Abstract
Epithelial spreading is a common and fundamental aspect of various developmental and disease-related processes such as epithelial closure and wound healing. A key challenge for epithelial tissues undergoing spreading is to increase their surface area without disrupting epithelial integrity. Here we show that orienting cell divisions by tension constitutes an efficient mechanism by which the enveloping cell layer (EVL) releases anisotropic tension while undergoing spreading during zebrafish epiboly. The control of EVL cell-division orientation by tension involves cell elongation and requires myosin II activity to align the mitotic spindle with the main tension axis. We also found that in the absence of tension-oriented cell divisions and in the presence of increased tissue tension, EVL cells undergo ectopic fusions, suggesting that the reduction of tension anisotropy by oriented cell divisions is required to prevent EVL cells from fusing. We conclude that cell-division orientation by tension constitutes a key mechanism for limiting tension anisotropy and thus promoting tissue spreading during EVL epiboly.
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Acknowledgements
We are grateful to B. Aiguy, S. Eaton, S. Grill, R. Hauschild and M. Sixt for advice, and the imaging and zebrafish facilities of the IST Austria and MPI-CBG for continuous help. We are particularly grateful to J-F. Joanny for discussions regarding the theory part of this work, and B. Baum for sharing data before publication. This work was supported by the IST Austria, MPI-CBG and a grant from the Fonds zur Förderung der wissenschaftlichen Forschung (FWF) (I930-B20) to C-P.H.
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P.C., M.B., J.R., T.R. and C-P.H. synergistically and equally developed the presented ideas and the experimental and theoretical approaches. P.C. performed the experiments; P.C. and M.B. did the data analysis; J.R. and T.R. developed the theory; M.B. contributed to the experimental work; N.M. contributed to the data analysis and interpretation.
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Integrated supplementary information
Supplementary Figure 1 EVL cell-shape changes.
a, Apical cell area of representative individual EVL cells (n = 30 cells, 6 embryos) as a function of time after sphere stage (4 hpf). (b,c), Apical-basal cell height (b), and volume (c), of individual EVL cells both before (full line) and after cell division (dashed line; n = 17 cells, 5 embryos) as a function of time after sphere stage (4 hpf).
Supplementary Figure 2 Cell-flow and anisotropic-tension profiles as predicted by the continuum theory.
(a) EVL cell-flow velocity νθ and (b) anisotropic tension /2 plotted as a function of θ for different degrees of epiboly progression and different values of the ratio of shear to bulk viscosity η/ζ. In order to minimize the number of potentially variable parameters, non-dimensionalized quantities are displayed; only simple global rescalings need to be performed to recover physical units. The cell-flow velocity is normalized by the reference velocity νθ = R(tm−t0)/ζ and tensions are normalized by the reference tension tm−t0. For all plots shown, we assume a vanishing substrate friction for simplicity (γ=0), consistent with previous work. The percentage of epiboly progression is simply related to the value of the opening angle by the relation % e.p. = (1−cos Θ)/2. Note that an underlying assumption of the model description is that cell-division orientation is biased by tension anisotropy in the tissue, such that the anisotropic tension distribution shown here corresponds also to the predicted pattern of cell-division orientation in the tissue, up to a global proportionality factor.
Supplementary Figure 3 Randomized cell-division orientation.
(a) Rose diagram of the orientation of cell-division axes at cytokinesis (yellow) for EVL cells dividing during the course of gastrulation in Dynein antibody-injected embryos (n = 291 divisions, 4 embryos); p = 0.37 (calculated by using division numbers). (b) Average tissue flow velocities within the EVL as a function of distance from the EVL margin for control (orange) and α-Dynein antibody-injected (blue) embryos at 30–50%, 50–60%, 60–70% and 70– 80 % epiboly stage; error bars, s.e.m.; n, number of embryos; control embryos for the α-Dynein antibody experiments were injected with the antibody supernatant/ascites. Number of independent experiments = 4 (a), 10 (b).
Supplementary Figure 4 Lineage relationship between fusing EVL cells.
(a) Time course of an exemplary EVL cell-fusion event (arrowhead) in a cell-division inhibitor-treated embryo from sphere stage (t = 0 min) onwards for cases where the fusing cells could be manually back-tracked to resolve their lineage. Note that one of the fusing cells undergoes a division (arrow) prior to the fusion. Lineage tracing of all fusing cells where a previous division could be detected (n = 5 fusions), shows that this division was not giving rise to both fusing cells, demonstrating that the fusing cells are not sisters. (b) Time courses of exemplary EVL cell-fusion events (arrowheads) in cell-division inhibitor-treated embryos from sphere stage (t = 0 min) onwards for cases where lineage tracing of the fusing cells was not successful. To determine the relationship of fusing cells in such cases (n = 16 fusions), we analyzed whether these cells display mid-bodies (arrows) with neighboring cells, indicative of a previous division. Only in one fusion event (n = 1; upper panel), a mid-body was found between the fusing EVL cells, suggesting that these cells are sisters. In all other fusion events (n = 15 fusions), the fusing EVL cells either showed mid-bodies with other EVL cells (n = 3, middle panel) or no mid-bodies at all (n = 12, lower panel), suggesting that they are not sisters. Cell membrane and spindle microtubules marking the mid-body were visualized by GPI–RFP and Tau-GFP, respectively; n, number of fusions, 5 embryos; scale bar, 20 μm. Number of independent experiments = 5 (a,b).
