Friction forces position the neural anlage

Abstract

During embryonic development, mechanical forces are essential for cellular rearrangements driving tissue morphogenesis. Here, we show that in the early zebrafish embryo, friction forces are generated at the interface between anterior axial mesoderm (prechordal plate, ppl) progenitors migrating towards the animal pole and neurectoderm progenitors moving in the opposite direction towards the vegetal pole of the embryo. These friction forces lead to global rearrangement of cells within the neurectoderm and determine the position of the neural anlage. Using a combination of experiments and simulations, we show that this process depends on hydrodynamic coupling between neurectoderm and ppl as a result of E-cadherin-mediated adhesion between those tissues. Our data thus establish the emergence of friction forces at the interface between moving tissues as a critical force-generating process shaping the embryo.

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Figure 1: Neurectoderm (ecto) and prechordal plate (ppl) morphogenesis during gastrulation.
Figure 2: Defective neurectoderm (ecto) morphogenesis in MZoep mutants.
Figure 3: Prechordal plate (ppl) velocity determines the effect of ppl on neurectoderm (ecto) morphogenesis.
Figure 4: Mesendoderm cell ingression is required for ppl affecting ecto cell movements.
Figure 5: Hydrodynamic model description of the influence of ppl on ecto cell flows through friction forces at the tissue interface.
Figure 6: E-cadherin-mediated friction forces between ppl and ecto determine ecto morphogenesis.
Figure 7: E-cadherin-mediated friction is sufficient to reorient ecto cell movements in vitro.
Figure 8: Friction forces trigger tissue deformations within the ecto.

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Acknowledgements

We would like to thank R. Hauschild for technical assistance and the scientific service units at the IST Austria for continuous support. This work was supported by grants from the OMAA Ernst Mach Predoctoral Scholarship to Z.Á., Cancer Research UK to M.T., the Spanish Ministry of Economy and Competitiveness ‘Centro de Excelencia Severo Ochoa 2013–2017’ and CERCA Programme/Generalitat de Catalunya to V.R., the European Union’s Seventh Framework Program/ERC Grant Agreement to B.H. (306589) and to T.V. (COLLMOT project; 227878), the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001317), the UK Medical Research Council (FC001317) and the Wellcome Trust (FC001317) to S.G. and G.S., and the Austrian Science Foundation (FWF): I930-B20 to C.-P.H.

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Authors

Contributions

M.S. and C.-P.H. conceived the project and designed the experiments; M.S. performed the experiments and data analysis with support from D.Č. for in-situ hybridizations and analysis, M.B. for CA-Mypt injections and EVL/YSL analysis and E.P. for immunohistochemistry.; Z.Á. developed computational tools for data analysis; S.S. and V.R. wrote scripts for data analysis; S.G. and G.S. developed the theory and S.G., S.S. and G.S. performed quantitative modelling; B.H. contributed to model interpretation; M.T. developed reagents and T.V. contributed to collective motion analysis and interpretation; M.S., G.S. and C.-P.H. wrote the paper.

Corresponding author

Correspondence to Carl-Philipp Heisenberg.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Fibronectin and interstitial fluid localization at the neurectoderm-to-prechordal plate interface during zebrafish gastrulation.

