Supplementary Figure 2: Prechordal plate and neurectoderm cell movements and neural plate positioning in wild type and MZoep mutant embryos.

(a) Fluorescent images of a wild type (wt) Tg(gsc:GFP) embryo showing neurectoderm nuclei (H2A-BFP, cyan) and gsc-expressing GFP-labeled prechordal plate (ppl) cells at a representative time point during gastrulation (t = 65 min, 7.1 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. (b) Correlation of ppl cell movements in a wt embryo at a representative time point during gastrulation (t = 111.7 min, 7.9 hpf); ppl cells are visualized as arrows in a 2D plot and color-coded corresponding 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. (c) Average degree of alignment of ppl cell movements in wt embryos (n = 5 embryos) 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. (d) Mean instantaneous cell speed and directionality of ppl cells in a wt embryo (n = 5 embryos) calculated from 6 to 8 hpf are plotted as bar graphs; error bars, s.e.m. (e) Schematic illustration of global neurectoderm velocity measurements at the dorsal side of the embryo; the neurectoderm was segmented into 100 × 200 μm sectors along the AV axis (V_AV); sectors were positioned and color-coded relative to the ppl leading edge (yellow dot), or fixed for cases without ppl cells; A1-3 and P1-3, sector anterior and posterior of the ppl leading edge, respectively; mean V_AV velocities in the different sectors were calculated for each time frame. (f) Mean movement velocities (μm min⁻¹) along the AV axis (V_AV) of neurectoderm cells in wt embryos (n = 6 embryos) plotted from 6 to 8 hpf (120 min); colors of curves correspond to respective sectors in (e); error bars, s.e.m. (g) Schematic illustration of global 3D movement correlation analysis between neurectoderm and ppl cells in defined sectors along the AV axis of the embryo. For 3D correlation calculations, neurectoderm cell velocities along the AV (V_AV), left-right (LR) (V_LR; see (e)) and dorsal-ventral (DV) axis (V_DV in sectors of 130 × 100 μm) were measured; sectors were positioned and color-coded relative to the ppl leading edge (yellow dot); A1-3 and P1-3, sector anterior and posterior of the ppl leading edge (yellow dot), respectively. (h) 3D movement correlation between leading edge ppl and adjacent neurectoderm cells in defined sectors along the AV axis of wt embryos (n = 6 embryos) plotted from 6 to 8 hpf (120 min); colors of curves correspond to respective sectors in (e) and (g); error bars, s.e.m. (i) Fluorescent images of a MZoep;Tg(dharma:EGFP) mutant embryo showing neurectoderm nuclei (H2A-BFP, cyan) and Dharma (dharma:EGFP, green, marked with asterisk) expression at the dorsal blastoderm margin at a representative time point during gastrulation (t = 74.22 min, 7.2 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. (j) Mean movement velocities (μm min⁻¹) of neurectoderm cells along the AV axis (V_AV) in MZoep mutant embryos (n = 4 embryos) plotted over from 6 to 8 hpf (120 min); colors of curves correspond to sectors outlined in (e); error bars, s.e.m. (k,l) Anterior neural anlage in wt (k) and MZoep mutant (l) embryos 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 (arrows); animal pole (dorsal down), dorsal (animal pole up) and lateral (dorsal right) views are shown; arrowheads mark the anterior most edge of the neural plate; Scale bars 200 μm. (m) Quantitative analysis of neural plate position during gastrulation in MZoep 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 (k,l) 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 < 0.001, (ns) non significant, P > 0.05; n (wt, bud) = 36, n (wt, 90%) = 36, n (wt, 80%) = 34, n (wt, 70%) = 29, n (MZoep, bud; P < 0.0001) = 24, n (MZoep, 90%; P < 0.0001) = 36, n (MZoep, 80%; P < 0.0001) = 20, n (MZoep, 70%; P < 0.358) = 18; box plot centre, median; red dot, mean; upper whisker, maximum; lower whisker, minimum.