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, 19 (4), 306-317

Friction Forces Position the Neural Anlage

Affiliations

Friction Forces Position the Neural Anlage

Michael Smutny et al. Nat Cell Biol.

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.

Figures

Figure 1
Figure 1. Neurectoderm (ecto) and prechordal plate (ppl) morphogenesis during gastrulation
(a,c) Bright-field/fluorescence images of a Tg(gsc:GFP) zebrafish embryo at 7.0hpf; GFP-labeled ppl leading edge cells are indicated (white arrowheads); rectangle in (c) marks magnified area in (e); dashed lines in (a) indicate axial mesendoderm (white), and in (c) ecto-to-ppl (white), yolk syncytial layer (YSL)-to-ppl (yellow), enveloping layer (EVL)-to-media (purple) and EVL-to-YSL (blue) interfaces; embryonic axes orientation as marked in (b,d) for same views. (b,d) Illustration of embryonic [anterior (ppl) and posterior axial mesendoderm (pm), paraxial mesoderm (pam) and ecto] and extra-embryonic [YSL, EVL, yolk) tissues, and their respective direction of movement during gastrulation at the dorsal side of the zebrafish embryo; arrows in (b,d) indicate animal-vegetal (A-V), left-right (L-R), and dorsal-ventral (D-V) embryonic axes. (e) Magnified view of the boxed area in (c) showing neighboring ppl (green) and overlying ecto (red pseudocolored) tissues; dashed lines as in (c). (f) Immunofluorescence confocal images of sagittal sections of the ecto-to-ppl interface at 7.5hpf stained for E-cadherin (upper panel) and merged with ppl progenitors expressing gsc:GFP and DAPI-stained nuclei (lower panel); arrows highlight E-cadherin accumulations at ecto-to-ppl interface, and asterisks mark ppl leading edge cells; blue dashed line indicates EVL-to-media interface, and yellow dashed line outlines ppl- and ecto-to-YSL interfaces; animal pole to the right. All embryos animal pole up; dorsal (a,b) and lateral (c,d,e,f) views with dorsal right; scale bars, 200µm (a,c), 100µm (e), and 20µm (f).
Figure 2
Figure 2. Defective neurectoderm (ecto) morphogenesis in MZoep mutants
(a,i) Brightfield/fluorescence images of Tg(gsc:GFP) wt (a) and MZoep mutant embryos (i) at the end of gastrulation (bud stage, 10hpf); arrowhead in (a) marks anterior edge of GFP (blue)-labeled ppl. (b,j) Anterior neurectoderm progenitor cells in a wt (b) and MZoep embryo (j) at bud stage (10hpf) visualized by whole-mount in situ hybridization of otx-2 mRNA. (c,k) 2D tissue flow map indicating average velocities of ecto movements along the animal-vegetal (AV) and left-right (LR) axis at the dorsal side of a wt (c; 7.1hpf) and MZoep embryo (k; 7.2hpf); local average ecto velocities color-coded ranging from 0 (blue) to 2 (red) µm/min; positions of all/leading edge ppl cells marked by black/green dots; boxed areas are used for measurements in (d,l). (d,l) Mean velocities along the AV axis (VAV) of ecto (red; right y-axis; boxed area in c,k) and underlying ppl leading edge cells (green, left y-axis) in wt (d; n=6 embryos) and MZoep embryos (l; n=4 embryos); 6-8hp; error bars, s.e.m.. (e) 3D directional correlation between ecto and ppl in a wt embryo at 7.1hpf; color-coded correlation ranging from 1 (red, highest) to -1 (white, lowest); red arrows indicate local averaged ecto velocities; boxed area was used for measurements in (f). (f) 3D average directional correlation between leading edge ppl and adjacent neurectoderm cells (black boxed area in e) used for local correlation (CL) calculation in wt embryos (n=6 embryos); 6-8hpf; error bars, s.e.m. (g,m) 2D tissue flow map of ecto cells showing time-averaged velocities (over 120min from 3 embryos) along the AV and LR axes at the dorsal side in wt (g) and MZoep embryos (m); black dot in (g) marks position of ppl leading edge. (h,n) Schematic of ecto (red), ppl (green), and enveloping layer (EVL)/yolk syncytial layer (YSL) movements (blue) in wt (h) and MZoep embryos (n); arrows indicate AV and LR embryonic axes. All embryos animal pole up; dorsal [b,j (dor) and h,n] and lateral [a,i and b,j(lat)] views with dorsal right; scale bars, 200µm (a,b,i,j) and 100µm (c,e,k,m).
Figure 3
Figure 3. Prechordal plate (ppl) velocity determines the effect of ppl on neurectoderm (ecto) morphogenesis.