(ac) Immunofluorescence confocal images of the neurectoderm (ecto)-to-prechordal plate (ppl) interface (white dashed line) in a wild type (wt) embryos at 6 (a), 8 (b), and 9 (c) hpf showing Fibronectin staining (pseudo-colored with Fire LUT) in maximum intens ity projections of dorsal views (top panels) and sagittal sections (middle panels); red dashed line outlines position of ppl leading edge cells; blue dashed line indicates ecto-to-EVL interface, and yellow dashed line shows YSL interface to ppl and ecto; bottom panels are sagittal sections of the ecto-to-ppl interface stained for F-actin (phalloidin) to mark this interface; double-sided arrows indicate animal (A) to vegetal (V) and dorsal (V) to ventral (V) embryo axes; asterisk labels ppl leading edge cell; Scale bar, 20 μm. (d) Multiphoton live cell images showing interstitial fluid (IF) accumulation (dextran-Alexa Fluor 647, left panel), F-actin localization (Tg(actb1:lifeact-GFP), middle panel) and a combination of those different labels (right panel) at the ecto-to-ppl interface (white dashed line) at 7 hpf; red arrows indicate extracellular cavities filled with IF at the ecto-to-ppl and ecto-to-YSL interfaces; white arrows indicate ecto-to-ppl cell-cell contacts devoid of IF accumulations; blue dashed line indicates ecto-to-EVL interface, and yellow dashed line shows YSL interface to ppl and ecto; double-sided arrows indicate AV and dorsal DV embryo axes; asterisk labels ppl leading edge cell; Scale bar, 20 μm. (e) Multiphoton live cell image of Tg(gsc:GFP) embryo (t = 120 min, 8 hpf) with pseudo-colored spots marking positions of nuclei within the axial mesendoderm (green); dorsal view with double-sided arrows indicating AP to VP and left (L) to right (R) embryo axes; color-code indicates mean total cell speeds of axial mesendoderm cells moving to the animal pole after internalization (cyan, 0–2 and yellow/magenta >2 μm min−1); position of anterior (ppl) and posterior mesendoderm marked; Scale bar, 50 μm. (f) Average instantaneous cell speeds in μm min−1 of internalized axial mesendoderm cells in wt embryos (n = 6 embryos) plotted along the normalized distance along the AV axis from anterior (0) to posterior (1); green dashed line marks position of transition from anterior (ppl) to posterior axial mesendoderm, error bars, s.e.m.

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 (VAV); 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 VAV velocities in the different sectors were calculated for each time frame. (f) Mean movement velocities (μm min−1) along the AV axis (VAV) 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 (VAV), left-right (LR) (VLR; see (e)) and dorsal-ventral (DV) axis (VDV 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 (VAV) 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.

Supplementary Figure 3 Prechordal plate cell movements and neural plate positioning in cyc and slb morphant embryos.

(a,e) Fluorescence images of a Tg(gsc:GFP) cyclops (cyc) (a) and silberblick (slb) morphant (e) embryo showing H2A-BFP expression (cyan) in all nuclei and GFP (green, white outline) expression in gsc-expressing prechordal plate (ppl) cells at a representative time point during gastrulation (a; t = 71.40 min, 7.2 hpf and e; 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; red line in (e) indicates widened ppl internalization zone; Scale bar, 100 μm. (b,f) Number of internalized ppl cells in Tg(gsc:GFP) cyc (b; blue curve, n = 3 embryos) and slb (f; blue curve, n = 3 embryos) morphant embryos (blue curve, n = 3 embryos) versus wt (green curve, n = 6 embryos) embryos plotted between 6 and 8 hpf (120 min); error bars, s.e.m. (c,g) Average degree of alignment of ppl cell movements in cyc (c) magenta curve/squares, n = 3 embryos) and slb morphant (g; magenta curve/dots, n = 3 embryos) versus wt (green curve/dots, see Supplementary Fig. 2c) embryos plotted from 6 to 8/8.3 hpf (120/140 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,h) Mean instantaneous ppl cell speed and directionality of cyc [d; gray bar graph, n = 4 embryos; P(speed) = 0.0061, P(dir) = 0.033] and slb morphant [gray bar graphs, n = 3 embryos, P(speed) = 0.0025, P(dir) < 0.0001] versus wt (white bar graph, see Supplementary Fig. 2d) embryos plotted as bar graphs; error bars, s.e.m.; Student’s t-test for all graphs; , P < 0.001, , P < 0.01; , P < 0.05. (i,j) Anterior neural plate anlage in cyc and slb morphant 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 most anterior edge of the neural plate; Scale bar 200 μm. (k) Quantitative analysis of neural plate position in cyc and slb morphant versus wt embryos during gastrulation. 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, j) 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 (slb, bud; P < 0.0001) = 23, n (slb, 90%; P < 0.0001) = 17, n (slb, 80%; P < 0.0001) = 20, n (slb, 70%; P = 0.134) = 16, n (cyc, bud; P < 0.0001) = 40, n (cyc, 90%; P < 0.0001) = 39, n (cyc, 80%; P < 0.0001) = 32, n (cyc, 70%; P = 0.851) = 27; red dots mark mean values; box plot centre, median; red dot, mean; upper whisker, maximum; lower whisker, minimum.

