|
Live-imaging of CM protrusion into the injured tissue during zebrafish heart regeneration. A Schematic of the experiment illustrating vibratome sectioning of cryoinjured hearts, slice culture, and live imaging. Created in BioRender. Beisaw, A. (2025) https://BioRender.com/ypwjv2r. B GFP staining in thin sections of Tg(myl7:LIFEACT-GFP) (left) and Tg(myl7:actn3b-EGFP) (right) ventricles at 10 dpci. LUT (look-up table) images depict GFP intensity color-coded according to the scale within the image. The LUT is linear and covers the full range of the data. Yellow boxes denote the cortical/trabecular CM focus area of time-lapse imaging. C Time-lapse imaging of Tg(myl7:LIFEACT-GFP)+ cortical CMs at 10 dpci. Yellow arrowhead follows a CM protrusion that displays a net positive migration into the injured area and the white arrowhead follows a CM protrusion that displays a net negative migration away from the injured area (IA). D Quantification of track displacement from CM protrusions at the border zone in Tg(myl7:LIFEACT-GFP)+ cortical CMs at 10 dpci (n = 114 protrusions from 6 ventricles) and Tg(myl7:actn3b-EGFP)+ trabecular CMs at 3 (n = 76 protrusions from 5 ventricles) and 10 (n = 84 protrusions from 4 ventricles) dpci. Data are presented as violin plots of all points with solid gray lines indicating the median and dotted gray lines indicating 25th and 75th percentile. P-values were calculated using a Kruskal–Wallis test with Dunnett’s multiple comparisons test. Source data are presented in the Source Data file. E Distribution of tracked CM protrusions with a net positive displacement (> 1 μm into the injured area), net negative displacement (> 1 μm away from the injured area), or net zero displacement (<1 μm) comparing t = 0 and t = 12 h of live-imaging in Tg(myl7:LIFEACT-GFP)+ cortical CMs at 10 dpci (n = 114 protrusions from 6 ventricles) and Tg(myl7:actn3b-EGFP)+ trabecular CMs at 3 (n = 76 protrusions from 5 ventricles) and 10 (n = 84 protrusions from 4 ventricles) dpci. Source data are presented in the Source Data file. F Immunostaining of myosin heavy chain (MHC) and embryonic CMHC (embCMHC) in thin sections of a ventricle at 10 dpci. The white dotted line denotes the outline of the wound. Blue arrowheads point to cortically-located CMs and the blue line outlines the increased invasion of cortically-located CMs into the injured tissue. G Quantification of total distance traveled by tracked CM protrusions in Tg(myl7:LIFEACT-GFP)+ cortical CMs at 10 dpci (n = 114 protrusions from 6 ventricles) and Tg(myl7:actn3b-EGFP)+ trabecular CMs at 3 (n = 76 protrusions from 5 ventricles) and 10 (n = 84 protrusions from 4 ventricles) dpci. Data are presented as violin plots of all points with solid gray lines indicating the median and dotted gray lines indicating 25th and 75th percentile. P-values were calculated using a Kruskal–Wallis test with Dunnett’s multiple comparisons test. Source data are presented in the Source Data file. H Maximum speed measured in tracked CM protrusions Tg(myl7:LIFEACT-GFP)+ cortical CMs at 10 dpci (n = 114 protrusions from 6 ventricles) and Tg(myl7:actn3b-EGFP)+ trabecular CMs at 3 (n = 76 protrusions from 5 ventricles) and 10 (n = 84 protrusions from 4 ventricles) dpci. Data are presented as violin plots of all points with solid gray lines indicating the median and dotted gray lines indicating 25th and 75th percentile. P-values were calculated using a Kruskal–Wallis test with Dunnett’s multiple comparisons test. Source data are presented in the Source Data file. Scale bars: 100 μm in (B and F), 20 μm in (C).
|