(a,f,k) Brightfield/fluorescence images of a Tg(gsc:GFP) wt (a,f; top panel), slb (a; bottom panel) and cyc morphant embryo (f, bottom panel) at 9hpf; Tg(gsc:GFP) embryo (k) injected at 3.3hpf with CA-Mypt and H2A-mCherry mRNAs into the YSL (top panel, schematic) at 8hpf; arrowheads mark GFP (green/blue)-labeled ppl leading edge. (b,g,l) 2D tissue flow map of time-averaged velocities of ecto cells (over 120min from 3 embryos) along animal-vegetal (AV) and left-right (LR) axes at the dorsal side of slb (b), cyc (g) and CA-Mypt injected embryos (l); black dots; ppl leading edge positions; boxed areas used for measurements in (c,h,m). (c,h,m) Mean velocities along the AV axis (VAV) of ecto (red; right y-axis; boxed areas in b,g,l) and underlying leading edge ppl cells (green, left y-axis) in slb (c; n=4 embryos), cyc (h; n=3 embryos) and CA-Mypt injected embryos (m; n=4 embryos); 6-8hpf; error bars (dashed lines), s.e.m. (d,i,n) 3D directional correlation map between ecto and ppl cells in a slb (d; 7.1hpf), cyc (i; 7.2hpf) and CA-Mypt-injected embryo (n; 6.6hpf); color-coded correlation ranging from 1 (red, highest) to -1 (white, lowest); red arrows; local averaged ecto velocities; boxed areas used for measurements in (e,j,o). (e,j,o) 3D average directional correlation between leading edge ppl and ecto cells (boxed areas in d,I,n) in slb (e; n=4 embryos), cyc (j; n=3 embryos) and CA-Mypt-injected embryos (o; n=4 embryos); 6-8hpf; error bars (dashed lines), s.e.m. (p) Anterior neural anlage and notochord labeled by otx2 (red arrows) and ntl expression (yellow arrows), respectively, in wt, slb, cyc and CA-Mypt-injected embryos at 9hpf; arrowheads denote anterior neural plate edge. (q) Angle (°) between the vegetal pole and neural plate anterior edge in wt, slb, cyc and CA-Mypt-injected embryos at 9hpf; student’s t-test (P value indicated) for all graphs; ***, P<0.001; *, P<0.05; n (embryos from 4 independent experiments) wt/cyc/slb/CA-Mypt=36(<0.001)/39(<0.001)/17(<0.0001)/22(0.0194); box plot centre, median; red dot, mean; upper whisker, maximum; lower whisker, minimum. (r) Schematic of ecto (red), ppl (green) and enveloping layer (EVL)/yolk syncytial layer (YSL) (blue/orange) movements in slb (f), cyc (l) and CA-Mypt-injected (r; magenta arrows;increased vortex flow) embryos; arrows;AV and LR axes. All embryos animal pole up; dorsal (f) and lateral (a,k,p) views with dorsal right; scale bars, 200µm (a,f,k,p) or 100µm (b,g,l,d,i,n).
Figure 4
Figure 4. Mesendoderm cell ingression is required for prechordal plate (ppl) affecting neurectoderm (ecto) cell movements.
(a,f) Schematic of ppl cells (green) transplanted at 6hpf into the dorsal side of a MZoep (a) or MZoep embryos injected with CA-Mypt mRNA into the YSL (f). (b,g) 2D tissue flow map indicating average velocities of ecto movements along the animal-vegetal (AV) (VAV) and left-right (LR) (VLR) axis at the dorsal side of a transplanted MZoep mutant (b; 6.6hpf) and transplanted MZoep embryo overexpressing CA-Mypt within the YSL (g; 6.7hpf); local average ecto velocites indicated by arrows color-coded ranging from 0 (blue) to 2 (red) µm/min; positions of all/leading edge transplanted ppl cells marked by black/green dots; boxed areas were used for measurements in (c,h). (c,h) Mean velocities along the AV axis (VAV) of ecto (red; boxed areas in b,g) and underlying ppl leading edge cells (green) in transplanted MZoep (c; n=3 embryos) and transplanted MZoep embryos overexpressing CA-Mypt within the YSL (h; n=3 embryos); 6-8hpf; vertical dashed line in (h) indicates start of vegetal-directed movements of ppl cells; error bars, s.e.m. (d,i) 3D directional correlation between leading edge ppl and ecto cells in a transplanted MZoep (d; 6.7hpf) and transplanted MZoep mutant embryo overexpressing CA-Mypt within the YSL (i; 6.7hpf); color-coded correlation ranging from 1 (red, highest) to -1 (white, lowest); red arrows indicate local averaged ecto velocities; boxed areas were used for measurements in (e,j). (e, j) 3D average directional correlation between leading edge ppl and adjacent ecto cells (boxed areas in d, i) in transplanted MZoep (e; n=3 embryos) and transplanted MZoep embryos overexpressing CA-Mypt within the YSL (j; n=3 embryos); 6-8hpf; vertical dashed line (j) as in (h); error bars, s.e.m. All scale bars, 100µm.
Figure 5
Figure 5. Hydrodynamic model description of the influence of prechordal plate (ppl) on neurectoderm (ecto) cell flows through friction forces at the tissue interface.
