature.com/scientificreports/ miR-126 administration displayed decreased Nkx2.5 expression in EE HH24 cells while no changes or increased is observed in HH17 PE/ST explants (Fig. 3A). On the other hand, concordant modulation is observed for Gata4 after miR-100, miR-125 and miR-146, as well as for Mef2c after miR-125, miR-146 and miR-223 (Fig. 3A). Importantly, up-regulation of terminally differentiation markers such as Tnnt2 and Mhy5 as also concordantly observed after miR-195 and miR-223, demonstrating that cardiomyogenesis is similarly enhanced in EE HH24 and PE/ST explants.
Epithelial-to mesenchymal transition also display concordant expression for Slug following microRNA administration in HH24 embryonic epicardial cells, in particular for miR-100, miR-125, miR-126 and miR-146 while opposite regulatory patterns are observed after miR-195 and miR-223 administration, respectively ( Supplementary Fig. 2). Similar discordant patterns are observed for Cdh5 expression except for miR-223 ( Supplementary Fig. 2). Curiously, no significant difference in cell migratory behavior was observed in time-lapse experiments (data not shown).
Expression of the fibrogenic marker Col1a1 display similar concordant patterns in EE HH24 and PE/ST explants after administration of miR-100 and miR-125, whereas discordant patterns were observed for all the other microRNAs tested ( Supplementary Fig. 2). Overall these data demonstrate that miR-195 and miR-223 maintain their potentially to enhance cardiomyogenesis in the embryonic epicardium and while the loose their ability to promote epithelial-to mesenchymal transition and enhance fibrogenesis in EE HH24 as compared to PE/ST explants. Thus, these data suggest a plausible therapeutic usage of miR-195 and miR-223 to enhance cardiomyogenesis without compromising putative adverse events such as EMT and fibrosis promotion. Observe that Bmp2 selectively enhances expression of miR-195 while no significant differences are observed after Fgf2 administration. HH17 PE were dissected from >30 embryos, treated with the corresponding microRNA mimics and/or Bmp/Fgf treatment, respectively, and subsequently pooled to perform RNA isolation. On each case, three-to-five distinct biological replicates were subsequently tested by qPCR. (2020) 10:9334 | https://doi.org/10.1038/s41598-020-66325-x www.nature.com/scientificreports www.nature.com/scientificreports/ In addition, we have also tested if these modulatory effects were similarly occurring in the PE HH17 cultured in hanging drop, to dissect if cell-matrix interactions are required or not for microRNA-mediated cardiac differentiation. For this purpose we assayed only those microRNAs displaying enhanced cardiomyogenesis in PE/ST explants and HH24 EE cultures, i.e. miR-195 and miR-223, and miR-100 as a negative control. Briefly, PE HH17 were dissected, set into hanging drops and concomitantly transfected with distinct microRNA mimics. After 24 h, RNA was isolated and cell lineage markers were assessed by qPCR. Our data demonstrate that administration of miR-100 does not enhance the expression of terminally differentiation cardiomyogenic marker cardiac troponin T while miR-195 and miR-223 significantly increased it (Fig. 3B), in line with previous data in PE HH17 cultured in collagen matrices (Fig. 2F) and HH24 EE cell cultures (Fig. 3A). Surprisingly, no enhancement was observed for Mhy5, probably due do time-specific differences in the onset of expression of these cardiomyogenic markers. Thus, our data revealed that cell-matrix interactions are not required for microRNA-mediated cardiomyogenesis.
Bmp and Fgf can distinctly modulate microRNA expression in the developing proepicardium.
