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Figure 1 The phosphoinositide 3-kinase (PI3K) pathway is required for cardiac fusion. Dorsal views, anterior to the top, of the myocardium labeled with myl7 (A–E) at 22 somite stage (s) or Tg(myl7:egfp) (A'–E') at 20s. In contrast to a ring of myocardial cells in DMSO-treated embryos (A, A'), in embryos treated with PI3K inhibitors LY294002 (LY, B, B'), Dactolisib (Dac, C, C'), or Pictilisib (Pic, D, D') at bud stage or injected with dnPI3K mRNA (750 pg) at the one-cell stage (E, E') cardiac fusion fails to occur properly with embryos displaying either cardia bifida (B, C) or fusion only at the posterior end (D, E). Graphs depict the percentage (F) and range (G) of cardiac fusion defects in control and PI3K-inhibited embryos. Dots represent the percent of embryos with cardiac defects per biological replicate. Total embryos analyzed n = 37 (DMSO), 31 (20 μM LY), 39 (40 μM Dac), 38 (50 μM Pic), and 86 (dnPI3K). Blue – cardiac ring/normal; orange – fusion only at posterior end/mild phenotype, red – cardia bifida/severe phenotype. (H) Representative immunoblot and ratiometric analysis of phosphorylated Akt (pAkt) to Akt protein levels in DMSO- and LY-treated embryos reveals a dose-dependent decrease in PI3K activation. Bar graphs indicate mean ± standard error of the mean (SEM), dots indicate pAKT/AKT ratio per biological replicate, normalized to DMSO. Three biological replicates per treatment. One-way analysis of variance (ANOVA) tests – letter changes indicate differences of p < 0.05 (F, H). Scale bars, 40 μm (A–E), 42 μm (A'–E'). Raw data and full p-values included in the source file.