(a) Illustration of 1D ecto flow description along the tissue midline axis; ppl domain exerts an animal-directed force on the ecto; the differential velocity v of ecto tissue equals 0 at tissue boundaries. (b,b1) 1D analysis of ecto flow velocity (vy) along the tissue midline axis in wt (b) and slb morphant (b1) embryos; predicted flow profile (red), experimentally obtained flow velocities in wt and slb morphant embryos subtracted by the flows in MZoep mutants (blue), non-subtracted flow profiles in wt and slb morphant embryos (green), and flows in MZoep mutants (purple) are shown; values of the 1D model parameters used for each experimental case are listed in Supplementary Table 1 (Supplementary Note). (c) Illustration of 2D ecto flow description within the experimental image plane; velocities at the up (U), down (D), right (R) and left (L) boundaries of the image plane are taken from experimental measurements; a uniform force density is exerted on the ecto within the ppl domain. (d-e1) 2D analysis of ecto flow velocities for wt (d,e) and slb morphant (d1,e1) embryos; upper panels show the vy velocities along the ecto tissue midline axis and lower panels (e, e1) the vy velocities along the mediolateral extent of the ecto; color labeling of curves as in (b,b1); values of the 2D model parameters used for each experimental case are listed in Supplementary Table 1 (Supplementary Note). (f-g3) 2D vector density plots for the theoretical and experimental ecto flow velocity fields of wt (f-f3) and slb morphant embryos (g-g3); subtracted flow fields for wt (f,f1) and slb morphant (g,g1) embryos; non-subtracted total flow velocity fields for wt (f2,f3) and slb morphant (g2,g3) embryos generated by adding corresponding experimental MZoep velocities to the theoretical flow profiles; direction (arrows) and color-coded velocities from 0 (white, lowest) to 2 (purple, highest). All error bars s.d.
Figure 6
Figure 6. E-cadherin-mediated friction forces between prechordal plate (ppl) and neurectoderm (ecto) determine ecto morphogenesis.
(a,b) Confocal images of leading edge (red dots) ppl donor cells expressing lifeact-GFP (actin, green) transplanted in host embryo labeled with Utrophin-Cherry (actin, purple) and H2A-mCherry (nuclei, purple); asterisks, ppl cells at YSL interface (yellow), between YSL and ecto (orange), and at ecto interface (white); dorsal view as maximal z-stack projection (a); dorsal (top) and sagittal (bottom) confocal sections with ppl protrusions (arrows) and interfaces to YSL (yellow dots) and ecto (white dots) indicated (b). (c,d) Average instantaneous velocities of migrating ppl cells in wt (c) and e-cadherin morphant embryo (d) along the AV and DV axis color-coded from 0 (blue) to 4 (red) µm/min. (e) Linear regression lines of binned mean velocities of ppl cells along the normalized radial distance of the DV axis from ventral (0) to dorsal (1) for wt (green; P=0.0006, n=6 embryos) and e-cadherin morphant embryos (blue; P=0.15; n= 4 embryos); P values from F-test with null hypothesis; P > 0.05, slope equals zero; error bars s.e.m. (f) Schematic illustrating ppl (yellow arrow) dragging ecto cells (white arrow) and friction forces slowing down ppl cells at the ppl-ecto interface (bottom), leading to a linear velocity gradient within ppl (top); Ff, friction force; E-cadherin, orange line/dots. (g) 2D tissue flow map indicating velocities of ecto cell movements along the AV (VAP) and left-right (LR) (VLR) axis at the dorsal side of a e-cadherin morphant embryo at 6.7hpf; local average ecto velocities indicated and color-coded from 0 (blue) to 2 (red) µm/min; positions of all/leading edge ppl cells, black/green dots; boxed area used for measurements in (h). (h) Mean velocities along the AV axis (VAV) of ecto (red; boxed area in g; right y-axis) and underlying ppl leading edge cells (green, left y-axis) in e-cadherin morphant embryos (n=4 embryos); 6-8hpf; error bars, s.e.m. (i) 3D directional correlation between leading edge ppl and adjacent ecto cells in a e-cadherin morphant embryo at 6.7hpf; correlation color-coded from 1 (red, highest) to -1 (white, lowest); red arrows, local averaged ecto velocities; boxed area used for measurements in (j). (j) 3D directional correlation values between leading edge ppl and adjacent ecto cells (boxed area in i) in e-cadherin morphant (n=4 embryos); 6-8hpf; error bars, s.e.m. Scale bars 20µm (a,b) and 100µm (g,i); arrows; AV and DV axes
Figure 7