Several growth factors members of the BMP and FGF families have been reported to distinctly modulate cell lineage specification in cardiogenic mesoderm into proepicardial and myocardial cells, respectively 14 . In particular, Bmp2 promotes differentiation of the septum transversum mesoderm into myocardial cells whereas Fgf2 enhances proepicardial lineage commitment, a process that is intricately regulated by a complex feed-back loop involving several other Bmp and Fgf family members 14 . We experimentally tested whether Bmp and Fgf signaling in the developing PE/ST influence the expression of distinct microRNAs with potential to modulate PE/ ST cell differentiation as reported above. HH17 PE/ST tissues were dissected and cultured in hanging drops. Administration of Bmp2 significantly increased expression of miR-195 while blocked expression of miR-100 and no significant differences were observed for miR-146 and miR-125. On the other hand, Fgf2 administration selectively blocked miR-125, miR-100, miR-125, miR-146 but enhanced miR-195 expression (Fig. 3C). These data illustrate that distinct administration of Bmp and Fgf signaling influence miRNA expression in the developing PE/ST tissues.
Search for common miRNA-mRNA pathways modulating cardiogenic lineage commitment. microRNAs can modulate multiple mRNA transcripts, ranging from hundreds to thousands targets 38 .
Distinct in silico algorisms can predict micro-mRNA interaction based on sequence complementary, biophysical interaction models and evolutionary conservation (i.e. TargetScan; http://www.targetscan.org/vert_72/ and MirWalk; http://mirwalk.umm.uni-heidelberg.de). We have demonstrated that over-expression of miR-195, miR-125, miR-146 and miR-223 respectively, in HH17 PE explants can enhance cardiomyocyte terminal differentiation. We therefore thought that they might share common targets governing these phenotypic changes. We selected all putative mRNA targets of miR-195, miR-125, miR-146 and miR-223 using MirWalk software and we identified all shared targets between these microRNAs. A total of 58 (1% all putative targets) mRNAs were identified for all four microRNAs, while 454 (8% all putative targets) were shared in three out of four microRNAs (Fig. 4A). We subsequently scrutinized all genes (512 target genes; 9% all putative targets) with previous cited reports playing a role in myogenesis, and selected short list of seven transcripts (Wnt5a, Smurf1, Sema5a, Smad3, Foxp1, Fosl2, RhoV) for further testing their implication in PE/ST-derived cardiomyogenesis. We then tested if these genes were modulated by miR-195, miR-125, miR-146 or miR-223 over-expression, respectively, in HH17 PE explants. In addition, miR-100 over-expression was also assayed as a negative control of cardiomyogenic inhibition and HH17 embryonic heart expression was also included to compare the relative expression of these genes in proepicardial and myocardial cells.
Comparative analyses of shared targets in the HH17 PE and embryonic heart demonstrate that Smurf1, Smad3, Foxp1, Fosl2 are enriched in the embryonic heart as compared to the PE, whereas Wnt5a Sema5a and RhoV display no significant differences (Fig. 4B-H). Over-expression of miR-100 selectively increased Smad3, decreased Wnt5a and Smurf1 while no significant differences were observed for Sema5a, Foxp1, Fosl2 and RhoV (Fig. 4B-H). Administration of miR-125 resulted in down-regulation of Wnt5a, up-regulation of Smad3, Foxp1 and RhoV, while no significant differences were observed for Smurf1, Sema5a and Fols2. Similarly, miR-146 over-expression resulted in down-regulation of Wnt5a, up-regulation of Fosl2, whereas all the other putative targets display no changes (Fig. 4B-H). Administration of miR-195 to HH17 PE explants resulted in up-regulation of Wnt5a, Smurf1, Smad3, Foxp1, Fosl2 and RhoV, but no significant changes were detected for Sema5a (Fig. 4B-H). Overall these data demonstrated that most of the shared targets are enriched in the HH17 PE. In addition, miR-125, miR-146 and miR-195 selectively modulate expression of these genes as predicted, except for Sema5a. Furthermore our data illustrate that miR-195 exerts up-regulation of multiple genes involved in early cardiomyogenesis, in line with our results demonstrating that miR-195 over-expression in HH17 PE explants enhances cardiomyogenesis, whereas miR-100 blocks several of them, in line with HH17 PE explants over-expression assays. Among those shared targets, up-regulation of Fosl2 and Smad3 is exerted by both miR-146 and miR-195, whereas up-regulation of RhoV and Foxp1 is exerted by miR-125 and miR-195, and Wnt5a and Smurf1 by miR-195 posing those genes as good candidates to explain the phenotypic consequences of driving cardiomyogenic differentiation upon microRNA over-expression.
