Foxf1 -mediated co-regulation of miR-495 and let-7c modulates epicardial cell migration and myocardial specification

Foxf1 -mediated co-regulation of miR-495 and let-7c modulates epicardial cell migration and myocardial specification | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Foxf1 -mediated co-regulation of miR-495 and let-7c modulates epicardial cell migration and myocardial specification Juan Manuel Castillo-Casas, Ángel Dueñas, Francisco Hernández-Torres, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5643113/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Jun, 2025 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted 5 You are reading this latest preprint version Abstract Background The heart is the first functional organ to develop in the vertebrate embryos. In mice, the primitive tubular heart begins beating at embryonic day (E) 8.0-E.8.5 and undergoes rightward looping to form the atrial and ventricular chambers. The proepicardium, a transient cell cluster at the sinus venous-lateral plate mesenchyme junction migrates onto the heart and gives rise to the embryonic epicardium, a squamous epithelium that plays a key role in cardiac development. Despite advances in understanding epicardial lineage contributions, the molecular mechanisms governing these processes remain poorly understood. Methods To characterize the transcriptional and post-transcriptional regulation of epicardial development, we performed RNA sequencing at two critical timepoints, proepicardium formation and embryonic epicardium establishment. We analysed differentially expressed coding and non-coding RNAs, focusing on microRNAs and their potential regulatory interactions. Results We identified a complex network involving differentially expressed mRNAs, microRNAs and lncRNAs between proepicardium and embryonic epicardium. Notably, with miR-495 and let-7c emerged as key regulators of epicardial cell migration, an essential process for proper epicardium formation and epicardial-derived cell migration. Our findings also reveal that these microRNAs not only regulate target gene expression but also modulate other microRNAs, suggesting a novel regulatory mechanism in epicardial development. Additionally, Foxf1 inhibition modulates let-7c , promoting the expression of key cardiogenic lineage markers in epicardial cells. Conclusion Our study highlights the role of Foxf1 in regulating miR-495 and let-7c , which in turn modulate epicardial cell migration and myocardial specification. These finding provide new insights into the intricate interplay between transcription factors and microRNAs in governing cardiogenesis. Transcription factors microRNAs epicardial cells cell migration cell lineage specification. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 BACKGROUND The heart is the first organ that becomes functional in the vertebrate embryo. In mice, the precardiac mesoderm forms a primitive tubular heart, beating at embryonic day (E) 8.0-E.8.5. This tubular structure undergoes a series of morphological changes, including rightward looping and thereafter configuring the prospective atrial and ventricular chambers (E9.5) ( 1 ). At E10.5, five distinct regions can be delineated in the embryonic heart, the inflow tract, the embryonic atrial chamber, the atrioventricular canal, the ventricular chambers and the outflow tract ( 2 ). From this stage onwards, each embryonic cardiac region will be separated into distinct left and right components, providing thus a double circuitry with distinct inlet and outlet connections ( 1 ). Besides the intrinsic cardiac progenitor cells, external cell populations also contribute to heart development i.e. the cardiac neural crest and the proepicardium and its derivatives ( 3 , 4 ). The proepicardium (PE) is a transitory cell cluster that develops at the junction between the sinus venosus (SV) and the posterior undifferentiated lateral plate mesenchyme (LPM) at E8.5-E9.0 in the mouse embryo ( 5 , 6 ). In chicken embryos, PE grows in size and villous projections extend towards the dorsal aspect of the cardiac inner curvature, ultimately contacting the atrioventricular (AV) junction and forming a tissue bridge ( 7 ). In mice, around E10.0 proepicardial villous projections attach to the heart ( 6 ) and proepicardial cells migrate and spread over the naked myocardium forming a single squamous epithelium which is termed the embryonic epicardium (EE), playing an essential role in cardiac development ( 5 , 8 ). The epicardium, the outermost layer of the heart, was long considered as an external cover devoid of any functional meaning, but recent studies discovered its essential contribution to the cardiac development and regeneration ( 9 – 12 ). The EE serves as a crucial source of epicardial-derived cells (EPDCs) that, after undergoing epithelial-to-mesenchymal transformation (EMT), migrate into the myocardial wall and differentiate into multiple cardiac cell types ( 13 , 14 ). EPDCs contribute to endothelial and smooth muscle cells in the coronary vasculature, cardiac fibroblasts ( 15 – 17 ), and to a lesser extent, atrioventricular cushion cells ( 18 – 20 ). More recently, a contribution to cardiac resident stem cells (mesenchymal-like) has also been reported ( 21 ) as well as to the cardiomyocyte lineage ( 22 – 24 ), yet this latter point remains highly controversial ( 25 , 26 ). Recent studies have enhanced our understanding of the molecular mechanisms driving PE and EE tissue formation ( 26 ). Signalling molecules such as Bmp and Fgf play pivotal roles in PE specification and cardiomyogenic differentiation ( 27 ). Transcription factors such as Wt1 are crucial for EMT and EPDCs maturation ( 11 , 28 – 32 ). Tbx18 has a role in epicardial EMT and subsequently in differentiation of EPDCs into smooth muscle cells and fibroblasts ( 33 , 34 ) while Tcf21 regulates proepicardial cell specification and maturation ( 35 ). Finally, while Gata4 is essential for PE formation, the precise contributions of other cardiac-enriched transcription factors such as Nkx2.5 , Isl1 , and Pitx2 remain unclear ( 36 – 38 ). Despite these advances, it is poorly understood how transcription factors and non-coding RNAs contribute to epicardial development. While transcriptional regulation plays a critical role in cardiac morphogenesis and cardiovascular cell differentiation, a growing body of evidence suggests that microRNAs, the most studied subtype of small non-coding RNAs, play crucial roles in gene regulation during embryonic development and tissue homeostasis ( 39 , 40 ) ( 41 , 42 ) microRNAs display temporal and spatial differential expression in both embryonic and adult tissues, where they fine-tune gene expression at the post-transcriptional level ( 43 ). In the context of cardiogenesis, several microRNAs have been implicated in cardiac differentiation, proliferation and morphogenesis. ( 44 – 48 ). Recent studies in our laboratory evidenced a microRNA differential expression during PE and EE formation in chicken, identifying miR-146 , miR-195 and miR-223 as potential regulators that selectively enhance cardiomyogenesis in PE and EE by modulating Smad3 and Smurf1 , in ex vivo conditions ( 48 ). Considering the species-specific differences in epicardium formation and the discovery of DE microRNAs in chicken, the functional role of microRNAs in PE and EE development in mice, as well as their potential application to enhance cardiogenesis remains elusive. Although significant progress has been made in understanding the cell lineage contribution of the EPDCs over the last decade, the molecular determinants that contribute to such cell fate decisions remains largely unknown. In this study, we carried out a comprehensive RNAseq analysis of coding and non-coding gene expression at two critical timepoints of PE and EE development in mouse embryos. Our data identified an intricate network of differentially expressed (DE) mRNAs, microRNAs and lncRNAs that regulate distinct biological pathways in PE vs . EE. We identified that Foxf1 transcription factor exerts a regulatory control over miR-495, miR-351 , and let-7c , thereby modulating epicardial cell migration and myocardial specification. These observations underscores the complex interplay between transcription factors and microRNAs in epicardial development, providing new insights into the molecular mechanisms that govern cardiogenesis during embryonic development. METHODS A comprehensive description of each procedure is detailed in the following sections. Supplementary Fig. 1 provides an overview of the experimental workflow, illustrating the key methodological steps. Mouse lines and tissue collection Previously described Wt1 GFP/+ mice were used in this study. The WT1 GFP knockin line in which the exon 1 of a Wt1 allele has been replaced by the GFP sequence was used as a reporter for active WT1 transcription ( 49 ). Pregnant Wt1 GFP/+ female mice were harvested to E9.5 and to E10.5, respectively. E9.5 PE were manually dissected, pooled and stored in buffer lysis for RNA isolation at -80°C until used. For flow cytometry analysis and sorting, dissected hearts from E10.5 embryos were placed in cytometry buffer (phosphate buffer saline [PBS] plus 2% fetal bovine serum [FBS] and 10 mM 4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid [HEPES]) and homogenized by repeated pipetting. Cell suspension was washed by pelleting at 400G during 5 minutes. Then, cells were incubated on ice in darkness with the fluorochrome conjugated antibodies: anti-CD31-APC (PECAM-1) (Mouse monoclonal anti-CD31 APC #Thermo Fisher, 17-0311-82) for general staining of the endothelium. 4',6-Diamidine-2'-phenylindole dihydrochloride (DAPI) staining was included to exclude dead cells. Negative controls (GFP negative littermates) and isotypic antibody allowed setting of the gates (FITC Rat IgG2a, k Isotype, #Biolegend). Epicardial cells were then sorted by GFP high fluorescence and lack of CD31staining (Supplementary Fig. 2) ( 50 ). Cells were sorted in a BD FACS Aria Fusion Cell Sorter. Data were analyzed with FlowJo TM10. After sorting, epicardial cells were pooled and stored in buffer lysis for RNA isolation at -80°C until used. At least 3–5 litters were used at each developmental stage until sufficient tissue was collected, which would guarantee optimal and sufficient RNA isolation for further sequencing. RNA-seq libraries preparation, sequencing and differential expression gene analysis For single-end microRNA libraries, 500 pg of total RNA were used to generate barcoded miRNA-seq single-end libraries using the Bioo NEXTflex Small RNA (BiooScientific). Briefly, 3´ and 5´ SR adapters were first ligated to the RNA sample. Next, reverse transcription followed by PCR amplification was used to enrich cDNA fragments with adapters at both ends. Adapter-ligated cDNA fragments from different samples were pooled and run in a 6% polyacrilamide gel. The 147 nt band, corresponding to the pooled miRNA libraries, was purified from the gel. Finally, the quantity and quality of the pooled miRNA libraries were determined using the Agilent 2100 Bioanalyzer High Sensitivity DNA chip. Both, mRNA microRNA libraries were sequenced on a HiSeq 2500 (Illumina) and processed with RTA v1.18.66.3. FastQ files for each sample were obtained using bcl2fastq v2.20.0.422 software (Illumina). For FastQC reads quality reports analysis, trimming of adaptors and alignment of sequences, fastq sequence reads were uploaded to the European version of the Galaxy platform ( 51 ). The quality of the reads was analyzed with FastQC Read Quality reports (Galaxy Version 0.74 + galaxy1) software, trimmed with Trim Galore software (Galaxy Version 0.6.7 + galaxy0) and aligned to the built-in mouse reference genome mm10 (GRCm38) with the RNA STAR Gapped-read mapper (Galaxy Version 2.7.10b + galaxy3) ( 52 ). For gene-expression analyses, bam files were downloaded from the Galaxy server and further analyzed with the different RStudio packages downloaded from the Bioconductor website ( http://bioconductor.org , accessed on 2 March 2023). Reads were assigned to mRNA and microRNA genes by using the “featureCounts” function of the “Rsubread” package, version 2.10.5 ( 53 ). In addition, mouse gencode.vM20.annotation.gff3 annotation file release M20, GRCm38.p6 ( https://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_mouse/release_M20/gencode.vM20.annotation.gff3.gz ) and mmu.gff3 chromosomal coordinates of Mus musculus microRNAs miRBase v22 ( https://www.mirbase.org/download/#:~:text=mml.gff3-,mmu.gff3,-osa.gff3 ) were used for mRNA and miRNA analysis, respectively. Uniquely mapped reads were used to calculate gene expression. The library size of each experimental point ranged from 18625406 to 26021292 sequences and from 601840 to 1251790 sequences for mRNA and miRNA analysis, respectively. Fastq files and abundance measurements of features were uploaded to Gene Expression Omnibus database with GEO accession number : GSE189344 Differential gene expression analysis was performed through package “edgeR-package” version 4.4.2 ( 54 ). The normLibSizes() function was used to normalize the library sizes by trimmed mean of M-values (TMM) method. Only transcripts detected in three transcriptomes were used in the analysis. All gene comparisons with an adjusted p-value 2 were considered differentially expressed under the experimental conditions. For miRNA-mRNA transcripts interaction analysis we used miRComb package ( 55 ). Gene Set Enrichment Analysis (GSEA)-based Gene Ontology (GO) analyses were conducted with the “clusterProfiler” package version 3.6.0 ( 56 , 57 ). The gene sets with a p-value < 0.05 were considered overrepresented under the experimental conditions. RNA isolation and qRT-PCR RNA samples from the same pools used for RNAseq libraries construction as well as additional isolated RNA samples were used; namely, those corresponding to E9.5 PE cells and E10.5 FACS sorted EE cells. All RT-qPCR experiments followed MIQE guidelines ( 58 ) and similarly as previously reported ( 59 , 60 ). Briefly, RNA from tissue samples was extracted and purified by using the Direct-zol™ RNA Miniprep kit (Zymo research) and the cell line RNA isolation was performed with ReliaPrep™ RNA Miniprep Systems kit (Promega), both according to manufacturer´s instructions. For mRNA/lncRNA expression measurements, 500ng of total RNA was used for retro-transcription with PrimeScript™ RT Master Mix (Takara), the resulting cDNA was diluted 1/5, both according to manufacturer’s guidelines. For microRNA expression analyses, 20 ng of total RNA was used for retro-transcription with with miRCURY LNA RT Kit (Qiagen), the resulting cDNA was diluted 1/40, following manufacturer´s guidelines. Negative controls, without reverse transcriptase, were performed for each sample to assess genomic contamination. Real-time PCR experiments were performed with 1 µL of cDNA, GoTaq qPCR Master Mix (Promega) and corresponding primer sets as described in Supplementary Table 1 . All qPCRs were performed using a CFX384TM thermocycler (Bio-Rad) following the manufacturer’s recommendations. For mRNA, the qPCR program consisted of 95°C for 30 s (initial denaturalization), followed by 40 cycles of 95°C for 5 s (denaturalization); 60°C for 10 s (annealing); 75°C for 7 s (extension). Finally, melting curves were determined by an initial step of 95°C for 5 s, followed by 0.5°C increments for 7 s from 65°C to 95°C. For microRNAs, the qPCR program consisted of 95°C for 10 min (initial denaturalization), followed by 40 cycles of 95°C for 5 s (denaturalization); 60°C for 1 min (annealing and extension). Finally, melting curves were determined by an initial step of 95°C for 5 s, followed by 0.5°C increments for 7 s from 65°C to 95°C. The relative level of expression of each gene was calculated as described by Livak & Schmittgen (2001) ( 61 ) using Gapdh as the internal control for mRNA expression analyses and 5S for microRNA expression analyses, respectively. Each PCR reaction was carried out in triplicate and repeated in at least three distinct biological samples to obtain representative means. Cell lines In this study four cell lines were used, immortalized embryonic endocardial MEVEC ( 62 ), muscle cardiac cell line HL1 (Sigma-Aldrich SCC065), mouse embryonic epicardial cell line MEC1 (Sigma-Aldrich SCC187) and epicardial EPIC ( 63 ). Each cell line was cultured following the manufacturer’s recommendations for 24hr at 37°C in a humidified atmosphere of 5% CO 2 at 4x10 4 cells per well in plates of 24 wells before transfection. Tissue explants isolation All experiments were performed with the approved consent of the Ethics Committee of the University of Jaén and Andalusian Regional Government (14/03/2022/038). Pregnant CD1 wild-type female mice were harvested to E10.5. E10.5 ventricles were manually dissected and cultured in DMEM/Glutamax culture medium. For embryonic epicardial cell isolation for qPCR analysis, the ventricles were dissected in Earle’s balanced salt solution (EBSS) (Gibco), and cultured in a 12-well plates with collagen type I gels (Sigma-Aldrich #C3867-1VL), as previously described ( 59 ), for 48 hours before transfection. Epicardial cells from transfected E10.5 ventricles were isolated, pooled and directly stored at -80°C until used. For confocal microscopy analyses, the ventricles were dissected in Earle’s balanced salt solution (EBSS) (Gibco), and cultured in a 4-chambered glass bottom dish with collagen treatment as previously described ( 59 ), 48 hours before transfection. Briefly, samples were fixed in freshly made 4% PFA and stored in PBS at 4°C until used. Each experimental condition was repeated at least three times with a minimum number of three explants per condition, respectively. microRNA mimics or anti-miR and siRNA transfections E10.5 ventricles were cultured for 48 hours at 37°C in a cell culture incubator before administration of miRNAs mimics (pre-miRNAs), anti-miRNAs or siRNAs, respectively, as previously described ( 60 ). Pre-miRNAs, anti-miRNA and siRNA transfections were carried out with Lipofectamine 2000 (Invitrogen), following the manufacturer’s guidelines. Briefly, 50nM of premiRNAs (microRNA precursor) or antimiRs (microRNA inhibitor) were applied to the explants (3 explants per well), and for siRNA transfection 60-80nM of siRNA were applied. These concentrations were selected based on preliminary experiments in which qRT-PCR was performed to assess transfection efficiency, adjusting the doses for each condition. After incubation, 24 hours for pre-miR or 48 hours for anti-miR and siRNA, explants were either processed for RT-qPCR or immunohistochemical (IHC) analyses. Negative controls, E10.5 ventricular explants, treated only with Lipofectamine were run in parallel. To perform IHC analyses, the explants were fixed in PFA 4% for 15 min at room temperature rinsed two times in PBS for 5 minutes and stored in PBS at 4°C. For RT-qPCR analysis, explant epicardial outgrowths were collected and stored at -80°C. Cell migration assays Mouse E10.5 ventricular explants were isolated from the developing embryo and the ventricular apex was dissected, plated upside down into coated collagen 4 chambered glass bottom dishes, incubated into DMEM Glutamax culture media for 48 hours as previously reported. At this stage, emerging epicardial outgrowths start to develop. Transfections with corresponding pre-miRNAs, anti-miRNAs, scrambled, siRNAs and negative controls, respectively, were carried out and cultures were allowed to develop for another 24/48 hours. Explants were rinsed in PBS for 5 min at room temperature and fixed in 4% PFA for 15 min at room temperature. After the fixation, the explants were rinsed two times in PBS for 5 min and incubated with Phalloidin FITC 1:1000 (Abcam) following the manufacturer’s recommendations. Finally, DAPI 1:2000 (Sigma) was incubated for 15 min at room temperature, rinsed two times in PBS for 5 min and stored in PBS in darkness at 4°C. Subsequently, representative images of each explant were collected using a Leica TCS SP5 II confocal scanning laser microscope, and the extension of the epicardial migration (i.e. cohesive, non-cohesive and total migration) was measured in ten different regions per image, using ImageJ software. Mean and SD values were subsequently plotted. Confocal Scanning Laser Microscopy analyses Immunofluorescence analyses were performed as previously reported ( 59 ). Briefly, control and experimental explants were collected after the corresponding treatment, rinsed in PBS for 10 min at room temperature, and fixed with 4% PFA at room temperature for 15 min. After fixation, the samples were rinsed three times (10 min each) in PBS at room temperature and then permeabilized with 0.02% Triton X-100, 50nM Nh 4 Cl and PBS for 10 min at room temperature. Non-specific binding sites were blocked with 0.2% Gelatin solution (Sigma-Aldrich) applied two times for 10 minutes. As primary antibodies, an anti-Wt1 (Santa Cruz) and anti-cTnT (Hytest) at 1:200 dilution in blocking solution was applied overnight at 4°C. Ventricle explants were rinsed 3 times in PBS for 10 min and incubated with secondary antibody anti-Goat 488 (Invitrogen) 1:100 dilution, 30 min at room temperature. Finally, ventricle explants were incubated with DAPI 1:2000 (Sigma) for 15 min at room temperature and rinsed two times in PBS for 5 min each. The explants were stored in PBS in darkness at 4°C until analysed using a Leica TCS SP5 II confocal scanning laser microscope. Statistical Analyses For statistical analyses of datasets, unpaired Student’s t-tests were used. Significance levels or P values are stated in each corresponding figure legend. P < 0.05 was considered statistically significant. RESULTS Coding and non-coding RNA differential expression in the PE to EE transition in mice To investigate gene expression changes during the transition from PE to EE, we performed RNAseq on manually dissected PE from E9.5 Wt1-GFP heterozygous mouse embryos (n∼20) and GFP + FACS-sorted EE cells from E10.5 Wt1-GFP embryonic hearts (n∼15). Each condition included three independent biological samples (PE: n = 7–8 per sample; EE: n = 3–5 hearts per sample). RNAseq libraries for microRNAs and mRNA/lncRNAs in these two distinct stages of epicardial development were constructed and sequenced, yielding an average of 5*10 6 reads (5,25*10 6 ± 1*10 6 ) for microRNA libraries and 35*10 6 reads (36,5*10 6 ± 2,5*10 6 ) for mRNA/lncRNA libraries. Alignment efficiency was approximately 85–90% for mRNAs of the total input resulting in the identification of ∼12500 genes, while microRNA reads alignment yielded lower inputs, 40–50% of the total and identified ∼200 microRNA expressed in both conditions. An exploratory analysis validated the similarity between PE E9.5 vs EE E10.5 RNAseq datasets (Supplementary Fig. 3). In order to identify those genes that might be involved in governing the transition between the PE and EE, we have identified those DE genes, including therein microRNAs, mRNAs and lncRNAs, using as selection criteria those genes displaying a log2 FC > 1 and FDR p < 0.05. This analysis identified 979 mRNAs up-regulated in the PE as compared to EE ( Table 1 ), whereas 886 display the opposite pattern, down-regulated in the PE as compared to the EE ( Table 2 ) (Fig. 1 A). RT-qPCR validation confirmed the differential expression of these mRNAs (Fig. 1 C-D). In this context, it is important to highlight that transcription factors such as Hnf4a , Hoxb1 and Prox1 are enriched in the PE at E9.5, whereas Spry1 , Hey2 and Itga1 are enriched in the EE at E10.5. Similarly, microRNA analysis identified 59 microRNAs highly expressed in PE as whereas 9 microRNAs were upregulated in EE ( Table 3 ) (Fig. 1 B). RT-qPCR validation confirmed the differential expression of these miRNAs, where in fact, miR-200a-3p, miR-200b-3p, miR-200c-3p, miR-429-3p and miR-495-3p displayed higher level of expression in PE, whereas let-7c-5p, miR-24-3p, miR-30a-3p, miR-30c-5p and miR-351-5p showed higher expression levels in EE (Fig. 1 E-F). LncRNAs display an averaged lower expression levels as compared to protein-coding RNAs. Even so, our RNAseq analyses identified 60 lncRNAs that are highly expressed in the PE ( Table 4) and 111 lncRNAs upregulated in EE ( Table 5 ). Similar to mRNAs and miRNAs, the differential expression of some of these lncRNAs was confirmed by RT-qPCR, i.e. Gm35409, Gm35533, 9030622O22Rik and 9030102K24Rik display high levels in PE and Gm13293, Gm42788 and 4833415N18Rik display high levels in EE (Fig. 1 G-H). These data demonstrate therefore an important contribution of miRNAs, mRNAs and lncRNAs in the PE to EE transition, yet their functional implications remain to be elucidated. Signalling pathway enrichment displays significant differences in mouse PE and EE differential expressed genes To provide a comprehensive analysis of the biological processes associated with DE genes profile in PE and EE stages, we performed a Gene Set Enrichment Analysis (GSEA). As depicted in Supplementary Fig. 4A , DE genes upregulated in EE display enhanced representation of myofilament and myosin complex, mitochondrial respiratory chain and actomyosin contractile and actin filaments in GSEA GO Cellular Compartment (CC) analyses. In contrast, DE genes downregulated in EE display enhanced representation of membrane and cell-cell contact pathways in GSEA GO CC analyses, suggesting a shift from intercellular communication in PE to muscle function and motility in EE. GSEA GO Molecular Function (MF) analysis further supported this distinction ( Supplementary Fig. 4B ). DE genes upregulated in EE display enhanced representation of chemokine receptor binding, tropomyosin and structural components of the muscle and muscle alpha-actinin binding, while those DE genes downregulated in EE display enhanced representation of cofactors and calcium ion binding and signalling receptor activity These data reinforce the role of DE genes in EE in muscle function and motility, whereas DE genes in PE remain more engaged with cell-cell signalling. Finally, the pathways revealed by GSEA GO Biological Pathway (BP) further support these findings ( Supplementary Fig. 4C ). Tissue-specific expression patterns of differentially expressed