Figure 7. E-cadherin-mediated friction is sufficient to reorient neurectoderm (ecto) cell movements in vitro.
(a) Illustration of parallel plate setup for application of friction on ecto cells in vitro; uncoated control or coated with E-cadherin/Fc (E-Fc) polystyrene beads were sheared uniaxial (- y) over a cluster of opposing moving ecto cells, (+ y) to create friction; fluorescent reference beads (red) absorbed to top plate were used to track position and movement of adjacent polystyrene beads; E-cadherin receptors (orange) mediating friction indicated. (b) Maximum projection confocal image of ecto cell cluster expressing GPI-GFP (membrane, green) and H2A-mCherry (nuclei, white) plated onto a fibronectin-coated dish; directions of cell/stage movement (+y; velocity ~0.5µm/min) and E-Fc-coated beads/top plate movement (-y; velocity ~1.5µm/min) indicated; position of cluster of beads above ecto cells outlined (orange dashed line). (c,d) 2D tissue flow map indicating average velocities of ecto cell movements along the Y (VY) and X (VX) axis after application of friction using control (c) or E-Fc-coated (d) beads at a representative time point; local average ecto velocities indicated and color-coded ranging from 0 (blue) to 2 (red) µm/min; positions of leading edge polystyrene beads are marked by green dots; boxed area was used for measurements in (e,f). (e,f) Mean velocities along the Y axis (Vy) of leading edge control (e; n=3 experiments) or E-Fc-coated (f; n=3 experiments) beads (green) and adjacent ecto cells (boxed area in c,d; red curve) plotted before (t = 0-10min) and after (t = 10-80min) application of friction; error bars, s.e.m. (g,h) 3D directional correlation between ecto cells and adjacent control (g) or E-Fc-coated beads (h) at a representative time point; correlation color-coded ranging from 1 (red, highest) to -1 (white, lowest); red arrows indicate local averaged ecto velocities; position of all/leading edge ppl cells marked by white/green dots; blue arrowhead indicates average velocity of ppl leading edge cells; boxed area was used for measurements in (i,j). (i,j) 3D average directional correlation between ecto cells (boxed area in g,h) and leading edge control (i; n=3 experiments) or E-Fc-coated beads (j; n=3 experiments) before (t = 0-10min) and after (t = 10-80min) application of friction. (k) Time-averaged tissue flow map (over 70 (10-80) min from 3 experiments) of ecto cell movements along the y (Vy) and x (Vx) axis after application of friction using E-Fc-coated beads; error bars, s.e.m. Scale bars, 100µm (b,c,d,g,h).
Figure 8
Figure 8. Friction forces trigger tissue deformations within the neurectoderm (ecto).
(a) Ecto tissue deformations along the AV and LR axes of wt (upper panels; n=3) and MZoep (lower panels; n=3) embryos plotted as time-averaged strain values for each domain (50x50µm); average normal strain rate is color coded according to amount of stretch [minimum green (0) to maximum red (10 x10-3s-1)] or compression [minimum green (0) to maximum blue (-10 x10-3s-1)]; tissue flows of ecto are indicated as time-averaged velocities; dashed line indicates ppl position and black dot marks ppl leading edge as reference point in wt and MZoep; rectangle outlines area used for defining sectors along the AV axis in (b). (b) Mean normal strain rates of ecto tissue along the AV (left panels) and LR (right panels) axes of wt (upper panels; n=3 embryos) and MZoep (lower panels; n=3 embryos) embryos in defined sectors (100x200µm) of the ecto (A1 and A2 anterior and P1 and P2 posterior of ppl leading edge; for detailed description refer to Supplementary Fig. 1e) as a function of time during gastrulation (plotted from 6.3-7.3 in 10min intervals); amount of stretch/compression within each sector is plotted along the y-axis; (c) Ecto tissue strain rate maps derived by subtraction of AV (left panel) and LR (right panel) time-averaged strain values of wt from MZoep mutant embryos (n=3 embryos); color-code as in (a); tissue flows of ecto are indicated as time-averaged velocities; black dot marks ppl leading edge as reference point. (d) Illustration of kind and direction of tissue deformation in the ecto derived from normal strain; arrows indicate direction of stretch or compression of a tissue domain along the AV and LR axes dependent on the direction and magnitude of ecto movements.

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