Tandon P , Miteva YV , Kuchenbrod LM , Cristea IM , Conlon FL .

DP1DA026192 NIDA NIH HHS , R01 DE018825 NIDCR NIH HHS, R01 HL 112618-01 NHLBI NIH HHS , R01 HL112618 NHLBI NIH HHS , P40 OD010997 NIH HHS , DP1 DA026192 NIDA NIH HHS

	Fig. 1. Conservation of epicardial markers in Xenopus. (A-L) In situ hybridization (ISH) for Tbx18 (A,D,G,J), Tcf21 (B,E,H,K) and Wt1 (C,F,I,L) in developing Xenopus embryos, showing ventral views of the anterior region (anterior left) (A-I) or transverse vibratome sections through the heart (anterior top) (J-L). Tbx18 expression was observed in the septum transversum (ST) (red arrowheads in A,D) earlier than that of Tcf21 (red arrowhead in E) and Wt1 (red arrowhead in F). Expression of all genes becomes restricted to proepicardial organ (PEO) by stage 40 (green arrowheads in G-L). cg, cement gland; h, heart. Scale bars: 2 mm in A-I; 100 μm in J-L.
	Fig. 2. Tcf21 is required for epicardial integrity and adhesion to the myocardial surface. (A-P) SEM images of hearts from control and Tcf21-depleted Xenopus embryos (A,C,E,G,I,K,M,O) with magnified views of the ventricular region (B,D,F,H,J,L,N,P). Blue dashed line indicates the extent of epicardial sheet migration over the ventricular surface. Ventral views of pericardial cavity with anterior at top (A-P). (Q-T′) Toluidine Blue staining (Q,R) (dorsal at top) and TEM images (S-T′) of transverse sections through the heart region demonstrating adhesions, or lack thereof, to the underlying myocardial surface (red arrows). The boxed regions in Q,R are magnified in S-T′. a, atrium; en, endocardium; ep, epicardium; m, myocardium; oft, outflow tract; peo, proepicardial organ; v/ven, ventricle. Scale bars: 50 μm in A,C,E,G,I,K,M,O; 25 μm in B,D,F,H,J,L,N,P; 5 μm in S,T; 2 μm in S′,T′.
	Fig. 3. Tcf21 associates with co-repressor proteins and is phosphorylated at multiple sites. (A) Experimental workflow for Tcf21 immunoaffinity purification (IP), and identification of Tcf21 interactions and post-translational modifications (PTMs). (B) Identification of Tcf21 peptide by CID MS/MS analysis. #916;M, ppmrefers to the difference between experimental and theoretical peptide masses in parts per million. (C) IP validation of interaction between Tcf21 and Ctbp2 in HEK293 cells. Both proteins run at ~50 kDa. WB, western blot. (D) Expression of Tcf21 protein interaction candidates, as identified by mass spectrometry, in Xenopus stage 45 cardiac tissue analyzed by RNA-seq read counts. Huwe1 and Tcf12 were not included in the RefSeq library used for alignment. (E) Enriched putative Tcf21 interactions, color-coded according to their GO annotations for biological processes. (F) Map of identified Tcf21 phosphorylation sites on serine residues.
	Fig. 4. Markers of epicardial precursor cells identified by high-throughput sequence analysis. (A-P) ISH of selected genes from the RNA-seq upregulated dataset showing expression within the PEO (arrowheads). Magnified lateral images of the heart region are shown (anterior right). (Q-FF) ISH of unique and known PE cell markers showing conserved expression patterns at earlier stages (stage 40) of epicardial development (ventral views, anterior top). cg, cement gland. Scale bars: 1 mm.
	Fig. 5. Tcf21 functions to promote epicardial maturation and epithelialization. Transverse sections (dorsal to the top) through the cardiac region of stage 46 CA:GFP transgenic embryos showing myocardium (green), cell nuclei stained with DAPI (blue) and immunohistochemical stains for (A-B″) aPKC, (C-D″) vimentin and (E-F″) laminin in red. Arrowheads highlight epicardial cells with increased or mislocalized staining compared with controls. Boxes indicates the regions magnified in A′-F′ and A″-F″. a, atrium; oft, outflow tract; ven, ventricle. Scale bars: 50 μm in A,C,E; 10 μm in A′,C′,E′; 5 μm in C″; 2.5 μm in A″,E″.
