Fig. 5

Itgb1b-Mediated Adhesion to Fn Promotes PLPM Cell Contact with Somitic Cells

(A) Confocal fluorescence images of transverse sections in the posterior regions of 16 hpf Tg(fli1a:Myr-GFP);(phldb1:mCherry) embryos immunostained with anti-GFP (green), anti-mCherry (blue), and anti-β-catenin (red) antibodies. Left, merged image of GFP, mCherry, and β-catenin; middle, merged image of GFP and β-catenin; right, β-catenin image. Boxed areas labeled with a and b are enlarged at the bottom.

(B) Confocal fluorescence images of 16 hpf WT embryos immunostained with anti-Fn antibody. Fn images in the xy-, xz-, and yz-planes. Blue and pink dotted lines on the xy-plane indicate the position of the corresponding xz- and yz-planes, respectively.

(C) Confocal fluorescence images of transverse sections in the posterior regions of 16 hpf Tg(fli1a:Myr-GFP) embryos immunostained with anti-GFP (green) and anti-Fn (white) antibodies. The merged images of GFP and Fn, GFP image, and Fn image corresponding to the boxed areas are enlarged at the second, third, and fourth rows, respectively.

(D) Expression pattern of itgb1b in the WT embryos at 15 hpf. Dorsal view of flat-mounted embryo with anterior to the top. The boxed area is enlarged below. Arrows indicate PLPM.

(E) Confocal stack fluorescence images of PLPM cell migration in itgb1b^mi371 mutant (upper) and fn1a/fn1b morphant (lower) with the Tg(fli1a:GFP) background at 14 hpf and their corresponding subsequent time-lapse images, obtained at the indicated time points (h:min). Dorsal views with anterior to the upper-left. Boxed areas are enlarged in the insets. Arrowheads indicate the cells that remained behind the group of migrating cells.

(F) Confocal fluorescence images of 16 hpf control MO-, itgb1b MO-, and fn1a/fn1b MO-injected Tg(Myr-fli1a:GFP) embryos immunostained with anti-GFP (green) and anti-Fn (white) antibodies are shown as in Figure 4B.

(G–I) Morphology of PLPM cells migrating along the somiteboundaries, as observed in (F). Cell spreading areas (G), cell length relative to the direction of cell migration (H), and cell length perpendicular to the direction of cell migration (I). Each dot represents an individual cell. Error bars indicate means ± SD. Control MO, n = 27; itgb1b MO, n = 24; fn1a/fn1b MO, n = 27. ^∗∗∗p < 0.001; n.s., not significant.

(J) Confocal fluorescence images of transverse sections in the posterior region of 16 hpf control MO-, itgb1b MO-, and fn1a/fn1b MO-injected Tg(fli1a:Myr-GFP);(phldb1:mCherry) embryos immunostained with anti-GFP (green), anti-mCherry (magenta), and anti-β-catenin (blue) antibodies. Left, the merged images of GFP, mCherry, and β-catenin; right, enlarged merged images (upper) and enlarged GFP images (lower) corresponding to the boxed areas.

(K) Contact length of PLPM cells with somitic cells in the embryos injected with control MO, itgb1b MO, and fn1a/fn1b MO, as observed in (J). Each dot represents an individual cell. Error bars indicate means ± SD. Control MO, n = 27; itgb1b MO, n = 24; fn1a/fn1b MO, n = 27. ^∗∗∗p < 0.001.

Scale bars: 50 μm (A and C, enlarged images in F (left)); 10 μm (enlarged images in A, C, and F (right), J); 25 μm (B); 200 μm (D); 100 μm (E and F).