genes in PE and EE further support different signalling pathway enrichment To further investigate the biological relevance of the DE genes between E9.5 PE and E10.5 EE, we analyzed their tissue-specific expression using the Genepaint database ( https://gp3.mpg.de ). Analyses of the top 10% downregulated DE genes (PE > EE) (∼90 genes) revealed that approximately 55% (47/85) of them displayed restricted liver expression at E14.5 days, 9% (8/85) were preferentially expressed in the endocardium and 8% (7/85) display expression in the epicardium. No detectable expression was observed for 23% (20/85) of the DE genes analysed and 11% (10/85) were not found on the Genepaint database ( Supplementary Fig. 5 ). Analyses of the top 10% downregulated DE genes (PE < EE) (∼100 genes) revealed that approximately 17% (17/101) were expressed in the epicardium, 12% (12/101) in the endocardium and 6% (6/101) within the myocardium. No detectable expression was observed for 32% (32/101) of the DE genes analyzed and 17% (17/101) were not found on the Genepaint database ( Supplementary Fig. 5). Overall, these data demonstrate a distinct bias on the preferential distribution of DE genes in the PE and EE stages. It is important to highlight in this context the large abundance of hepatic specific genes in the E9.5 PE fraction and a relatively low abundance of epicardial restricted genes. On the other hand, it is equally surprising that a small but consistent number of DE genes with enhanced expression in the EE 10.5 are mostly myocardial restricted. MicroRNA-mRNA regulatory networks reveal distinct transcriptional pathways involved in mouse PE to EE transition Previous studies have identified microRNA-mRNA cross-talk correlations by searching for opposite patterns between mRNA and microRNAs in distinct experimental conditions ( 64 – 66 ). Using miRComb software, we have searched for all putative candidate microRNAs that target each of the DE mRNAs with high expression in the PE as compared to the EE as depicted in Fig. 1 I. Nine distinct microRNAs with enhanced expression in the EE ( let7c-5p, miR-351-5p, miR-30c-5p, miR-780-3p, miR-677-5p, miR-5112, miR-320-3p, miR-483-5p and miR-6236 ) display complementary pattern to mRNAs with the opposite pattern (PE > EE). Let7c-5p, miR-351-5p and miR-30c-5p ( let-7c, miR-351 and miR-30c , respectively, will be used throughout this text) display a wide range of interactions, supporting a more relevant functional role, as compared to miR-780-3p, miR-677-5p, miR-5112, miR-320-3p, miR-483-5p and miR-6236 (Fig. 1 I, Supplementary Fig. 6 ). On the other hand, 60 distinct microRNAs with enhanced expression in the PE display a complementary pattern to mRNAs with the opposite pattern (PE < EE). miR-495-5p, miR-200b-3p and miR-181c-5p ( miR-495, miR-200b and miR-181c , respectively, will be used throughout this text) are the three microRNAs that display a larger range of interactions, respectively, as compared to the other DE microRNAs (Fig. 1 I, Supplementary Fig. 6 ). Overall, these data identify novel microRNA-mRNA predicted interactions that might be functionally important during PE/EE development. We have centered our attention on those three DE microRNAs that display a larger number of mRNA interactions in each developmental stage, i.e. let-7c, miR-351 and miR-30c in PE EE. Biological theme comparison of the molecular function of the putative DE-mRNA targets identified by these microRNAs revealed that let-7c is primarily involved in RNA polymerase/transcription factor DNA binding (Supplementary Fig. 7) while miR-30c is involved in receptor and cell-cell signalling as well as in ion channel regulation. miR-495 is primarily involved in RNA polymerase, transcription factor DNA binding and GAG binding, while miR-200b in proteoglycan, GAG, calcium binding and GTPase receptor activity. Moreover, miR-181c is primarily involved in protein heterodimerization, nuclear receptor, transcription factor and steroid/hormone activity as well as on protein phosphatase and collagen binding. Similar findings are observed in CC and BP biological theme comparisons (data not shown). Importantly, there was a minimal overlap on the predicted targets between these key microRNAs, reinforcing the idea that they modulate different signaling pathways. Let-7c, miR-351 and miR-30c display only one shared target ( Prtg ) ( Supplementary Fig. 8A ), while miR-495, miR-200b and miR-181c display equally uncommon shared targets ( Nfib, Mbnl2, Kat2b and Nr3c1 ). However, an increased number of targets are shared between miR-495 and miR-200b (12 genes; Fn1, Rnd3, Elf2, Rapgef2, Plxna4, Gpm6a, Psd3, Amotl2, Arl4a, Hapln1, Dusp1, Vegfa ) and between miR-495 and miR-181c (11 genes; Col16a1, Acer3, Sox6, Dmxl2, Dusp6, Sept8, Gpr22, Akap6, Adamts5, Aqp4, Mcc ), suggesting common functional roles in signalling pathways ( Supplementary Fig. 8B ). Additionally, it worth mentioning that several shared DE mRNAs target of PE > EE miRNAs have a role modulating cell migration in other different biological context, i.e. Nfib ( 67 – 69 ), Mbnl2 ( 70 ), Nr3c1 ( 71 , 72 ), Fn1 ( 73 , 74 ) and Rnd3 ( 75 , 76 ), a key process during PE/EE development. Differentially expressed microRNA-mRNA predicted interactions are distinctly validated in EPIC and MEC epicardial cells To dissect the regulatory mechanisms driven by DE-microRNAs, we performed gain-of-function assays of different microRNAs in two distinct epicardial cell lines, MEC1 and EPIC, respectively. MEC1 cells retain the morphology of early primary epithelial epicardial cells and express epicardium-specific markers including epicardin ( Tcf21 ), Tbx18 and Krt18 , while EPIC cells continuously proliferate and expand, acquiring a characteristic mesenchymal phenotype and expressing mesenchymal markers such as Sox9 ( 63 , 77 ). Overexpression of PE-enriched microRNAs miR-181c, miR-200b and miR-495 in EPIC cells resulted in significant downregulation of predicted targets, including miR-181c reduced Nr3c1 , miR-200b suppressed Rnd3 and Fn1 , while miR-495 decreased Mbln2 and Nfib ( Fig. 2 A ). In MEC1 cells, a similar pattern was observed for Mbln2 downregulated by miR-495 and Fn1 by miR-200b , but Nr3c1, Nfib or Rnd3 were not significantly affected ( Fig. 2 C ) . The combinatorial effect of these three microRNAs (preMix) led to a decreased expression of Rnd3 and Hapln1 in both epicardial cell lines, while Nr3c1 and Nfib were downregulated exclusively in EPIC ( Fig. 2 A,C ) . To uncover if the functional role of microRNAs also occurs in other cardiovascular cell types, RT-qPCR analyses were also performed after DE microRNAs gain-of-function experiments in HL1 cardiomyocytes and MEVEC endocardial cells (Supplementary Fig. 9A,B) . Interestingly, our data demonstrated that neither Mbln2 nor Nfib were downregulated in HL1 cardiomyocytes or MEVEC endocardial cells, while Fn1 was impaired by all the three microRNAs , and Nr3c1 and Rnd3 were downregulated in HL1 cardiomyocytes. However, the preMix had a greater impact on target regulation in HL1 cardiomyocytes and MEVEC endocardial cells (Supplementary Fig. 9A,B) . In conclusion, it is worth highlighting that EPIC epicardial cells recapitulate most of the microRNA-mRNA target predicted interactions after miRNAs gain-of-function, except for Hapln1 , supporting the notion that miRNAs PE > EE molecular regulation is cell type-specific. In the same regard, loss-of-function assays were executed for EE-enriched microRNAs, let-7c, miR-30c and miR-351 , in EPIC and MEC1 epicardial cells ( Fig. 2 B,D ) . Our RT-qPCR analysis led us to identify that in the EPIC cell line miR-351 inhibition modulated the up-regulation of Prtg , whereas Nr6a1 was up-regulated after the inhibition of miR-30c and Hic2 after let7c or miR-351 inhibition, respectively. On the other hand, neither Peg10 , Fbxo32, Trim71 nor Ccnjl expression were boosted after the loss-of-function of these microRNAs in EPIC cells ( Fig. 2 B ) . In the MEC1 cell line is relevant to point out that Fbxo32 and Trim71 were significantly up-regulated after let-7c, miR-30c and miR-351 inhibition in MEC1 cell line ( Fig. 2 D ) , while Peg10 was up-regulated after miR-30c and miR-351 inhibition. Additionally, Prtg and Ccnjl were up-regulated after miR-30c and let-7c inhibition, respectively, in MEC1 cells. Curiously, Nr6a1 and Hic2 expression was not increased, in some cases ( miR-351 ) even significantly decreased, after these microRNA inhibition ( Fig. 2 D ) . In this case is noteworthy that the combinatorial effect of these three microRNAs (antiMix) does not have any relevant effect on the up-regulation of the mentioned target genes except for Trim71 in MEC1 and Peg10 in EPIC cells ( Fig. 2 B,D ) . To summarize, our data demonstrate that MEC1 cells recapitulate most of the microRNA-mRNA target predicted interaction after miRNAs loss-of-function as compared to EPIC cells. Differentially expressed microRNA-mRNA predicted interactions are not recapitulated in ex vivo E10.5 epicardial outgrowths To further dissect the regulatory mechanisms driven by DE-microRNAs, we performed gain- and loss-of-function assays of the PE > EE and PE < EE microRNAs, in mouse E10.5 epicardial explants. After 24 hours of culture, Wt1 + epicardial cells (EEx) were transfected with microRNAs and collected after ventricular tissue removal (Supplementary Fig. 9C) . Gain-of-function experiments with PE > EE microRNAs miR-181c, miR-200b, miR-495 demonstrated that epicardial expression of Mbln2 and Hapln1 was significantly decreased in all individual conditions, while Nfib and Fn1 were negatively regulated after miR-181c and miR-495 administration. Additionally, Rnd3 was down-regulated by miR-181c and miR-200b , whereas Nr3c1 down-regulation was specific to miR-200b. Finally, Mbln2 , Hapln1 and Fn1 expression was significantly decreased after premix treatment ( Fig. 2 E ) . Comparing these findings with the previous in vitro experimental assays, EPIC cells recapitulated key interaction seen in EEx, such miR-181c -mediated Nfib suppression and miR-200b -driven regulation of Nr3c1 and Rnd3 . Finally miR-495 controls Mbln2, Nfib and Fn1 . However, discrepancies were observed, including miR-181c downregulating nearly all the predicted target genes except for Nr3c1 ex vivo but not in vitro . Such discrepancies might be related to the myocardium-epicardium interaction ex vivo and/or the differential cell behaviour of in vitro epicardial cell lines vs E10.5 ex vivo epicardial explants. Similarly, loss-of-function assays of the three PE < EE microRNAs, let-7c, miR-30c and miR-351 in EEx revealed that Trim71 expression was up-regulated after let-7c and miR-30c inhibition, in line with the observations in MEC1. However, Prtg and Nr6a1 were down-regulated in all individual experimental conditions. The combinatorial effect of antiMix leads exclusively to Nr6a1 up-regulation ( Fig. 2 F ) . These results demonstrate intriguing discrepancies in the regulatory effects of DE microRNAs in both MEC1/EPIC cells and EEx from E10.5 explants, being needed to underscore the importance of considering cell-specific contexts in understanding the functional implications of the microRNAs in embryonic epicardial cells ex vivo . MiR-495, let-7c , and miR-351 regulate epicardial cell migration Epicardial cell migration is fundamental for heart development, as PE cells move by direct contact or through cell aggregates to cover the myocardium. This process begins at the atrioventricular canal region and, expands to form a continuous epithelial layer that eventually covers the entire heart. As the epicardium matures, EMT facilitates EPDC proliferation, migration, and differentiation, supporting coronary vasculature and ventricular growth. To assess the impact of microRNAs on this process, we performed gain- or loss-of-function assays in E10.5 ventricular explants and analyzed three different aspects. Total cell migration , representing all cellular migration from the explant ventricular border to the outermost individual cell of the culture; Cohesive cell migration , when only considering those collective migrating cells from the explant ventricular border; and finally, non-cohesive cell migration , representing only those individual migrating cells from the outermost periphery of the cohesive migration ( Fig. 3 A ) . Our data demonstrated that total cell migration was impaired after miR-495 overexpression and enhanced after preMix treatment, while no significant outgrowth differences were noted for miR-181c and miR-200b experimental conditions. Cohesive migration was repressed after miR-495 and preMix treatment while non-cohesive migration was enhanced after miR-200b and miR-495 gain-of-function, as well as, preMix treatment ( Fig. 3 A-D ) . In this regard, we observed that epicardial cells within the non-cohesive outgrowth exhibit a reduced size compared to controls following miR-495 gain-of-function, with a concurrent increase in Myh9 protein expression in MEC1 epicardial cells in vitro , whereas no differences are observed in F-actin polymerization (Supplementary Fig. 10A-B) . Thus, these data demonstrate that differential expression of distinct microRNAs can selectively modulate total, cohesive and non-cohesive epicardial cell migration. As previously mentioned, our RT-qPCR analysis unveiled a specific explant epicardial cells regulatory miRNA-mRNA crosstalk ( Fig. 2 E ). To further elucidate the role of the PE < EE DE-mRNAs in epicardial cell migration, E10.5 cardiac explants were transfected with Mbln2 , Nfib , Nr3c1 , Rnd3 and Fn1 siRNAs. Our epicardial cell migration analysis evidenced that total cell migration was enhanced after Nfib, Nr3c1 and Fn1 inhibition and decreased after siRnd3 treatment, while no significant outgrowth differences were noted for siMbln2 . Cohesive migration was promoted after loss-of-function of all mRNAs, except for Nfib , while non-cohesive migration was enhanced only by Nfib loss-of-function, and decreased after Rnd3 and Mbln2 inhibition, whereas Nr3c1 and Fn1 did not exerted any effect on non-cohesive cell migration ( Fig. 3 E-H ) . Thus, our data demonstrate that DE-mRNAs in PE vs. EE can selectively modulate total, cohesive and non-cohesive epicardial cell migration. A similar analysis was performed for those PE < EE DE-microRNAs, where cell migration was assessed through loss-of-function experiments targeting individual microRNAs let-7c , miR-30c , miR-351 and antiMix. Our results demonstrated that total cell migration was decreased after antilet-7c, antimiR-351 and antiMix treatment, while no significant outgrowth differences were observed after antimiR-30c administration. Cohesive migration was repressed only after antiMix treatment without significant effect mediated by individual conditions, and finally, non-cohesive migration was impaired by miR-30c loss-of-function, as well as, antiMix treatment ( Fig. 3 I-L ) . These results align with our findings that epicardial cells in the non-cohesive outgrowth do not exhibit a reduced area compared to controls following let-7c loss-of-function, and no significant difference in Myh9 protein expression is observed in MEC1 epicardial cells in vitro , although F-actin polymerization is impaired (Supplementary Fig. 10A-C) . Therefore, these data further underscore the functional role of DE-microRNAs in epicardial cell migration. Importantly, it should be highlighted that miR-495, let-7c and miR-351 exert significant effects on epicardial cell migration, underscoring the importance of their precise regulation for proper epicardium formation and EPDCs migration. MiR-181c, miR-200b , and let-7c administration promotes cardiomyogenic cell specification in vitro but not ex vivo As outlined previously, after the embryonic epicardium covers the naked myocardium, it subsequently undergoes EMT leading to EPDCs that thereafter differentiate into distinct cell types such as cardiac fibroblasts, vascular smooth muscle cells, pericytes, endothelial and fat cells and possibly, also into cardiomyocytes. To dissect the role of DE microRNAs during cell lineage specification, epicardial cells (EPIC and MEC1) were overexpressed with miR-181c, miR-200b, miR-495 and preMix, respectively ( Fig. 4 A,C, and Supplementary Fig. 11) . Linage specific markers of epicardium ( Wt1, Tcf21 and Tbx18 ), myocardium ( Gata4, Nkx2.5, Srf, Tnnt2 and Myh6 ) and endocardium ( Pecam1, Tie2 and Postn as an endocardial derivative marker) as well as markers for EMT ( Cdh5, Snail1, Snail and Prrx1 ), fibrogenesis ( Col1a1, Col3a1, Fn1 and Sox9 ) and angio-vasculogenesis ( Ang1, Ang2, Efnb2 and Flt1 ) were analysed by RT-qPCR. miR-181c gain-of-function did not evidence enhancement of early myocardial markers (i.e. Gata4, Nkx2.5 ), except for Srf in MEC1, but upregulated the cardiomyocyte terminal differentiation marker Tnnt2 , in both epicardial cell lines. Moreover, endocardial and epicardial lineage markers were slightly increased such as Tie2 , and Postn in epicardial MEC1 and EPIC cells and Tcf21 only in epicardial MEC1 cells ( Fig. 4 A ) . In the same line, miR-495 overexpression promoted the expression of endocardial lineage specification markers as revealed by upregulation of Tie2 in both epicardial cell lines and Postn in MEC1 cells. Additionally, miR-495 promotes the expression of the epicardial marker Tcf21 in MEC1 as well as the cardiomyocyte terminal differentiation marker Tnnt2 in EPIC. Finally, miR-200b did not exert any modulation on endocardial, myocardial and epicardial markers in EPIC cells, nonetheless the overexpression of miR-200b in MEC1 leads to an increment of myocardial markers such as Srf, Myh6 and Tnnt2 ( Fig. 4 A ) . When the three microRNAs are overexpressed together, it can be observed an increment of myocardial markers, for instance, early cardiogenic transcription factor as Nkx2.5 is upregulated in both epicardial cells similarly as Srf , whereas Gata4 is increased only in EPIC. Finally, epicardial markers as Tcf21 and Wt1 are upregulated in MEC1 and EPIC epicardial cells, respectively ( Fig. 4 A ) . Therefore, these data demonstrate that DE microRNAs in PE > EE can distinctly modulate epicardial-derived lineage specification. Given the role of the epicardium during cardiogenesis, in order to have further insight of the molecular mechanisms driven by the DE microRNAs, we analysed by RT-qPCR, the expression levels of different molecular markers related with EMT, fibrogenesis and angiogenesis after gain-of-function experiments. For EMT markers all three DE-microRNAs exerted a marked repression of Cdh5, Snail1, Snail2 , and Prrx1 in MEC1, except for miR-200b which only promoted Cdh5 gene expression. However, in EPIC cells, the gain-of-function of the miR-181c promoted the expression of Snail1 , and miR-495 induced Snail1 and Cdh5 expression, while miR-200b did not exert any significant effect over EMT markers ( Fig. 4 C ) . For angio-vasculogenesis markers analysis, no significant effect was observed after microRNA gain-of-function except for angiopoietins, i.e. Ang1 in MEC1, mediated by miR-495 and Ang2 in EPIC, regulated by miR-181c and miR-495 . Notwithstanding, miR-181c and miR-495 overexpression slightly promoted the expression of fibrogenic markers such as Col1a1 and Col3a1 in EPIC and MEC1 epicardial cell lines, respectively ( Fig. 4 C ) . Finally, EMT, angio-vasculogenesis and fibrotic molecular markers were upregulated by combinatorial miRNA overexpression, i.e.: Ang2, Efnb2, Snail2, Fn1 and Sox9 in EPIC epicardial cells, and Ang1, Ang2, Cdh5, Col1a1, Col3a1 and Fn1 in MEC1 ( Fig. 4 C ) . In sum, these results support the notion that miR-181c and miR-495 , but not miR-200b , could modulate cell lineage specification promoting epicardial, endocardial and fibrogenic markers, whereas miR-181c and miR-200b promote myocardial markers expression in MEC1 epicardial cells (Supplementary Fig. 11) . Similar to DE-miRNAs in PE > EE, miR-let7c, miR-30c , and miR-351 loss-of-function experiments were performed in epicardial EPIC and MEC1 cells. Specific cardiac cell lineage and biological processes markers were studied ( Fig. 4 B,D, Supplementary Fig. 12) . In this analysis, we observed that anti-let7c consistently promoted upregulation of early cardiogenic lineage markers in both epicardial cell lines, such as Gata4 and Srf , whereas Nkx2.5 and terminal differentiation markers such as Myh6 and Tnnt2 were only upregulated in MEC1 ( Fig. 4 B ) . In addition, the epicardial cell lineage markers are slightly promoted in EPIC cells through increment of Tbx18 and Wt1 expression as well as Tcf21 in MEC1. Finally, endocardial markers revealed that loss-of-function of let-7c induced endocardial cell specification in EPIC cell line endorsed by Post1 and Tie2 upregulation. In the same scenario of loss-of-function, miR-30c and miR-351 modulated myocardial and epicardial markers in EPIC leading to the upregulation of Gata4, Nkx2.5 and Tnnt2 , as well as Tbx18 , however, the modulation of these miRNAs in MEC1 cells did not have any impact on cardiogenic markers except for Srf expression. Finally, we evidenced that miR-351 inhibition has an impact on the promotion of endocardial linage specification markers in both epicardial cells endorsed by Pecam1, Postn and Tie2 expression, and the loss-of-function of miR-30c modulates Pecam1 and Postn in MEC1 epicardial cells ( Fig. 4 B ). To get further insights into the functional role of the DE-miRNAs PE < EE other biological processes such as, EMT, fibrogenesis and angio-vasculogenesis representative markers were analysed in a loss-of-function model for let-7c, miR-30c and miR-351 , respectively. Our RT-qPCR data demonstrated that all of them were significantly downregulated in MEC1 epicardial cells ( Fig. 4 D ) . However, loss-of-function of DE-miRNAs, let-7c and miR-351 in EPIC cells, promoted the expression fibrogenic markers such as Col1a1, Col3a1 and Fn1 . Moreover, antimiR-351 but no anti-let7c nor antimiR-30c promoted the expression of angio-vasculogenic markers as Flt-1 . Finally, the antiMix treatment in both epicardial cells modulate EMT markers by the upregulation of Snail1 in MEC1 and Snail2 in MEC1 and EPIC cells ( Fig. 4 D ) . In sum, these results support the notion that let-7c can modulate cell lineage specification promoting myocardial markers while miR-351 promotes endocardial lineage specification in epicardial cells. Moreover, these two miRNAs have a marked effect on the modulation of cardiac fibrogenic markers in EPIC epicardial cells (Supplementary Fig. 12) . Furthermore, let-7c loss-of-function but no miR-181c and miR-200b gain-of-function, increases Tnnt2 protein expression levels as observed in MEC1 epicardial cells ( Fig. 4 E ) . Since we have previously demonstrated the dynamic modulation of the lineage specification markers in epicardial cell lines by these microRNAs, we sought to investigate if the expression of epicardial, myocardial and/or endocardial markers were induced ex vivo in EEx. For this purpose, gain- and loss-of function of miR-181c , miR-200b and let-7c were performed in mouse E10.5 epicardial explants. RT-qPCR analysis evidenced that overexpression of miR-181c did not promote myocardial, endocardial or epicardial markers ex vivo . Moreover, miR-200b overexpression repressed Tnnt2 and Mhy6 expression in mouse EEx from E10.5 epicardial explants and similar effects were observed after let-7c inhibition ex vivo ( Fig. 4 F ) . Thus, these data suggest that the function of these three microRNAs in the embryonic epicardial cell specification is limited, underscoring the potential influence of other molecular factors as well as the neighbouring myocardial and endocardial tissues. Differentially expressed microRNAs cross-talk modulates epicardial cell specification Despite our comprehensive understanding of the fundamental principles underlying miRNA biogenesis and function, novel and unexpected aspects within these processes underscore the complexity of miRNA regulation. To get further insight of the functional regulation of the DE microRNAs, we conducted a RT-qPCR analysis of their mutual molecular regulation. In this regard, we analysed the expression levels of each microRNA following individual microRNA gain- or loss-of-function in MEC1 and EPIC epicardial cells as well as in embryonic epicardial cells ( Fig. 5 A-F ) . As expected, our results evidenced that miR-181c, miR-200b and miR-495 administration were upregulated following miRNA gain-of-function in all epicardial cells, respectively ( Fig. 5 A,B,E ) . Moreover, overexpression of miR-181c enhanced miR-200b and miR-495 expression and similarly, miR-495 gain-of-function promoted miR-181c and miR-200b expression in epicardial cells ( Fig. 5 A,B,E ) . Notably, overexpression of miR-200b led to a decreased expression of miR-181c and miR-495 in MEC1 but not in EPIC or EEx ( Fig. 5 A,B,E ) . Similarly, analyses of loss-of-function assays also demonstrated a downregulation of let-7c , miR-30c and miR-351 in all epicardial cells, as expected ( Fig. 5 C,D,F ) . However, loss-of-function experiments targeting let-7c resulted in elevated expression of miR-30c in MEC1 but not in EPIC epicardial cells, alongside increased miR-351 expression in both cell types, a trend also observed in EEx ( Fig. 5 C,D,F ) . Comparable results were noted upon miR-30c inhibition, with upregulation observed in let-7c in MEC1, EPIC, and EEx, and miR-351 only in MEC1 and EEx. Finally, after antimiR-351 treatment, let-7c expression in MEC1 and miR-30c expression in MEC1 and EEx were elevated ( Fig. 5 C,D,F ) . Therefore, these data demonstrate a microRNA cross-talk regulation between differentially expressed microRNAs, a process that is also cell-type specific. To further analyse microRNA cross-talk, we performed in vitro experiments blocking transcription by using α-amanitin and we found that miR-30c and miR-351 were upregulated when let-7c is inhibited, indicating that this microRNA acts by enhancing these two microRNAs at post-transcriptional levels (data not shown). Since we have previously observed that the inhibition of let-7c displays an increment of miR-30c and miR-351 expression in MEC1 and EEx, we sought to investigate the molecular implications of this regulatory feedback in cardiomyogenic cell lineage markers. miR-30c and