Fig. 4

snai1b activation in Notch-negative cardiomyocytes undergoing delamination and trabeculation.

a At 4 dpf, myocardial Notch signaling (TP1) develops at the outer curvature of compact (cortical) layer, and discontinuation was observed when trabeculation sprouting occurs (dashed outlines in magnified sub-panels). Endocardial Notch mediates the ErbB2 signaling in trabecular cardiomyocytes (CMs) to activate Notch in the adjacent CMs and inhibit their delamination. On average, 1.3 snai1b⁺ CMs were found per ventricle (see (e), n = 6). b After ISO treatment, the gap between Notch-positive (⁺) CMs widened, and snai1b activation (arrowheads, via in situ hybridization) was observed in the Notch-negative (⁻) CMs in the compact layer (29.5%) and trabeculation (50%), and a small number of apically delaminated CMs (3.4%) (see Fig. S3a–c). On average, 12.6 snai1b⁺ CMs were found per ventricle (see e, f, n = 7). c Co-treatment with ErbB2 inhibitor, PD168393, reduced trabeculation (see Fig. S3d) but did not inhibit snai1b activation (arrowheads, see e, f). d Schematic summarizing the Notch and snai1b expression pattern in (a–c). e, f At 4 dpf, ISO treatment led to a 10-fold increase in the total number of snai1b⁺ CMs per ventricle. ErbB2 inhibitor (PD) co-treatments at 55 hpf (n = 5) or 72 hpf (n = 7) did not change the total number of snai1b⁺ CMs. Rather, they reduced the total trabecular population from 50 to 37% and 39%, respectively. All values in panel e are displayed with mean and standard error of mean (SEM). p-value is displayed for each comparison. Ordinary one-way ANOVA followed by Šídák’s multiple comparisons test on the means was applied to determine statistical significance. Source data are provided as a Source Data file. Anatomic labels: V ventricle, A atrium, AV atrioventricular canal.