NC-Cm cells regulate Notch signaling during trabeculation. a–f Ventricle sections of a 3 dpf embryo from a sox10:tagRFP transgenic line crossed with the Notch reporter Tp1:GFP line, immunostained for the cardiomyocyte marker MF20. NC-derived lineages (tagRFP) did not show high levels of Notch response (GFP). Arrows indicate trabeculating NC-Cms that were next to a Notch-activated cardiomyocyte (arrowheads). Scale bar = 10 µm. g qPCR gene expression of isolated 4 dpf hearts from NC-Cm-ablated and control siblings. Values are delta delta Ct computations. Bars represent mean of four biological replicates. Points are individual experiments. Delta Ct values used to compute standard T test significance (P-values shown) between NC-Cm ablated and control delta Ct values. h–k Jag2b fluorescent in situ hybridization in a magnified ventricle section of 3 dpf NC-Cm lineage-labeled embryos. Scale bar = 5 µm. Image is representative of N = 17 embryos imaged and analyzed in probe positive in situs compared with probe negative embryos imaged and analyzed. l Quantification of jag2b fluorescent in situ signal in individual NC-Cm and Cm ventricle cells. Relative intensity was computed by the average intensity of all jag2b expression in the heart cells and then comparing with individual NC-Cm tRFP + and Cm GFP + cell intensities. Circles are individual cells from N = 3 embryos from three independent in situ experiments. Bar = mean. Standard T test was used for P-value. m Schematic model in which NC-Cms provide spatial patterning of trabeculation using Notch signaling components. During the transition to initiate trabeculae, NC-Cms provide a significant source of jag2b expression that triggers its neighbor Cm to repress protrusion and trabeculation initiation. This results in evenly, spatially distinct trabeculae branches at 5dpf. However, without NC-Cms and their major source of jag2b expression (although not exclusive as indicated by the remaining Cm expressing jag2b at 3dpf), more Cms are poised to protrude and create trabeculae, yielding conjoined branches and poor trabeculae spacing by 5 dpf
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