miR-351 gain-of-function experiments were conducted in MEC1 and EEx ( Fig. 5 G,H ) . Our results evidenced that Tnnt2 expression levels are not increased following modulation of miR-30c nor miR-351 in both MEC1 and EEx, whereas Myh6 expression levels were upregulated specifically after miR-351 overexpression in MEC1 epicardial cells ( Fig. 5 G,H ). Thus, the cardiogenic role exerted by the inhibition of let-7c is not mediated by the modulation of these microRNAs, as only miR-351 modulates Myh6 expression. Overall, our comprehensive analysis demonstrates that gain- or loss-of-function of one microRNA had a notable effect on the expression of other microRNAs, evidencing the intricate interplay between DE-microRNAs in regulating epicardial cell behaviour and highlighting their potential functional role in cardiac development. Foxf1 modulates epicardial cell specification into myocardial and endothelial lineages via let7c and miR-30c regulation in vitro As we have previously mentioned, several mRNAs were significantly differentially expressed in PE (E9.5) and EE (E10.5). Among them, we have observed that there are 47 transcription factors (~ 4.6% of total DE mRNAs) with enhanced expression in PE, i.e, Tbx5, Sox18, Prox1 , Foxf1 (Table 1) , whereas 22 (~ 2.4%) display the opposite pattern, down-regulated in the PE as compared to the EE, e.g. Tbx18, Sox9, Lhx9, Foxc1 ( Table 2) . FOX (Forkhead box) proteins are a family of transcription factors that play important roles in regulating gene expression that govern cardiogenesis. Basal expression analysis for Foxc1 and Foxf1 in EPIC, MEC1, MEVEC, HL1 and EEx revealed that Foxc1 is abundantly expressed in EEx compared with the similar expression evidenced in EPIC and MEC1, while Foxf1 is significantly downregulated in EEx and EPIC compared with MEC1 (Supplementary Fig. 13A). Since Foxf1 is expressed in the PE and Foxc1 in EE we investigated whether these DE transcription factors, i.e. Foxc1 and Foxf1 , could modulate the expression of our DE-microRNAs in MEC1 epicardial cells. Foxc1 inhibition resulted in decreased miR-495 and miR-351 expression, while loss-of-function of both Foxc1 and Foxf1 led to reduced let-7c and increased miR-30c expression ( Fig. 6 A ). We subsequently examined whether these DE transcription factors exhibit also cross-talk regulation, similar as the DE microRNAs. Inhibition of Foxc1 in MEC1 cells resulted in decreased expression of the Foxf1 transcription factor, while inhibition of Foxf1 in MEC1 cells led to upregulation of Foxc1 expression ( Fig. 6 B ) . To further elucidate the intricate regulatory network involving our DE-miRNAs, we assessed the expression levels of Foxc1 and Foxf1 in MEC1 epicardial cells overexpressed with the DE-miRNAs in PE > EE. No significant differences in expression levels were observed for Foxc1 after miR-181c , miR-200b and miR-495 administration while Foxf1 expression was decreased after premiRs treatment ( Fig. 6 C ) . Conversely, inhibition of let-7c consistently upregulated Foxc1 expression, exerting the opposite effect over Foxf1 . Similarly, loss-of-function of miR-351 resulted in decreased expression of Foxc1 and increased expression of Foxf1 . However, inhibition of miR-30c did not yield significant differences ( Fig. 6 D ) . Finally, we aimed to elucidate the involvement of the DE-transcription factors in epicardial cell lineage specification and migration. We analysed the expression levels of epicardial, myocardial and endocardial markers following loss-of-function of Foxc1 and Foxf1 in MEC1 epicardial cells. Our findings revealed that inhibition of Foxc1 represses epicardial lineage specification markers such as Wt1, Tbx18 and Tcf21 , similar to those observed after Foxf1 inhibition except for Tcf21 that displayed no significant differences ( Fig. 6 E,F ) . Moreover, loss-of-function of Foxc1 resulted in increased expression levels of Tnnt2 and Tie2 , while Foxf1 inhibition promoted the expression of myocardial markers such as Gata4, Myh6, Srf and Tnnt2 , as well as endocardial markers such as Pecam1, Tie2 and Postn . ( Fig. 6 E,F ) . Finally, epicardial cell migration in MEC1 following Foxf1 loss-of-function lead to similar results as those observed for let7c inhibition in vitro , with no significant difference in Myh9 protein expression, although F-actin polymerization was impaired (Supplementary Fig. 13B) . In conclusion, our study unveiled complex regulatory networks orchestrated by DE-microRNAs and DE-transcription factors. These findings offer valuable insights into the regulatory mechanisms governing epicardial cell specification and cardiac development, particularly mediated by Foxf1, let-7c and miR-30c . DISCUSSION In this study, we provide novel insights into the dynamic regulation of microRNAs and their functional role in proepicardial (PE) and embryonic epicardial (EE) formation in mice. Our findings reveal distinct microRNA expression patterns that selectively influence key cellular processes, including cell-cell signaling, migration, and lineage specification during epicardial development. Specifically, we demonstrate that Foxf1 modulates the expression of miR-495, miR-351, and let-7c, which in turn regulate epicardial cell migration and myocardial specification. Notably, our results suggest a previously unrecognized microRNA-microRNA regulatory network, shedding light on the intricate molecular crosstalk driving epicardial development. These discoveries provide new mechanistic evidence of the transcriptional and post-transcriptional regulation orchestrating PE and EE formation, with potential implications for cardiac regenerative strategies. microRNAs are short non-coding RNAs with tissue-specific expression that exert regulatory roles over different cellular processes ranging from embryonic development to pathological response ( 64 – 66 ). In the cardiovascular system, several laboratories including ours, have yielded substantial evidence elucidating the microRNA differential expression during cardiogenesis ( 44 – 47 , 78 ). Moreover, Dueñas et al. (2020) recently evidenced a microRNA differential expression during PE and EE formation in chicken ( 48 ), however, the functional role of microRNAs in PE and EE development in mice and their application to enhance cardiogenesis remains elusive. In this study, we provide evidence about the microRNA differential expression pattern during PE and EE formation and their functional implications in mice. We found a large subset of microRNAs and mRNAs that display increasing expression in PE vs . EE, suggesting a plausible role in cell-cell signaling during proepicardial cell specification, differentiation and vesicles formation for direct contact and attachment of the proepicardial cells to naked myocardium in mice ( 5 , 8 , 38 , 79 – 83 ). On the other hand, a small subset of microRNAs displays increased expression, as well as high mRNA expression levels in EE vs. PE, supporting a modulatory role in the coordination of epicardial-derived signals involved in muscle function, coronary development, as well as myocardial growth ( 84 – 87 ). Thus, these data revealed that differential microRNA signatures can selectively influence different signaling pathways during PE and EE formation. The PE derives from the LPM and is formed at the venous pole of the heart during embryonic development in E9.5 mice. Lumenized vesicles from the proepicardial surface attach to the naked myocardium subsequently forming the embryonic epicardium at E10.5 ( 5 , 8 , 88 ). Epithelial cells undergo EMT forming EPDCs that migrate into the myocardium, proliferate and differentiate into different cell types, including coronary endothelial cells, smooth muscle cells and cardiac fibroblasts, whereas their contribution to cardiomyocytes remain controversial ( 16 , 19 , 23 – 25 , 50 , 89 – 98 ). Several laboratories have identified microRNA-mRNA cross-talk correlation during cardiac development (see recent reviews; ( 99 , 100 )). Brønnum et al. ( 101 ) have elucidated that miR-21 modulates epicardium development and EPDCs fate-decision, through the established interplay between Pdcd4 and Spry1 , whereas Pontemezzo et al. ( 85 ) reported that miR-200c after Tfg-ß administration impact on the epithelial-to-mesenchymal transition process in epicardial cells. Our RNAseq analysis evidenced a complementary expression pattern of microRNAs and putative mRNA targets in PE vs . EE. Those microRNAs with enhanced expression in PE such as, miR-495-5p, miR-200b-3p and miR-181c-5p , have a plausible role blocking mRNA target expression (i.e. Mbln2, Nr3c1, Nfib, Rnd3, Hapln1 , and Fn1 ) during the morphogenetic induction of proepicardial to embryonic epicardial cells transition. Herein, we demonstrate that miR-495 regulates Mbln2 in epicardial cells but not in myocardial nor endocardial cells, providing novel mechanistic insight into the role of microRNAs in this context. In addition, microRNAs upregulated in EE, such as let7c-5p, miR-351-5p , and miR-30c-5p , are repressing inductive signals derived from mRNAs targets (i.e. Prtg, Nr6a1, Peg10, Fbxo32, Trim71, Hic2 and Ccnjl ) during EMT and epicardial cell specification process, illustrated by the fact that let-7c regulates Trim71 in epicardial cells. In summary, these findings further support the notion that tight regulation of microRNA-mRNA interaction plays a crucial role in coordinating cellular processes, i.e. cellular migration and lineage specification during PE and EE formation ( 4 , 10 , 23 , 27 , 34 , 35 , 95 , 102 – 107 ). Moreover, our results highlight the dynamic rewiring of miRNA-mRNA interaction during cardiogenesis, which modulate mRNA target expression in a cell-specific manner in epicardium, myocardium and endocardium. These differences may arise from variation in target mRNA expression, the presence of RNA-binding proteins that modulate microRNA function, or competing endogenous RNAs, as previously reported for microRNAs in other biological contexts ( 108 – 110 ). These findings underscore the complexity of miRNA-mediated gene regulation in epicardial, reflecting the intricate nature of post-transcriptional networks across different cellular context during cardiac development. The discrepancies between individual miRNAs and the combinatorial conditions likely stem from interaction within broader miRNA networks producing non-linear outcomes. Additionally, cell-specific factors in MEC1 and EPIC cells contribute to these differences, emphasizing the dynamic regulation of gene expression during cardiac development. microRNAs can modulate multiple biological processes, including cell migration in homeostatic and pathological conditions ( 111 – 115 ). Cell migration is a key biological process during PE formation, such PE cells initially migrate onto the heart to establish the embryonic epicardium primarily at ventricular level, subsequently across the atrial chamber. Following EMT, EPDCs migrate into the myocardium to support coronary vasculature and ventricular development ( 116 ). We provide herein evidences that let-7c and miR-351 in the EE have an important role in controlling epicardial cell migration, in line with previous reports in other biological contexts ( 117 – 119 ). Additionally, miR-495 modulates epicardial cell migration, partially mediated by Nr3c1 , in line with recent reports in pathological conditions ( 71 , 72 , 120 – 123 ). Similarly, we evidenced that Rnd3 is involved in epicardial cell migration, although is not coordinated with miR-181c and miR-200b modulation ( 75 , 76 ). These findings underscore the functional role of microRNAs and mRNAs in regulating epicardial cell migration, a highly relevant process in PE and EE formation, through the differential expression of cytoskeletal proteins. Given that defective migration of PE and EE cells can lead to severe congenital heart defects by disrupting coronary vasculature, myocardial growth, EMT and valve formation, our findings emphasize the pivotal role of miRNA-mRNA interactions in ensuring the precise regulation of epicardial cell migration, which is essential for proper cardiac morphogenesis As extensively documented, the epicardium plays a key role during cardiogenesis since a subset of EPDCs undergo EMT, migrate into the myocardium and differentiate into different cardiovascular lineages, i.e. coronary vascular smooth muscle cells, cardiac fibroblasts, endothelial cells, contributing to complete heart formation. Furthermore, it has been suggested that epicardial progenitors can also contribute to the cardiomyocyte lineage, although this statement remains controversial ( 50 , 93 , 97 , 98 ). In our study, we explored the plausible contribution of microRNAs in the process of epicardial cell lineage specification. Our findings indicate that the administration of miR-181c and miR-200b , as well as the inhibition of let-7c facilitates epicardial cell specification into the myocardial cell lineage in vitro but these effects were not observed ex vivo in the explant model. This discrepancy arises from the fundamental differences between the simplified in vitro environment and the more complex, physiologically relevant conditions of the explant model. Hence, these data support the notion that miR-181c , miR-200b and let-7c modulate myocardial specification of embryonic epicardial cells, underscoring the potential impact of molecular regulation induced by the neighbouring myocardial and endocardial tissues. Additionally, the induction of fibrotic markers in the epicardial cells observed after loss-of-function of let-7c suggests that this microRNA plays a broader role in modulating epicardial cell fate. This finding implies that let-7c may influence fibrotic processes during cardiac development, consistent with its established roles in regulating fibrosis in other biological contexts ( 124 , 125 ). During embryogenesis, extracellular information is needed for cells to make decisions during development and differentiation ( 126 , 127 ). Tight cross-talk between different signaling pathways such as TGF-β/BMP, Wnt/Wg, Hedgehog (Hh), Notch, and mitogen-activated protein kinases (MAPK) have been thoroughly described ( 128 – 133 ). Similarly, cross-talk between transcription factors has been evidenced, e.g. Gata4-Tbx5 controls cardiac septum formation ( 134 ) and Tbx5-Nkx2.5 interaction promotes cardiomyocyte differentiation ( 135 ). Nevertheless, there is scarce evidence regarding the plausible microRNA cross-talk that could modulate the maturation and expression of other microRNAs ( 136 ). Our analysis demonstrated that DE microRNAs in PE and EE regulate the expression of other microRNAs in epicardial cells at post-transcriptional levels. Therefore, this is, to the best of our knowledge the first evidence that microRNAs can regulate the expression of other microRNAs, supporting thus functional implications for PE and EE development. Given the role of let-7c in terminal myocardial differentiation and its role in the regulation of miR-30c and miR-351 expression, we evidenced that only miR-351 promotes myocardial terminal differentiation in epicardial cells, promoting Myh6 expression. These findings elucidate that in spite of the intricate interplay among DE microRNAs in PE vs . EE, governing epicardial cell behaviour, the terminal myocardial differentiation exerted by let-7c is not solely mediated by the EE > PE microRNAs interplay. Cardiac-specific transcription factors such as Nkx2.5 ( 137 – 139 ), Mef2c ( 140 – 142 ), Pitx2 ( 143 – 146 ), Srf ( 147 , 148 ) and Fox ( 149 – 153 ), are fundamental in both cardiogenesis and the development of PE and EE ( 37 , 38 , 154 , 155 ). Moreover, Tbx18 is highly expressed in PE and essential in epicardium and coronary vasculature development ( 33 , 104 ). Tcf21 and Tbx5 are essential for mature proepicardial cells to establish contact with the myocardium and properly form the epicardium ( 35 , 156 ). Wt1 is crucial for EMT of epicardial cells ( 28 ). These transcription factors exert transcriptional control over multiple downstream targets, including both coding and non-coding RNAs and miRNAs, particularly those pivotal for heart development. In our RNAseq analysis, Foxf1 displays enhanced expression in PE, whereas Foxc1 shows an opposite expression pattern, with high expression in EE. Our data analysis revealed that Foxc1 and Foxf1 exert transcriptional control over the DE microRNAs in PE and EE during development, in line with previous report demonstrating similar transcription factor-miRNA transcriptional regulation i.e. Pitx2 -miRNAs in a skeletal-muscle context ( 60 ). In PE and EE formation and specification, it is worth highlighting that Foxf1 controls let-7c and miR-30c expression. In addition, DE microRNAs in PE vs. EE, i.e. miR-495 and let-7c modulate Foxc1 and Foxf1 expression and similar to those observed effects for these microRNAs, both transcription factors exhibit a cross-talk modulation. Overall these findings reveal a complex transcription factor vs . microRNA regulation in PE and EE formation and specification in epicardial cells during cardiogenesis ( Fig. 7 ) . Understanding of the molecular mechanisms driving PE and EE formation and specification has greatly advanced over the last decade including the functional role of microRNAs (see recent reviews ( 157 – 159 )). Regarding the tight molecular regulation of transcription factors and DE microRNAs in PE vs. EE, and the previously observed implication of microRNAs during epicardial cell specification, we evidenced that Foxc1 modulates the expression of myocardial and endocardial markers (i.e. Tnnt2 and Tie2 ), whereas Foxf1 controls early cardiogenesis (i.e. Gata4 and Srf ), as well as cardiomyocyte terminal differentiation (i.e. Tnnt2 and Myh6 ). Therefore, these observation supports the notion that transcription factors Foxc1 and Foxf1 modulate EE specification, being essential during cardiogenesis as previously reported ( 149 – 153 , 160 – 164 ). Therefore, mechanistically this study evidenced that Foxf1 controls epicardial cell specification towards cardiomyocytes by modulating let-7c ( Fig. 7 ) . In summary, we provide herein evidence that PE and EE formation and specification are biological processes tightly regulated by DE microRNAs and mRNAs during cardiogenesis. We demonstrated that Foxf1 transcription factor modulates miR-495, miR-351 and let-7c expression and these microRNAs regulate epicardial cell migration and myocardial specification, hinting the essential co-regulatory role of transcription factor vs. microRNA for cardiogenesis during embryonic development ( Fig. 7 ) . Despite the valuable insights provided by this study, some limitations should be acknowledged. While our RNA sequencing data suggest potential microRNA-mRNA regulatory interactions, further exploration to fully elucidate the regulatory networks that govern cardiogenesis would strengthen our findings. Additionally, future studies employing genetic loss- and gain-of-function models in mice would be necessary to confirm the role of specific microRNAs in PE and EE development. Lastly, although our study suggests potential translational applications for cardiac regenerative medicine, additional research is needed to determine whether modulating these microRNA pathways could enhance epicardial cell contribution to cardiac repair in postnatal or adult hearts. CONCLUSIONS Our study highlights the intricate regulatory mechanisms orchestrated by differentially expressed microRNAs and mRNAs during the formation and specification of the PE and EE in cardiogenesis. We provide novel evidence that specific microRNAs, such as miR-495 and let-7c , play crucial roles in modulating epicardial cell migration and myocardial specification. The transcription factor Foxf1 regulates let-7c expression, thereby promoting key developmental processes as myocardial lineage specification from epicardial cells. Our findings underscore the complexity and importance of the microRNA-mRNA interaction networks and their co-regulatory roles with transcription factors in governing cardiogenesis. This study advances our understanding of the molecular mechanisms underlying heart development and highlights potential therapeutic targets. Abbreviations Anti-miRNA microRNA inhibitor AV Atrioventricular cDNA Complementary DNA DE Differentially expressed Dapi 4',6-Diamidine-2'-phenylindole dihydrochloride E8.5 Embryonic day 8.5 E9.5 Embryonic day 8.5 E10.5 Embryonic day 10.5 EBSS Earle’s balance salt solution EE Embryonic epicardium EEx Wt1 + epicardial cells EMT Epithelial to mesenchymal transition EPDCs Epicardial derived cells FBS Fetal bovine serum FC Fold change GEO Gene Expression Omnibus GO Gene ontology GSEA Gene Set Enrichment Analysis HEPES 4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid IHC Immunohistochemical analyses lncRNA Long non-coding RNA LPM Lateral plate mesenchyme PBS Phosphate buffer saline PE Proepicardium Pre-miRNAs microRNA precursor RT-qPCR Reverse transcriptase-quantitative polymerase chain reaction SV Sinus venosus TMM Trimmed mean of M-values Declarations Ethics approval This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of the University of Jaén (code 14/03/2022/038). Consent for publication Not applicable Availability of data and material RNAseq data were uploaded into Gene Expresssion Onmibus platform with accession number GSE189344. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE189344 Competing interests The authors declare that they have no competing interest. Funding This work was supported by grants of the Ministerio de Innovación y Ciencia of the Spanish Government to DF (PID2022-138163OB-C32) and of the Consejería de Universidad, Investigación e Innovación of the Junta de Andalucia Regional Council to DF (ProyExcel_00409). Author´s contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by JM C-C, A D, F H-T, R C, R M-C, A D, R A, E VdL. The first draft of the manuscript was written by EL-V and all authors commented on previous versions of the manuscript. 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Molecular mechanism of cardiac differentiation in P19 embryonal carcinoma cells regulated by Foxa2. Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2013 Apr;38(4):356–64. Tables Tables 1-5 are available in the Supplementary Files section. Supplementary Files SupplementaryFigure1.pdf SupplementaryFigure2.pdf SupplementaryFigure3.pdf SupplementaryFigure4.pdf SupplementaryFigure5.pdf SupplementaryFigure6.pdf SupplementaryFigure7.pdf SupplementaryFigure8.pdf SupplementaryFigure9.pdf SupplementaryFigure10.pdf SupplementaryFigure11.pdf SupplementaryFigure12.pdf SupplementaryFigure13.pdf Table1PEEERNAseq.pdf Table2PEEERNAseq.pdf Table3PEEERNAseq.pdf Table4PEEERNAseq.pdf Table5PEEERNAseq.pdf SUPPLEMENTARYFIGURELEGENDS.docx Cite Share Download PDF Status: Published Journal Publication published 25 Jun, 2025 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted Editorial decision: Accept as is 30 Apr, 2025 Reviewers agreed at journal 29 Mar, 2025 Reviewers invited by journal 29 Mar, 2025 Editor assigned by journal 26 Mar, 2025 First submitted to journal 26 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Experimentales","correspondingAuthor":true,"prefix":"","firstName":"Estefanía","middleName":"","lastName":"Lozano-Velasco","suffix":""}],"badges":[],"createdAt":"2024-12-14 11:00:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5643113/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5643113/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00018-025-05735-4","type":"published","date":"2025-06-25T15:57:36+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79652955,"identity":"5dc5401a-f317-473a-8c3c-dfafbf805229","added_by":"auto","created_at":"2025-04-01 08:16:36","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":698830,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePanel A\u003c/strong\u003e. Volcano plot of mRNA expression profile. Red dots represent significant DE genes in PE E9.5 \u003cem\u003evs.\u003c/em\u003e EE E10.5, right side downregulated mRNAs in PE9.5, left side upregulated mRNAs in PE9.5. Green dots represent mRNAs that were not DE genes in PE E9.5 \u003cem\u003evs.\u003c/em\u003eEE E10.5. \u003cstrong\u003ePanel B.\u003c/strong\u003e Volcano plot of miRNA expression profile. Red dots represent significant DE genes in PE E9.5 \u003cem\u003evs.\u003c/em\u003e EE E10.5, right side downregulated miRNAs in PE9.5, left side upregulated miRNAs in PE10.5. Green dots represent miRNAs that were not DE genes in PE E9.5 \u003cem\u003evs.\u003c/em\u003eEE E10.5. \u003cstrong\u003ePanel C. \u003c/strong\u003eRT-qPCR\u003cstrong\u003e \u003c/strong\u003eanalysis for\u003cstrong\u003e \u003c/strong\u003emRNA validation of DE genes in PE\u0026gt;EE (i.e. \u003cem\u003eHnf4a\u003c/em\u003e, \u003cem\u003eHoxb1\u003c/em\u003e, and \u003cem\u003eProx1\u003c/em\u003e) in PE9.5 and EE10.5 mouse tissues, demonstrating high expression in PE \u003cem\u003evs.\u003c/em\u003e EE (n=3). \u003cstrong\u003ePanel D. \u003c/strong\u003eRT-qPCR\u003cstrong\u003e \u003c/strong\u003eanalysis for\u003cstrong\u003e \u003c/strong\u003emRNA validation of DE genes in PE\u0026lt;EE (i.e. \u003cem\u003eSpry1\u003c/em\u003e, \u003cem\u003eHey2\u003c/em\u003e, and \u003cem\u003eItga\u003c/em\u003e) in PE9.5 and EE10.5 mouse tissues, demonstrating high expression in EE \u003cem\u003evs.\u003c/em\u003ePE (n=3). \u003cstrong\u003ePanel E. \u003c/strong\u003eRT-qPCR\u003cstrong\u003e \u003c/strong\u003eanalysis for\u003cstrong\u003e \u003c/strong\u003emiRNA validation of DE genes in PE\u0026gt;EE (i.e. \u003cem\u003emiR-200, miR-200b, miR-200c, miR-429 \u003c/em\u003eand\u003cem\u003e miR-495) \u003c/em\u003ein PE9.5 and EE10.5 mouse tissues, demonstrating high expression in PE \u003cem\u003evs.\u003c/em\u003e EE (n=3). \u003cstrong\u003ePanel F. \u003c/strong\u003eRT-qPCR\u003cstrong\u003e \u003c/strong\u003eanalysis for\u003cstrong\u003e \u003c/strong\u003emiRNA validation of DE genes in PE\u0026lt;EE (i.e. \u003cem\u003elet-7c, miR-24, miR-30a, miR-30c\u003c/em\u003e and \u003cem\u003emiR-351\u003c/em\u003e) in PE9.5 and EE10.5 mouse tissues, demonstrating high expression in EE \u003cem\u003evs.