Smad3 and Smurf1, but not Fols2, are essential for miR-195 driven cardiomyogenesis in PE/ ST explants. In order to test the functional role of these genes in miR-195 promoted cardiomyogenesis, loss-of-function experiments were performed in presence or absence of miR-195. siRNAs were successfully designed and validated against Fols2, Smad3 and Smurf1 while failed for Wnt5a and Foxp1 silencing either on the design itself or validation (data not shown). As illustrated in Fig. 5A, successful inhibition was obtained for Smad3, Smurf1 and Fols2, respectively. Interestingly, co-administration of miR-195 mimics and the corresponding siRNA, rescued Smad3 and Smurf1 but not Fols2 expression. Subsequently we tested if siRNA silencing leads Scientific RepoRtS | (2020) 10:9334 | https://doi.org/10.1038/s41598-020-66325-x www.nature.com/scientificreports www.nature.com/scientificreports/ to impair cardiomyogenesis in PE/ST explants and if silencing was rescued by miR-195 administration by measuring early (Mef2c, Gata4 and Nkx2.5) and terminally (Tnnt2) differentiation cardiomyocyte markers by qPCR. Our data demonstrate that Smad3 silencing significantly blocked the expression of Mef2c, Gata4, Nkx2.5 and Tnnt2, while miR-195 administration in this setting of Smad3 silencing only partially rescued expression of Nkx2. 5 and Gata4 but without reaching control levels (Fig. 5B). Similarly, Smurf1 siRNA significantly down-regulates Nkx2.5, Gata4 and Tnnt2, but surprisingly up-regulates Mef2c. miR-195 administration on Smurf1 siRNA treated explants partially rescued Mef2c to basal control levels but it was unable to recover Nkx2.5, Gata4 and Tnnt2  Smurf1, Smad3, Foxp1, Fols2 and RhoV, while no significant differences were observed for Sema5a. On the contrary, miR-100 over-expression lead to significant downregulation of Wnt5a, Smurf1, up-regulation of Smad3 and no significant differences of Sema5a, Foxp1, Fosl2 and RhoV. HH17 PE were dissected from >30 embryos, treated with the correspoding microRNA mimics and subsequently pooled to performed RNA isolation. On each case, three distinct biological replicates were subsequently tested by qPCR.
RNAs are emerging as novel molecules playing essential roles in gene expression regulation in multiple biological contexts [39][40][41] . Several studies have provided evidence that lncRNAs can play regulatory roles affecting neighboring genes [42][43][44] . We have identified nine distinct annotated lncRNAs neighboring key regulatory factors (Bmp2, Bmp4,  Wt1, Fgf2, Fgf8, Tcf21) involved in PE development in the chicken genome (Fig. 6A) and we have assessed their expression in PE as compared to age-matched developing heart. Three distinct patterns were observed, those displaying no significant differences (Bmp2_33140, Bmp2_53839, Wt1_74077), those with decreased expression in PE (Fgf2_56708, Tcf21_48334) and those with increased expression in PE (Wt1_76127, Bmp4_53170 and Fgf8_57126) as compared to HH17 embryonic heart (Fig. 6B). We subsequently assessed if those lncR-NAs with enhanced expression in PE were modulated by cardiomyogenic enhancing signals provided by Bmp administration or PE signals provided by Fgf administration 14 . We observed that Wt1_76127 was significantly down-regulated by Fgf8 while up-regulated by Bmp4 administration. Similarly, Bmp4_53170 was down-regulated by Fgf8 while up-regulated by both Bmp2 and Bmp4. On the other hand, Fgf8_57126 was up-regulated by Fgf2 and Fgf8 while significantly down-regulated by Bmp2 and Bmp4 (Fig. 6D). Overall these data demonstrate that these lncRNAs are complementary modulate by Fgf and Bmp signaling suggesting a plausible role in PE/ST derived cardiomyogenesis. In addition, we also tested if thymosin β4, a epicardial to myocardial priming agent 15 respectively. HH17 PE and HH17 embryonic hearts were collected from >30 embryos and pooled to performed RNA isolation. In all cases, three distinct biological replicates of pooled HH17 PE/ST and HH17 heart samples were subsequently tested by qPCR (panel A). HH17 PE/ST were dissected from >30 embryos, treated with thymosine beta4 (panel C), the correspoding Bmp/Fgf growth factor (panel D), and/or microRNA mimics treatment (panel E), respectively and subsequently pooled to performed RNA isolation. On each case, three distinct biological replicates were subsequently tested by qPCR.
