The myocardium utilizes Pdgfra-PI3K signaling to steer towards the midline during heart tube formation

Figures

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  Fig. 1.. The PI3K pathway is required for cardiac fusion. A-E 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 (750pg) 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). F, G 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 (20μM LY), 39 (40μM Dac), 38 (50μM Pic), 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.…

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  Fig 2:. PI3K is required in the myocardium throughout cardiac fusion. A, B 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 (clear). The myl7 promoter restricts dnPI3K expression to the myocardium in Tg(myl7:dnPI3K) embryos. D-G Dorsal view of the myocardium labeled…

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  Fig 3:. PI3K signaling regulates the medial movement and velocity of the myocardium during cardiac fusion. A-D Time points from a representative time-lapse video of myocardial cells visualized with the Tg(myl7:egfp) transgene in embryos treated with DMSO (A, B, Video-1) or 20μM LY (C, D, Video-2) from bud - 22s. 3D reconstructions of confocal slices (A, C) reveal the changes in conformation and location of the myocardium at 3 major stages of cardiac fusion: early medial movement towards the embryonic midline (A-A’, C-C’), posterior merging of bilateral populations (A”, C”) and anterior merging to form a ring (A”’, C”’). Representative tracks (B, D) show the paths of a subset of myocardial cells over ~2.5 hr timelapse. Yellow dots indicate the starting point of each track. E-H Graphs depict box-whisker plots of the velocity (E), efficiency index (F) and angle of movement (H) of myocardial cells. The direction of movement is visualized by rose plots (G). Myocardial cells in PI3K-inhibited (LY-treated) embryos show an overall direction of movement that is angular (60–90 degrees) and is slower than in DMSO-treated embryos. Scale bar, 60μm. 96 and 125 cells were analyzed from five DMSO- and…

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  Fig 4:. Myocardial membrane protrusions are misdirected in PI3K-inhibited embryos. A-B”” timepoints from representative timelapse videos (see Video-4) of myocardial cells whose membrane has been labeled with myl7:lck-eGFP (black), medial to the right, in a DMSO- (A-A””) or a 20μM LY- (B-B””) treated embryo. Red arrowheads indicate representative protrusions, which are oriented medially, coincident with the direction of movement in DMSO-treated embryos (A-A””) but are oriented in all directions in LY-treated embryos (B-B””). C, D Rose (C) and Bar (D) graphs displaying the orientation of membrane protrusions in DMSO- (left) or LY- (right) treated embryos. The length of each radial bar in (C) represents the percentage of protrusions in each bin. Bar graph displays the total percentage of forward or backward protrusions. Forward protrusions: 270–90 degrees, pink. Backward protrusions: 90–270 degrees, black. n = 425 protrusions from 11 cells in 5 embryos (DMSO), and 480 protrusions from 11 cells in 4 embryos (20μM LY). Fisher’s exact test, P value < 0.0001. Error bars, mean ± SEM. Scale bar, 30 μm. Raw data and full p-values included in the source file.

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  Fig 5:. Pdgfra activates and genetically interacts with PI3K signaling to regulate cardiac fusion. A, B Immunoblot and ratiometric analysis of phosphorylated Akt (pAkt) compared to total Akt levels reveals reduced pAkt levels in loss-of-function pdgfrask16 heterozygous (−/+) or homozygous (−/−) mutant embryos at 22s (A), and elevated pAkt levels at 22s when PDGF signaling is activated with the hs:pdgfaa transgene (B). Bar graphs display averages from three separate experiments. C-E Dorsal views, anterior to the top, of the myocardium labeled with myl7 at 22s. In contrast to a normal ring of myocardial cells in wild-type embryos treated with 10μM LY starting at bud stage (C) or pdgfra heterozygous embryos (D), when pdgfra heterozygous mutants are exposed to 10μM LY, cardiac fusion is defective with embryos displaying severe phenotypes such as cardia bifida (E). F Bar graph depicts the average distribution of cardiac fusion defects in DMSO-treated wild-type and pdgfra heterozygous mutants as well as 10μM LY-treated wild-type and pdgfra heterozygous mutant embryos. The total number of embryos examined over three separate biological replicates are 47 (DMSO, +/+), 25 (DMSO, −/+), 36 (10μM LY,…