\u003c/em\u003e PE (n=3). \u003cstrong\u003ePanel G. \u003c/strong\u003eRT-qPCR\u003cstrong\u003e \u003c/strong\u003eanalysis for\u003cstrong\u003e \u003c/strong\u003elncRNA validation of DE genes in PE\u0026gt;EE (i.e. \u003cem\u003eGm35409, Gm35533, 9030622O22Rik \u003c/em\u003eand\u003cem\u003e 9030102K24Rik) \u003c/em\u003ein PE9.5 and EE10.5 mouse tissues, demonstrating high expression in PE \u003cem\u003evs.\u003c/em\u003e EE (n=3). \u003cstrong\u003ePanel H. \u003c/strong\u003eRT-qPCR\u003cstrong\u003e \u003c/strong\u003eanalysis for\u003cstrong\u003e \u003c/strong\u003elncRNA validation of DE genes in PE\u0026lt;EE (i.e. \u003cem\u003eGm13293, Gm42788,\u003c/em\u003e and \u003cem\u003e4833415N18Rik\u003c/em\u003e) in PE9.5 and EE10.5 mouse tissues, demonstrating high expression in EE \u003cem\u003evs.\u003c/em\u003e PE (n=3). \u003cstrong\u003ePanel I. \u003c/strong\u003eSchematic representation of putative microRNAs-mRNAs interactions of DE genes mRNA in PE E9.5 \u003cem\u003evs.\u003c/em\u003e EE E10.5. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Binder11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5643113/v1/9fda9c9d10dca388049fdb22.jpg"},{"id":79653743,"identity":"1414a6b4-94fc-457f-a049-3d94e90ce7de","added_by":"auto","created_at":"2025-04-01 08:24:36","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":751912,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePanel A\u003c/strong\u003e. RT-qPCR analysis for mRNA targets after microRNA gain-of-function (\u003cem\u003emiR-181c, miR-200b, miR-495\u003c/em\u003e and \u003cem\u003epreMix\u003c/em\u003e) in EPIC epicardial cells. Observe that selective mRNA downregulation is achieved after microRNA administration. Note that \u003cem\u003eNr3c1\u003c/em\u003eand \u003cem\u003eNfib\u003c/em\u003e are significantly decreased after all individual or combinatorial microRNA treatments (n=3). \u003cstrong\u003ePanel B\u003c/strong\u003e. RT-qPCR analysis for mRNA targets after microRNA loss-of-function (\u003cem\u003elet-7c, miR-30c, miR-351\u003c/em\u003e and \u003cem\u003eantiMix\u003c/em\u003e) in EPIC epicardial cells. Selective mRNA upregulation is achieved for \u003cem\u003ePrtg\u003c/em\u003e, \u003cem\u003eNr6a1\u003c/em\u003e and \u003cem\u003eHic2\u003c/em\u003e after individual microRNAs knockdown (n=3). \u003cstrong\u003ePanel C\u003c/strong\u003e. RT-qPCR analysis for mRNA targets after microRNA gain-of-function (\u003cem\u003emiR-181c, miR-200b, miR-495\u003c/em\u003e and \u003cem\u003epreMix\u003c/em\u003e) in MEC1 epicardial cells. Observe that only \u003cem\u003eMbln2\u003c/em\u003e and \u003cem\u003eHapln1\u003c/em\u003e are downregulated after \u003cem\u003emiR-495\u003c/em\u003e administration and Fn1 after miR-200b treatment (n=3). \u003cstrong\u003ePanel D\u003c/strong\u003e. RT-qPCR analysis for mRNA targets after microRNA loss-of-function (\u003cem\u003elet-7c, miR-30c, miR-351\u003c/em\u003e and \u003cem\u003eantiMix\u003c/em\u003e) in MEC1 epicardial cells. Note that Fbxo32 and Trim71 are up-regulated after all individual microRNA inhibition (n=3). \u003cstrong\u003ePanel E\u003c/strong\u003e. RT-qPCR analysis for mRNA targets after microRNA gain-of-function (\u003cem\u003emiR-181c, miR-200b, miR-495\u003c/em\u003e and \u003cem\u003epreMix\u003c/em\u003e) in EEx from E10.5 epicardial explants. Observe that \u003cem\u003eMbln2\u003c/em\u003e and \u003cem\u003eHapln1\u003c/em\u003e are decreased after all individual or combinatorial microRNA treatments. Note also that selective mRNA downregulation is achieved after microRNA administration for \u003cem\u003eNr3c1\u003c/em\u003e, \u003cem\u003eNfib\u003c/em\u003e, \u003cem\u003eRnd3\u003c/em\u003e and \u003cem\u003eFn1 \u003c/em\u003e(n=3). \u003cstrong\u003ePanel F\u003c/strong\u003e. RT-qPCR analysis for mRNA targets after microRNA loss-of-function (\u003cem\u003elet-7c, miR-30c, miR-351\u003c/em\u003e and \u003cem\u003eantiMix\u003c/em\u003e) in EEx from EE10.5 epicardial explants. Observe a selective up-regulation of Trim71 after let-7c and miR-30c inhibition (n=3). *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Binder12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5643113/v1/97d4d0a9415a327ac9c1932b.jpg"},{"id":79652956,"identity":"f7f30ff5-9216-4c50-971e-fc7246e829f7","added_by":"auto","created_at":"2025-04-01 08:16:36","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1070420,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePanel A\u003c/strong\u003e. Migration assays after DE microRNAs in PE\u0026gt;EE \u003cem\u003emiR-181c\u003c/em\u003e, \u003cem\u003emiR-200b, miR-495\u003c/em\u003e and \u003cem\u003epreMix\u003c/em\u003e administration, respectively. Quantitative cell migration analysis guided by the schematic representation of cell migration quantification; \u003cem\u003eTotal cell migration\u003c/em\u003e, cellular migration from ventricular explant border to the outermost individual cell of the culture; \u003cem\u003eCohesive cell migration\u003c/em\u003e, collective migration from ventricular explant border; \u003cem\u003eNon-cohesive cell migration\u003c/em\u003e, individual cell migration from the outermost periphery of the cohesive migration. \u003cstrong\u003ePanel B,C,D\u003c/strong\u003eQuantitative cell migration for DE microRNAs in PE\u0026gt;EE; \u003cem\u003emiR-181c\u003c/em\u003e, \u003cem\u003emiR-200b, miR-495\u003c/em\u003eand \u003cem\u003epreMix \u003c/em\u003eadministration\u003cem\u003e. \u003c/em\u003eNote that \u003cem\u003emiR-495\u003c/em\u003e administration impairs total cell migration followed by cohesive migration repression (n=5). \u003cstrong\u003ePanel E.\u003c/strong\u003e Migration assays after DE mRNAs in PE\u0026lt;EE \u003cem\u003eNfib, Nr3c1\u003c/em\u003e, \u003cem\u003eRnd3, Mbln2\u003c/em\u003eand \u003cem\u003eFn1\u003c/em\u003e silencing. \u003cstrong\u003ePanel F,G,H\u003c/strong\u003e Quantitative cell migration for DE mRNAs in PE\u0026lt;EE; \u003cem\u003eNfib, Nr3c1\u003c/em\u003e, \u003cem\u003eRnd3, Mbln2\u003c/em\u003e and \u003cem\u003eFn1 \u003c/em\u003einhibition. Note that \u003cem\u003eNr3c1 \u003c/em\u003eloss-of-function enhances total cell migration followed by cohesive migration increment (n=5). \u003cstrong\u003ePanel I\u003c/strong\u003eMigration assays after DE microRNAs in PE\u0026lt;EE \u003cem\u003elet-7c, miR-30c, miR-351\u003c/em\u003e and \u003cem\u003eantiMix \u003c/em\u003eadministration, respectively. \u003cstrong\u003ePanel J,K,L\u003c/strong\u003e Quantitative cell migration for DE microRNAs in PE\u0026lt;EE \u003cem\u003elet-7c, miR-30c, miR-351\u003c/em\u003e and \u003cem\u003eMix \u003c/em\u003einhibition\u003cem\u003e. \u003c/em\u003eNote that \u003cem\u003elet-7c and miR-351\u003c/em\u003e loss-of-function administration impair total cell migration without cohesive and non-cohesive migration affection (n=5). *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Binder13.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5643113/v1/89ce4df1a1b2f5fb06156c31.jpg"},{"id":79652957,"identity":"e5731957-e714-419c-9f99-ce5364ddbba5","added_by":"auto","created_at":"2025-04-01 08:16:36","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":808228,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePanel A,B\u003c/strong\u003e Analysis of lineage specific markers expression, i.e. myocardial (\u003cem\u003eGata4, Myh6, Nkx2.5, Tnnt2, Srf\u003c/em\u003e), epicardial (\u003cem\u003eTcf21, Tbx18, Wt1\u003c/em\u003e), and endocardial (\u003cem\u003ePecam1, Postn, Tie2\u003c/em\u003e) after \u003cem\u003emiR-181c\u003c/em\u003e, \u003cem\u003emiR-200b, miR-495\u003c/em\u003e and \u003cem\u003epreMix \u003c/em\u003eadministration or \u003cem\u003elet-7c, miR-30c, miR-351\u003c/em\u003e and anti\u003cem\u003eMix \u003c/em\u003einhibition, respectively (n=3). \u003cstrong\u003ePanel C,D\u003c/strong\u003e Analysis of biological processes markers, i.e. angiogenesis (\u003cem\u003eAng1, Ang2, Efnb2, Flt1\u003c/em\u003e), EMT (\u003cem\u003eCdh5, Snail1, Snail2\u003c/em\u003e), and fibrosis (\u003cem\u003eCol1a1, Col3a1, Fn1, Sox9\u003c/em\u003e) in MEC1 and EPIC epicardial cells after \u003cem\u003emiR-181c\u003c/em\u003e, \u003cem\u003emiR-200b, miR-495\u003c/em\u003e and \u003cem\u003epreMix \u003c/em\u003eadministration or \u003cem\u003elet-7c, miR-30c, miR-351\u003c/em\u003e and anti\u003cem\u003eMix \u003c/em\u003einhibition, respectively (n=3). \u003cstrong\u003ePanel E.\u003c/strong\u003e Representative images of immunohistochemical analyses of cardiac troponinT (cTnt) in MEC1 epicardial cells, after administration of \u003cem\u003epremiR-181c, premiR-200b\u003c/em\u003e, and \u003cem\u003eanti-let7c\u003c/em\u003e as compared to controls. Observe that there is a significant difference in the expression of cTnt after \u003cem\u003elet-7c\u003c/em\u003einhibition (n=5). \u003cstrong\u003ePanel F.\u003c/strong\u003e RT-qPCR analyses of selected cardiogenic markers (Tnnt2, Myh6, Tcf21, Tie2) in EEx from E10.5 epicardial explants after \u003cem\u003epremiR-181c, premiR-200b\u003c/em\u003e, and \u003cem\u003eanti-let7c\u003c/em\u003eadministration (n=5). *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Binder14.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5643113/v1/b97cd4e087f41cba7baee743.jpg"},{"id":79653741,"identity":"57cb9af3-05bd-4c3b-a685-03606188472f","added_by":"auto","created_at":"2025-04-01 08:24:36","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":558889,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePanels A, B, E \u003c/strong\u003eRT-qPCR analysis for microRNA expression after microRNA gain-of-function (\u003cem\u003emiR-181c, miR-200b, miR-495\u003c/em\u003e) (n=3). \u003cstrong\u003ePanel A\u003c/strong\u003e MEC1 epicardial cells. \u003cstrong\u003ePanel B\u003c/strong\u003e EPIC epicardial cells. \u003cstrong\u003ePanel E\u003c/strong\u003eEEx from E10.5 epicardial explants. Observe that microRNA upregulation is achieved after \u003cem\u003emiR-181c \u003c/em\u003eand\u003cem\u003e miR-495\u003c/em\u003e administration in all epicardial cell types and for \u003cem\u003emiR-200b\u003c/em\u003eonly in E10.5 epicardial cells. \u003cstrong\u003ePanels C, D, F \u003c/strong\u003eRT-qPCR analysis for microRNA expression after microRNA loss-of-function (\u003cem\u003elet-7c, miR-30c, miR-351\u003c/em\u003e) (n=3). \u003cstrong\u003ePanel C\u003c/strong\u003e MEC1 epicardial cells. \u003cstrong\u003ePanel D\u003c/strong\u003e EPIC epicardial cells. \u003cstrong\u003ePanel F\u003c/strong\u003e EEx from E10.5 epicardial explants. Observe that let-7c inhibition leads to high expression levels of \u003cem\u003emiR-30c \u003c/em\u003eand\u003cem\u003e miR-351\u003c/em\u003e in both epicardial cell types. \u003cstrong\u003ePanel G,H\u003c/strong\u003e RT-qPCR analysis of myocardial lineage markers (\u003cem\u003eMyh6, Tnnt2)\u003c/em\u003ein MEC1 and EEx from E10.5 epicardial cells after \u003cem\u003emiR-30c \u003c/em\u003eand\u003cem\u003e miR-351 \u003c/em\u003egain-of-function. Observe that \u003cem\u003emiR-351\u003c/em\u003e promotes \u003cem\u003eMyh6\u003c/em\u003e expression only in MEC1 epicardial cells (n=5). *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Binder15.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5643113/v1/937d0ec9d728d3c9b81972ad.jpg"},{"id":79652960,"identity":"fd57b548-57a3-4887-a3c4-cb9ceed91397","added_by":"auto","created_at":"2025-04-01 08:16:36","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":558716,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePanel A. \u003c/strong\u003eRT-qPCR analysis of DE microRNAs (\u003cem\u003emiR-495, let-7c, miR-30c, miR-351\u003c/em\u003e) after \u003cem\u003eFoxc1\u003c/em\u003e and \u003cem\u003eFoxf1\u003c/em\u003e siRNA treatment in MEC1 epicardial cells. Observe a differential microRNA regulation. Note also a significant downregulation of \u003cem\u003elet-7\u003c/em\u003e and upregulation of \u003cem\u003emiR-30c\u003c/em\u003e after \u003cem\u003eFoxf1\u003c/em\u003e inhibition (n=3). \u003cstrong\u003ePanel B. \u003c/strong\u003eRT-qPCR analysis of \u003cem\u003eFoxc1\u003c/em\u003e and \u003cem\u003eFoxf1 \u003c/em\u003eexpression\u003cem\u003e \u003c/em\u003eafter \u003cem\u003eFoxc1\u003c/em\u003e and \u003cem\u003eFoxf1\u003c/em\u003e siRNA treatment in MEC1 epicardial cells, respectively (n=3). \u003cstrong\u003ePanel C. \u003c/strong\u003eRT-qPCR analysis of \u003cem\u003eFoxc1\u003c/em\u003e and \u003cem\u003eFoxf1 \u003c/em\u003eexpression\u003cem\u003e \u003c/em\u003eafter DE microRNAs in PE\u0026gt;EE treatment (\u003cem\u003emiR-181c, miR-200b, miR-495\u003c/em\u003e) in MEC1 epicardial cells. Observe that \u003cem\u003eFoxf1\u003c/em\u003e is downregulated after pre-miRNA administration (n=3). \u003cstrong\u003ePanel D. \u003c/strong\u003eRT-qPCR analysis of \u003cem\u003eFoxc1\u003c/em\u003e and \u003cem\u003eFoxf1 \u003c/em\u003eexpression\u003cem\u003e \u003c/em\u003eafter DE microRNAs in PE\u0026lt;EE treatment (\u003cem\u003elet-7c, miR-30c, miR-351\u003c/em\u003e) in MEC1 epicardial cells. Note that \u003cem\u003eFoxf1\u003c/em\u003e is downregulated only after let-7c inhibition (n=3). \u003cstrong\u003ePanel E,F. \u003c/strong\u003eRT-qPCR expression analysis of cardiogenic markers; epicardial (\u003cem\u003eWt1, Tbx18, Tcf21\u003c/em\u003e), myocardial (\u003cem\u003eGata4, Myh6, Srf, Tnnt2\u003c/em\u003e) and endocardial (\u003cem\u003ePecam1, Postn, Tie2\u003c/em\u003e) in MEC1 epicardial cells after \u003cem\u003eFoxc1\u003c/em\u003e and \u003cem\u003eFoxf1\u003c/em\u003e siRNA treatment, respectively. Observe that selective regulation is achieved after \u003cem\u003eFoxc1\u003c/em\u003e and \u003cem\u003eFoxf1\u003c/em\u003e inhibition. Note also that myocardial and endocardial lineage markers are upregulated after \u003cem\u003eFoxf1\u003c/em\u003einhibition (n=3). *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Binder16.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5643113/v1/84e80cb15859bab6bddd0b5d.jpg"},{"id":79653744,"identity":"f0775a8c-1617-41db-b334-0547f055bb04","added_by":"auto","created_at":"2025-04-01 08:24:37","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":491823,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the functional role of \u003cem\u003eFoxf1\u003c/em\u003e, \u003cem\u003emiR-495\u003c/em\u003e and \u003cem\u003elet-7c\u003c/em\u003e in epicardial cell migration and specification. The figure illustrates the intricated molecular network regulating epicardial cell migration and specification, highlighting the molecular interplay between the transcription factors \u003cem\u003eFoxf1\u003c/em\u003e and \u003cem\u003eFoxc1\u003c/em\u003e, as well as their regulatory effect on \u003cem\u003emiR-495\u003c/em\u003e and \u003cem\u003elet-7c\u003c/em\u003e. This intricate molecular interplay manifests functionally in both epicardial cell migration and epicardial cell specification into cardiogenic lineage as highlighted in color. Additionally, in the background, the diagram also reflects the molecular crosstalk among distinct microRNAs and the role of miR495 and let-7c in epicardial cell lineage specification into epicardium and endocardium, along with their involvement in other biological processes such as EMT, angiogenesis and fibrosis (Grey). Pointed arrows denote positive regulation, blunt arrows negative regulation and dashed lines no significant regulation.\u003c/p\u003e","description":"","filename":"Binder17.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5643113/v1/188af3a6d246556aa318381e.jpg"},{"id":85686255,"identity":"969071b7-a8b4-4f04-9629-4d898dfef0f6","added_by":"auto","created_at":"2025-06-30 16:05:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7183900,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5643113/v1/021b470a-fdf2-4cf1-b123-1759d5b02156.pdf"},{"id":79652963,"identity":"1e1bab60-0b3e-4693-82da-f0895c206fa1","added_by":"auto","created_at":"2025-04-01 08:16:37","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1862658,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5643113/v1/5f77b4ed3349db4028c0cbcd.pdf"},{"id":79652962,"identity":"042e2f9a-d17a-4fec-9edc-78bb8521c7fd","added_by":"auto","created_at":"2025-04-01 08:16:36","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":654445,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5643113/v1/030fa4f3c92ef0c57526ca2e.pdf"},{"id":79654062,"identity":"b2ddc145-53a5-4b45-a716-21b58317b0ea","added_by":"auto","created_at":"2025-04-01 08:32:36","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":58828,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5643113/v1/fee463ff81bb8661f2c0ad8e.pdf"},{"id":79652979,"identity":"f92922c2-2e58-48d4-92f7-4129016774f4","added_by":"auto","created_at":"2025-04-01 08:16:37","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":2236552,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5643113/v1/30297c5481ae5837a5a3a839.pdf"},{"id":79652965,"identity":"1b9ecb8a-1c4b-4587-8ef0-0940b070431f","added_by":"auto","created_at":"2025-04-01 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08:16:37","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":98719,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure7.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5643113/v1/d79c7f2b40836f79d67b8012.pdf"},{"id":79652968,"identity":"68cee8ac-dce0-42ae-9c92-21cc4607207f","added_by":"auto","created_at":"2025-04-01 08:16:37","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":535904,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure8.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5643113/v1/fb32c5d02a64d60ed8f84a74.pdf"},{"id":79654066,"identity":"1b2e3da9-4329-4139-9359-16a63395c5b4","added_by":"auto","created_at":"2025-04-01 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08:16:37","extension":"pdf","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":441159,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure11.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5643113/v1/604587da8a549dd989aff0eb.pdf"},{"id":79653746,"identity":"bc597ae4-0c62-47aa-9a85-c872791e6dc2","added_by":"auto","created_at":"2025-04-01 08:24:37","extension":"pdf","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":449268,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure12.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5643113/v1/682423ec81ed0b38833121da.pdf"},{"id":79654065,"identity":"54585207-24b8-4b4f-af10-477b53b6c4cb","added_by":"auto","created_at":"2025-04-01 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08:24:37","extension":"pdf","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":177506,"visible":true,"origin":"","legend":"","description":"","filename":"Table2PEEERNAseq.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5643113/v1/5742e3e373f7f109750e9678.pdf"},{"id":79653745,"identity":"fc8d853f-b1df-4637-b397-042e1ec24d0c","added_by":"auto","created_at":"2025-04-01 08:24:37","extension":"pdf","order_by":16,"title":"","display":"","copyAsset":false,"role":"supplement","size":64386,"visible":true,"origin":"","legend":"","description":"","filename":"Table3PEEERNAseq.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5643113/v1/5941d36fafd6842651bfbfd1.pdf"},{"id":79652976,"identity":"f3be6302-f295-43fd-bc44-df8d71b31ae1","added_by":"auto","created_at":"2025-04-01 08:16:37","extension":"pdf","order_by":17,"title":"","display":"","copyAsset":false,"role":"supplement","size":54462,"visible":true,"origin":"","legend":"","description":"","filename":"Table4PEEERNAseq.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5643113/v1/d426b0b9413cd500b8b09ab0.pdf"},{"id":79653753,"identity":"f1b7fa52-53de-44f8-b514-6aa9be4bf0b8","added_by":"auto","created_at":"2025-04-01 08:24:37","extension":"pdf","order_by":18,"title":"","display":"","copyAsset":false,"role":"supplement","size":60980,"visible":true,"origin":"","legend":"","description":"","filename":"Table5PEEERNAseq.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5643113/v1/e4003ea1facc3639d92d1a64.pdf"},{"id":79652974,"identity":"9a826c02-21f3-4e60-9c39-387f0c9057eb","added_by":"auto","created_at":"2025-04-01 08:16:37","extension":"docx","order_by":19,"title":"","display":"","copyAsset":false,"role":"supplement","size":16691,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLEMENTARYFIGURELEGENDS.docx","url":"https://assets-eu.researchsquare.com/files/rs-5643113/v1/c0a10f124a8108de716df728.docx"}],"financialInterests":"","formattedTitle":"Foxf1 -mediated co-regulation of miR-495 and let-7c modulates epicardial cell migration and myocardial specification","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eThe heart is the first organ that becomes functional in the vertebrate embryo. In mice, the precardiac mesoderm forms a primitive tubular heart, beating at embryonic day (E) 8.0-E.8.5. This tubular structure undergoes a series of morphological changes, including rightward looping and thereafter configuring the prospective atrial and ventricular chambers (E9.5) (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). At E10.5, five distinct regions can be delineated in the embryonic heart, the inflow tract, the embryonic atrial chamber, the atrioventricular canal, the ventricular chambers and the outflow tract (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). From this stage onwards, each embryonic cardiac region will be separated into distinct left and right components, providing thus a double circuitry with distinct inlet and outlet connections (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Besides the intrinsic cardiac progenitor cells, external cell populations also contribute to heart development i.e. the cardiac neural crest and the proepicardium and its derivatives (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). The proepicardium (PE) is a transitory cell cluster that develops at the junction between the sinus venosus (SV) and the posterior undifferentiated lateral plate mesenchyme (LPM) at E8.5-E9.0 in the mouse embryo (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). In chicken embryos, PE grows in size and villous projections extend towards the dorsal aspect of the cardiac inner curvature, ultimately contacting the atrioventricular (AV) junction and forming a tissue bridge (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). In mice, around E10.0 proepicardial villous projections attach to the heart (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) and proepicardial cells migrate and spread over the naked myocardium forming a single squamous epithelium which is termed the embryonic epicardium (EE), playing an essential role in cardiac development (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe epicardium, the outermost layer of the heart, was long considered as an external cover devoid of any functional meaning, but recent studies discovered its essential contribution to the cardiac development and regeneration (\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). The EE serves as a crucial source of epicardial-derived cells (EPDCs) that, after undergoing epithelial-to-mesenchymal transformation (EMT), migrate into the myocardial wall and differentiate into multiple cardiac cell types (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). EPDCs contribute to endothelial and smooth muscle cells in the coronary vasculature, cardiac fibroblasts (\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), and to a lesser extent, atrioventricular cushion cells (\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). More recently, a contribution to cardiac resident stem cells (mesenchymal-like) has also been reported (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) as well as to the cardiomyocyte lineage (\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), yet this latter point remains highly controversial (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Recent studies have enhanced our understanding of the molecular mechanisms driving PE and EE tissue formation (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Signalling molecules such as Bmp and Fgf play pivotal roles in PE specification and cardiomyogenic differentiation (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Transcription factors such as \u003cem\u003eWt1\u003c/em\u003e are crucial for EMT and EPDCs maturation (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan additionalcitationids=\"CR29 CR30 CR31\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). \u003cem\u003eTbx18\u003c/em\u003e has a role in epicardial EMT and subsequently in differentiation of EPDCs into smooth muscle cells and fibroblasts (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e) while \u003cem\u003eTcf21\u003c/em\u003e regulates proepicardial cell specification and maturation (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Finally, while \u003cem\u003eGata4\u003c/em\u003e is essential for PE formation, the precise contributions of other cardiac-enriched transcription factors such as \u003cem\u003eNkx2.5\u003c/em\u003e, \u003cem\u003eIsl1\u003c/em\u003e, and \u003cem\u003ePitx2\u003c/em\u003e remain unclear (\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Despite these advances, it is poorly understood how transcription factors and non-coding RNAs contribute to epicardial development.\u003c/p\u003e \u003cp\u003eWhile transcriptional regulation plays a critical role in cardiac morphogenesis and cardiovascular cell differentiation, a growing body of evidence suggests that microRNAs, the most studied subtype of small non-coding RNAs, play crucial roles in gene regulation during embryonic development and tissue homeostasis (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e) (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e) microRNAs display temporal and spatial differential expression in both embryonic and adult tissues, where they fine-tune gene expression at the post-transcriptional level (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). In the context of cardiogenesis, several microRNAs have been implicated in cardiac differentiation, proliferation and morphogenesis. (\u003cspan additionalcitationids=\"CR45 CR46 CR47\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Recent studies in our laboratory evidenced a microRNA differential expression during PE and EE formation in chicken, identifying \u003cem\u003emiR-146\u003c/em\u003e, \u003cem\u003emiR-195\u003c/em\u003e and \u003cem\u003emiR-223\u003c/em\u003e as potential regulators that selectively enhance cardiomyogenesis in PE and EE by modulating \u003cem\u003eSmad3\u003c/em\u003e and \u003cem\u003eSmurf1\u003c/em\u003e, in \u003cem\u003eex vivo\u003c/em\u003e conditions (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Considering the species-specific differences in epicardium formation and the discovery of DE microRNAs in chicken, the functional role of microRNAs in PE and EE development in mice, as well as their potential application to enhance cardiogenesis remains elusive.