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Fig. 1 Multiple secreted Wnt inhibitors sFrps and Dkks are induced following cardiac injury. (A) Expression levels of wnt inhibitors (dkk1b, dkk2, dkk3b, sfrp1a, sfrp1b, and sfrp2) and wnt ligands (wnt2ba, wnt4a, wnt6b, and wnt8a) during cardiac regeneration. raldh2 was used as a positive control (Kikuchi et al., 2011). Data are mean ± SEM from three biological replicates and three technical replicates. Student’s t-test, **P < 0.01. (B‒U) Confocal images displaying sFrp1, Dkk3, Dkk1b, and sFrp2 expression in uninjured and injured hearts at 3, 7, and 14 dpa. Dotted line indicates amputation line. Brackets indicate amputation plane. Scale bar, 100 µm. (B‒E) Small numbers of epicardial cells adjacent to the injury site induce sFrp1 expression (arrows) at 3 dpa, whereas sFrp1 expression is little detectable in uninjured ventricles. At 7 dpa, sFrp1 expression is expanded throughout the ventricular epicardium and detectable within the regenerate (arrows). By 14 dpa, sFrp1 expression is largely restricted to the epicardial sheet surrounding the regenerate (arrows). (F‒I) Dkk3 is little detectable in uninjured ventricles. At 3 dpa, Dkk3 is expressed in epicardial cells adjacent to the injury region. By 7 dpa, Dkk3 expression is enhanced and expanded in the epicardium enclosing the ventricle and the wound (arrows). Dkk3 expression remains in the epicardial sheet surrounding the regenerate (arrows) by 14 dpa. (J–Q) Whereas Dkk1b expression is not detectable in the uninjured ventricle in Tg(dkk1b:egfp) animals (J and N), some CMs express Dkk1b at the apical edge of the wound at 3 dpa (K and O). Enhanced Dkk1b expression is detectable in the apical edge cells of the regenerating myocardium at 7 dpa (L and P). Dkk1b expression remains in a limited number of CMs within the regenerate at 14 dpa (M and Q). (R‒U) sFrp2 expression is detectable in the wounded heart at 3 dpa, enhanced at the apical cell edge cells of the injured myocardium at 7 dpa, and gradually reduced by 14 dpa. Faint expression of sFrp2 expression is detected in uninjured hearts (R).