	Fig. 6. Epicardial cell migration is regulated by Tcf21. (A,B) Brightfield images of control (A) and Tcf21-depleted (B) cardiac explant on collagen gel. The dashed line indicates the extent of epicardial outgrowth after 16 hours of culture. (C,D) Imaris tracking software depicts individual epicardial cell movements during culture, comparing control (C) and Tcf21-depleted (D) epicardial behavior. (E,F) A significantly increased rate of epicardial cell migration (E) and total cell outgrowth (F) in a 48-hour culture period was observed in Tcf21-depleted as compared with control embryos. ****P<0.0001, two-tailed unpaired non-parametric Mann-Whitney test. Mean s.d. from a total of three independent experiments with the number of (E) migrating cells from 27 control and 34 Tcf21-depleted explants or (F) hearts assayed indicated in parentheses. epic, epicardial cells; oft, outflow tract; ven, ventricle. Scale bar: 200 μm.
	Fig. 7. Tcf21 is required for correct specification of precursor PE cells and for epicardial maturation. (A-F‴) Transverse sections through the cardiac region of stage 46 CA:GFP transgenic Xenopus embryos stained for cytokeratin (red) and with DAPI (blue), with the myocardium expressing GFP under the cardiac actin promoter. Punctate cytokeratin staining is observed in migrating PE cells (arrowheads). (G-J′) ISH of stage 46 embryos showing the PEO markers (red arrowheads) Lhx9 and Itga4; lateral views with head facing right. G′-J′ are magnifications from G-J. (K-R) ISH of Tbx18 and Tcf21 in stage 46 embryos, showing lateral magnified views of hearts (K,L,O,P; anterior right) and transverse gelatin vibratome sections (M,N,Q,R; dorsal to top). Red arrowheads indicate PEO expression, green arrowheads migrating epicardial cell expression. Note the thickened and more rounded appearance of the Tbx18-expressing layer in Tcf21-depleted embryos (N). (S-V) ISH of Tbx18 and Tcf21; ventral images of younger, stage 40 embryos (anterior right). Arrowheads indicate PEO expression and duplication/expansion thereof in Tcf21-depleted embryos (U,V). (W) The number of migrating epicardial cells from Tcf21-depleted cardiac explants (17 control hearts, 23 Tcf21-depleted hearts, two independent experiments) is significantly increased compared with control (****P<0.0001, two-tailed unpaired non-parametric Mann-Whitney test). Mean s.d. (X-Y″) The increased number of DAPI-stained nuclei in Tcf21-depleted explants, compared with controls, is not due to an increase in proliferation as shown by the absence of phospho-Histone H3 staining. a, atrium; oft, outflow tract; peo, proepicardium; v, ventricle. Scale bars: 50 μm in A; 10 μm in C′; 500 μm in K; 100 μm in M; 2 mm in G,S; 1 mm in G′,X.
	Model for the role of Tcf21 in proepicardial cell specification and maturation. In control embryos, PE cells migrate onto the heart surface and mature into the epithelial-like epicardium (top panel). In the absence of Tcf21, PE cell numbers are increased and these cells retain their precursor cell characteristics upon migration onto the heart (bottom panel).
	Fig. S1. Conserved epicardial development in Xenopus. (A-F9) SEM images of ventral views of Xenopus epicardial development at (A) stage 39, pre-PEO attachment, (B) stage 41, (C) stage 42, (D) stage 43, (E) stage 44, (F) stage 46. Red arrowheads denote attached PEO (B,D) and the white dashed line the extent of epicardial sheet migration over the heart surface (C9-E9). A9-F9 are magnified views of the ventricular surface. (G-J) ST-specific photoconversion of Kikume in stage 40 embryo (G) results in the migration of red fluorescent cells (white arrowheads) onto the myocardial surface at stage 43 (H-J); ventral view of cardiac region, hearts outlined with white dashed line. (K-P) Bmp2 expression detected by ISH. Lateral (K,L) and transverse (M-P) views of stage 45 embryos showing Bmp2 localized to the atrioventricular sulcus near the outflow tract (AVS/OFT) and the inflow tract-ventricular sulcus (IFT/ VS). Hearts outlined with red dashed lines. e, eye; h, heart; gb, gall bladder; li/st, liver/septum transversum; oft, outflow tract; ven, ventricle. Scale bars: 1 mm in H,K,M; 250 μm in G; 500 μm in O.
