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. 2023 Nov 3;12:e85930. doi: 10.7554/eLife.85930

Figure 2. Phosphoinositide 3-kinase (PI3K) is required in the myocardium throughout cardiac fusion.

Graphical representation of the PI3K inhibitor addition (A) and wash-out (B) experiments used to determine the developmental stage over which PI3K is required. In (A) LY is added to embryos at different developmental stages and incubated until 22s, when cardiac fusion is assessed. In (B), LY is added at bud stage and washed-out at different developmental stages, after which embryos are incubated in normal media till 22s, when cardiac fusion is assessed. Bar graphs indicate the average proportion of embryos displaying different phenotypes. Blue – cardiac ring/normal; orange – fusion only at posterior end/mild phenotype, red – cardia bifida/severe phenotype. n = 45 embryos per treatment condition from three biological replicates. (C) Schematic outlines experimental design to test requirement for PI3K in the myocardium. Pink – cells with the Tg(myl7:dnPI3K) transgene. F0 animals are mosaic for the transgene, while all cells in F1 embryos either have the transgene (pink) or do not (white). The myl7 promoter restricts dnPI3K expression to the myocardium in Tg(myl7:dnPI3K) embryos. (D–G) Dorsal view of the myocardium labeled with myl7 in embryos at 22s from four different founder pairs (D–D', E–E', F–F', G–G'). F1 embryos without the Tg(myl7:dnPI3K) transgene (as determined by genotyping) display normal cardiac fusion (D–G, n = 23/24, 16/16, 16/16, 16/16, per founder pair), while F1 siblings with the Tg(myl7:dnPI3K) transgene display cardiac fusion defects (D'–G', n = 6/6, 13/13, 11/11, 13/13), indicating that PI3K signaling is required in myocardial cells. (H) Graph indicating the average % of wild-type and Tg(myl7:dnPI3K)+ embryos with cardiac fusion defects. Letter difference indicates a significant Fisher’s exact test, p = 5.56 × 10−31. Scale bar, 40 μm.

Figure 2—source data 1. Statistical source data for Figure 2.

Figure 2.

Figure 2—figure supplement 1. The morphology of endoderm is not compromised in phosphoinositide 3-kinase (PI3K)-inhibited embryos.

Figure 2—figure supplement 1.

Dorsal views, anterior to the top, of the anterior endoderm labeled with axial (A–C) or the Tg(sox17:eGFP) transgene (E–J) at 30s (A–G) or 22s (I, J). Embryos incubated with either DMSO (A, E, I), 15 μM LY (B, F), or 25 μM LY (C, G, J) from the bud stage to 30s (A–H) or 22s (I–J) show no observable difference in the appearance or width of the anterior endoderm. Box-whisker plots display median width of the anterior endoderm from D, H, respectively. n = 47 (axial) and 42 (Tg(sox17:egfp)) embryos per inhibitor concentration from three separate incubations. Yellow lines: width of the endodermal sheet. Purple dots (D, H) indicate individual embryos. No letter differences indicate p-value >0.05 as tested by one-way analysis of variance (ANOVA). High-resolution confocal images of the endoderm at 22s (I, J) further reveals no changes in continuity of the endoderm layer. Three-dimensional reconstructions of the anterior endoderm in DMSO- and 25 μM LY-treated embryos (I, J). Magnifications of I, J with XZ and YZ transverse slices (I’, J’). Tg(sox17:eGFP) transgene = green, blue = DAPI. Scale bars, 60 (A–C), 50 (E–G), 70.6 (I, J) μm. Raw data and full p-values included in the source file.
Figure 2—figure supplement 1—source data 1. Statistical source data for Figure 2—figure supplement 1D, H.
Figure 2—figure supplement 2. Phosphoinositide 3-kinase (PI3K) activity in myocardial cells.

Figure 2—figure supplement 2.

(A–B) Three-dimensional confocal reconstructions of the myocardium at 20s in DMSO- (A) and LY- (B) treated Tg(myl7:lck-egfp) embryos in which a myl7:PH-mkate2 plasmid was injected to mosaically visualize PI3K activity. PH-mkate2 translocates to the membrane when PI3K activity produces PIP3. In DMSO-treated embryos PH-mkate2 was often found at the membrane enriched asymmetrically (A'–A''') indicating PI3K activity in the myocardium, while in LY-treated embryos PH-mkate2 was defuse in the cytoplasm of myocardial cells (B''' arrowhead) or enriched in subcellular organelles (B''' arrow). A'–A''', B'–B''' are magnifications of A, B, respectively, showing both lck-emgfp and PH-mkate2 (A', B'), PH-mkate2 only (A'', B''), or PH-mkate2 intensities as a heat-map (A''', B'''). (C–E) Graphs depict the average fluorescent intensity for lck-emGFP (green) and PH-mkate2 (red) at different points across the length of a labeled cell, starting with the side of highest mkate2 fluorescence, in DMSO- (C) and LY- (D) treated embryos. The fluorescent intensity of mkate2 normalized to emGFP fluorescence at each point along the line (E) reveals an asymmetrical enrichment of PH-mkate2 at the membrane (0–20%) in DMSO-treated embryos, compared to LY-treated embryos in which PH-mkate2 is enrich in the cytoplasm, the middle of the cell (20–70%). 17 and 13 cells from four DMSO- and four LY-treated embryos were analyzed. Scale bars, 30 μm. Raw data and full p-values included in the source file.
Figure 2—figure supplement 2—source data 1. Statistical source data for Figure 2—figure supplement 2C–E.
Figure 2—video 1. PH-mkate2 is localized asymmetrically at the membrane of myocardial cells in DMSO-treated embryos, but is found in the cytoplasm and subcellular organelles in LY-treated embryos.
Download video file (212.3KB, mp4)
Representative time-lapse movie of myocardial cells expressing myl7:PH-mkate2 at 20 s in DMSO- (A) and 20 µM LY- (B) treated embryos. Time-lapse images are a three-dimensional reconstruction of confocal slices taken at 3:30 min intervals, beginning at 20 s.