Visualization of LPM cells in the Tg(fli1a:EGFP)^y1 zebrafish embryos at 17 hpf

(A and B) DIC (A) and confocal stack fluorescence (B) images showing lateral view of the Tg(fli1a:EGFP)^y1 embryo at 17 hpf (16 somite stage). Note that EGFP fluorescence labels the anterior LPM and posterior LPM (B).

(C–E) Confocal stack fluorescence images showing dorsal view of the Tg(fli1a:EGFP)^y1 embryo. Anterior (C), middle (D), and posterior (E) parts of the embryos are shown.

ALPM, anterior lateral plate mesoderm; PLPM, posterior lateral plate mesoderm. Scale bars, 100 μm.

Preparation of the dechorionated zebrafish embryos at 17 hpf

(A) Set up zebrafish to breed in the separate breeding tank (step 1).

(B) Initiate breeding by removing the divider separating the male and female fish (step 2).

(C) Rinse the embryos on the strainer with 0.03% sea salt solution (step 5).

(D) Cleaned embryos on the strainer.

(E) The embryos transferred into a 10-cm petri dish containing E3 medium (step 6).

(F and G) The 17 hpf embryos transferred into a beaker containing E3 medium (step 8b).

(H) The embryos treated with the Pronase solution (step 8d). Note that the chorions start to break open (arrow).

(I) Dechorionated embryos in a 10-cm petri dish containing E3 medium (step 8g).

LPM cells adhering to the fibronectin-coated dish

(A) FluoView FV1000 confocal upright microscope system.

(B) The cells derived from 17 hpf embryos were left to adhere to the fibronectin-coated dish for 13 h and then stained with anti-GFP (green) and anti-vinculin (red) antibodies. Arrow indicates GFP-positive LPM cell. Scale bar, 10 μm.

Sequence of image processing by ImageJ for analyzing the number and size of focal adhesions

Scale bar, 10 μm.

Rap1b regulates integrin-mediated adhesion of LPM cells to fibronectin-coated dishes

(A) Cells dissociated from 17 hpf wild type (upper) and rap1b^ncv124 (lower) Tg(fli1a:GFP)^y1 embryos. Merged images of GFP (green)/vinculin (red)/F-actin (blue), those of GFP (green)/vinculin (red), and vinculin (red) and F-actin (blue) images are shown, as indicated. Scale bars, 10 μm.

(B) Number of vinculin-marked focal adhesions per cell, as observed in (A).

(C) Total area of vinculin-marked focal adhesions per cell, as observed in (A).

(D) Number of vinculin-marked focal adhesions, as observed in (A). Data are shown as means ± s.d. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (B)-(D) reuse parts of a figure from Rho et al. (Rho et al., 2019).