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.

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

(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.

Supplementary Figure 6 Effect of external friction on one-dimensional neurectoderm flow profile.

(a) For capturing the flow profile induced solely by prechordal plate (ppl) cells, MZoep mutants devoid of ppl cells were used to measure unperturbed epiboly movements, and those movements were subtracted from the overall neurectoderm flow field in wt embryos. This allowed decomposing the neurectoderm flow field and obtaining the ppl-induced movement alterations only. In the 2D description, neurectoderm flows exclusively within the experimental image plane (red square) were taken into account. (b) Theoretical 1D flow profile when the external friction coefficient ξ0 between neurectoderm and tissues other than the ppl, such as the yolk cell and/or EVL, is varied. In case the external friction coefficient is increased, the range of flow triggered by ppl cells is decreased (blue: orange: , green: , all curves: × min−1). (c) Experimental velocities in wt embryos (blue dots) compared to theoretical flow profiles for the parameter settings as used in Fig. 5 (red dotted line, × min−1, ), and for zero external friction (green line, × min−1, ). The experimental velocity profile in wt embryos is well explained by either a small () or vanishing (ξ0 = 0) external friction coefficient.

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; Ff, 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 (VAP) and left-right (LR) (VLR) 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 (VAV) 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.

Supplementary Figure 8 Alterations in ectoderm movements upon application of E-cadherin mediated friction ex vivo and shear-strain-induced neurectoderm tissue deformation in vivo.

(a) Bright-field/fluorescence image showing setup of magnetic polystyrene beads (20 μm diameter) and fluorescent reference beads (red, 4 μm diameter) attached to a glass plate used to apply friction onto ectoderm cells; dashed line outlines shape of polystyrene cluster; Scale bars, 100 μm and 20 μm for magnified area. (b) Western Blot analysis showing detection of E-cadherin ectodomain (80 kDa) eluted from magnetic polystyrene beads coupled to E-cadherin-Fc Chimera (E-Fc) or uncoated control beads; molecular weight markers, 100 and 200 kDa. (c) Section of maximum projection confocal image (see Fig. 7a; t = 19.33 min) showing top plate with fluorescent reference beads and selected beads are highlighted (red arrows in xy and yz cross-section); cross-section (yz; red rectangle) shows the position of E-Fc-coated beads (outlined in orange) at the ectoderm cell interface (yellow dashed line); direction of beads movement (top plate; −y; velocity 1.5 μm min−1) is indicated; Scale bars, 100 μm in xy and 20 μm in yz. (d) Shear strain-induced neurectoderm tissue deformations of wild type (wt, upper panels; n = 3 embryos) and MZoep (lower panels; n = 3 embryos) embryos plotted as time-averaged strain values for each domain (50 × 50 μm); average shear strain rate is color-coded according to amount of plane distortion [minimum green (0) to maximum red (5 × 10−3 s−1)]; tissue flows of neurectoderm are indicated as time-averaged velocities; dashed line indicates ppl and black dot marks ppl leading edge as reference point in wt and MZoep; rectangle outlines area used for defining sectors along the animal-vegetal (AV) axis in (e). (e) Mean shear strain rates of neurectoderm tissue of wt (upper panels; n = 3 embryos) and MZoep (lower panels; n = 3 embryos) in defined sectors (100 × 200 μm) are plotted along the AV axis over time of gastrulation (plotted from 6.3 to 7.3 hpf in 10 min intervals); sectors were positioned and color-coded relative to the ppl leading edge (anterior A1-2 and posterior P1-2 of the ppl leading edge; for detailed description refer to Supplementary Fig. 1e); amount of plane distortion [minimum green (0) to maximum red (10 × 10−3 s−1)] is plotted along the y-axis; (f) Neurectoderm tissue strain rate maps derived by subtraction of time-averaged shear strain values of wt from MZoep embryos (n = 3 embryos); color-code as in (f); tissue flows of neurectoderm are indicated as time-averaged velocities; dashed line indicates ppl and black dot marks ppl leading edge as reference point. (g) Illustration of shear strain tissue deformation in the neurectoderm; arrows indicate direction of plane distortion of a tissue domain along the AV and left-right (LR) axis dependent on the direction and magnitude of neurectoderm movements; shear strain-induced domain angle of plane distortion can shrink (positive value) or enlarge (negative value).