Supplementary Figure 5 EVL junction remodeling.
Rose diagrams of the orientations of appearing (green) and disappearing (red) junctions within the EVL of control (left, 6 embryos) and α-Dynein antibody-injected (right, 4 embryos) embryos during the course of gastrulation; n, number of junctions; p(control, appearing junctions) = 5.3E-12; p (control, disappearing junctions) = 1.4E-05; p (α-Dynein antibody-injection, appearing junctions) = 1.8E-09; p (α-Dynein antibody-injection, disappearing junctions) = 4.5E-09; p (α-Dynein antibody-injection vs. control, disappearing junctions) = 1.31E-06 (calculated by using junction numbers). Number of independent experiments = 10.
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EVL cell divisions.
Time-lapse of the EVL in a wild-type embryo expressing GPI–RFP to outline EVL cells and cell divisions marked in yellow; t = 0 min corresponds to sphere stage (4 hpf). Scale bar, 100 μm. (MOV 1513 kb)
UV laser cuts to map EVL tissue tension.
Time-lapses of cortical laser cuts of the apical actomyosin cortex perpendicular (red) and parallel (green) to the EVL margin and at the animal pole (blue), in Tg(actb2:myl12.1-eGFP) embryos at 65% epiboly also expressing GPI–RFP to outline EVL cells. Red, green and blue lines (100 μm length) mark the position of the perpendicular, parallel and animal cuts, respectively. Scale bar, 20 μm. (MOV 805 kb)
Ectopic EVL tissue tension re-orients the mitotic spindle.
Time-lapse of the alignment of the cell division axis with the axis of induced tension in a Tg(actb2:myl12.1-mCherry) embryo at 40% epiboly also expressing Tau-GFP to mark spindle microtubules. Tension was induced orthogonally to the initial axis of the spindle (yellow) by creating two constricting wounds in the EVL. Scale bar, 20 μm. (MOV 1282 kb)
EVL cells do not round up during mitosis.
Time-lapse of a typical EVL cell division (arrow) in a wild-type embryo at 50% epiboly expressing GPI-GFP to outline EVL cells. Scale bar, 20 μm. (MOV 371 kb)
Myosin II activity is required for proper positioning of the mitotic spindle to the cell long axis.
Time-lapse of dividing EVL cells in Tg(actb2:myl12.1- mCherry) embryos between 30–50% epiboly. Embryos also express Tau-mCherry to mark spindle microtubules and were treated with either the myosin II-inhibitor Blebbistatin (right) or its inactive enantiomer (left). Scale bar, 20 μm. (MOV 960 kb)
Tension-oriented cell divisions release anisotropic tension within the EVL.
Time-lapse of exemplary cortical laser cuts of the apical actomyosin cortex perpendicular (blue) or parallel (orange) to the axis of induced tension either in the presence (right) or absence (left) of an EVL cell division (white contour) oriented along the axis of tension. Blue and orange lines (50 μm length) indicate were the cuts will be performed. Tg(actb2:myl12.1-eGFP) embryos at 30–40% epiboly. Scale bar, 20 μm. (MOV 807 kb)
Tension-oriented cell divisions facilitate EVL spreading.
Time-lapse of the spreading displacement of an EVL cell (white cell contour) in Tg(actb2:myl12.1-eGFP) embryos at 30–40% epiboly upon induction of ectopic tension either in the presence (right) or absence (left) of a cell division oriented along the axis of tension. Red crosses mark the ablation sites where wounds were induced. Scale bar, 20 μm. (MOV 830 kb)
EVL cells fuse when EVL cell divisions are inhibited.
Time-lapse of a exemplary EVL cell fusion (arrowhead) in a cell division inhibitor-treated embryo, expressing both Tau-mCherry and GPI–RFP to mark the spindle microtubules and plasma membrane, respectively. Scale bar, 20 μm. (MOV 740 kb)
EVL cells fuse in cylindrically deformed embryos.
Time-lapse of an exemplary EVL cell fusion event (arrowheads) in a cylindrical Tg(actb2:GFP-utrCH) embryo from sphere stage (t = 0 min) onwards. Arrows point at a cell division, which gives rise to a daughter cell that subsequently fuses with another unrelated cell. Cell membrane and nuclei were marked by GPI–RFP and H2A-Cherry, respectively. Scale bar, 20 μm. (MOV 711 kb)
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Campinho, P., Behrndt, M., Ranft, J. et al. Tension-oriented cell divisions limit anisotropic tissue tension in epithelial spreading during zebrafish epiboly. Nat Cell Biol 15, 1405–1414 (2013). https://doi.org/10.1038/ncb2869
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DOI: https://doi.org/10.1038/ncb2869
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