Supplementary Figure 5: Movement of transplanted prechordal plate cells in MZoep mutant embryos. | Nature Cell Biology

Supplementary Figure 5: Movement of transplanted prechordal plate cells in MZoep mutant embryos.

From: Friction forces position the neural anlage

Supplementary Figure 5

(a,h) Schematic illustration of a MZoep;Tg(dharma:EGFP) (a) and a MZoep;Tg(dharma:EGFP) mutant embryo that was injected with CA-Mypt mRNA into the YSL at high stage (h; 3.3 hpf) transplanted with prechordal plate (ppl) cells (green) into the dorsal side at 60% epiboly (6 hpf); asterisk marks position of dorsal marker Dharma; orange arrows indicate reduced vegetal-directed movement of EVL margin (h); AP, animal pole; VP, vegetal pole; L, left; R, right. (b,i) Bright-field/fluorescence image of a MZoep;Tg(dharma:EGFP) mutant embryo at 90% epiboly (9 hpf) and a MZoep;Tg(dharma:EGFP) mutant embryo at 80% epiboly (8 hpf) that overexpresses CA-Mypt and the nuclei marker H2A-mCherry (red) within the YSL containing transplanted GFP-labeled ppl cells from Tg(gsc:GFP) donor (b) and Tg(gsc:GFP-CAAX) donor (i) embryos; ppl cell nuclei are marked by H2A-mCherry (i; red, co-localizes with green ppl cells); dashed white line indicates position of transplanted ppl progenitors; arrowhead points at anterior edge of ppl cells; asterisk marks dharma:EGFP signal at the dorsal side of the embryo; dorsal (animal pole up, top panel) and lateral (dorsal right, bottom panel) views; Scale bar, 200 μm. (c,j) Fluorescence images of representative time points during gastrulation (c; t = 47.19 min, 6.8 hpf and j; t = 56.39 min, 6.9 hpf) showing a MZoep;Tg(dharma:EGFP) (c) and a MZoep; Tg(dharma:EGFP) mutant embryo which overexpresses CA-Mypt and the nuclei marker H2A-mCherry (magenta) within the YSL (j) containing transplanted gsc-expressing GFP-labeled ppl cells (white outline) from Tg(gsc:GFP) donor (c) and Tg(gsc:GFP-CAAX) donor (j) embryos; all nuclei are marked by H2A-mCherry (c; magenta) and H2A-BFP (j; cyan) expression, and the dorsal side of the embryos is marked by dharma:EGFP expression (green, asterisk); dorsal and sagittal (dorsal up) sections through the embryo (yellow tags in upper panel mark sagittal section plane in lower panel); animal pole (AP) and vegetal pole (VP) indicated by arrows; Scale bar, 100 μm. (d) Protrusion orientation of ppl cells transplanted into MZoep mutants: top panel, fluorescence image of ppl cells with cytoplasm in green (gsc:GFP) and nuclei in cyan (H2A-BFP); animal pole up; Scale bar, 20 μm. Bottom panel, polar plot or protrusion orientation of transplanted ppl cells (n = 48 cells from 2 embryos) with 0° = animal pole, 180° = vegetal pole. (e) Number of ppl cells transplanted into MZoep mutant embryos (n = 3 embryos) plotted from 6 to 8 hpf (120 min); error bars, s.e.m. (f) Directional correlation of transplanted ppl cell movements in a MZoep mutant embryo at a representative time point during gastrulation (t = 83.5 min, 7.4 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 5th cell is plotted; AP, animal pole; VP, vegetal pole; Scale bar, 50 μm. (g) Average degree of alignment of transplanted ppl cell movements in MZoep mutant embryos (magenta curve/squares, n = 3 embryos) versus endogenous ppl cell movements in wt embryos (green curve/dots, see Supplementary Fig. 2c) 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. (k) Mean neurectoderm cell velocities along the animal-vegetal (AV) axis (VAV) (measurement area indicated by black box in Supplementary Fig. 2e) in MZoep mutant embryos (red curve, n = 3 embryos) and MZoep mutant embryos overexpressing CA-Mypt in the YSL (black curve, n = 3 embryos); error bars, s.e.m.

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