Given the plausible role of these lncRNAs in PE/ST derived cardiomyogenesis, we tested if those microR-NAs enhancing (miR-195 and miR-223) or blocking (miR-23 and miR-27) cardiomyogenesis are capable of modulating their expression. PE/ST explants treated with miR-23 and miR-27 significantly inhibited expression of Wt1_76127, but did not modify expression of Bmp4_53170 and Fgf_57126. On the other hand, miR-195 significantly enhanced Wt1_76127 while inhibited Bmp4_53170, but no differences were observed for Fgf8_57126. miR-223 administration significantly blocked Wt1_76127 while Bmp4_53170 and Fgf8_57126 display no significant differences (Fig. 6E). These data demonstrate that microRNAs can regulate these lncRNAs and furthermore, microRNAs promoting vs inhibiting cardiomyogenesis display complementary regulatory roles, particularly on Wt1_76127 and Bmp4_53170, further reinforcing their plausible role in PE/ST derived cardiomyogenesis.

Discussion
Differential expression of microRNAs have been widely reported in distinct biological settings including homeostatic and pathological contexts [45][46][47] . Within the cardiovascular system, several studies have provided evidences of the differential expression of microRNAs during cardiogenesis 48,49 . However, to date, microRNA profiling of the proepicardium and/or epicardium is still missing. We provide herein evidence that multiple microRNAs display differential expression during the process of PE and epicardium formation. A large subset of microRNAs display increasing expression, supporting a plausible role blocking or inhibiting the expression of mRNA target genes during PE to embryonic epicardial transition. On the other hand, a small subset display decreased expression supporting a role in releasing repression of inductive signals while a similar subset display transition peak expression in HH24 as compared to HH32 embryonic epicardium, suggesting a plausible modulatory role in this transition, probably affecting thus epicardial to mesenchymal transition onset [50][51][52] . Thus, these data provide an entry site to start dissecting the functional roles of microRNAs during epicardial development.
Seminal evidences on the functional role of microRNAs in epicardial development was provided by Singh et al. 25 by selective deletion of Dicer, a ribonuclease involved in microRNA maturation, in the embryonic epicardium. However, understanding of the functional role of discrete microRNAs in the epicardium have only been provided for miR-31 and miR-21, both of them directing fibrogenic EMT by distinctly modulating Islet1 51 and Pcd4/Spry1 53 expression, respectively. Importantly, to the best of our knowledge this is first evidence reporting cardiomyocyte cell fate modulation of the PE/ST. A significant enhancement of cardiomyocyte terminal differentiation was provided by administration of miR-223 and miR-195 mimics, a weaker activation was provided by miR-125 and miR-146 while only activation of early cardiogenic markers but not terminal differentiation was obtained for miR-126. On the other hand, miR-23 and miR-27 selectively inhibited cardiomyogenesis while miR-100 and miR-21 essentially displayed not significant enhancement. These data therefore evidence the differential microRNA modulation of PE/ST cardiomyogenesis.