\u003c/p\u003e \u003cp\u003eAlthough significant progress has been made in understanding the cell lineage contribution of the EPDCs over the last decade, the molecular determinants that contribute to such cell fate decisions remains largely unknown. In this study, we carried out a comprehensive RNAseq analysis of coding and non-coding gene expression at two critical timepoints of PE and EE development in mouse embryos. Our data identified an intricate network of differentially expressed (DE) mRNAs, microRNAs and lncRNAs that regulate distinct biological pathways in PE \u003cem\u003evs\u003c/em\u003e. EE. We identified that \u003cem\u003eFoxf1\u003c/em\u003e transcription factor exerts a regulatory control over \u003cem\u003emiR-495, miR-351\u003c/em\u003e, and \u003cem\u003elet-7c\u003c/em\u003e, thereby modulating epicardial cell migration and myocardial specification. These observations underscores the complex interplay between transcription factors and microRNAs in epicardial development, providing new insights into the molecular mechanisms that govern cardiogenesis during embryonic development.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003eA comprehensive description of each procedure is detailed in the following sections. \u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e provides an overview of the experimental workflow, illustrating the key methodological steps.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMouse lines and tissue collection\u003c/h2\u003e \u003cp\u003ePreviously described Wt1\u003csup\u003eGFP/+\u003c/sup\u003e mice were used in this study. The WT1\u003csup\u003eGFP\u003c/sup\u003e knockin line in which the exon 1 of a Wt1 allele has been replaced by the GFP sequence was used as a reporter for active WT1 transcription (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). Pregnant Wt1\u003csup\u003eGFP/+\u003c/sup\u003e female mice were harvested to E9.5 and to E10.5, respectively. E9.5 PE were manually dissected, pooled and stored in buffer lysis for RNA isolation at -80\u0026deg;C until used. For flow cytometry analysis and sorting, dissected hearts from E10.5 embryos were placed in cytometry buffer (phosphate buffer saline [PBS] plus 2% fetal bovine serum [FBS] and 10 mM 4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid [HEPES]) and homogenized by repeated pipetting. Cell suspension was washed by pelleting at 400G during 5 minutes. Then, cells were incubated on ice in darkness with the fluorochrome conjugated antibodies: anti-CD31-APC (PECAM-1) (Mouse monoclonal anti-CD31 APC #Thermo Fisher, 17-0311-82) for general staining of the endothelium. 4',6-Diamidine-2'-phenylindole dihydrochloride (DAPI) staining was included to exclude dead cells. Negative controls (GFP negative littermates) and isotypic antibody allowed setting of the gates (FITC Rat IgG2a, k Isotype, #Biolegend). Epicardial cells were then sorted by GFP high fluorescence and lack of CD31staining \u003cb\u003e(Supplementary Fig.\u0026nbsp;2)\u003c/b\u003e (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Cells were sorted in a BD FACS Aria Fusion Cell Sorter. Data were analyzed with FlowJo TM10. After sorting, epicardial cells were pooled and stored in buffer lysis for RNA isolation at -80\u0026deg;C until used. At least 3\u0026ndash;5 litters were used at each developmental stage until sufficient tissue was collected, which would guarantee optimal and sufficient RNA isolation for further sequencing.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRNA-seq libraries preparation, sequencing and differential expression gene analysis\u003c/h3\u003e\n\u003cp\u003e \u003c/p\u003e \u003cp\u003eFor single-end microRNA libraries, 500 pg of total RNA were used to generate barcoded miRNA-seq single-end libraries using the Bioo NEXTflex Small RNA (BiooScientific). Briefly, 3\u0026acute; and 5\u0026acute; SR adapters were first ligated to the RNA sample. Next, reverse transcription followed by PCR amplification was used to enrich cDNA fragments with adapters at both ends. Adapter-ligated cDNA fragments from different samples were pooled and run in a 6% polyacrilamide gel. The 147 nt band, corresponding to the pooled miRNA libraries, was purified from the gel. Finally, the quantity and quality of the pooled miRNA libraries were determined using the Agilent 2100 Bioanalyzer High Sensitivity DNA chip. Both, mRNA microRNA libraries were sequenced on a HiSeq 2500 (Illumina) and processed with RTA v1.18.66.3. FastQ files for each sample were obtained using bcl2fastq v2.20.0.422 software (Illumina).\u003c/p\u003e \u003cp\u003eFor FastQC reads quality reports analysis, trimming of adaptors and alignment of sequences, fastq sequence reads were uploaded to the European version of the Galaxy platform (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). The quality of the reads was analyzed with FastQC Read Quality reports (Galaxy Version 0.74\u0026thinsp;+\u0026thinsp;galaxy1) software, trimmed with Trim Galore software (Galaxy Version 0.6.7\u0026thinsp;+\u0026thinsp;galaxy0) and aligned to the built-in mouse reference genome mm10 (GRCm38) with the RNA STAR Gapped-read mapper (Galaxy Version 2.7.10b\u0026thinsp;+\u0026thinsp;galaxy3) (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor gene-expression analyses, bam files were downloaded from the Galaxy server and further analyzed with the different RStudio packages downloaded from the Bioconductor website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioconductor.org\u003c/span\u003e\u003cspan address=\"http://bioconductor.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, accessed on 2 March 2023). Reads were assigned to mRNA and microRNA genes by using the \u0026ldquo;featureCounts\u0026rdquo; function of the \u0026ldquo;Rsubread\u0026rdquo; package, version 2.10.5 (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). In addition, mouse gencode.vM20.annotation.gff3 annotation file release M20, GRCm38.p6 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_mouse/release_M20/gencode.vM20.annotation.gff3.gz\u003c/span\u003e\u003cspan address=\"https://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_mouse/release_M20/gencode.vM20.annotation.gff3.gz\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and mmu.gff3 chromosomal coordinates of Mus musculus microRNAs miRBase v22 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.mirbase.org/download/#:~:text=mml.gff3-,mmu.gff3,-osa.gff3\u003c/span\u003e\u003cspan address=\"https://www.mirbase.org/download/#:~:text=mml.gff3-,mmu.gff3,-osa.gff3\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were used for mRNA and miRNA analysis, respectively. Uniquely mapped reads were used to calculate gene expression. The library size of each experimental point ranged from 18625406 to 26021292 sequences and from 601840 to 1251790 sequences for mRNA and miRNA analysis, respectively. Fastq files and abundance measurements of features were uploaded to Gene Expression Omnibus database with GEO accession number : GSE189344\u003c/p\u003e \u003cp\u003eDifferential gene expression analysis was performed through package \u0026ldquo;edgeR-package\u0026rdquo; version 4.4.2 (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). The normLibSizes() function was used to normalize the library sizes by trimmed mean of M-values (TMM) method. Only transcripts detected in three transcriptomes were used in the analysis. All gene comparisons with an adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and an abslog2 fold change (FC)\u0026thinsp;\u0026gt;\u0026thinsp;2 were considered differentially expressed under the experimental conditions. For miRNA-mRNA transcripts interaction analysis we used miRComb package (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). Gene Set Enrichment Analysis (GSEA)-based Gene Ontology (GO) analyses were conducted with the \u0026ldquo;clusterProfiler\u0026rdquo; package version 3.6.0 (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). The gene sets with a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered overrepresented under the experimental conditions.\u003c/p\u003e\n\u003ch3\u003eRNA isolation and qRT-PCR\u003c/h3\u003e\n\u003cp\u003eRNA samples from the same pools used for RNAseq libraries construction as well as additional isolated RNA samples were used; namely, those corresponding to E9.5 PE cells and E10.5 FACS sorted EE cells. All RT-qPCR experiments followed MIQE guidelines (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e) and similarly as previously reported (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). Briefly, RNA from tissue samples was extracted and purified by using the Direct-zol\u0026trade; RNA Miniprep kit (Zymo research) and the cell line RNA isolation was performed with ReliaPrep\u0026trade; RNA Miniprep Systems kit (Promega), both according to manufacturer\u0026acute;s instructions. For mRNA/lncRNA expression measurements, 500ng of total RNA was used for retro-transcription with PrimeScript\u0026trade; RT Master Mix (Takara), the resulting cDNA was diluted 1/5, both according to manufacturer\u0026rsquo;s guidelines. For microRNA expression analyses, 20 ng of total RNA was used for retro-transcription with with miRCURY LNA RT Kit (Qiagen), the resulting cDNA was diluted 1/40, following manufacturer\u0026acute;s guidelines. Negative controls, without reverse transcriptase, were performed for each sample to assess genomic contamination. Real-time PCR experiments were performed with 1 \u0026micro;L of cDNA, GoTaq qPCR Master Mix (Promega) and corresponding primer sets as described in \u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e. All qPCRs were performed using a CFX384TM thermocycler (Bio-Rad) following the manufacturer\u0026rsquo;s recommendations. For mRNA, the qPCR program consisted of 95\u0026deg;C for 30 s (initial denaturalization), followed by 40 cycles of 95\u0026deg;C for 5 s (denaturalization); 60\u0026deg;C for 10 s (annealing); 75\u0026deg;C for 7 s (extension). Finally, melting curves were determined by an initial step of 95\u0026deg;C for 5 s, followed by 0.5\u0026deg;C increments for 7 s from 65\u0026deg;C to 95\u0026deg;C. For microRNAs, the qPCR program consisted of 95\u0026deg;C for 10 min (initial denaturalization), followed by 40 cycles of 95\u0026deg;C for 5 s (denaturalization); 60\u0026deg;C for 1 min (annealing and extension). Finally, melting curves were determined by an initial step of 95\u0026deg;C for 5 s, followed by 0.5\u0026deg;C increments for 7 s from 65\u0026deg;C to 95\u0026deg;C. The relative level of expression of each gene was calculated as described by Livak \u0026amp; Schmittgen (2001) (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e) using \u003cem\u003eGapdh\u003c/em\u003e as the internal control for mRNA expression analyses and \u003cem\u003e5S\u003c/em\u003e for microRNA expression analyses, respectively. Each PCR reaction was carried out in triplicate and repeated in at least three distinct biological samples to obtain representative means.\u003c/p\u003e\n\u003ch3\u003eCell lines\u003c/h3\u003e\n\u003cp\u003eIn this study four cell lines were used, immortalized embryonic endocardial MEVEC (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e), muscle cardiac cell line HL1 (Sigma-Aldrich SCC065), mouse embryonic epicardial cell line MEC1 (Sigma-Aldrich SCC187) and epicardial EPIC (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e). Each cell line was cultured following the manufacturer\u0026rsquo;s recommendations for 24hr at 37\u0026deg;C in a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e at 4x10\u003csup\u003e4\u003c/sup\u003e cells per well in plates of 24 wells before transfection.\u003c/p\u003e\n\u003ch3\u003eTissue explants isolation\u003c/h3\u003e\n\u003cp\u003e All experiments were performed with the approved consent of the Ethics Committee of the University of Ja\u0026eacute;n and Andalusian Regional Government (14/03/2022/038). Pregnant CD1 wild-type female mice were harvested to E10.5. E10.5 ventricles were manually dissected and cultured in DMEM/Glutamax culture medium. For embryonic epicardial cell isolation for qPCR analysis, the ventricles were dissected in Earle\u0026rsquo;s balanced salt solution (EBSS) (Gibco), and cultured in a 12-well plates with collagen type I gels (Sigma-Aldrich #C3867-1VL), as previously described (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e), for 48 hours before transfection. Epicardial cells from transfected E10.5 ventricles were isolated, pooled and directly stored at -80\u0026deg;C until used. For confocal microscopy analyses, the ventricles were dissected in Earle\u0026rsquo;s balanced salt solution (EBSS) (Gibco), and cultured in a 4-chambered glass bottom dish with collagen treatment as previously described (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e), 48 hours before transfection. Briefly, samples were fixed in freshly made 4% PFA and stored in PBS at 4\u0026deg;C until used. Each experimental condition was repeated at least three times with a minimum number of three explants per condition, respectively.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003emicroRNA mimics or anti-miR and siRNA transfections\u003c/h2\u003e \u003cp\u003eE10.5 ventricles were cultured for 48 hours at 37\u0026deg;C in a cell culture incubator before administration of miRNAs mimics (pre-miRNAs), anti-miRNAs or siRNAs, respectively, as previously described (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). Pre-miRNAs, anti-miRNA and siRNA transfections were carried out with Lipofectamine 2000 (Invitrogen), following the manufacturer\u0026rsquo;s guidelines. Briefly, 50nM of premiRNAs (microRNA precursor) or antimiRs (microRNA inhibitor) were applied to the explants (3 explants per well), and for siRNA transfection 60-80nM of siRNA were applied. These concentrations were selected based on preliminary experiments in which qRT-PCR was performed to assess transfection efficiency, adjusting the doses for each condition. After incubation, 24 hours for pre-miR or 48 hours for anti-miR and siRNA, explants were either processed for RT-qPCR or immunohistochemical (IHC) analyses. Negative controls, E10.5 ventricular explants, treated only with Lipofectamine were run in parallel. To perform IHC analyses, the explants were fixed in PFA 4% for 15 min at room temperature rinsed two times in PBS for 5 minutes and stored in PBS at 4\u0026deg;C. For RT-qPCR analysis, explant epicardial outgrowths were collected and stored at -80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell migration assays\u003c/h3\u003e\n\u003cp\u003eMouse E10.5 ventricular explants were isolated from the developing embryo and the ventricular apex was dissected, plated upside down into coated collagen 4 chambered glass bottom dishes, incubated into DMEM Glutamax culture media for 48 hours as previously reported. At this stage, emerging epicardial outgrowths start to develop. Transfections with corresponding pre-miRNAs, anti-miRNAs, scrambled, siRNAs and negative controls, respectively, were carried out and cultures were allowed to develop for another 24/48 hours. Explants were rinsed in PBS for 5 min at room temperature and fixed in 4% PFA for 15 min at room temperature. After the fixation, the explants were rinsed two times in PBS for 5 min and incubated with Phalloidin FITC 1:1000 (Abcam) following the manufacturer\u0026rsquo;s recommendations. Finally, DAPI 1:2000 (Sigma) was incubated for 15 min at room temperature, rinsed two times in PBS for 5 min and stored in PBS in darkness at 4\u0026deg;C. Subsequently, representative images of each explant were collected using a Leica TCS SP5 II confocal scanning laser microscope, and the extension of the epicardial migration (i.e. cohesive, non-cohesive and total migration) was measured in ten different regions per image, using ImageJ software. Mean and SD values were subsequently plotted.\u003c/p\u003e\n\u003ch3\u003eConfocal Scanning Laser Microscopy analyses\u003c/h3\u003e\n\u003cp\u003eImmunofluorescence analyses were performed as previously reported (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). Briefly, control and experimental explants were collected after the corresponding treatment, rinsed in PBS for 10 min at room temperature, and fixed with 4% PFA at room temperature for 15 min. After fixation, the samples were rinsed three times (10 min each) in PBS at room temperature and then permeabilized with 0.02% Triton X-100, 50nM Nh\u003csub\u003e4\u003c/sub\u003eCl and PBS for 10 min at room temperature. Non-specific binding sites were blocked with 0.2% Gelatin solution (Sigma-Aldrich) applied two times for 10 minutes. As primary antibodies, an anti-Wt1 (Santa Cruz) and anti-cTnT (Hytest) at 1:200 dilution in blocking solution was applied overnight at 4\u0026deg;C. Ventricle explants were rinsed 3 times in PBS for 10 min and incubated with secondary antibody anti-Goat 488 (Invitrogen) 1:100 dilution, 30 min at room temperature. Finally, ventricle explants were incubated with DAPI 1:2000 (Sigma) for 15 min at room temperature and rinsed two times in PBS for 5 min each. The explants were stored in PBS in darkness at 4\u0026deg;C until analysed using a Leica TCS SP5 II confocal scanning laser microscope.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analyses\u003c/h2\u003e \u003cp\u003eFor statistical analyses of datasets, unpaired Student\u0026rsquo;s t-tests were used. Significance levels or P values are stated in each corresponding figure legend. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCoding and non-coding RNA differential expression in the PE to EE transition in mice\u003c/h2\u003e \u003cp\u003eTo investigate gene expression changes during the transition from PE to EE, we performed RNAseq on manually dissected PE from E9.5 Wt1-GFP heterozygous mouse embryos (n\u0026sim;20) and GFP\u0026thinsp;+\u0026thinsp;FACS-sorted EE cells from E10.5 Wt1-GFP embryonic hearts (n\u0026sim;15). Each condition included three independent biological samples (PE: n\u0026thinsp;=\u0026thinsp;7\u0026ndash;8 per sample; EE: n\u0026thinsp;=\u0026thinsp;3\u0026ndash;5 hearts per sample). RNAseq libraries for microRNAs and mRNA/lncRNAs in these two distinct stages of epicardial development were constructed and sequenced, yielding an average of 5*10\u003csup\u003e6\u003c/sup\u003e reads (5,25*10\u003csup\u003e6\u003c/sup\u003e\u0026plusmn; 1*10\u003csup\u003e6\u003c/sup\u003e) for microRNA libraries and 35*10\u003csup\u003e6\u003c/sup\u003e reads (36,5*10\u003csup\u003e6\u003c/sup\u003e \u0026plusmn; 2,5*10\u003csup\u003e6\u003c/sup\u003e) for mRNA/lncRNA libraries. Alignment efficiency was approximately 85\u0026ndash;90% for mRNAs of the total input resulting in the identification of \u0026sim;12500 genes, while microRNA reads alignment yielded lower inputs, 40\u0026ndash;50% of the total and identified \u0026sim;200 microRNA expressed in both conditions. An exploratory analysis validated the similarity between PE E9.5 vs EE E10.5 RNAseq datasets \u003cb\u003e(Supplementary Fig.\u0026nbsp;3).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn order to identify those genes that might be involved in governing the transition between the PE and EE, we have identified those DE genes, including therein microRNAs, mRNAs and lncRNAs, using as selection criteria those genes displaying a log2 FC\u0026thinsp;\u0026gt;\u0026thinsp;1 and FDR p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. This analysis identified 979 mRNAs up-regulated in the PE as compared to EE (\u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e), whereas 886 display the opposite pattern, down-regulated in the PE as compared to the EE (\u003cb\u003eTable\u0026nbsp;2\u003c/b\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). RT-qPCR validation confirmed the differential expression of these mRNAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D). In this context, it is important to highlight that transcription factors such as \u003cem\u003eHnf4a\u003c/em\u003e, \u003cem\u003eHoxb1\u003c/em\u003e and \u003cem\u003eProx1\u003c/em\u003e are enriched in the PE at E9.5, whereas \u003cem\u003eSpry1\u003c/em\u003e, \u003cem\u003eHey2\u003c/em\u003e and \u003cem\u003eItga1\u003c/em\u003e are enriched in the EE at E10.5.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSimilarly, microRNA analysis identified 59 microRNAs highly expressed in PE as whereas 9 microRNAs were upregulated in EE (\u003cb\u003eTable\u0026nbsp;3\u003c/b\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). RT-qPCR validation confirmed the differential expression of these miRNAs, where in fact, \u003cem\u003emiR-200a-3p, miR-200b-3p, miR-200c-3p, miR-429-3p\u003c/em\u003e and \u003cem\u003emiR-495-3p\u003c/em\u003e displayed higher level of expression in PE, whereas \u003cem\u003elet-7c-5p, miR-24-3p, miR-30a-3p, miR-30c-5p\u003c/em\u003e and \u003cem\u003emiR-351-5p\u003c/em\u003e showed higher expression levels in EE (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-F).\u003c/p\u003e \u003cp\u003eLncRNAs display an averaged lower expression levels as compared to protein-coding RNAs. Even so, our RNAseq analyses identified 60 lncRNAs that are highly expressed in the PE (\u003cb\u003eTable\u0026nbsp;4)\u003c/b\u003e and 111 lncRNAs upregulated in EE (\u003cb\u003eTable\u0026nbsp;5\u003c/b\u003e). Similar to mRNAs and miRNAs, the differential expression of some of these lncRNAs was confirmed by RT-qPCR, i.e. \u003cem\u003eGm35409, Gm35533, 9030622O22Rik and 9030102K24Rik\u003c/em\u003e display high levels in PE and \u003cem\u003eGm13293, Gm42788\u003c/em\u003e and \u003cem\u003e4833415N18Rik\u003c/em\u003e display high levels in EE (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-H). These data demonstrate therefore an important contribution of miRNAs, mRNAs and lncRNAs in the PE to EE transition, yet their functional implications remain to be elucidated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSignalling pathway enrichment displays significant differences in mouse PE and EE differential expressed genes\u003c/h2\u003e \u003cp\u003eTo provide a comprehensive analysis of the biological processes associated with DE genes profile in PE and EE stages, we performed a Gene Set Enrichment Analysis (GSEA). As depicted in \u003cb\u003eSupplementary Fig.\u0026nbsp;4A\u003c/b\u003e, DE genes upregulated in EE display enhanced representation of myofilament and myosin complex, mitochondrial respiratory chain and actomyosin contractile and actin filaments in GSEA GO Cellular Compartment (CC) analyses. In contrast, DE genes downregulated in EE display enhanced representation of membrane and cell-cell contact pathways in GSEA GO CC analyses, suggesting a shift from intercellular communication in PE to muscle function and motility in EE. GSEA GO Molecular Function (MF) analysis further supported this distinction (\u003cb\u003eSupplementary Fig.\u0026nbsp;4B\u003c/b\u003e). DE genes upregulated in EE display enhanced representation of chemokine receptor binding, tropomyosin and structural components of the muscle and muscle alpha-actinin binding, while those DE genes downregulated in EE display enhanced representation of cofactors and calcium ion binding and signalling receptor activity These data reinforce the role of DE genes in EE in muscle function and motility, whereas DE genes in PE remain more engaged with cell-cell signalling. Finally, the pathways revealed by GSEA GO Biological Pathway (BP) further support these findings (\u003cb\u003eSupplementary Fig.\u0026nbsp;4C\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTissue-specific expression patterns of differentially expressed genes in PE and EE further support different signalling pathway enrichment\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the biological relevance of the DE genes between E9.5 PE and E10.5 EE, we analyzed their tissue-specific expression using the Genepaint database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gp3.mpg.de\u003c/span\u003e\u003cspan address=\"https://gp3.mpg.de\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Analyses of the top 10% downregulated DE genes (PE\u0026thinsp;\u0026gt;\u0026thinsp;EE) (\u0026sim;90 genes) revealed that approximately 55% (47/85) of them displayed restricted liver expression at E14.5 days, 9% (8/85) were preferentially expressed in the endocardium and 8% (7/85) display expression in the epicardium. No detectable expression was observed for 23% (20/85) of the DE genes analysed and 11% (10/85) were not found on the Genepaint database (\u003cb\u003eSupplementary Fig.\u0026nbsp;5\u003c/b\u003e). Analyses of the top 10% downregulated DE genes (PE\u0026thinsp;\u0026lt;\u0026thinsp;EE) (\u0026sim;100 genes) revealed that approximately 17% (17/101) were expressed in the epicardium, 12% (12/101) in the endocardium and 6% (6/101) within the myocardium. No detectable expression was observed for 32% (32/101) of the DE genes analyzed and 17% (17/101) were not found on the Genepaint database (\u003cb\u003eSupplementary Fig.\u0026nbsp;5).