Supplementary Figure 7: Prechordal plate cell movements and neural plate positioning in e-cadherin morphant embryos.

(a) Brightfield/fluorescence image of a wild type (wt) Tg(gsc:GFP) (top panel) and e-cadherin (e-cad) morphant embryo (bottom panel) with gsc-expressing GFP-labeled prechordal plate progenitor (ppl) cells (green, white outline) at 80% epiboly; dorsal views, animal pole up; the increasing distance between the margins of the enveloping layer (EVL; red dashed line) and deep cell/neurectoderm (blue dashed line) shows (neur)ectoderm epiboly delay in e-cad morphant embryos; Scale bar, 200 μm. (b) Fluorescence images of a Tg(gsc:GFP) e-cad morphant embryo showing H2A-mCherry (magenta) expression in all nuclei and GFP (green) expression in ppl cells (white outline) at a representative time point during gastrulation (t = 80.30 min, 7.3 hpf); dorsal and sagittal (dorsal up) sections through the embryo (yellow tags in upper panel mark sagittal section plane in lower panel); red and blue dashed lines as in (A); animal pole (AP) and vegetal pole (VP) indicated by arrows; Scale bar, 100 μm. (c) Number of internalized ppl cells in Tg(gsc:GFP) e-cad morphant (blue curve, n = 4 embryos) versus wt (green curve) embryos plotted from 6 to 8 hpf (120 min); error bars, s.e.m. (d) Correlation of ppl cell movements in a e-cad morphant embryo at a representative time point during gastrulation (t = 80 min, 7.3 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. (e) Average degree of alignment of ppl movements in e-cad morphant (magenta curve/squares, n = 3) versus wt (green curve/dots, see Supplementary Fig. 2c) embryos 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. (f) Mean ppl instantaneous speed and directionality in e-cad morphant [gray bar graphs, n = 4 embryos; P(speed) = 0.0362; P(dir) = 0.222] versus wt (white bar graphs, see Supplementary Fig. 2d) embryos plotted as bar graphs; error bars, s.e.m; Student’s t-test for all graphs; ^∗, P < 0.05; (ns) non significant, P > 0.05. (g) Model of friction generation under E-cadherin reduced conditions (compare with wt in Fig. 6f) in e-cadherin morphant embryo leads to decreased friction at the ppl-to-neurectoderm (ecto) interface and to non-graded velocities within the ppl (left panel; F_f, friction force; orange dashes indicate remaining cadherin); reduced E-cadherin-mediated adhesion between ppl and neurectoderm leads to loss of frictional drag and vegetal-directed movements (red arrow) of neurectoderm cells (right panel; yellow arrows indicate ppl movement); double-sided arrows indicate embryonic axes, animal (A) to vegetal (V), dorsal (D) to ventral (V). (h) 2D tissue flow map indicating velocities (μm min⁻¹) of neurectoderm (ectoderm) cell movements along the AV (V_AP) and left-right (LR) (V_LR) axis at the dorsal side of a MZoep embryo overexpressing CA-Mypt within the YSL and transplanted with e-cad morphant ppl cells (t = 41.40 min, 6.7 hpf) at a representative time point; average velocity vector for each defined area is indicated and color-coded ranging from 0 (blue) to 2 (red) μm min⁻¹; positions of all/leading edge ppl cells are marked by black/green dots; black boxed area was used for mean velocity measurements in (i); Scale bar, 100 μm. (i) Mean movement velocities (μm min⁻¹) along the AV axis (V_AV) of ppl leading edge progenitor cells (green curve, left y-axis) and neurectoderm (ecto) cells positioned above the ppl leading edge (black boxed area in h; red curve, right y-axis) in MZoep embryos overexpressing CA-Mypt within the YSL and transplanted with e-cad morphant ppl cells (n = 4 embryos) plotted from 6 to 8 hpf; vertical dashed line indicates start of vegetal-directed movements of ppl cells; error bars, s.e.m. (j) 3D directional correlation values between leading edge ppl and adjacent neurectoderm (ecto) cells in MZoep embryo overexpressing CA-Mypt within the YSL and transplanted with e-cad morphant ppl cells (t = 41.40 min, 6.7 hpf) at a representative time point during gastrulation; degree of correlation is color-coded ranging rom 1 (red, highest) to −1 (white, lowest); average neurectoderm velocities for each defined area are marked; black boxed area was used for local correlation measurements in (k); Scale bar, 100 μm. (k) 3D directional correlation values between leading edge ppl and adjacent neurectoderm (ecto) cells (black boxed area in j) in MZoep embryos overexpressing CA-Mypt within the YSL and transplanted with e-cad morphant ppl cells (n = 4 embryos) plotted from 6 to 8 hpf; error bars, s.e.m. (l) Anterior neural anlage in e-cad morphant embryos marked by whole-mount in situ hybridization of otx2 mRNA expression at consecutive stage during gastrulation from 70% to 90% epiboly (7–9 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 arrowheads mark the most anterior edge of the neural plate; Scale bar, 200 μm. (m) Quantitative analysis of neural plate position during gastrulation in e-cad morphant 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 (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, 90%) = 36, n (wt, 80%) = 34, n (wt, 70%) = 29, n (e-cad, 90%; P < 0.0001) = 30, n (e-cad, 80%; P < 0.0001) = 37, n (e-cad, 70%; P = 0.00036) = 41; box plot centre, median; red dot, mean; upper whisker, maximum; lower whisker, minimum.