Supplementary information

Supplementary Information

Supplementary Information (PDF 1623 kb)

Live cell imaging of cell movements in wt embryo.

Multiphoton time-lapse imaging of a wild type (wt) Tg(gsc:GFP) embryo with gsc-expressing GFP-labeled prechordal plate progenitor (ppl) cells (green) and neurectoderm cells at the dorsal side of the embryo from 6 to 8 hpf (123 min); all nuclei were labeled with histone H2A-BFP; animal/vegetal pole, up/down. (AVI 2027 kb)

Life cell imaging of cell movements in MZoep mutant embryo.

Multiphoton time-lapse imaging of a MZoep;Tg(dharma:EGFP) mutant embryo (Dharma:EGFP signal green) showing neurectoderm cells at the dorsal side of the embryo from 6 to 8.1 hpf (129 min); all nuclei were labeled with histone H2A-BFP; animal/vegetal pole, up/down. (AVI 2429 kb)

2D velocities of neurectoderm cells in wt embryo.

Tissue flow map indicating velocity vectors of neurectoderm cell movements along the animal-vegetal (AV) (VAV) and left-right (LR) (VLR) axis at the dorsal side of a wild type (wt) embryo between 6 to 8 hpf (117 min); average velocity vector for each defined area is indicated and color-coded ranging from 0 (blue) to 2 (red) μm min−1; position of all/leading edge prechordal plate (ppl) cells are indicated as black/green dots; xy-axes in μm; time in mins; animal/vegetal pole, up/down. (AVI 1464 kb)

3D correlation of neurectoderm and prechordal plate (pp) cell movements in wt embryo.

Movement correlation between neurectoderm and underlying (ppl) cells at the dorsal side of a wild type (wt) embryo between 6 to 8 hpf (118 min); degree of correlation is color-coded ranging from 1 (red, highest correlation) to −1 (white, lowest correlation); average neurectoderm movement velocities and direction for each defined area are indicated by arrows; position of all/leading edge ppl cells are indicated as white/green dots; blue arrow marks movement direction of ppl leading edge cells; xy-axes in μm; time in mins; animal/vegetal pole, up/down. (AVI 1698 kb)

2D velocities of neurectoderm cells in MZoep mutant embryo.

Tissue flow map indicating velocity vectors of neurectoderm cell movements along the animal-vegetal (AV) (VAV) and left-right (LR) (VLR) axis at the dorsal side of a MZoep mutant embryo between 6 to 8 hpf (121 min); average velocity vector for each defined area is indicated and color-coded ranging from 0 (blue) to 2 (red) μm min−1; xy-axes in μm; time in mins; animal/vegetal pole, up/down. (AVI 1303 kb)

3D correlation of neurectoderm and prechordal plate (pp) cell movements in CA-Mypt injected embryo.

Movement correlation between neurectoderm and underlying prechordal plate (ppl) cells at the dorsal side of a wt embryo overexpressing CA-Mypt in the YSL between 6 to 8 hpf (118 min); degree of correlation is color-coded ranging from 1 (red, highest correlation) to −1 (white, lowest correlation); average neurectoderm movement velocities and direction for each defined area are indicated by arrows; position of all/leading edge ppl cells are indicated as white/green dots; blue arrow marks movement direction of ppl leading edge cells; xy-axes in μm; time in mins; animal/vegetal pole, up/down. (AVI 1458 kb)

2D velocities of neurectoderm cells in ppl-transplanted MZoep mutant embryo.

