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

Figures

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  Fig. 1. Panel A. Volcano plot of mRNA expression profile. Red dots represent significant DE genes in PE E9.5 vs. 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 vs. EE E10.5. Panel B. Volcano plot of miRNA expression profile. Red dots represent significant DE genes in PE E9.5 vs. 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 vs. EE E10.5. Panel C. RT-qPCR analysis for mRNA validation of DE genes in PE > EE (i.e. Hnf4a, Hoxb1, and Prox1) in PE9.5 and EE10.5 mouse tissues, demonstrating high expression in PE vs. EE (n = 3). Panel D. RT-qPCR analysis for mRNA validation of DE genes in PE < EE (i.e. Spry1, Hey2, and Itga) in PE9.5 and EE10.5 mouse tissues, demonstrating high expression in EE vs. PE (n = 3). Panel E. RT-qPCR analysis for miRNA validation of DE genes in PE > EE (i.e. miR-200, miR-200b, miR-200c, miR-429 and miR-495) in PE9.5 and EE10.5 mouse tissues, demonstrating high expression in PE vs. EE (n = 3). Panel F. RT-qPCR analysis for miRNA validation of DE genes in PE < EE…

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  Fig. 2. Panel A. RT-qPCR analysis for mRNA targets after microRNA gain-of-function (miR-181c, miR-200b, miR-495 and preMix) in EPIC epicardial cells. Observe that selective mRNA downregulation is achieved after microRNA administration. Note that Nr3c1 and Nfib are significantly decreased after all individual or combinatorial microRNA treatments (n = 3). Panel B. RT-qPCR analysis for mRNA targets after microRNA loss-of-function (let-7c, miR-30c, miR-351 and antiMix) in EPIC epicardial cells. Selective mRNA upregulation is achieved for Prtg, Nr6a1 and Hic2 after individual microRNAs knockdown (n = 3). Panel C. RT-qPCR analysis for mRNA targets after microRNA gain-of-function (miR-181c, miR-200b, miR-495 and preMix) in MEC1 epicardial cells. Observe that only Mbln2 and Hapln1 are downregulated after miR-495 administration and Fn1 after miR-200b treatment (n = 3). Panel D. RT-qPCR analysis for mRNA targets after microRNA loss-of-function (let-7c, miR-30c, miR-351 and antiMix) in MEC1 epicardial cells. Note that Fbxo32 and Trim71 are up-regulated after all individual microRNA inhibition (n = 3). Panel E. RT-qPCR analysis for mRNA targets after microRNA gain-of-function (miR-181c, miR-200b,…

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  Fig. 3. Panel A. Migration assays after DE microRNAs in PE > EE miR-181c, miR-200b, miR-495 and preMix administration, respectively. Quantitative cell migration analysis guided by the schematic representation of cell migration quantification; Total cell migration, cellular migration from ventricular explant border to the outermost individual cell of the culture; Cohesive cell migration, collective migration from ventricular explant border; Non-cohesive cell migration, individual cell migration from the outermost periphery of the cohesive migration. Panel B, C, D Quantitative cell migration for DE microRNAs in PE > EE; miR-181c, miR-200b, miR-495 and preMix administration. Note that miR-495 administration impairs total cell migration followed by cohesive migration repression (n = 5). Panel E. Migration assays after DE mRNAs in PE < EE Nfib, Nr3c1, Rnd3, Mbln2 and Fn1 silencing. Panel F, G, H Quantitative cell migration for DE mRNAs in PE < EE; Nfib, Nr3c1, Rnd3, Mbln2 and Fn1 inhibition. Note that Nr3c1 loss-of-function enhances total cell migration followed by cohesive migration increment (n = 5). Panel I Migration assays after DE microRNAs in PE < EE let-7c, miR-30c, miR-351 and antiMix…