Previous studies reported the involvement of miR-23 and miR-27 in both cardiac development and pathology 48,54-58 , while miR-100 has only been reported as a protective agent of cardiomyocyte apoptosis 59 . On the other hand, miR-223 and miR-195 have been reported in distinct cardiac pathologies [60][61][62][63][64][65][66][67] but no evidences on their functional role during cardiac development have been described so far. Furthermore, scarce evidences on the role of miR-125 68 and/or miR-14 69 in cardiac development and pathology have been reported. On the other hand, miR-126 represents a vascular specific microRNA and miR-126 deficient zebrafish are embryonic lethal 70 . Furthermore, additional functional roles for miR-126 in the vasculature have been extensively reported [71][72][73][74] . Importantly a functional role in cardiomyocytes, particularly in apoptosis, is recently emerging [75][76][77] . Our data demonstrate that a more enhanced cardiomyogenic differentiation is exerted by miR-195 and miR-223 as compared to miR-125, miR-146 and miR-126, while miR-23 and miR-27 blocked such cardiomyogenic differentiation. Furthermore, our findings open up the possibility of exploring these microRNAs as therapeutic tools to enhance cardiomyogenesis.
Regulation of cardiac transcription factors such as Mef2c, Gata4 and Nkx2.5 by microRNAs have been reported in different biological contexts [78][79][80] . In striated muscle, miR-27 and miR-125 distinctly regulate Mef2c in cardiac and skeletal muscle cells 31,48 . Curiously, miR-223 downregulation leads to Mef2c upregulation in leukemia 81 while no evidences have been reported for miR-195 and/or miR-146 modulating the expression of these early cardiomyogenic differentiation markers in striated muscle. In this study we demonstrate for the first time the regulatory role of these microRNAs modulating expression of early and terminally differentiation cardiomyogenic markers in both PE/ST and epicardial cell cultures, enhancing thus their potential therapeutic usage.
An integral developmental process linked to PE and epicardium morphogenesis is driven by an epithelial to mesenchymal transition that provides mechanistic clues to these cells favouring their integration into the embryonic myocardium and subsequently differentiation into distinct cell types, such as fibroblasts smooth muscle cells and endothelial cells 52 . In this study we further investigated how administration of these microRNAs influence EMT and fibrogenic differentiation. Our data demonstrate that miR-195 and miR-23 can selectively down-regulate expression of EMT inducers such as Snail and Slug and up-regulate Cdh1 expression without modulating Cdh2 and Chd5, in line with previous reports in other biological contexts 72,82 . On the other hand, all the other microRNAs tested (i.e. miR-21, miR-27, miR-100, miR-125, miR-126, miR-223 and miR-146) resulted in Snail and/or Slug up-regulation while effects on Cdh expression is not always concomitant. Several of these microRNAs have been reported to promote EMT 83,84 while other can either promote or inhibit it in different biological contexts [85][86][87][88][89][90][91][92][93][94] , in line with our findings. Importantly, we provide evidence for the first time on the involvement of miR-125 and miR-146 in EMT regulation. Our data suggest that up-regulation of EMT inducers and subsequent cytoskeletal remodeling represent uncoupled events in this setting, in line with previous reports Scientific RepoRtS | (2020) 10:9334 | https://doi.org/10.1038/s41598-020-66325-x www.nature.com/scientificreports www.nature.com/scientificreports/ during AV EMT modulation by microRNAs 28 , alternatively that additional time is required to see such transcriptional changes in Cdh expression or that transcriptional overriding effects by microRNAs over-expression is occurring 37 . Thus, additional experiments are required to fully elucidate this apparently discordant findings. Furthermore, our data demonstrate that EMT is not required to PE/ST cardiomyogenic differentiation, since administration of miR-223 can simultaneously induce both processes. i.e. EMT and cardiomyogenesis.