Tissue flow map indicating velocity vectors of neurectoderm cell movements along the animal-vegetal (AV) (VAV) and left-right (LR) (VLR) axis at the dorsal side of a transplanted MZoep mutant embryo between 6 to 7.5 hpf (91 min); average velocity vector for each defined area is indicated and color-coded ranging from 0 (blue) to 2 (red) μm min−1; position of all/leading edge transplanted prechordal plate (ppl) cells are indicated as black /green dots; xy-axis in μm; time in mins; animal/vegetal pole, up/down. (AVI 1296 kb)

2D velocities of neurectoderm cells in ppl-transplanted and CA-Mypt injected MZoep mutant embryo.

Tissue flow map indicating velocity vectors of neurectoderm cell movements along the animal-vegetal (AV) (VAV) and left-right (LR) (VLR) axis at the dorsal side of a MZoep embryo overexpressing CA-Mypt within the YSL between 6 to 8 hpf (120 min); average velocity vector for each defined area is indicated and color-coded ranging from 0 (blue) to 2 (red) μm min−1; position of all/leading edge transplanted prechordal plate (ppl) cells are indicated as black /green dots; xy-axis in μm; time in mins; animal/vegetal pole, up/down. (AVI 1380 kb)

Arrangement of leading and trailing prechordal plate (ppl) cells in wild type (wt) embryo.

Consecutive z-sections of a fluorescent imaging stack showing lifeact-GFP (F-actin) expressing ppl cells transplanted into the ppl leading edge of a wt embryo expressing Utrophin-mCherry (F-actin) and H2A-mCherry (nuclei); section starts at the ppl-neurectoderm interface and progresses through the leading edge ppl to the ppl-YSL interface; animal pole to the left; z-section taken from movie 16 at t = 12.36 min; Scale bar, 20 μm. (AVI 1481 kb)

Life cell imaging of leading and trailing prechordal plate (ppl) cells in wild type (wt) embryo.

Fluorescence time-lapse imaging of lifeact-GFP (F-actin) expressing ppl cells transplanted into the ppl leading edge of a wt embryo expressing Utrophin-mCherry (F-actin) and H2A-mCherry (nuclei) starting at 70% epiboly (7 hpf); dorsal (top, animal pole left) and sagittal (bottom, animal pole left) sections through the embryo with dual (left side) and single (right side) color label; time in mins; Scale bar, 20 μm. (AVI 1821 kb)

2D velocities of neurectoderm cells in e-cadherin morphant embryo.

Tissue flow map indicating velocity vectors of neurectoderm cell movements along the animal-vegetal (AV) (VAV) and lefty-right (LR) (VLR) axis at the dorsal side of a e-cadherin morphant embryo between 6 to 8 hpf (120 min); average velocity vector for each defined area is indicated and color-coded ranging from 0 (blue) to 2 (red) μm min−1; position of all/leading edge prechordal plate (ppl) cells are indicated as black /green dots; xy-axis in μm; time in mins; animal/vegetal pole, up/down. (AVI 1580 kb)

3D correlation of neurectoderm and prechordal plate (ppl) cell movements in e-cadherin morphant embryo.

3D movement correlation between neurectoderm and underlying prechordal plate (ppl) cells at the dorsal side of e-cadherin morphant embryo 6 to 8 hpf (120 min); degree of correlation is color-coded ranging from 1 (red, highest correlation) to −1 (white, lowest correlation); average neurectoderm movement velocities and direction for each defined area are indicated by arrows; position of all/leading edge ppl cells are indicated as white/green dots; blue arrow marks movement direction of ppl leading edge cells; xy-axes in μm; time in mins; animal/vegetal pole, up/down. (AVI 1783 kb)

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Smutny, M., Ákos, Z., Grigolon, S. et al. Friction forces position the neural anlage. Nat Cell Biol 19, 306–317 (2017). https://doi.org/10.1038/ncb3492

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