\u003c/b\u003e Overall, these data demonstrate a distinct bias on the preferential distribution of DE genes in the PE and EE stages. It is important to highlight in this context the large abundance of hepatic specific genes in the E9.5 PE fraction and a relatively low abundance of epicardial restricted genes. On the other hand, it is equally surprising that a small but consistent number of DE genes with enhanced expression in the EE 10.5 are mostly myocardial restricted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMicroRNA-mRNA regulatory networks reveal distinct transcriptional pathways involved in mouse PE to EE transition\u003c/h2\u003e \u003cp\u003ePrevious studies have identified microRNA-mRNA cross-talk correlations by searching for opposite patterns between mRNA and microRNAs in distinct experimental conditions (\u003cspan additionalcitationids=\"CR65\" citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e). Using miRComb software, we have searched for all putative candidate microRNAs that target each of the DE mRNAs with high expression in the PE as compared to the EE as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI. Nine distinct microRNAs with enhanced expression in the EE (\u003cem\u003elet7c-5p, miR-351-5p, miR-30c-5p, miR-780-3p, miR-677-5p, miR-5112, miR-320-3p, miR-483-5p and miR-6236\u003c/em\u003e) display complementary pattern to mRNAs with the opposite pattern (PE\u0026thinsp;\u0026gt;\u0026thinsp;EE). \u003cem\u003eLet7c-5p, miR-351-5p\u003c/em\u003e and \u003cem\u003emiR-30c-5p\u003c/em\u003e (\u003cem\u003elet-7c, miR-351\u003c/em\u003e and \u003cem\u003emiR-30c\u003c/em\u003e, respectively, will be used throughout this text) display a wide range of interactions, supporting a more relevant functional role, as compared to \u003cem\u003emiR-780-3p, miR-677-5p, miR-5112, miR-320-3p, miR-483-5p and miR-6236\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI, \u003cb\u003eSupplementary Fig.\u0026nbsp;6\u003c/b\u003e). On the other hand, 60 distinct microRNAs with enhanced expression in the PE display a complementary pattern to mRNAs with the opposite pattern (PE\u0026thinsp;\u0026lt;\u0026thinsp;EE). \u003cem\u003emiR-495-5p, miR-200b-3p\u003c/em\u003e and \u003cem\u003emiR-181c-5p\u003c/em\u003e (\u003cem\u003emiR-495, miR-200b\u003c/em\u003e and \u003cem\u003emiR-181c\u003c/em\u003e, respectively, will be used throughout this text) are the three microRNAs that display a larger range of interactions, respectively, as compared to the other DE microRNAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI, \u003cb\u003eSupplementary Fig.\u0026nbsp;6\u003c/b\u003e). Overall, these data identify novel microRNA-mRNA predicted interactions that might be functionally important during PE/EE development.\u003c/p\u003e \u003cp\u003eWe have centered our attention on those three DE microRNAs that display a larger number of mRNA interactions in each developmental stage, i.e. \u003cem\u003elet-7c, miR-351\u003c/em\u003e and \u003cem\u003emiR-30c\u003c/em\u003e in PE\u0026thinsp;\u0026lt;\u0026thinsp;EE and \u003cem\u003emiR-495, miR-200b\u003c/em\u003e and \u003cem\u003emiR-181c\u003c/em\u003e in PE\u0026thinsp;\u0026gt;\u0026thinsp;EE. Biological theme comparison of the molecular function of the putative DE-mRNA targets identified by these microRNAs revealed that \u003cem\u003elet-7c\u003c/em\u003e is primarily involved in RNA polymerase/transcription factor DNA binding \u003cb\u003e(Supplementary Fig.\u0026nbsp;7)\u003c/b\u003e while \u003cem\u003emiR-30c\u003c/em\u003e is involved in receptor and cell-cell signalling as well as in ion channel regulation. \u003cem\u003emiR-495\u003c/em\u003e is primarily involved in RNA polymerase, transcription factor DNA binding and GAG binding, while \u003cem\u003emiR-200b\u003c/em\u003e in proteoglycan, GAG, calcium binding and GTPase receptor activity. Moreover, \u003cem\u003emiR-181c\u003c/em\u003e is primarily involved in protein heterodimerization, nuclear receptor, transcription factor and steroid/hormone activity as well as on protein phosphatase and collagen binding. Similar findings are observed in CC and BP biological theme comparisons (data not shown). Importantly, there was a minimal overlap on the predicted targets between these key microRNAs, reinforcing the idea that they modulate different signaling pathways. \u003cem\u003eLet-7c, miR-351\u003c/em\u003e and \u003cem\u003emiR-30c\u003c/em\u003e display only one shared target (\u003cem\u003ePrtg\u003c/em\u003e) (\u003cb\u003eSupplementary Fig.\u0026nbsp;8A\u003c/b\u003e), while \u003cem\u003emiR-495, miR-200b\u003c/em\u003e and \u003cem\u003emiR-181c\u003c/em\u003e display equally uncommon shared targets (\u003cem\u003eNfib, Mbnl2, Kat2b\u003c/em\u003e and \u003cem\u003eNr3c1\u003c/em\u003e). However, an increased number of targets are shared between \u003cem\u003emiR-495\u003c/em\u003e and \u003cem\u003emiR-200b\u003c/em\u003e (12 genes; \u003cem\u003eFn1, Rnd3, Elf2, Rapgef2, Plxna4, Gpm6a, Psd3, Amotl2, Arl4a, Hapln1, Dusp1, Vegfa\u003c/em\u003e) and between \u003cem\u003emiR-495\u003c/em\u003e and \u003cem\u003emiR-181c\u003c/em\u003e (11 genes; \u003cem\u003eCol16a1, Acer3, Sox6, Dmxl2, Dusp6, Sept8, Gpr22, Akap6, Adamts5, Aqp4, Mcc\u003c/em\u003e), suggesting common functional roles in signalling pathways (\u003cb\u003eSupplementary Fig.\u0026nbsp;8B\u003c/b\u003e). Additionally, it worth mentioning that several shared DE mRNAs target of PE\u0026thinsp;\u0026gt;\u0026thinsp;EE miRNAs have a role modulating cell migration in other different biological context, i.e. Nfib (\u003cspan additionalcitationids=\"CR68\" citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e), Mbnl2 (\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e), Nr3c1 (\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e), Fn1 (\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e) and Rnd3 (\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e), a key process during PE/EE development.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDifferentially expressed microRNA-mRNA predicted interactions are distinctly validated in EPIC and MEC epicardial cells\u003c/h2\u003e \u003cp\u003eTo dissect the regulatory mechanisms driven by DE-microRNAs, we performed gain-of-function assays of different microRNAs in two distinct epicardial cell lines, MEC1 and EPIC, respectively. MEC1 cells retain the morphology of early primary epithelial epicardial cells and express epicardium-specific markers including epicardin (\u003cem\u003eTcf21\u003c/em\u003e), \u003cem\u003eTbx18\u003c/em\u003e and \u003cem\u003eKrt18\u003c/em\u003e, while EPIC cells continuously proliferate and expand, acquiring a characteristic mesenchymal phenotype and expressing mesenchymal markers such as \u003cem\u003eSox9\u003c/em\u003e (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e). Overexpression of PE-enriched microRNAs \u003cem\u003emiR-181c, miR-200b\u003c/em\u003e and \u003cem\u003emiR-495\u003c/em\u003e in EPIC cells resulted in significant downregulation of predicted targets, including \u003cem\u003emiR-181c\u003c/em\u003e reduced \u003cem\u003eNr3c1\u003c/em\u003e, \u003cem\u003emiR-200b\u003c/em\u003e suppressed \u003cem\u003eRnd3 and Fn1\u003c/em\u003e, while \u003cem\u003emiR-495\u003c/em\u003e decreased \u003cem\u003eMbln2\u003c/em\u003e and \u003cem\u003eNfib\u003c/em\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e).\u003c/b\u003e In MEC1 cells, a similar pattern was observed for \u003cem\u003eMbln2\u003c/em\u003e downregulated by \u003cem\u003emiR-495\u003c/em\u003e and \u003cem\u003eFn1\u003c/em\u003e by \u003cem\u003emiR-200b\u003c/em\u003e, but \u003cem\u003eNr3c1, Nfib\u003c/em\u003e or \u003cem\u003eRnd3\u003c/em\u003e were not significantly affected \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. The combinatorial effect of these three microRNAs (preMix) led to a decreased expression of \u003cem\u003eRnd3\u003c/em\u003e and \u003cem\u003eHapln1\u003c/em\u003e in both epicardial cell lines, while \u003cem\u003eNr3c1\u003c/em\u003e and \u003cem\u003eNfib\u003c/em\u003e were downregulated exclusively in EPIC \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA,C\u003cb\u003e)\u003c/b\u003e. To uncover if the functional role of microRNAs also occurs in other cardiovascular cell types, RT-qPCR analyses were also performed after DE microRNAs gain-of-function experiments in HL1 cardiomyocytes and MEVEC endocardial cells \u003cb\u003e(Supplementary Fig.\u0026nbsp;9A,B)\u003c/b\u003e. Interestingly, our data demonstrated that neither \u003cem\u003eMbln2\u003c/em\u003e nor \u003cem\u003eNfib\u003c/em\u003e were downregulated in HL1 cardiomyocytes or MEVEC endocardial cells, while \u003cem\u003eFn1\u003c/em\u003e was impaired by \u003cem\u003eall the three microRNAs\u003c/em\u003e, and \u003cem\u003eNr3c1\u003c/em\u003e and \u003cem\u003eRnd3\u003c/em\u003e were downregulated in HL1 cardiomyocytes. However, the preMix had a greater impact on target regulation in HL1 cardiomyocytes and MEVEC endocardial cells \u003cb\u003e(Supplementary Fig.\u0026nbsp;9A,B)\u003c/b\u003e. In conclusion, it is worth highlighting that EPIC epicardial cells recapitulate most of the microRNA-mRNA target predicted interactions after miRNAs gain-of-function, except for \u003cem\u003eHapln1\u003c/em\u003e, supporting the notion that miRNAs PE\u0026thinsp;\u0026gt;\u0026thinsp;EE molecular regulation is cell type-specific.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the same regard, loss-of-function assays were executed for EE-enriched microRNAs, \u003cem\u003elet-7c, miR-30c\u003c/em\u003e and \u003cem\u003emiR-351\u003c/em\u003e, in EPIC and MEC1 epicardial cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB,D\u003cb\u003e)\u003c/b\u003e. Our RT-qPCR analysis led us to identify that in the EPIC cell line \u003cem\u003emiR-351\u003c/em\u003e inhibition modulated the up-regulation of \u003cem\u003ePrtg\u003c/em\u003e, whereas \u003cem\u003eNr6a1\u003c/em\u003e was up-regulated after the inhibition of \u003cem\u003emiR-30c\u003c/em\u003e and \u003cem\u003eHic2\u003c/em\u003e after \u003cem\u003elet7c\u003c/em\u003e or \u003cem\u003emiR-351\u003c/em\u003e inhibition, respectively. On the other hand, neither \u003cem\u003ePeg10\u003c/em\u003e, \u003cem\u003eFbxo32, Trim71\u003c/em\u003e nor \u003cem\u003eCcnjl\u003c/em\u003e expression were boosted after the loss-of-function of these microRNAs in EPIC cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. In the MEC1 cell line is relevant to point out that \u003cem\u003eFbxo32\u003c/em\u003e and \u003cem\u003eTrim71\u003c/em\u003e were significantly up-regulated after \u003cem\u003elet-7c, miR-30c\u003c/em\u003e and \u003cem\u003emiR-351\u003c/em\u003e inhibition in MEC1 cell line \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e, while \u003cem\u003ePeg10\u003c/em\u003e was up-regulated after \u003cem\u003emiR-30c\u003c/em\u003e and \u003cem\u003emiR-351\u003c/em\u003e inhibition. Additionally, \u003cem\u003ePrtg\u003c/em\u003e and \u003cem\u003eCcnjl\u003c/em\u003e were up-regulated after \u003cem\u003emiR-30c\u003c/em\u003e and \u003cem\u003elet-7c\u003c/em\u003e inhibition, respectively, in MEC1 cells. Curiously, \u003cem\u003eNr6a1\u003c/em\u003e and \u003cem\u003eHic2\u003c/em\u003e expression was not increased, in some cases (\u003cem\u003emiR-351\u003c/em\u003e) even significantly decreased, after these microRNA inhibition \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. In this case is noteworthy that the combinatorial effect of these three microRNAs (antiMix) does not have any relevant effect on the up-regulation of the mentioned target genes except for \u003cem\u003eTrim71\u003c/em\u003e in MEC1 and \u003cem\u003ePeg10\u003c/em\u003e in EPIC cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB,D\u003cb\u003e)\u003c/b\u003e. To summarize, our data demonstrate that MEC1 cells recapitulate most of the microRNA-mRNA target predicted interaction after miRNAs loss-of-function as compared to EPIC cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDifferentially expressed microRNA-mRNA predicted interactions are not recapitulated in\u003c/b\u003e \u003cb\u003eex vivo\u003c/b\u003e \u003cb\u003eE10.5 epicardial outgrowths\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo further dissect the regulatory mechanisms driven by DE-microRNAs, we performed gain- and loss-of-function assays of the PE\u0026thinsp;\u0026gt;\u0026thinsp;EE and PE\u0026thinsp;\u0026lt;\u0026thinsp;EE microRNAs, in mouse E10.5 epicardial explants. After 24 hours of culture, Wt1\u003csup\u003e+\u003c/sup\u003e epicardial cells (EEx) were transfected with microRNAs and collected after ventricular tissue removal \u003cb\u003e(Supplementary Fig.\u0026nbsp;9C)\u003c/b\u003e. Gain-of-function experiments with PE\u0026thinsp;\u0026gt;\u0026thinsp;EE microRNAs \u003cem\u003emiR-181c, miR-200b, miR-495\u003c/em\u003e demonstrated that epicardial expression of \u003cem\u003eMbln2\u003c/em\u003e and \u003cem\u003eHapln1\u003c/em\u003e was significantly decreased in all individual conditions, while \u003cem\u003eNfib\u003c/em\u003e and \u003cem\u003eFn1\u003c/em\u003e were negatively regulated after \u003cem\u003emiR-181c\u003c/em\u003e and \u003cem\u003emiR-495\u003c/em\u003e administration. Additionally, \u003cem\u003eRnd3\u003c/em\u003e was down-regulated by \u003cem\u003emiR-181c\u003c/em\u003e and \u003cem\u003emiR-200b\u003c/em\u003e, whereas \u003cem\u003eNr3c1\u003c/em\u003e down-regulation was specific to \u003cem\u003emiR-200b.\u003c/em\u003e Finally, \u003cem\u003eMbln2\u003c/em\u003e, \u003cem\u003eHapln1\u003c/em\u003e and \u003cem\u003eFn1\u003c/em\u003e expression was significantly decreased after premix treatment \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e. Comparing these findings with the previous \u003cem\u003ein vitro\u003c/em\u003e experimental assays, EPIC cells recapitulated key interaction seen in EEx, such \u003cem\u003emiR-181c\u003c/em\u003e-mediated \u003cem\u003eNfib\u003c/em\u003e suppression and \u003cem\u003emiR-200b\u003c/em\u003e-driven regulation of \u003cem\u003eNr3c1\u003c/em\u003e and \u003cem\u003eRnd3\u003c/em\u003e. Finally \u003cem\u003emiR-495\u003c/em\u003e controls \u003cem\u003eMbln2, Nfib\u003c/em\u003e and \u003cem\u003eFn1\u003c/em\u003e. However, discrepancies were observed, including \u003cem\u003emiR-181c\u003c/em\u003e downregulating nearly all the predicted target genes except for \u003cem\u003eNr3c1 ex vivo\u003c/em\u003e but not \u003cem\u003ein vitro\u003c/em\u003e. Such discrepancies might be related to the myocardium-epicardium interaction \u003cem\u003eex vivo\u003c/em\u003e and/or the differential cell behaviour of \u003cem\u003ein vitro\u003c/em\u003e epicardial cell lines vs E10.5 \u003cem\u003eex vivo\u003c/em\u003e epicardial explants.\u003c/p\u003e \u003cp\u003eSimilarly, loss-of-function assays of the three PE\u0026thinsp;\u0026lt;\u0026thinsp;EE microRNAs, \u003cem\u003elet-7c, miR-30c\u003c/em\u003e and \u003cem\u003emiR-351\u003c/em\u003e in EEx revealed that \u003cem\u003eTrim71\u003c/em\u003e expression was up-regulated after \u003cem\u003elet-7c\u003c/em\u003e and \u003cem\u003emiR-30c\u003c/em\u003e inhibition, in line with the observations in MEC1. However, \u003cem\u003ePrtg\u003c/em\u003e and \u003cem\u003eNr6a1\u003c/em\u003e were down-regulated in all individual experimental conditions. The combinatorial effect of antiMix leads exclusively to \u003cem\u003eNr6a1\u003c/em\u003e up-regulation \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eThese results demonstrate intriguing discrepancies in the regulatory effects of DE microRNAs in both MEC1/EPIC cells and EEx from E10.5 explants, being needed to underscore the importance of considering cell-specific contexts in understanding the functional implications of the microRNAs in embryonic epicardial cells \u003cem\u003eex vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMiR-495, let-7c\u003c/b\u003e, \u003cb\u003eand\u003c/b\u003e \u003cb\u003emiR-351\u003c/b\u003e \u003cb\u003eregulate epicardial cell migration\u003c/b\u003e\u003c/p\u003e \u003cp\u003eEpicardial cell migration is fundamental for heart development, as PE cells move by direct contact or through cell aggregates to cover the myocardium. This process begins at the atrioventricular canal region and, expands to form a continuous epithelial layer that eventually covers the entire heart. As the epicardium matures, EMT facilitates EPDC proliferation, migration, and differentiation, supporting coronary vasculature and ventricular growth. To assess the impact of microRNAs on this process, we performed gain- or loss-of-function assays in E10.5 ventricular explants and analyzed three different aspects. \u003cem\u003eTotal cell migration\u003c/em\u003e, representing all cellular migration from the explant ventricular border to the outermost individual cell of the culture; \u003cem\u003eCohesive cell migration\u003c/em\u003e, when only considering those collective migrating cells from the explant ventricular border; and finally, \u003cem\u003enon-cohesive cell migration\u003c/em\u003e, representing only those individual migrating cells from the outermost periphery of the cohesive migration \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Our data demonstrated that total cell migration was impaired after \u003cem\u003emiR-495\u003c/em\u003e overexpression and enhanced after preMix treatment, while no significant outgrowth differences were noted for \u003cem\u003emiR-181c\u003c/em\u003e and \u003cem\u003emiR-200b\u003c/em\u003e experimental conditions. Cohesive migration was repressed after \u003cem\u003emiR-495\u003c/em\u003e and preMix treatment while non-cohesive migration was enhanced after \u003cem\u003emiR-200b\u003c/em\u003e and \u003cem\u003emiR-495\u003c/em\u003e gain-of-function, as well as, preMix treatment \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D\u003cb\u003e)\u003c/b\u003e. In this regard, we observed that epicardial cells within the non-cohesive outgrowth exhibit a reduced size compared to controls following miR-495 gain-of-function, with a concurrent increase in Myh9 protein expression in MEC1 epicardial cells \u003cem\u003ein vitro\u003c/em\u003e, whereas no differences are observed in F-actin polymerization \u003cb\u003e(Supplementary Fig.\u0026nbsp;10A-B)\u003c/b\u003e. Thus, these data demonstrate that differential expression of distinct microRNAs can selectively modulate total, cohesive and non-cohesive epicardial cell migration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs previously mentioned, our RT-qPCR analysis unveiled a specific explant epicardial cells regulatory miRNA-mRNA crosstalk \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE\u003cb\u003e).\u003c/b\u003e To further elucidate the role of the PE\u0026thinsp;\u0026lt;\u0026thinsp;EE DE-mRNAs in epicardial cell migration, E10.5 cardiac explants were transfected with \u003cem\u003eMbln2\u003c/em\u003e, \u003cem\u003eNfib\u003c/em\u003e, \u003cem\u003eNr3c1\u003c/em\u003e, \u003cem\u003eRnd3\u003c/em\u003e and \u003cem\u003eFn1\u003c/em\u003e siRNAs. Our epicardial cell migration analysis evidenced that total cell migration was enhanced after \u003cem\u003eNfib, Nr3c1\u003c/em\u003e and \u003cem\u003eFn1\u003c/em\u003e inhibition and decreased after \u003cem\u003esiRnd3\u003c/em\u003e treatment, while no significant outgrowth differences were noted for \u003cem\u003esiMbln2\u003c/em\u003e. Cohesive migration was promoted after loss-of-function of all mRNAs, except for \u003cem\u003eNfib\u003c/em\u003e, while non-cohesive migration was enhanced only by \u003cem\u003eNfib\u003c/em\u003e loss-of-function, and decreased after \u003cem\u003eRnd3\u003c/em\u003e and \u003cem\u003eMbln2\u003c/em\u003e inhibition, whereas \u003cem\u003eNr3c1\u003c/em\u003e and \u003cem\u003eFn1\u003c/em\u003e did not exerted any effect on non-cohesive cell migration \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-H\u003cb\u003e)\u003c/b\u003e. Thus, our data demonstrate that DE-mRNAs in PE \u003cem\u003evs.\u003c/em\u003e EE can selectively modulate total, cohesive and non-cohesive epicardial cell migration.\u003c/p\u003e \u003cp\u003eA similar analysis was performed for those PE\u0026thinsp;\u0026lt;\u0026thinsp;EE DE-microRNAs, where cell migration was assessed through loss-of-function experiments targeting individual microRNAs \u003cem\u003elet-7c\u003c/em\u003e, \u003cem\u003emiR-30c\u003c/em\u003e, \u003cem\u003emiR-351\u003c/em\u003e and antiMix. Our results demonstrated that total cell migration was decreased after \u003cem\u003eantilet-7c, antimiR-351\u003c/em\u003e and antiMix treatment, while no significant outgrowth differences were observed after \u003cem\u003eantimiR-30c\u003c/em\u003e administration. Cohesive migration was repressed only after antiMix treatment without significant effect mediated by individual conditions, and finally, non-cohesive migration was impaired by \u003cem\u003emiR-30c\u003c/em\u003e loss-of-function, as well as, antiMix treatment \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI-L\u003cb\u003e)\u003c/b\u003e. These results align with our findings that epicardial cells in the non-cohesive outgrowth do not exhibit a reduced area compared to controls following \u003cem\u003elet-7c\u003c/em\u003e loss-of-function, and no significant difference in Myh9 protein expression is observed in MEC1 epicardial cells \u003cem\u003ein vitro\u003c/em\u003e, although F-actin polymerization is impaired \u003cb\u003e(Supplementary Fig.\u0026nbsp;10A-C)\u003c/b\u003e. Therefore, these data further underscore the functional role of DE-microRNAs in epicardial cell migration.\u003c/p\u003e \u003cp\u003eImportantly, it should be highlighted that \u003cem\u003emiR-495, let-7c\u003c/em\u003e and \u003cem\u003emiR-351\u003c/em\u003e exert significant effects on epicardial cell migration, underscoring the importance of their precise regulation for proper epicardium formation and EPDCs migration.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMiR-181c, miR-200b\u003c/b\u003e, \u003cb\u003eand\u003c/b\u003e \u003cb\u003elet-7c\u003c/b\u003e \u003cb\u003eadministration promotes cardiomyogenic cell specification\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003ebut not\u003c/b\u003e \u003cb\u003eex vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAs outlined previously, after the embryonic epicardium covers the naked myocardium, it subsequently undergoes EMT leading to EPDCs that thereafter differentiate into distinct cell types such as cardiac fibroblasts, vascular smooth muscle cells, pericytes, endothelial and fat cells and possibly, also into cardiomyocytes. To dissect the role of DE microRNAs during cell lineage specification, epicardial cells (EPIC and MEC1) were overexpressed with \u003cem\u003emiR-181c, miR-200b, miR-495\u003c/em\u003e and preMix, respectively \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA,C, \u003cb\u003eand Supplementary Fig.\u0026nbsp;11)\u003c/b\u003e. Linage specific markers of epicardium (\u003cem\u003eWt1, Tcf21\u003c/em\u003e and \u003cem\u003eTbx18\u003c/em\u003e), myocardium (\u003cem\u003eGata4, Nkx2.5, Srf, Tnnt2\u003c/em\u003e and \u003cem\u003eMyh6\u003c/em\u003e) and endocardium (\u003cem\u003ePecam1, Tie2\u003c/em\u003e and \u003cem\u003ePostn\u003c/em\u003e as an endocardial derivative marker) as well as markers for EMT (\u003cem\u003eCdh5, Snail1, Snail\u003c/em\u003e and \u003cem\u003ePrrx1\u003c/em\u003e), fibrogenesis (\u003cem\u003eCol1a1, Col3a1, Fn1\u003c/em\u003e and \u003cem\u003eSox9\u003c/em\u003e) and angio-vasculogenesis (\u003cem\u003eAng1, Ang2, Efnb2\u003c/em\u003e and \u003cem\u003eFlt1\u003c/em\u003e) were analysed by RT-qPCR. \u003cem\u003emiR-181c\u003c/em\u003e gain-of-function did not evidence enhancement of early myocardial markers (i.e. \u003cem\u003eGata4, Nkx2.5\u003c/em\u003e), except for \u003cem\u003eSrf\u003c/em\u003e in MEC1, but upregulated the cardiomyocyte terminal differentiation marker \u003cem\u003eTnnt2\u003c/em\u003e, in both epicardial cell lines. Moreover, endocardial and epicardial lineage markers were slightly increased such as \u003cem\u003eTie2\u003c/em\u003e, and \u003cem\u003ePostn\u003c/em\u003e in epicardial MEC1 and EPIC cells and \u003cem\u003eTcf21\u003c/em\u003e only in epicardial MEC1 cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. In the same line, \u003cem\u003emiR-495\u003c/em\u003e overexpression promoted the expression of endocardial lineage specification markers as revealed by upregulation of \u003cem\u003eTie2\u003c/em\u003e in both epicardial cell lines and \u003cem\u003ePostn\u003c/em\u003e in MEC1 cells. Additionally, miR-495 promotes the expression of the epicardial marker \u003cem\u003eTcf21\u003c/em\u003e in MEC1 as well as the cardiomyocyte terminal differentiation marker \u003cem\u003eTnnt2\u003c/em\u003e in EPIC. Finally, \u003cem\u003emiR-200b\u003c/em\u003e did not exert any modulation on endocardial, myocardial and epicardial markers in EPIC cells, nonetheless the overexpression of \u003cem\u003emiR-200b\u003c/em\u003e in MEC1 leads to an increment of myocardial markers such as \u003cem\u003eSrf, Myh6\u003c/em\u003e and \u003cem\u003eTnnt2\u003c/em\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. When the three microRNAs are overexpressed together, it can be observed an increment of myocardial markers, for instance, early cardiogenic transcription factor as \u003cem\u003eNkx2.5\u003c/em\u003e is upregulated in both epicardial cells similarly as \u003cem\u003eSrf\u003c/em\u003e, whereas \u003cem\u003eGata4\u003c/em\u003e is increased only in EPIC. Finally, epicardial markers as \u003cem\u003eTcf21\u003c/em\u003e and \u003cem\u003eWt1\u003c/em\u003e are upregulated in MEC1 and EPIC epicardial cells, respectively \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Therefore, these data demonstrate that DE microRNAs in PE\u0026thinsp;\u0026gt;\u0026thinsp;EE can distinctly modulate epicardial-derived lineage specification.