Fig 1: Tbx18 Lineage Analysis after Cardiac Injury(A–K) Tbx18MerCreMer;R26RtdTomato (A–F and H–) and Tbx18MerCreMer;R26RlacZ (G) mice were injected with tamoxifen after apex resection.(A–F) Immunostaining was performed on Tbx18MerCreMer;R26RtdTomato hearts at 30 dps (A–D) and 60 dps (E and F). Arrows in (A), (B), (E), and (F) indicate tdTomato and cTNT double-positive cells in the apex at 30 dps (A and B) and 60 dps (E and F). Arrows in (C) and (D) indicate non-myocardial Tbx18 lineage (cTnT-) in the remote area (C) or in the apex of sham group (D).(B–D and F) Right-bottom corner images are high magnification of the areas indicated by arrows.(G) X-gal staining of Tbx18MerCreMer;R26RlacZ hearts revealed Tbx18-derived cardiomyocytes (arrows in G4–G6) in the apex at 60 dps. (G2), (G3), and (G4) are high magnification of the square areas in G1. (G5) and (G6) are high magnification of the square areas in (G4). Unnotched arrows (G2–G4) indicate X-gal negative cells.(H–K) Immunostaining on Tbx18MerCreMer;R26RtdTomato hearts at 21 dps (H) and 60 dps (I). tdTomato signals (arrows) are co-localized with SM-MHC in the apex at 21 dps and 60 dps (H and I). PECAM staining (unnotched arrows in J and K) is not co-localized with tdTomato (arrows) in the regenerative coronary at 21 dps and 60 dps. Right bottom corner images (H-K) are high magnification of areas indicated by notched arrows. Scale bar, 50 µm (white), 100 µm (black) and 20 µm (yellow).See also Figure S6.
Fig 2: Accumulated Tbx18-Expressing Cells in the Injured Region(A) DAPI staining at 3, 7, 14, 21, 30, and 60 dps.(B and C) Robust Tbx18-expressing cells (Tbx18H2B-GFP-positive) in the injury site during repair (arrows) with high density at 3–21 dps (B1–B4 and C1–C4).(D and E) Immunostaining in the injured site at 21 dps (D) and 60 dps (E). Tbx18H2B-GFP is not co-expressed with cTNT (D1 and E1) but is co-expressed with SM-MHC (D2 and E2), a-SMA (D3 and E3), and PDGFRß (D4 and E4). Tbx18H2B-GFP is not co-expressed with PECAM in the endothelial cells (D5 and E5). The top right corner images in (D) and (E) are high magnification of the areas outlined in each panel. Scale bar, 1mm (white) and 100 µm (yellow).See also Figures S4 and S5.
Fig 3: Activation of RA signaling pathway by RA biases the differentiation of hiPSC toward atrial cardiomyocytes. (A) RA was introduced on day 5 at the concentrations indicated, the effect on the expression of NPPA, MYL7, COUPTFII, KCNJ5, and CX40 was analyzed by qPCR. (B) 2 µM RA was introduced on day 5 after the differentiation, and the expression of SHOX2, TBX18, TBX3, HCN4, ISL1, CX30.2, CACNB1, CACNA1A, KCNN4, KCNK2, KCND2, and SCN3B was quantitated by qPCR on day 21. The expression was normalized to that of GAPDH. Data are presented as ‘Mean±SD’ from at least 3 indepen-dent experiments with duplicate on each sample, with ns denoting non -significant, *denoting p<0.05, and **denoting p<0.01.
Fig 4: Inhibition of RA signaling pathway by BMS biases the differentiation of hiPSC toward Sinoatrial node-like cells. (A) BMS was introduced on day 5 at the concentrations indicated, the effect on the expression of NPPA, MYL7, COUPTFII, KCNJ5, and CX40 was analyzed by qPCR. (B) 5 μM BMS was introduced on day 5 after the differentiation, and the expression of SHOX2, TBX18, TBX3, HCN4, ISL1, CX30.2, CACNB1, CACNA1A, KCNN4, KCNK2, KCND2, and SCN3B was quantitated by qPCR on day 21. The expression was normalized to that of GAPDH. Data are presented as ‘Mean±SD’ from at least 3 independent experiments with duplicate on each sample, with ns denoting non-significant, *denoting p<0.05, **denoting p<0.01, and ***denoting p<0.001.
Fig 5: Inhibition of RA signaling pathway by BMS biases the differentiation of hiPSC to Sinoatrial node-like cells. BMS at 5 µM was introduced on day 5 after the differentiation, and the expression of COUPTFII (A, B), TBX18 (C, D), and TBX3 (E, F) was evaluated by IF. Representative images and corresponding quantitations showing that inhibition of RA signaling pathway by BMS decreases the percentage of COUPTFII (A, B) positive cardio-myocytes and increases the percentage of TBX18-(C, D) and TBX3-positive (E, F) cardiomyocytes. Scale bars=100 µm (400× magnification).
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