Supplementary Figure 4: Prechordal plate cell movements and neural plate positioning in wild type embryos overexpressing CA-Mypt within the yolk syncytial layer.

(a) Schematic illustration of CA-Mypt and H2A-mCherry mRNA injection into the yolk syncytial layer (YSL) of an embryo at high stage (3.3 hpf). (b) Confocal images of the enveloping layer (EVL)/YSL epiboly progression in F-actin labeled Tg(actb1:GFP-UtrCH) wild type (wt) control (lower panel) and embryos injected with constitutively active myosin II phosphatase mRNA into the YSL (CA-Mypt, upper panel) at 8 hpf; both embryos were co-injected with H2A-mCherry mRNA into the YSL to mark YSL nuclei. (c) Quantification of the average advancement (μm min⁻¹) of the EVL margin of wt control and CA-Mypt injected embryos between 7 and 9 hpf; Student’s t-test; ^∗∗∗, P < 0.001; n = 4 embryos; error bars, s.e.m. (d) Fluorescence images of a Tg(gsc:GFP) embryo overexpressing CA-Mypt and H2A-mCherry (magenta, arrows) within the YSL, also showing H2A-BFP expression within all nuclei and GFP-expression in gsc-expressing prechordal plate (ppl) progenitors (green, white outline) at a representative time point during gastrulation (t = 75 min, 6.25 hpf); dorsal and sagittal (dorsal up) sections through the embryo (yellow tags in upper panel mark sagittal section plane in lower panel); animal (AP) and vegetal pole (VP) indicated by arrows; Scale bar, 100 μm. (e) Number of internalized ppl cells in Tg(gsc:GFP) embryos overexpressing CA-Mypt within the YSL (blue curve, n = 4 embryos) versus wt embryos (green curve) plotted from 6 to 8 hpf (120 min); error bars, s.e.m. (f) Directional correlation of ppl cell movements in a wt embryo overexpressing CA-Mypt within the YSL at a representative time point during gastrulation (t = 77.40 min, 6.8 hpf); ppl cells are visualized as arrows in a 2D plot and color-coded according to their 3D correlation values between 1 (red, maximum correlation) and −1 (blue, minimum correlation); every 3rd cell is plotted; AP, animal pole; VP, vegetal pole; Scale bar, 50 μm. (g) Average degree of alignment of ppl movements in embryos overexpressing CA-Mypt within the YSL (magenta curve/squares, n = 3 embryos) versus wt embryos (green curve/dots, see Supplementary Fig. 1c) plotted from 6 to 8 hpf (120 min); the order parameter corresponds to the degree of alignment ranging from 0 (disordered movement) to 1 (highly ordered movement); error bars, s.e.m. (h) Mean ppl cell instantaneous speed and directionality in CA-Mypt injected [gray bar graphs, n = 4 embryos; P(speed) = 0.323, P(dir) = 0.702] versus wt (white bar graphs, see Supplementary Fig. 2d) embryos plotted over 120 min (6 to 8 hpf) as bar graphs; error bars, s.e.m.; Student’s t-test for all graphs; ns (not-significant), P > 0.05. (i) Anterior neural anlage in embryos overexpressing CA-Mypt within the YSL marked by whole-mount in situ hybridization of otx2 mRNA expression at consecutive stages of gastrulation from 70% epiboly to bud stage (7–10 hpf); posterior axial mesoderm was detected by no tail (ntl) mRNA expression (yellow arrows); animal pole (dorsal down), dorsal (animal pole up) and lateral (dorsal right) views are shown; red arrowhead marks the most anterior edge of the neural plate; 200 μm. (j) Quantitative analysis of neural plate position during gastrulation in embryos overexpressing CA-Mypt in the YSL versus wt embryos. The angle (°) between the vegetal pole and the anterior border of the otx2 expression domain was measured for embryos at different stages during gastrulation (i) and plotted as box-whisker graphs; n, embryos analyzed from 4 independent experiments; Student’s t-test (P value indicated) for all graphs comparing same stages; ^∗∗, P < 0.01; ^∗, P < 0.05, (ns) non significant, P > 0.05; n (wt, bud) = 36, n (wt, 90%) = 36, n (wt, 70%) = 29, n (CA-Mypt, bud; P = 0.49) = 16, n (CA-Mypt, 90%; P = 0.0259) = 22, n (CA-Mypt, 80%; P = 0.0016) = 34, n (CA-Mypt, 70%; P = 0.0016) = 12; red dots mark mean values; box plot centre, median; red dot, mean; upper whisker, maximum; lower whisker, minimum.