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGiven the role of the epicardium during cardiogenesis, in order to have further insight of the molecular mechanisms driven by the DE microRNAs, we analysed by RT-qPCR, the expression levels of different molecular markers related with EMT, fibrogenesis and angiogenesis after gain-of-function experiments. For EMT markers all three DE-microRNAs exerted a marked repression of \u003cem\u003eCdh5, Snail1, Snail2\u003c/em\u003e, and \u003cem\u003ePrrx1\u003c/em\u003e in MEC1, except for \u003cem\u003emiR-200b\u003c/em\u003e which only promoted \u003cem\u003eCdh5\u003c/em\u003e gene expression. However, in EPIC cells, the gain-of-function of the \u003cem\u003emiR-181c\u003c/em\u003e promoted the expression of \u003cem\u003eSnail1\u003c/em\u003e, and \u003cem\u003emiR-495\u003c/em\u003e induced \u003cem\u003eSnail1\u003c/em\u003e and \u003cem\u003eCdh5\u003c/em\u003e expression, while \u003cem\u003emiR-200b\u003c/em\u003e did not exert any significant effect over EMT markers \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. For angio-vasculogenesis markers analysis, no significant effect was observed after microRNA gain-of-function except for angiopoietins, i.e. \u003cem\u003eAng1\u003c/em\u003e in MEC1, mediated by \u003cem\u003emiR-495\u003c/em\u003e and Ang2 in EPIC, regulated by \u003cem\u003emiR-181c\u003c/em\u003e and \u003cem\u003emiR-495\u003c/em\u003e. Notwithstanding, \u003cem\u003emiR-181c\u003c/em\u003e and \u003cem\u003emiR-495\u003c/em\u003e overexpression slightly promoted the expression of fibrogenic markers such as \u003cem\u003eCol1a1\u003c/em\u003e and \u003cem\u003eCol3a1\u003c/em\u003e in EPIC and MEC1 epicardial cell lines, respectively \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. Finally, EMT, angio-vasculogenesis and fibrotic molecular markers were upregulated by combinatorial miRNA overexpression, i.e.: \u003cem\u003eAng2, Efnb2, Snail2, Fn1\u003c/em\u003e and \u003cem\u003eSox9\u003c/em\u003e in EPIC epicardial cells, and \u003cem\u003eAng1, Ang2, Cdh5, Col1a1, Col3a1\u003c/em\u003e and \u003cem\u003eFn1\u003c/em\u003e in MEC1 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. In sum, these results support the notion that \u003cem\u003emiR-181c\u003c/em\u003e and \u003cem\u003emiR-495\u003c/em\u003e, but not \u003cem\u003emiR-200b\u003c/em\u003e, could modulate cell lineage specification promoting epicardial, endocardial and fibrogenic markers, whereas \u003cem\u003emiR-181c\u003c/em\u003e and \u003cem\u003emiR-200b\u003c/em\u003e promote myocardial markers expression in MEC1 epicardial cells \u003cb\u003e(Supplementary Fig.\u0026nbsp;11)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eSimilar to DE-miRNAs in PE\u0026thinsp;\u0026gt;\u0026thinsp;EE, \u003cem\u003emiR-let7c, miR-30c\u003c/em\u003e, and \u003cem\u003emiR-351\u003c/em\u003e loss-of-function experiments were performed in epicardial EPIC and MEC1 cells. Specific cardiac cell lineage and biological processes markers were studied \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB,D, \u003cb\u003eSupplementary Fig.\u0026nbsp;12)\u003c/b\u003e. In this analysis, we observed that \u003cem\u003eanti-let7c\u003c/em\u003e consistently promoted upregulation of early cardiogenic lineage markers in both epicardial cell lines, such as \u003cem\u003eGata4\u003c/em\u003e and \u003cem\u003eSrf\u003c/em\u003e, whereas \u003cem\u003eNkx2.5\u003c/em\u003e and terminal differentiation markers such as \u003cem\u003eMyh6\u003c/em\u003e and \u003cem\u003eTnnt2\u003c/em\u003e were only upregulated in MEC1 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. In addition, the epicardial cell lineage markers are slightly promoted in EPIC cells through increment of \u003cem\u003eTbx18\u003c/em\u003e and \u003cem\u003eWt1\u003c/em\u003e expression as well as \u003cem\u003eTcf21\u003c/em\u003e in MEC1. Finally, endocardial markers revealed that loss-of-function of \u003cem\u003elet-7c\u003c/em\u003e induced endocardial cell specification in EPIC cell line endorsed by \u003cem\u003ePost1\u003c/em\u003e and \u003cem\u003eTie2\u003c/em\u003e upregulation.\u003c/p\u003e \u003cp\u003eIn the same scenario of loss-of-function, \u003cem\u003emiR-30c\u003c/em\u003e and \u003cem\u003emiR-351\u003c/em\u003e modulated myocardial and epicardial markers in EPIC leading to the upregulation of \u003cem\u003eGata4, Nkx2.5\u003c/em\u003e and \u003cem\u003eTnnt2\u003c/em\u003e, as well as \u003cem\u003eTbx18\u003c/em\u003e, however, the modulation of these miRNAs in MEC1 cells did not have any impact on cardiogenic markers except for \u003cem\u003eSrf\u003c/em\u003e expression. Finally, we evidenced that \u003cem\u003emiR-351\u003c/em\u003e inhibition has an impact on the promotion of endocardial linage specification markers in both epicardial cells endorsed by \u003cem\u003ePecam1, Postn\u003c/em\u003e and \u003cem\u003eTie2\u003c/em\u003e expression, and the loss-of-function of \u003cem\u003emiR-30c\u003c/em\u003e modulates \u003cem\u003ePecam1\u003c/em\u003e and \u003cem\u003ePostn\u003c/em\u003e in MEC1 epicardial cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo get further insights into the functional role of the DE-miRNAs PE\u0026thinsp;\u0026lt;\u0026thinsp;EE other biological processes such as, EMT, fibrogenesis and angio-vasculogenesis representative markers were analysed in a loss-of-function model for \u003cem\u003elet-7c, miR-30c\u003c/em\u003e and \u003cem\u003emiR-351\u003c/em\u003e, respectively. Our RT-qPCR data demonstrated that all of them were significantly downregulated in MEC1 epicardial cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. However, loss-of-function of DE-miRNAs, \u003cem\u003elet-7c\u003c/em\u003e and \u003cem\u003emiR-351\u003c/em\u003e in EPIC cells, promoted the expression fibrogenic markers such as \u003cem\u003eCol1a1, Col3a1\u003c/em\u003e and \u003cem\u003eFn1\u003c/em\u003e. Moreover, \u003cem\u003eantimiR-351\u003c/em\u003e but no \u003cem\u003eanti-let7c\u003c/em\u003e nor \u003cem\u003eantimiR-30c\u003c/em\u003e promoted the expression of angio-vasculogenic markers as \u003cem\u003eFlt-1\u003c/em\u003e. Finally, the antiMix treatment in both epicardial cells modulate EMT markers by the upregulation of \u003cem\u003eSnail1\u003c/em\u003e in MEC1 and \u003cem\u003eSnail2\u003c/em\u003e in MEC1 and EPIC cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. In sum, these results support the notion that \u003cem\u003elet-7c\u003c/em\u003e can modulate cell lineage specification promoting myocardial markers while \u003cem\u003emiR-351\u003c/em\u003e promotes endocardial lineage specification in epicardial cells. Moreover, these two miRNAs have a marked effect on the modulation of cardiac fibrogenic markers in EPIC epicardial cells \u003cb\u003e(Supplementary Fig.\u0026nbsp;12)\u003c/b\u003e. Furthermore, \u003cem\u003elet-7c\u003c/em\u003e loss-of-function but no \u003cem\u003emiR-181c\u003c/em\u003e and \u003cem\u003emiR-200b\u003c/em\u003e gain-of-function, increases Tnnt2 protein expression levels as observed in MEC1 epicardial cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eSince we have previously demonstrated the dynamic modulation of the lineage specification markers in epicardial cell lines by these microRNAs, we sought to investigate if the expression of epicardial, myocardial and/or endocardial markers were induced \u003cem\u003eex vivo\u003c/em\u003e in EEx. For this purpose, gain- and loss-of function of \u003cem\u003emiR-181c\u003c/em\u003e, \u003cem\u003emiR-200b\u003c/em\u003e and \u003cem\u003elet-7c\u003c/em\u003e were performed in mouse E10.5 epicardial explants. RT-qPCR analysis evidenced that overexpression of \u003cem\u003emiR-181c\u003c/em\u003e did not promote myocardial, endocardial or epicardial markers \u003cem\u003eex vivo\u003c/em\u003e. Moreover, \u003cem\u003emiR-200b\u003c/em\u003e overexpression repressed \u003cem\u003eTnnt2\u003c/em\u003e and \u003cem\u003eMhy6\u003c/em\u003e expression in mouse EEx from E10.5 epicardial explants and similar effects were observed after \u003cem\u003elet-7c\u003c/em\u003e inhibition \u003cem\u003eex vivo\u003c/em\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e. Thus, these data suggest that the function of these three microRNAs in the embryonic epicardial cell specification is limited, underscoring the potential influence of other molecular factors as well as the neighbouring myocardial and endocardial tissues.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eDifferentially expressed microRNAs cross-talk modulates epicardial cell specification\u003c/h2\u003e \u003cp\u003eDespite our comprehensive understanding of the fundamental principles underlying miRNA biogenesis and function, novel and unexpected aspects within these processes underscore the complexity of miRNA regulation. To get further insight of the functional regulation of the DE microRNAs, we conducted a RT-qPCR analysis of their mutual molecular regulation. In this regard, we analysed the expression levels of each microRNA following individual microRNA gain- or loss-of-function in MEC1 and EPIC epicardial cells as well as in embryonic epicardial cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-F\u003cb\u003e)\u003c/b\u003e. As expected, our results evidenced that \u003cem\u003emiR-181c, miR-200b\u003c/em\u003e and \u003cem\u003emiR-495\u003c/em\u003e administration were upregulated following miRNA gain-of-function in all epicardial cells, respectively \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA,B,E\u003cb\u003e)\u003c/b\u003e. Moreover, overexpression of \u003cem\u003emiR-181c\u003c/em\u003e enhanced \u003cem\u003emiR-200b\u003c/em\u003e and \u003cem\u003emiR-495\u003c/em\u003e expression and similarly, miR-495 gain-of-function promoted \u003cem\u003emiR-181c\u003c/em\u003e and \u003cem\u003emiR-200b\u003c/em\u003e expression in epicardial cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA,B,E\u003cb\u003e)\u003c/b\u003e. Notably, overexpression of \u003cem\u003emiR-200b\u003c/em\u003e led to a decreased expression of \u003cem\u003emiR-181c\u003c/em\u003e and miR-495 in MEC1 but not in EPIC or EEx \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA,B,E\u003cb\u003e)\u003c/b\u003e. Similarly, analyses of loss-of-function assays also demonstrated a downregulation of \u003cem\u003elet-7c\u003c/em\u003e, \u003cem\u003emiR-30c\u003c/em\u003e and \u003cem\u003emiR-351\u003c/em\u003e in all epicardial cells, as expected \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC,D,F\u003cb\u003e)\u003c/b\u003e. However, loss-of-function experiments targeting \u003cem\u003elet-7c\u003c/em\u003e resulted in elevated expression of \u003cem\u003emiR-30c\u003c/em\u003e in MEC1 but not in EPIC epicardial cells, alongside increased \u003cem\u003emiR-351\u003c/em\u003e expression in both cell types, a trend also observed in EEx \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC,D,F\u003cb\u003e)\u003c/b\u003e. Comparable results were noted upon \u003cem\u003emiR-30c\u003c/em\u003e inhibition, with upregulation observed in \u003cem\u003elet-7c\u003c/em\u003e in MEC1, EPIC, and EEx, and \u003cem\u003emiR-351\u003c/em\u003e only in MEC1 and EEx. Finally, after \u003cem\u003eantimiR-351\u003c/em\u003e treatment, \u003cem\u003elet-7c\u003c/em\u003e expression in MEC1 and \u003cem\u003emiR-30c\u003c/em\u003e expression in MEC1 and EEx were elevated \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC,D,F\u003cb\u003e)\u003c/b\u003e. Therefore, these data demonstrate a microRNA cross-talk regulation between differentially expressed microRNAs, a process that is also cell-type specific. To further analyse microRNA cross-talk, we performed \u003cem\u003ein vitro\u003c/em\u003e experiments blocking transcription by using α-amanitin and we found that \u003cem\u003emiR-30c\u003c/em\u003e and \u003cem\u003emiR-351\u003c/em\u003e were upregulated when \u003cem\u003elet-7c\u003c/em\u003e is inhibited, indicating that this microRNA acts by enhancing these two microRNAs at post-transcriptional levels (data not shown).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSince we have previously observed that the inhibition of \u003cem\u003elet-7c\u003c/em\u003e displays an increment of \u003cem\u003emiR-30c\u003c/em\u003e and \u003cem\u003emiR-351\u003c/em\u003e expression in MEC1 and EEx, we sought to investigate the molecular implications of this regulatory feedback in cardiomyogenic cell lineage markers. \u003cem\u003emiR-30c\u003c/em\u003e and \u003cem\u003emiR-351\u003c/em\u003e gain-of-function experiments were conducted in MEC1 and EEx \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG,H\u003cb\u003e)\u003c/b\u003e. Our results evidenced that \u003cem\u003eTnnt2\u003c/em\u003e expression levels are not increased following modulation of \u003cem\u003emiR-30c\u003c/em\u003e nor \u003cem\u003emiR-351\u003c/em\u003e in both MEC1 and EEx, whereas \u003cem\u003eMyh6\u003c/em\u003e expression levels were upregulated specifically after \u003cem\u003emiR-351\u003c/em\u003e overexpression in MEC1 epicardial cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG,H\u003cb\u003e).\u003c/b\u003e Thus, the cardiogenic role exerted by the inhibition of \u003cem\u003elet-7c\u003c/em\u003e is not mediated by the modulation of these microRNAs, as only \u003cem\u003emiR-351\u003c/em\u003e modulates \u003cem\u003eMyh6\u003c/em\u003e expression.\u003c/p\u003e \u003cp\u003eOverall, our comprehensive analysis demonstrates that gain- or loss-of-function of one microRNA had a notable effect on the expression of other microRNAs, evidencing the intricate interplay between DE-microRNAs in regulating epicardial cell behaviour and highlighting their potential functional role in cardiac development.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFoxf1\u003c/b\u003e \u003cb\u003emodulates epicardial cell specification into myocardial and endothelial lineages via\u003c/b\u003e \u003cb\u003elet7c\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003emiR-30c\u003c/b\u003e \u003cb\u003eregulation\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAs we have previously mentioned, several mRNAs were significantly differentially expressed in PE (E9.5) and EE (E10.5). Among them, we have observed that there are 47 transcription factors (~\u0026thinsp;4.6% of total DE mRNAs) with enhanced expression in PE, i.e, \u003cem\u003eTbx5, Sox18, Prox1\u003c/em\u003e, \u003cem\u003eFoxf1\u003c/em\u003e \u003cb\u003e(Table\u0026nbsp;1)\u003c/b\u003e, whereas 22 (~\u0026thinsp;2.4%) display the opposite pattern, down-regulated in the PE as compared to the EE, e.g. \u003cem\u003eTbx18, Sox9, Lhx9, Foxc1\u003c/em\u003e (\u003cb\u003eTable\u0026nbsp;2)\u003c/b\u003e. FOX (Forkhead box) proteins are a family of transcription factors that play important roles in regulating gene expression that govern cardiogenesis. Basal expression analysis for \u003cem\u003eFoxc1\u003c/em\u003e and \u003cem\u003eFoxf1\u003c/em\u003e in EPIC, MEC1, MEVEC, HL1 and EEx revealed that \u003cem\u003eFoxc1\u003c/em\u003e is abundantly expressed in EEx compared with the similar expression evidenced in EPIC and MEC1, while \u003cem\u003eFoxf1\u003c/em\u003e is significantly downregulated in EEx and EPIC compared with MEC1 \u003cb\u003e(Supplementary Fig.\u0026nbsp;13A).\u003c/b\u003e Since \u003cem\u003eFoxf1\u003c/em\u003e is expressed in the PE and \u003cem\u003eFoxc1\u003c/em\u003e in EE we investigated whether these DE transcription factors, i.e. \u003cem\u003eFoxc1\u003c/em\u003e and \u003cem\u003eFoxf1\u003c/em\u003e, could modulate the expression of our DE-microRNAs in MEC1 epicardial cells. \u003cem\u003eFoxc1\u003c/em\u003e inhibition resulted in decreased \u003cem\u003emiR-495\u003c/em\u003e and \u003cem\u003emiR-351\u003c/em\u003e expression, while loss-of-function of both \u003cem\u003eFoxc1\u003c/em\u003e and \u003cem\u003eFoxf1\u003c/em\u003e led to reduced \u003cem\u003elet-7c\u003c/em\u003e and increased \u003cem\u003emiR-30c\u003c/em\u003e expression \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cb\u003e).\u003c/b\u003e We subsequently examined whether these DE transcription factors exhibit also cross-talk regulation, similar as the DE microRNAs. Inhibition of \u003cem\u003eFoxc1\u003c/em\u003e in MEC1 cells resulted in decreased expression of the \u003cem\u003eFoxf1\u003c/em\u003e transcription factor, while inhibition of \u003cem\u003eFoxf1\u003c/em\u003e in MEC1 cells led to upregulation of \u003cem\u003eFoxc1\u003c/em\u003e expression \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. To further elucidate the intricate regulatory network involving our DE-miRNAs, we assessed the expression levels of \u003cem\u003eFoxc1\u003c/em\u003e and \u003cem\u003eFoxf1\u003c/em\u003e in MEC1 epicardial cells overexpressed with the DE-miRNAs in PE\u0026thinsp;\u0026gt;\u0026thinsp;EE. No significant differences in expression levels were observed for \u003cem\u003eFoxc1\u003c/em\u003e after \u003cem\u003emiR-181c\u003c/em\u003e, \u003cem\u003emiR-200b\u003c/em\u003e and \u003cem\u003emiR-495\u003c/em\u003e administration while \u003cem\u003eFoxf1\u003c/em\u003e expression was decreased after premiRs treatment \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. Conversely, inhibition of \u003cem\u003elet-7c\u003c/em\u003e consistently upregulated \u003cem\u003eFoxc1\u003c/em\u003e expression, exerting the opposite effect over \u003cem\u003eFoxf1\u003c/em\u003e. Similarly, loss-of-function of \u003cem\u003emiR-351\u003c/em\u003e resulted in decreased expression of \u003cem\u003eFoxc1\u003c/em\u003e and increased expression of \u003cem\u003eFoxf1\u003c/em\u003e. However, inhibition of \u003cem\u003emiR-30c\u003c/em\u003e did not yield significant differences \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. Finally, we aimed to elucidate the involvement of the DE-transcription factors in epicardial cell lineage specification and migration. We analysed the expression levels of epicardial, myocardial and endocardial markers following loss-of-function of \u003cem\u003eFoxc1\u003c/em\u003e and \u003cem\u003eFoxf1\u003c/em\u003e in MEC1 epicardial cells. Our findings revealed that inhibition of \u003cem\u003eFoxc1\u003c/em\u003e represses epicardial lineage specification markers such as \u003cem\u003eWt1, Tbx18\u003c/em\u003e and \u003cem\u003eTcf21\u003c/em\u003e, similar to those observed after \u003cem\u003eFoxf1\u003c/em\u003e inhibition except for \u003cem\u003eTcf21\u003c/em\u003e that displayed no significant differences \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE,F\u003cb\u003e)\u003c/b\u003e. Moreover, loss-of-function of \u003cem\u003eFoxc1\u003c/em\u003e resulted in increased expression levels of \u003cem\u003eTnnt2\u003c/em\u003e and \u003cem\u003eTie2\u003c/em\u003e, while \u003cem\u003eFoxf1\u003c/em\u003e inhibition promoted the expression of myocardial markers such as \u003cem\u003eGata4, Myh6, Srf\u003c/em\u003e and \u003cem\u003eTnnt2\u003c/em\u003e, as well as endocardial markers such as \u003cem\u003ePecam1, Tie2 and Postn\u003c/em\u003e. \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE,F\u003cb\u003e)\u003c/b\u003e. Finally, epicardial cell migration in MEC1 following \u003cem\u003eFoxf1\u003c/em\u003e loss-of-function lead to similar results as those observed for \u003cem\u003elet7c\u003c/em\u003e inhibition \u003cem\u003ein vitro\u003c/em\u003e, with no significant difference in Myh9 protein expression, although F-actin polymerization was impaired \u003cb\u003e(Supplementary Fig.\u0026nbsp;13B)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn conclusion, our study unveiled complex regulatory networks orchestrated by DE-microRNAs and DE-transcription factors. These findings offer valuable insights into the regulatory mechanisms governing epicardial cell specification and cardiac development, particularly mediated by \u003cem\u003eFoxf1, let-7c\u003c/em\u003e and \u003cem\u003emiR-30c\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, we provide novel insights into the dynamic regulation of microRNAs and their functional role in proepicardial (PE) and embryonic epicardial (EE) formation in mice. Our findings reveal distinct microRNA expression patterns that selectively influence key cellular processes, including cell-cell signaling, migration, and lineage specification during epicardial development. Specifically, we demonstrate that Foxf1 modulates the expression of miR-495, miR-351, and let-7c, which in turn regulate epicardial cell migration and myocardial specification. Notably, our results suggest a previously unrecognized microRNA-microRNA regulatory network, shedding light on the intricate molecular crosstalk driving epicardial development. These discoveries provide new mechanistic evidence of the transcriptional and post-transcriptional regulation orchestrating PE and EE formation, with potential implications for cardiac regenerative strategies.\u003c/p\u003e \u003cp\u003emicroRNAs are short non-coding RNAs with tissue-specific expression that exert regulatory roles over different cellular processes ranging from embryonic development to pathological response (\u003cspan additionalcitationids=\"CR65\" citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e). In the cardiovascular system, several laboratories including ours, have yielded substantial evidence elucidating the microRNA differential expression during cardiogenesis (\u003cspan additionalcitationids=\"CR45 CR46\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e). Moreover, \u003cem\u003eDue\u0026ntilde;as et al.\u003c/em\u003e (2020) recently evidenced a microRNA differential expression during PE and EE formation in chicken (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e), however, the functional role of microRNAs in PE and EE development in mice and their application to enhance cardiogenesis remains elusive. In this study, we provide evidence about the microRNA differential expression pattern during PE and EE formation and their functional implications in mice. We found a large subset of microRNAs and mRNAs that display increasing expression in PE \u003cem\u003evs\u003c/em\u003e. EE, suggesting a plausible role in cell-cell signaling during proepicardial cell specification, differentiation and vesicles formation for direct contact and attachment of the proepicardial cells to naked myocardium in mice (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan additionalcitationids=\"CR80 CR81 CR82\" citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e). On the other hand, a small subset of microRNAs displays increased expression, as well as high mRNA expression levels in EE \u003cem\u003evs.\u003c/em\u003e PE, supporting a modulatory role in the coordination of epicardial-derived signals involved in muscle function, coronary development, as well as myocardial growth (\u003cspan additionalcitationids=\"CR85 CR86\" citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e). Thus, these data revealed that differential microRNA signatures can selectively influence different signaling pathways during PE and EE formation.\u003c/p\u003e \u003cp\u003eThe PE derives from the LPM and is formed at the venous pole of the heart during embryonic development in E9.5 mice. Lumenized vesicles from the proepicardial surface attach to the naked myocardium subsequently forming the embryonic epicardium at E10.5 (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e). Epithelial cells undergo EMT forming EPDCs that migrate into the myocardium, proliferate and differentiate into different cell types, including coronary endothelial cells, smooth muscle cells and cardiac fibroblasts, whereas their contribution to cardiomyocytes remain controversial (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan additionalcitationids=\"CR90 CR91 CR92 CR93 CR94 CR95 CR96 CR97\" citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e). Several laboratories have identified microRNA-mRNA cross-talk correlation during cardiac development (see recent reviews; (\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e, \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e)). Br\u0026oslash;nnum et al. (\u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e) have elucidated that miR-21 modulates epicardium development and EPDCs fate-decision, through the established interplay between \u003cem\u003ePdcd4\u003c/em\u003e and \u003cem\u003eSpry1\u003c/em\u003e, whereas Pontemezzo et al. (\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e) reported that miR-200c after Tfg-\u0026szlig; administration impact on the epithelial-to-mesenchymal transition process in epicardial cells. Our RNAseq analysis evidenced a complementary expression pattern of microRNAs and putative mRNA targets in PE \u003cem\u003evs\u003c/em\u003e. EE. Those microRNAs with enhanced expression in PE such as, \u003cem\u003emiR-495-5p, miR-200b-3p\u003c/em\u003e and \u003cem\u003emiR-181c-5p\u003c/em\u003e, have a plausible role blocking mRNA target expression (i.e. \u003cem\u003eMbln2, Nr3c1, Nfib, Rnd3, Hapln1\u003c/em\u003e, and \u003cem\u003eFn1\u003c/em\u003e) during the morphogenetic induction of proepicardial to embryonic epicardial cells transition. Herein, we demonstrate that \u003cem\u003emiR-495\u003c/em\u003e regulates \u003cem\u003eMbln2\u003c/em\u003e in epicardial cells but not in myocardial nor endocardial cells, providing novel mechanistic insight into the role of microRNAs in this context. In addition, microRNAs upregulated in EE, such as \u003cem\u003elet7c-5p, miR-351-5p\u003c/em\u003e, and \u003cem\u003emiR-30c-5p\u003c/em\u003e, are repressing inductive signals derived from mRNAs targets (i.e. \u003cem\u003ePrtg, Nr6a1, Peg10, Fbxo32, Trim71, Hic2\u003c/em\u003e and \u003cem\u003eCcnjl\u003c/em\u003e) during EMT and epicardial cell specification process, illustrated by the fact that \u003cem\u003elet-7c\u003c/em\u003e regulates \u003cem\u003eTrim71\u003c/em\u003e in epicardial cells. In summary, these findings further support the notion that tight regulation of microRNA-mRNA interaction plays a crucial role in coordinating cellular processes, i.e. cellular migration and lineage specification during PE and EE formation (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e, \u003cspan additionalcitationids=\"CR103 CR104 CR105 CR106\" citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e107\u003c/span\u003e). Moreover, our results highlight the dynamic rewiring of miRNA-mRNA interaction during cardiogenesis, which modulate mRNA target expression in a cell-specific manner in epicardium, myocardium and endocardium. These differences may arise from variation in target mRNA expression, the presence of RNA-binding proteins that modulate microRNA function, or competing endogenous RNAs, as previously reported for microRNAs in other biological contexts (\u003cspan additionalcitationids=\"CR109\" citationid=\"CR108\" class=\"CitationRef\"\u003e108\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e110\u003c/span\u003e). These findings underscore the complexity of miRNA-mediated gene regulation in epicardial, reflecting the intricate nature of post-transcriptional networks across different cellular context during cardiac development. The discrepancies between individual miRNAs and the combinatorial conditions likely stem from interaction within broader miRNA networks producing non-linear outcomes. Additionally, cell-specific factors in MEC1 and EPIC cells contribute to these differences, emphasizing the dynamic regulation of gene expression during cardiac development.