Importantly, we demonstrate herein that a single microRNA can exert different regulatory aspects in both PE/ST explants and HH24 EE cell cultures. miR-23 can block cardiomyogenic differentiation and EMT while promotes fibrogenic differentiation, miR-195 enhances cardiomyogenesis while blocking EMT but promoting fibrogenic differentiation while, miR-223 can promote all three developmental processes, providing thus a therapeutic potential for cardiomyogenic regeneration.
To understand the molecular mechanisms that drive promotion of cardiomyogenic terminal differentiation by miR-223, miR-195, miR-125 and miR-146 administration, we search for common shared putative targets. A short list of seven genes (Wnt5a, Smurf1, Sema5a, Smad3, Foxp1, Fosl2 and RhoV) previously involved in myogenesis [95][96][97][98][99] were assessed, demonstrating that miR-195 overexpression lead to up-regulation of of all these genes, except Sema5a, further supporting their plausible involvement in miR-195 driven cardiomyogenesis in PE/ST explants. Furthermore, silencing of Smad3 and Smurf1 lead to significant down-regulation of early and terminally differentiation markers. Importantly, application of miR-195 in siSmad3 and siSmurf1 treated PE/ST explants was unable to rescue the expression of cardiomyogenic lineage markers. Thus, these data demonstrate for the first time that miR-195 application modulates expression of Smad3 and Smurf1, factors that are essential to promote cardiomyogenesis. It remains unclear whether Smad3 and Smurf1 up-regulation by miR-195 is a direct or an indirect effect. Future experiments will be designed to unravel the molecular mechanisms, although it is important to highlight that microRNAs can directly increase mRNA stability 100 . Overall, these data provide novel insights into the molecular mechanisms whereby miR-195 administration exerts increased cell differentiation into the cardiomyogenic lineage, i.e. by regulating the expression of Smad3 and Smurf1.
Long non coding RNAs represents a novel emerging class of non coding RNAs with highly diverse cellular functions 101 . Tissue-specific expression of lncRNAs has been widely reported in distinct biological settings, including the cardiovascular system 102 . Seminal studies by Klatenhoff et al. 103 reported the functional role of Braveheart, a mesoderm-restricted lncRNA essential for normal lateral plate mesoderm formation and thus cardiac development. Similarly, the function role of handful set of lncRNAs have been reported such as Fendrr, Carmen, Upperhand and Tbx5ua 42,104-106 . However, to date, no lncRNAs has been reported during PE and epicardium formation. We provide herein a systematic analyses of lncRNAs neighboring key growth factors and transcription factors involved in PE and epicardium development and we identify three lncRNAs with enhanced expression in the PE. Secondly, we demonstrate that all three of them and distinctly regulated by cardiomyogenic inductive signals such as thymosin β4 15 and Bmp 14 administration as well as by repressive signals, i.e. Fgf signaling. These data support a plausible role for these lncRNAs in PE/ST cardiomyogenic differentiation, however additional experiments are required to dissect their functional role in this context. Regulatory effects of lncRNAs upon microRNAs has been widely reported in the cardiovascular system 107 as well as in other biological settings [108][109][110][111][112][113] . However, evidence of microRNA regulation of lncRNAs is still scarce. We provide herein evidences for the first time that microRNAs can modulate the expression of lncRNAs. miR-195 administration exerts opposite regulatory effects as compared to miR-23 and miR-27 supporting a role for these lncRNAs in PE/ST miR-195 driven cardiomyogenesis. Surprisingly, miR-223 did not affect the expression of these lncRNAs, suggesting a microRNA-specific modulation. In sum, our data opened up new pathways to dissect the functional role of microRNAs and lncRNAs in PE/ST development and their plausible application to enhance myocardial formation.