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Fig. S1. In situ hybridization of cxcl12b and cxcr4a in the regenerating heart. (A-F) In situ hybridization of cxcl12b (A,C,E) and cxcr4a (B,D,F) at 3 day (A,B), 5 day (C,D) and 7 day (E,F) post-amputation of the wildtype zebrafish heart. Expression of cxcl12b and cxcr4a were not detected. Scale bar: 50 mm. Fig. S2. Anti-Mef2 antibody is specific to CMs. (A-C) Anti-Mef2 staining (A), anti-GFP staining and merged image (C) of sections from the cmlc2a-EGFP fish heart. Seven sections from three hearts were examined, and 3216 Mef2-positive cells were all positive for cmlc2a-EGFP signal. (D-F) Anti-Mef2 staining (D), anti-GFP staining (E) and merged image (F) of sections from the fli1-EGFP fish heart. Five sections from four hearts were examined, and 2268 Mef2-positive cells were all negative for fli1-EGFP signal. Fig. S3. cxcl12a is expressed in the epicardial cells. (A,B) In situ hybridization for wt1b (A) and anti-DsRed2 staining (B) of adjacent sections from cxcl12a-DsRed2 reporter fish. (C,C9) A neighboring section, which is doubly stained for wt1b mRNA and anti-DsRed2. C9 shows higher magnification image of the boxed area in C. Arrowheads indicate signals where wt1b signals and Dsred2 signals overlap. Dotted line indicates the amputation plane. Shown are representative images from three independent experiments. (D-F) MHC staining (D), antiDsRed2 staining (E) and merged image (F) from cxcl12a-DsRed2 reporter fish. Shown are representative images of multiple sections from two hearts. (G-I) fli1-EGFP reporter signal (G), cxcl12a-DsRed2 reporter signal (H) and merged image (I) from fli1-EGFP; cxcl12a-DsRed2 fish. Shown are representative images of multiple sections from two hearts. All images are from 3 dpa hearts. Scale bar: 50 mm. Fig. S4. FC131 effectively blocks directed migration of primordial germ cells in zebrafish. (A-D) In situ hybridization of vasa to visualize PGCs in control (A,C) and FC131-treated (B,D) zebrafish embryos at 18 hours post-fertilization. FC131 treatment was carried out between 8 and 18 hours post-fertilization. Lateral views (A,B) and ventral views (C,D) of typical samples from more than 50 embryos are shown. Black arrowheads point to PGCs localized at the region where the gonad develops, and white arrowheads point to mis-localized PGCs. Scale bar: 250 mm. (E) Dose-dependent activity of FC131. FC131 at 0, 4, 20, 40 and 400 nM were tested for PCG migration. Vertical axis represents percentage of embryos that exhibited abnormal PCG migration. Fig. S5. AFOG and MHC staining. (A-B9) MHC (brown) and acid fuchsin orange G (AFOG) staining of 14 dpa hearts of control fish (A,A9) and cxcr4b–/– fish (B,B9). Fibrin is visualized as red (arrows in A9 and B9). Small amount of fibrin is present similarly in control and cxcr4–/– fish at 14 dpa. Fig. S6. CXCR4 antagonist treatment did not affect cell proliferation. Quantitation of the number of proliferating cells in control and CXCR4 antagonist-treated hearts at 7, 14, 21 and 30 dpa. Vertical axis represents the number of Pcna-positive, proliferating cells per section. Same sections analyzed in Fig. 5C were used. P values at each time point are shown. Fig. S7. CXCR4 antagonist treatment did not induce cell death. (A-D) Apoptotic cells detected using the TUNEL method (green, arrowhead) and CM marker Mef2 (magenta) were simultaneously detected. Control (A,C) and CXCR4 antagonist-treated heart (B,D) at 7 dpa (A,B) and 14 dpa (C,D) were analyzed. Shown are typical images from three hearts at each time point. Dotted lines indicate the amputation plane. TUNEL-positive CMs were barely detectable in both control and Cxcr4-inhibited heart. Scale bar: 50 mm. Fig. S8. Photoconversion of the cmlc2a-Kaede heart. (A,B) Schematic representation of the cmlc2a-Kaede construct (A) and heart specific expression of green fluorescence of Kaede in 48 hours post fertilization cmlc2aKaede embryo (B). (C-H) Photoconversion of adult cmlc2a-Kaede heart. Bright field (C,F), green fluorescence (D,G) and red fluorescence (E,H) are shown, before (C-E) and after (F-H) photoconversion. Green fluorescence of Kaede (D) was converted to red fluorescence (H) by irradiation. Dotted line indicates the exposed heart. Fig. S9. CXCR4 antagonist-treatment does not affect epicardial gene expression. (A-D) aldh1a2 mRNA expression (A,B) and fli1-EGFP reporter signal (C,D) in adjacent sections from the control (A,C) and CXCR4 antagonist-treated (B,D) hearts at 3 dpa. Arrows and arrowheads in A and B indicate epicardial expression of aldh1a2 and fli1-EGFP-positive endocardial aldh1a2 expression, respectively. (E,F) aldh1a2 expression in the control (E) and CXCR4 antagonist-treated (F) hearts at 7 dpa. Arrows indicate aldh1a2 expression. (G-J9) wt1b expression in the control (G,G9,I,I9) and CXCR4 antagonist-treated (H,H9,J,J9) hearts at 3 dpa (G-H9) and 7 dpa (I-J9). Arrowheads indicate expression of wt1b. G9,H9,I9,J9 show higher magnification of the boxed areas in G,H,I,J. Dotted lines indicate the amputation planes. Scale bar: 50 mm. (K) Quantitation of wt1bexpressing epicardial tissue in the regenerating area. The ratio of the length of wt1b-expressing epicardial tissue (represented by blue lines in G,H,I,J), compared with the length of the surface of regenerating area was quantitated. n=3 at each time point.