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. 2021 Jul 6;12:4155. doi: 10.1038/s41467-021-24414-z

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a Schematic of experimental design to isolate epicardial cells (EPDC) of indicated genotypes for bulk RNA-seq. Embryonic day (E)12.5 hearts were removed from Mrtf-a^−/−;Mrtf-b^fl/fl, and Srf^fl/fl mice and incubated with adenovirus (ad) expressing GFP to label epicardium (green), and either ad/βgalactosidase (βgal) or ad/Cre. Hearts were cultured ex vivo in TGFβ2 [2 ng/mL] and PDGF-BB [20 ng/mL] to induce EMT, and GFP⁺ epicardial cells were collected using FACS. GFP⁻ cells were collected as non-EPDCs. cTNT, cardiac troponin T (red), and DAPI staining were utilized to visualize nuclei (blue). Scale bar, 50 μm. Refer to Supplementary Fig. 8e for FACS sequential gating and enrichment of epicardial cells. b Principal component analysis (PCA) of GFP⁻ non-EPDCs and GFP⁺ EPDCs from control, Mrtf-a;Mrtf-b double knockout (MRTF^epiDKO) or Srf knockout (SRF^epiKO) mice. c Analysis of differentially expressed genes (DEGs) reveals biological processes that are enriched between MRTF^epiDKO and control EPDCs. GO-term enrichment significance was determined using Fisher exact test. d Heat map highlighting the relative expression of genes involved in axon guidance signaling in EPDCs of the indicated genotype. Relative (Rel.) expression represents the scaled expression of rlog normalized data. e Expression of genes encoding axon guidance cues was validated by qRT-PCR. Values are represented as a fold change in expression relative to non-EPDCs (dashed line at 1). n represents samples acquired from independent embryos, which were analyzed in 1 experiment. n = 3 non-EPDCs, n = 4 for Efna5, Sema3d, Slit3, Wnt5a, and n = 3 for Slit2 in Control EPDCs; n = 3 MRTF^epiDKO EPDCs were analyzed. Data are presented as mean values ± SEM. Statistical significance was determined by a two-sample unpaired Student’s t-test. TGFβ2 transforming growth factor beta-2, PDGF-BB platelet-derived growth factor BB, m-tdTom membrane tdTomato, m-eGFP membrane-enhanced green fluorescent protein, TAM tamoxifen.