\u003c/p\u003e \u003cp\u003emicroRNAs can modulate multiple biological processes, including cell migration in homeostatic and pathological conditions (\u003cspan additionalcitationids=\"CR112 CR113 CR114\" citationid=\"CR111\" class=\"CitationRef\"\u003e111\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR115\" class=\"CitationRef\"\u003e115\u003c/span\u003e). Cell migration is a key biological process during PE formation, such PE cells initially migrate onto the heart to establish the embryonic epicardium primarily at ventricular level, subsequently across the atrial chamber. Following EMT, EPDCs migrate into the myocardium to support coronary vasculature and ventricular development (\u003cspan citationid=\"CR116\" class=\"CitationRef\"\u003e116\u003c/span\u003e). We provide herein evidences that \u003cem\u003elet-7c\u003c/em\u003e and \u003cem\u003emiR-351\u003c/em\u003e in the EE have an important role in controlling epicardial cell migration, in line with previous reports in other biological contexts (\u003cspan additionalcitationids=\"CR118\" citationid=\"CR117\" class=\"CitationRef\"\u003e117\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR119\" class=\"CitationRef\"\u003e119\u003c/span\u003e). Additionally, \u003cem\u003emiR-495\u003c/em\u003e modulates epicardial cell migration, partially mediated by \u003cem\u003eNr3c1\u003c/em\u003e, in line with recent reports in pathological conditions (\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan additionalcitationids=\"CR121 CR122\" citationid=\"CR120\" class=\"CitationRef\"\u003e120\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR123\" class=\"CitationRef\"\u003e123\u003c/span\u003e). Similarly, we evidenced that \u003cem\u003eRnd3\u003c/em\u003e is involved in epicardial cell migration, although is not coordinated with \u003cem\u003emiR-181c\u003c/em\u003e and \u003cem\u003emiR-200b\u003c/em\u003e modulation (\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e). These findings underscore the functional role of microRNAs and mRNAs in regulating epicardial cell migration, a highly relevant process in PE and EE formation, through the differential expression of cytoskeletal proteins. Given that defective migration of PE and EE cells can lead to severe congenital heart defects by disrupting coronary vasculature, myocardial growth, EMT and valve formation, our findings emphasize the pivotal role of miRNA-mRNA interactions in ensuring the precise regulation of epicardial cell migration, which is essential for proper cardiac morphogenesis\u003c/p\u003e \u003cp\u003eAs extensively documented, the epicardium plays a key role during cardiogenesis since a subset of EPDCs undergo EMT, migrate into the myocardium and differentiate into different cardiovascular lineages, i.e. coronary vascular smooth muscle cells, cardiac fibroblasts, endothelial cells, contributing to complete heart formation. Furthermore, it has been suggested that epicardial progenitors can also contribute to the cardiomyocyte lineage, although this statement remains controversial (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e, \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e, \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e). In our study, we explored the plausible contribution of microRNAs in the process of epicardial cell lineage specification. Our findings indicate that the administration of \u003cem\u003emiR-181c\u003c/em\u003e and \u003cem\u003emiR-200b\u003c/em\u003e, as well as the inhibition of \u003cem\u003elet-7c\u003c/em\u003e facilitates epicardial cell specification into the myocardial cell lineage \u003cem\u003ein vitro\u003c/em\u003e but these effects were not observed \u003cem\u003eex vivo\u003c/em\u003e in the explant model. This discrepancy arises from the fundamental differences between the simplified \u003cem\u003ein vitro\u003c/em\u003e environment and the more complex, physiologically relevant conditions of the explant model. Hence, these data support the notion that \u003cem\u003emiR-181c\u003c/em\u003e, \u003cem\u003emiR-200b\u003c/em\u003e and \u003cem\u003elet-7c\u003c/em\u003e modulate myocardial specification of embryonic epicardial cells, underscoring the potential impact of molecular regulation induced by the neighbouring myocardial and endocardial tissues. Additionally, the induction of fibrotic markers in the epicardial cells observed after loss-of-function of \u003cem\u003elet-7c\u003c/em\u003e suggests that this microRNA plays a broader role in modulating epicardial cell fate. This finding implies that \u003cem\u003elet-7c\u003c/em\u003e may influence fibrotic processes during cardiac development, consistent with its established roles in regulating fibrosis in other biological contexts (\u003cspan citationid=\"CR124\" class=\"CitationRef\"\u003e124\u003c/span\u003e, \u003cspan citationid=\"CR125\" class=\"CitationRef\"\u003e125\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDuring embryogenesis, extracellular information is needed for cells to make decisions during development and differentiation (\u003cspan citationid=\"CR126\" class=\"CitationRef\"\u003e126\u003c/span\u003e, \u003cspan citationid=\"CR127\" class=\"CitationRef\"\u003e127\u003c/span\u003e). Tight cross-talk between different signaling pathways such as TGF-β/BMP, Wnt/Wg, Hedgehog (Hh), Notch, and mitogen-activated protein kinases (MAPK) have been thoroughly described (\u003cspan additionalcitationids=\"CR129 CR130 CR131 CR132\" citationid=\"CR128\" class=\"CitationRef\"\u003e128\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR133\" class=\"CitationRef\"\u003e133\u003c/span\u003e). Similarly, cross-talk between transcription factors has been evidenced, e.g. Gata4-Tbx5 controls cardiac septum formation (\u003cspan citationid=\"CR134\" class=\"CitationRef\"\u003e134\u003c/span\u003e) and Tbx5-Nkx2.5 interaction promotes cardiomyocyte differentiation (\u003cspan citationid=\"CR135\" class=\"CitationRef\"\u003e135\u003c/span\u003e). Nevertheless, there is scarce evidence regarding the plausible microRNA cross-talk that could modulate the maturation and expression of other microRNAs (\u003cspan citationid=\"CR136\" class=\"CitationRef\"\u003e136\u003c/span\u003e). Our analysis demonstrated that DE microRNAs in PE and EE regulate the expression of other microRNAs in epicardial cells at post-transcriptional levels. Therefore, this is, to the best of our knowledge the first evidence that microRNAs can regulate the expression of other microRNAs, supporting thus functional implications for PE and EE development. Given the role of \u003cem\u003elet-7c\u003c/em\u003e in terminal myocardial differentiation and its role in the regulation of \u003cem\u003emiR-30c\u003c/em\u003e and \u003cem\u003emiR-351\u003c/em\u003e expression, we evidenced that only \u003cem\u003emiR-351\u003c/em\u003e promotes myocardial terminal differentiation in epicardial cells, promoting \u003cem\u003eMyh6\u003c/em\u003e expression. These findings elucidate that in spite of the intricate interplay among DE microRNAs in PE \u003cem\u003evs\u003c/em\u003e. EE, governing epicardial cell behaviour, the terminal myocardial differentiation exerted by \u003cem\u003elet-7c\u003c/em\u003e is not solely mediated by the EE\u0026thinsp;\u0026gt;\u0026thinsp;PE microRNAs interplay.\u003c/p\u003e \u003cp\u003eCardiac-specific transcription factors such as \u003cem\u003eNkx2.5\u003c/em\u003e (\u003cspan additionalcitationids=\"CR138\" citationid=\"CR137\" class=\"CitationRef\"\u003e137\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR139\" class=\"CitationRef\"\u003e139\u003c/span\u003e), \u003cem\u003eMef2c\u003c/em\u003e (\u003cspan additionalcitationids=\"CR141\" citationid=\"CR140\" class=\"CitationRef\"\u003e140\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR142\" class=\"CitationRef\"\u003e142\u003c/span\u003e), \u003cem\u003ePitx2\u003c/em\u003e (\u003cspan additionalcitationids=\"CR144 CR145\" citationid=\"CR143\" class=\"CitationRef\"\u003e143\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR146\" class=\"CitationRef\"\u003e146\u003c/span\u003e), \u003cem\u003eSrf\u003c/em\u003e (\u003cspan citationid=\"CR147\" class=\"CitationRef\"\u003e147\u003c/span\u003e, \u003cspan citationid=\"CR148\" class=\"CitationRef\"\u003e148\u003c/span\u003e) and \u003cem\u003eFox\u003c/em\u003e (\u003cspan additionalcitationids=\"CR150 CR151 CR152\" citationid=\"CR149\" class=\"CitationRef\"\u003e149\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR153\" class=\"CitationRef\"\u003e153\u003c/span\u003e), are fundamental in both cardiogenesis and the development of PE and EE (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR154\" class=\"CitationRef\"\u003e154\u003c/span\u003e, \u003cspan citationid=\"CR155\" class=\"CitationRef\"\u003e155\u003c/span\u003e). Moreover, \u003cem\u003eTbx18\u003c/em\u003e is highly expressed in PE and essential in epicardium and coronary vasculature development (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e104\u003c/span\u003e). \u003cem\u003eTcf21\u003c/em\u003e and \u003cem\u003eTbx5\u003c/em\u003e are essential for mature proepicardial cells to establish contact with the myocardium and properly form the epicardium (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR156\" class=\"CitationRef\"\u003e156\u003c/span\u003e). \u003cem\u003eWt1\u003c/em\u003e is crucial for EMT of epicardial cells (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). These transcription factors exert transcriptional control over multiple downstream targets, including both coding and non-coding RNAs and miRNAs, particularly those pivotal for heart development. In our RNAseq analysis, \u003cem\u003eFoxf1\u003c/em\u003e displays enhanced expression in PE, whereas \u003cem\u003eFoxc1\u003c/em\u003e shows an opposite expression pattern, with high expression in EE. Our data analysis revealed that \u003cem\u003eFoxc1\u003c/em\u003e and \u003cem\u003eFoxf1\u003c/em\u003e exert transcriptional control over the DE microRNAs in PE and EE during development, in line with previous report demonstrating similar transcription factor-miRNA transcriptional regulation i.e. \u003cem\u003ePitx2\u003c/em\u003e-miRNAs in a skeletal-muscle context (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). In PE and EE formation and specification, it is worth highlighting that \u003cem\u003eFoxf1\u003c/em\u003e controls \u003cem\u003elet-7c\u003c/em\u003e and \u003cem\u003emiR-30c\u003c/em\u003e expression. In addition, DE microRNAs in PE \u003cem\u003evs.\u003c/em\u003e EE, i.e. \u003cem\u003emiR-495\u003c/em\u003e and \u003cem\u003elet-7c\u003c/em\u003e modulate \u003cem\u003eFoxc1\u003c/em\u003e and \u003cem\u003eFoxf1\u003c/em\u003e expression and similar to those observed effects for these microRNAs, both transcription factors exhibit a cross-talk modulation. Overall these findings reveal a complex transcription factor \u003cem\u003evs\u003c/em\u003e. microRNA regulation in PE and EE formation and specification in epicardial cells during cardiogenesis \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUnderstanding of the molecular mechanisms driving PE and EE formation and specification has greatly advanced over the last decade including the functional role of microRNAs (see recent reviews (\u003cspan additionalcitationids=\"CR158\" citationid=\"CR157\" class=\"CitationRef\"\u003e157\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR159\" class=\"CitationRef\"\u003e159\u003c/span\u003e)). Regarding the tight molecular regulation of transcription factors and DE microRNAs in PE \u003cem\u003evs.\u003c/em\u003e EE, and the previously observed implication of microRNAs during epicardial cell specification, we evidenced that \u003cem\u003eFoxc1\u003c/em\u003e modulates the expression of myocardial and endocardial markers (i.e. \u003cem\u003eTnnt2\u003c/em\u003e and \u003cem\u003eTie2\u003c/em\u003e), whereas \u003cem\u003eFoxf1\u003c/em\u003e controls early cardiogenesis (i.e. \u003cem\u003eGata4\u003c/em\u003e and \u003cem\u003eSrf\u003c/em\u003e), as well as cardiomyocyte terminal differentiation (i.e. \u003cem\u003eTnnt2\u003c/em\u003e and \u003cem\u003eMyh6\u003c/em\u003e). Therefore, these observation supports the notion that transcription factors \u003cem\u003eFoxc1\u003c/em\u003e and \u003cem\u003eFoxf1\u003c/em\u003e modulate EE specification, being essential during cardiogenesis as previously reported (\u003cspan additionalcitationids=\"CR150 CR151 CR152\" citationid=\"CR149\" class=\"CitationRef\"\u003e149\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR153\" class=\"CitationRef\"\u003e153\u003c/span\u003e, \u003cspan additionalcitationids=\"CR161 CR162 CR163\" citationid=\"CR160\" class=\"CitationRef\"\u003e160\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR164\" class=\"CitationRef\"\u003e164\u003c/span\u003e). Therefore, mechanistically this study evidenced that \u003cem\u003eFoxf1\u003c/em\u003e controls epicardial cell specification towards cardiomyocytes by modulating \u003cem\u003elet-7c\u003c/em\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eIn summary, we provide herein evidence that PE and EE formation and specification are biological processes tightly regulated by DE microRNAs and mRNAs during cardiogenesis. We demonstrated that \u003cem\u003eFoxf1\u003c/em\u003e transcription factor modulates \u003cem\u003emiR-495, miR-351 and let-7c\u003c/em\u003e expression and these microRNAs regulate epicardial cell migration and myocardial specification, hinting the essential co-regulatory role of transcription factor \u003cem\u003evs.\u003c/em\u003e microRNA for cardiogenesis during embryonic development \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eDespite the valuable insights provided by this study, some limitations should be acknowledged. While our RNA sequencing data suggest potential microRNA-mRNA regulatory interactions, further exploration to fully elucidate the regulatory networks that govern cardiogenesis would strengthen our findings. Additionally, future studies employing genetic loss- and gain-of-function models in mice would be necessary to confirm the role of specific microRNAs in PE and EE development. Lastly, although our study suggests potential translational applications for cardiac regenerative medicine, additional research is needed to determine whether modulating these microRNA pathways could enhance epicardial cell contribution to cardiac repair in postnatal or adult hearts.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eOur study highlights the intricate regulatory mechanisms orchestrated by differentially expressed microRNAs and mRNAs during the formation and specification of the PE and EE in cardiogenesis. We provide novel evidence that specific microRNAs, such as \u003cem\u003emiR-495\u003c/em\u003e and \u003cem\u003elet-7c\u003c/em\u003e, play crucial roles in modulating epicardial cell migration and myocardial specification. The transcription factor \u003cem\u003eFoxf1\u003c/em\u003e regulates \u003cem\u003elet-7c\u003c/em\u003e expression, thereby promoting key developmental processes as myocardial lineage specification from epicardial cells. Our findings underscore the complexity and importance of the microRNA-mRNA interaction networks and their co-regulatory roles with transcription factors in governing cardiogenesis. This study advances our understanding of the molecular mechanisms underlying heart development and highlights potential therapeutic targets.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAnti-miRNA\u0026nbsp; \u0026nbsp; \u0026nbsp;microRNA inhibitor\u003c/p\u003e\n\u003cp\u003eAV\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Atrioventricular\u003c/p\u003e\n\u003cp\u003ecDNA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Complementary DNA\u003c/p\u003e\n\u003cp\u003eDE\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Differentially expressed\u003c/p\u003e\n\u003cp\u003eDapi\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;4\u0026apos;,6-Diamidine-2\u0026apos;-phenylindole dihydrochloride\u003c/p\u003e\n\u003cp\u003eE8.5 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Embryonic day 8.5\u003c/p\u003e\n\u003cp\u003eE9.5\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Embryonic day 8.5\u003c/p\u003e\n\u003cp\u003eE10.5\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Embryonic day 10.5\u003c/p\u003e\n\u003cp\u003eEBSS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Earle\u0026rsquo;s balance salt solution\u003c/p\u003e\n\u003cp\u003eEE \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Embryonic epicardium\u003c/p\u003e\n\u003cp\u003eEEx\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Wt1\u003csup\u003e+\u003c/sup\u003e epicardial cells\u003c/p\u003e\n\u003cp\u003eEMT\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Epithelial to mesenchymal transition\u003c/p\u003e\n\u003cp\u003eEPDCs\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Epicardial derived cells\u003c/p\u003e\n\u003cp\u003eFBS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Fetal bovine serum\u003c/p\u003e\n\u003cp\u003eFC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Fold change\u003c/p\u003e\n\u003cp\u003eGEO\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Gene Expression Omnibus\u003c/p\u003e\n\u003cp\u003eGO \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Gene ontology\u003c/p\u003e\n\u003cp\u003eGSEA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Gene Set Enrichment Analysis\u003c/p\u003e\n\u003cp\u003eHEPES\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid\u003c/p\u003e\n\u003cp\u003eIHC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Immunohistochemical analyses\u003c/p\u003e\n\u003cp\u003elncRNA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Long non-coding RNA\u003c/p\u003e\n\u003cp\u003eLPM\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Lateral plate mesenchyme\u003c/p\u003e\n\u003cp\u003ePBS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Phosphate buffer saline\u003c/p\u003e\n\u003cp\u003ePE \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Proepicardium\u003c/p\u003e\n\u003cp\u003ePre-miRNAs\u0026nbsp; \u0026nbsp; \u0026nbsp;microRNA precursor\u003c/p\u003e\n\u003cp\u003eRT-qPCR\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Reverse transcriptase-quantitative polymerase chain reaction\u003c/p\u003e\n\u003cp\u003eSV\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Sinus venosus\u003c/p\u003e\n\u003cp\u003eTMM \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Trimmed mean of M-values\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics approval\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of the University of Ja\u0026eacute;n (code 14/03/2022/038).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAvailability of data and material\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNAseq data were uploaded into Gene Expresssion Onmibus platform with accession number GSE189344. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE189344\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants of the Ministerio de Innovaci\u0026oacute;n y Ciencia of the Spanish Government to DF (PID2022-138163OB-C32) and of the Consejer\u0026iacute;a de Universidad, Investigaci\u0026oacute;n e Innovaci\u0026oacute;n of the Junta de Andalucia Regional Council to DF (ProyExcel_00409).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthor\u0026acute;s contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. \u0026nbsp;Material preparation, data collection and analysis were performed by JM C-C, A D, F H-T, R C, R M-C, A D, R A, E VdL. The first draft of the manuscript was written by EL-V and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgments\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the excellent technical support of the CICT-Universidad de Ja\u0026eacute;n.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe would like to thank Jose Luis de la Pompa (CNIC, Madrid) for sharing MEVEC cells. We would like to thank Jose Mar\u0026iacute;a P\u0026eacute;rez Pomares (UMA, M\u0026aacute;laga) for sharing EPIC cells.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFranco D, Christoffels VM, Campione M. Homeobox transcription factor Pitx2: The rise of an asymmetry gene in cardiogenesis and arrhythmogenesis. 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IJMS. 2022 Mar 16;23(6):3220. \u003c/li\u003e\n\u003cli\u003eSanchez-Fernandez C, Rodriguez-Outeiri\u0026ntilde;o L, Matias-Valiente L, Ram\u0026iacute;rez De Acu\u0026ntilde;a F, Franco D, Ar\u0026aacute;nega AE. Understanding Epicardial Cell Heterogeneity during Cardiogenesis and Heart Regeneration. JCDD. 2023 Sep 1;10(9):376. \u003c/li\u003e\n\u003cli\u003eCarmona R, L\u0026oacute;pez-S\u0026aacute;nchez C, Garcia-Martinez V, Garcia-L\u0026oacute;pez V, Mu\u0026ntilde;oz-Ch\u0026aacute;puli R, Lozano-Velasco E, Franco D. Novel Insights into the Molecular Mechanisms Governing Embryonic Epicardium Formation. JCDD. 2023 Oct 24;10(11):440. \u003c/li\u003e\n\u003cli\u003eHarrelson Z, Kaestner KH, Evans SM. \u003cem\u003eFoxa2\u003c/em\u003e mediates critical functions of prechordal plate in patterning and morphogenesis and is cell autonomously required for early ventral endoderm morphogenesis. 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Molecular mechanism of cardiac differentiation in P19 embryonal carcinoma cells regulated by Foxa2. Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2013 Apr;38(4):356\u0026ndash;64. \u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1-5 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Transcription factors, microRNAs, epicardial cells, cell migration, cell lineage specification.","lastPublishedDoi":"10.21203/rs.3.rs-5643113/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5643113/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe heart is the first functional organ to develop in the vertebrate embryos. In mice, the primitive tubular heart begins beating at embryonic day (E) 8.0-E.8.5 and undergoes rightward looping to form the atrial and ventricular chambers. The proepicardium, a transient cell cluster at the sinus venous-lateral plate mesenchyme junction migrates onto the heart and gives rise to the embryonic epicardium, a squamous epithelium that plays a key role in cardiac development. Despite advances in understanding epicardial lineage contributions, the molecular mechanisms governing these processes remain poorly understood.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eTo characterize the transcriptional and post-transcriptional regulation of epicardial development, we performed RNA sequencing at two critical timepoints, proepicardium formation and embryonic epicardium establishment. We analysed differentially expressed coding and non-coding RNAs, focusing on microRNAs and their potential regulatory interactions.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe identified a complex network involving differentially expressed mRNAs, microRNAs and lncRNAs between proepicardium and embryonic epicardium. Notably, with \u003cem\u003emiR-495 and let-7c\u003c/em\u003e emerged as key regulators of epicardial cell migration, an essential process for proper epicardium formation and epicardial-derived cell migration. Our findings also reveal that these microRNAs not only regulate target gene expression but also modulate other microRNAs, suggesting a novel regulatory mechanism in epicardial development. Additionally, \u003cem\u003eFoxf1\u003c/em\u003e inhibition modulates \u003cem\u003elet-7c\u003c/em\u003e, promoting the expression of key cardiogenic lineage markers in epicardial cells.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur study highlights the role of \u003cem\u003eFoxf1\u003c/em\u003e in regulating \u003cem\u003emiR-495\u003c/em\u003e and \u003cem\u003elet-7c\u003c/em\u003e, which in turn modulate epicardial cell migration and myocardial specification. These finding provide new insights into the intricate interplay between transcription factors and microRNAs in governing cardiogenesis.\u003c/p\u003e","manuscriptTitle":"Foxf1 -mediated co-regulation of miR-495 and let-7c modulates epicardial cell migration and myocardial specification","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-01 08:16:32","doi":"10.21203/rs.3.rs-5643113/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept as is","date":"2025-04-30T14:39:38+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-03-29T15:47:44+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-29T12:41:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-27T01:32:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellular and Molecular Life Sciences","date":"2025-03-26T06:10:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6be80682-b406-4a94-bd73-1ee1fca5a27f","owner":[],"postedDate":"April 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-30T16:04:06+00:00","versionOfRecord":{"articleIdentity":"rs-5643113","link":"https://doi.org/10.1007/s00018-025-05735-4","journal":{"identity":"cellular-and-molecular-life-sciences","isVorOnly":false,"title":"Cellular and Molecular Life Sciences"},"publishedOn":"2025-06-25 15:57:36","publishedOnDateReadable":"June 25th, 2025"},"versionCreatedAt":"2025-04-01 08:16:32","video":"","vorDoi":"10.1007/s00018-025-05735-4","vorDoiUrl":"https://doi.org/10.1007/s00018-025-05735-4","workflowStages":[]},"version":"v1","identity":"rs-5643113","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5643113","identity":"rs-5643113","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}