	Fig. S2. Analysis of Wt1-depleted embryos. (A-H) Brightfield images of live embryos at (A,B) stage 28, (C,D) stage 40, (E,F) stage 43, (G,H) stage 46. (I) Quantification of pericardial edema observed in embryos at stages 44-46; total embryos in brackets, two independent experiments, P<0.0001 by two-tailed Fisher exact test. Likelihood ratio c2 N=362, DF=1=259.856; P<0.0001, Pearson c2 N=362, DF=1=234.094; P<0.0001. (J-M) SEM images of stage 45 hearts depicting normal epicardial sheet migration over the heart in both (J,L) control and (K,M) Wt1-depleted embryos. Boxed areas indicate the regions of the ventricular surface magnified in L,M. Scale bar: 10 μm.
	Fig. S3. Validation of Tcf21-MO specificity. (A) Schematic showing relative MO positioning on Xenopus Tcf21 mRNA; numbers denote nucleotide base pair positions. Validation of MO-specific Tcf21 translation inhibition by V5 western blot on stage 12 embryos; representative blot from four independent experiments. Shp2 protein provided a loading control, in vitro translated Tcf21-V5 a positive control. Molecular weight standards are included to right of blot.
	Fig. S3. Validation of Tcf21-MO specificity. (B-J) Rescue of embryonic Tcf21-overexpression phenotype (stunted AP axis development, membrane ruffling, reduced ocular development, prominent cement gland) by (D,G,J) Tcf21-MO injection relative to (C,F,I) control 5-base mismatch MO and (B,E,H) uninjected embryos; representative images from two independent experiments. mGFP RNA provided an RNA injection control (H-J); anterior to left of panel. (K-N) In situ hybridization on whole control and Tcf21-depleted embryos (stage 26-28) demonstrating correct specification of heart mesoderm by (K,M) Nkx2.5 and (L,N) Tbx5 expression. bHLH, basic helix-loop-helix domain; hp, heart primordium. Scale bars: 10 mm in H; 2 mm in K-N.
	Fig. S7. RNA-seq validation of cardiac transcriptome by RT-PCR. (A) Gene Ontology (GO) term analysis on Tcf21-depleted upregulated RNA-seq dataset ≥1.8 fold (GOrilla and ReviGO). (B) RT-PCR of stage 45 cardiac cDNA from control and Tcf21- depleted embryos from independent experiments to validate RNA-seq candidates. T, PCR control lane.
	Fig. S8. Distribution of aPKC in the PEO resembles that in Tcf21-depleted epicardial cells. Immunohistochemical staining of transverse cardiac sections showing that staining of aPKC and its accumulation in the cytosol of attached PEO cells (arrowheads) in (A) control embryos resembles that in (B) Tcf21-depleted PEO cells and Tcf21-depleted migrating epicardial cells (see Fig. 5B0). m, myocardium. Scale bar: 10 μm.
	Fig. S9. Double-transgenic hearts used in epicardial migration assay. Cardiac explants were taken from double CA:eGFP and CMV:dsRED embryos to conduct epicardial migration live assays for more accurate tracking by Imaris software. (A) Brightfield, (B) GFP, (C) dsRED and (D) overlay. Confirmation that migrating cells are not cardiomyocytes is by the absence of GFP expression (B). Scale bar: 100 μm.
	Fig. S10. F-actin stress fiber enrichment in migrating epicardial explant cultures. (A-D) Phalloidin staining of epicardial cells in cardiac explant cultures from control (A,B) and Tcf21-depleted (C,D) embryos. White outline depicts extent of epicardial migration after 48 hours. Magnified images of boxed views (B,D). (E) Two-tailed unpaired non-parametric Mann-Whitney statistical test analysis showing increased phalloidin-enriched epicardial area between control and Tcf21-depleted explant cultures, P=0.0823. (F) RT-PCR from explant cultures after removal of the heart, confirming that migrating cells express known epicardial genes Tbx18 and Lhx9 in both control and Tcf21-depleted cultures.

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