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  Fig. 4. Panel A, B Analysis of lineage specific markers expression, i.e. myocardial (Gata4, Myh6, Nkx2.5, Tnnt2, Srf), epicardial (Tcf21, Tbx18, Wt1), and endocardial (Pecam1, Postn, Tie2) after miR-181c, miR-200b, miR-495 and preMix administration or let-7c, miR-30c, miR-351 and antiMix inhibition, respectively (n = 3). Panel C, D Analysis of biological processes markers, i.e. angiogenesis (Ang1, Ang2, Efnb2, Flt1), EMT (Cdh5, Snail1, Snail2), and fibrosis (Col1a1, Col3a1, Fn1, Sox9) in MEC1 and EPIC epicardial cells after miR-181c, miR-200b, miR-495 and preMix administration or let-7c, miR-30c, miR-351 and antiMix inhibition, respectively (n = 3). Panel E. Representative images of immunohistochemical analyses of cardiac troponinT (cTnt) in MEC1 epicardial cells, after administration of premiR-181c, premiR-200b, and anti-let7c as compared to controls. Observe that there is a significant difference in the expression of cTnt after let-7c inhibition (n = 5). Panel F. RT-qPCR analyses of selected cardiogenic markers (Tnnt2, Myh6, Tcf21, Tie2) in EEx from E10.5 epicardial explants after premiR-181c, premiR-200b, and anti-let7c administration (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001,…

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  Fig. 5. Panels A, B, E RT-qPCR analysis for microRNA expression after microRNA gain-of-function (miR-181c, miR-200b, miR-495) (n = 3). Panel A MEC1 epicardial cells. Panel B EPIC epicardial cells. Panel E EEx from E10.5 epicardial explants. Observe that microRNA upregulation is achieved after miR-181c and miR-495 administration in all epicardial cell types and for miR-200b only in E10.5 epicardial cells. Panels C, D, F RT-qPCR analysis for microRNA expression after microRNA loss-of-function (let-7c, miR-30c, miR-351) (n = 3). Panel C MEC1 epicardial cells. Panel D EPIC epicardial cells. Panel F EEx from E10.5 epicardial explants. Observe that let-7c inhibition leads to high expression levels of miR-30c and miR-351 in both epicardial cell types. Panel G, H RT-qPCR analysis of myocardial lineage markers (Myh6, Tnnt2) in MEC1 and EEx from E10.5 epicardial cells after miR-30c and miR-351 gain-of-function. Observe that miR-351 promotes Myh6 expression only in MEC1 epicardial cells (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

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  Fig. 6. Panel A. RT-qPCR analysis of DE microRNAs (miR-495, let-7c, miR-30c, miR-351) after Foxc1 and Foxf1 siRNA treatment in MEC1 epicardial cells. Observe a differential microRNA regulation. Note also a significant downregulation of let-7 and upregulation of miR-30c after Foxf1 inhibition (n = 3). Panel B. RT-qPCR analysis of Foxc1 and Foxf1 expression after Foxc1 and Foxf1 siRNA treatment in MEC1 epicardial cells, respectively (n = 3). Panel C. RT-qPCR analysis of Foxc1 and Foxf1 expression after DE microRNAs in PE > EE treatment (miR-181c, miR-200b, miR-495) in MEC1 epicardial cells. Observe that Foxf1 is downregulated after pre-miRNA administration (n = 3). Panel D. RT-qPCR analysis of Foxc1 and Foxf1 expression after DE microRNAs in PE < EE treatment (let-7c, miR-30c, miR-351) in MEC1 epicardial cells. Note that Foxf1 is downregulated only after let-7c inhibition (n = 3). Panel E, F. RT-qPCR expression analysis of cardiogenic markers; epicardial (Wt1, Tbx18, Tcf21), myocardial (Gata4, Myh6, Srf, Tnnt2) and endocardial (Pecam1, Postn, Tie2) in MEC1 epicardial cells after Foxc1 and Foxf1 siRNA treatment, respectively. Observe that selective regulation is achieved after Foxc1 and…

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  Fig. 7. Schematic representation of the functional role of Foxf1, miR-495 and let-7c 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 Foxf1 and Foxc1, as well as their regulatory effect on miR-495 and let-7c. 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