Fig. 2

A Precardiac Field Is Established during Regeneration and Supports CM Proliferation (A) The precardiac marker hand2 is expressed in cells coating the apical edge of the existing muscle at 3 dpa (violet stains). At 4 dpa, nkx2.5 and tbx20 are also expressed in cells distributed along the resection plane. By contrast, no new CM differentiation events are detected by anti-DsRed staining in cmlc2:nRFP zebrafish at 3 dpa (see Figure 1) and only sparse events at 4 dpa (Table S1). The pink background represents myocardial autofluorescence, which aids visualization of positive signals. (B) nkx2.5, tbx20, hand2, tbx5, and Mef2 (red nuclear stain) are each present in a domain of enhanced expression at the apical edge of the regenerate (brackets), an area of most recent myocardial differentiation events as assessed in the cmlc2:nRFP; cmlc2:EGFP strain. The ISH pattern of cmlc2 at 7 dpa is also shown to indicate differentiated muscle. (C) By 14 dpa, freshly differentiated RFP^cyto cells in cmlc2:nRFP; cmlc2:EGFP hearts appear smaller and remain at the apical edge as regeneration proceeds (brackets). Enhanced nkx2.5, tbx20, and hand2 expression remains detectable in this region. (D) Seven (left) and 14 dpa (right) ventricles from cmlc2:nRFP zebrafish stained for DsRed immunoreactivity (red) and BrdU incorporation (green). At 7 dpa, BrdU label is detectable only in newly differentiated RFP^cyto cells (arrowheads). By 14 dpa, BrdU is also detectable in RFP^nuc CMs (arrows). Scale bar = 100 μm.