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Forces Driving Epithelial Spreading in Zebrafish Gastrulation

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Science  12 Oct 2012:
Vol. 338, Issue 6104, pp. 257-260
DOI: 10.1126/science.1224143

Embryonic Cell Sorting and Movement

Differential cell adhesion has long been thought to drive cell sorting. Maître et al. (p. 253, published online 23 August) show that cell sorting in zebrafish gastrulation is triggered by differences in the ability of cells to modulate cortex tension at cell-cell contacts, thereby controlling contact expansion. Cell adhesion functions in this process by mechanically coupling the cortices of adhering cells at their contacts, allowing cortex tension to control contact expansion. In zebrafish epiboly the enveloping cell layer (EVL)—a surface epithelium formed at the animal pole of the gastrula—gradually spreads over the entire yolk cell to engulf it at the end of gastrulation. Behrndt et al. (p. 257) show that an actomyosin ring connected to the epithelial margin triggers EVL spreading both by contracting around its circumference and by generating a pulling force through resistance against retrograde actomyosin flow.

Abstract

Contractile actomyosin rings drive various fundamental morphogenetic processes ranging from cytokinesis to wound healing. Actomyosin rings are generally thought to function by circumferential contraction. Here, we show that the spreading of the enveloping cell layer (EVL) over the yolk cell during zebrafish gastrulation is driven by a contractile actomyosin ring. In contrast to previous suggestions, we find that this ring functions not only by circumferential contraction but also by a flow-friction mechanism. This generates a pulling force through resistance against retrograde actomyosin flow. EVL spreading proceeds normally in situations where circumferential contraction is unproductive, indicating that the flow-friction mechanism is sufficient. Thus, actomyosin rings can function in epithelial morphogenesis through a combination of cable-constriction and flow-friction mechanisms.

In zebrafish epiboly, the enveloping cell layer (EVL) surface epithelium formed at the animal pole of the gastrula (1) spreads over the entire yolk cell to completely engulf it at the end of gastrulation. EVL movements are independent of epiboly movements of the deep cells below (2), but require contact between the EVL and the yolk syncytial layer (YSL) to which the EVL is connected at its margin (3, 4). Contractile actomyosin rings drive many fundamental morphogenetic processes (59), and it has been proposed that in zebrafish a contractile actomyosin ring within the YSL, a thin cytoplasmic layer at the surface of the yolk cell, drives EVL epiboly by pulling on the edge of the EVL (4, 10, 11). However, the force-generating mechanisms by which this ring drives EVL epiboly movements remain elusive.

To investigate the physical mechanisms driving EVL epiboly, we first examined the global distribution of actin and myosin-2 within the embryo (movies S1 and S2). Just after the onset of epiboly, when the EVL covers 40% of the spherical yolk cell (40% epiboly; 5 hours post-fertilization, hpf), we observed an accumulation of both actin and myosin-2 within the YSL in a band-like structure close to the margin of the EVL (Fig. 1A). This initially broad actomyosin band (85.8 ± 11.8 μm; errors denote error of the mean at 95% confidence unless noted otherwise) decreases its width during EVL epiboly progression, eventually forming a tight circumferential ring-like structure (32.2 ± 3.5 μm) at 70 to 80% epiboly (7.5 to 8.5 hpf; Fig. 1, A to C).

Fig. 1

Actomyosin ring morphogenesis and function. (A and B) In Tg(actb1:myl12.1-eGFP) embryos at 40% epiboly (A), an initially diffuse and broad actomyosin band (orange bar) narrows along the AV axis to form a distinct cable-like structure at 70% epiboly (B). Scale bar, 100 μm. (C) Quantification of myosin-2 peak fluorescence intensity and width of the actomyosin ring during epiboly (supplementary material; fig. S1). Error bars correspond to error of the mean at 95% confidence unless noted otherwise. (D and E) Local disruption of the actomyosin ring in Tg(actb1:myl12.1-eGFP) embryos at 60% epiboly by consecutive UV-laser ablation [white dashed rectangle, (D)] reduces advancement of the adjacent EVL margin [dark blue, (E)]. Scale bar, 50 μm. (F) Quantification of advancement rate of the EVL margin adjacent (dark blue) and farther away (light blue) from the ablation site by particle image velocimetry show a 22.6 ± 6.3% reduction. P = 0.000015.

We then asked if the YSL actomyosin ring drives EVL epiboly movements. Previous studies on the function of the mitogen-activated protein kinase (MAPK) pathway components traf2/nika (4) and mapkapk2 (11) suggested that actomyosin contractility within the YSL affects EVL epiboly movements. To directly test whether YSL actomyosin contractility drives EVL epiboly, we locally disrupted the YSL actomyosin ring close to the EVL margin in 60% epiboly-stage embryos (6.5 hpf) with an ultraviolet (UV) laser cutter (12). This caused considerable delays in epiboly movements of the EVL margin directly adjacent to the ablation site (Fig. 1, D to F, and movie S3), suggesting that the YSL actomyosin ring is required for EVL epiboly movements and that its functional requirement is locally restricted.

We next sought to understand how YSL actomyosin ring contractility drives EVL epiboly movements. One possibility is that the ring actively generates tension along its circumference, which due to the spherical geometry of the embryo gives rise to a force pulling on the EVL margin (supplementary material). To test for the presence of circumferential tension within the YSL actomyosin ring, we used a UV laser cutter (12) to sever the actomyosin network along a 20-μm-long line 20 μm away from the EVL/YSL border in an orientation perpendicular to the EVL margin (Fig. 2A and movie S4). Perpendicular cuts resulted in rapid recoil velocities of the actomyosin network at 60 to 70% epiboly that decayed exponentially over time (12) (6.5 to 7.5 hpf; vcut = 19.1 ± 1.5 μm/s, τ = 1.7 ± 0.2 s, N = 40; fig. S2D). Repeating this experiment at different stages of EVL epiboly revealed that initial recoil velocities increase over the course of gastrulation (Fig. 2B), suggesting that circumferential tension within the actomoysin ring becomes larger during EVL epiboly progression.

Fig. 2

Actomyosin ring tension and flow. (A) Tg(actb1:myl12.1-eGFP) embryos at 65% epiboly where the actomyosin ring was cut along a 20-μm line perpendicular (red) and parallel (green) to the EVL margin. Scale bars, 10 μm. (B) Initial laser cut recoil velocities at different stages of epiboly for perpendicular (red) and parallel cuts (green) as obtained by exponentially fitting the quantified recoil velocity curves (fig. S2, A to E). Error bars represent the 95% confidence interval of the fit result. (C) Cortical flows within the actomyosin ring of a Tg(actb1:myl12.1-eGFP) embryo injected with lifeact-RFP mRNA labeling myosin-2 (left) and F-actin (right), respectively, at 60% epiboly (movie S8). The EVL margin (blue line) and the two-dimensional (2D) vector field of actomyosin flow (yellow arrows) are shown. Scale bar, 20 μm. (D) Average profile of cortical flow in embryos at 60 to 70% epiboly (N = 22). Negative velocities correspond to flow toward the animal pole. Inhibiting myosin-2 activity by treatment with blebbistatin reduces recoil velocities for both parallel and perpendicular cuts as well as retrograde flow, indicating that cortical tension and flow are due to myosin-2 activity (fig. S4).

Because of the spherical geometry of the yolk cell, circumferential active tension will result in a net force pulling on the EVL margin, which is zero when the ring is at the equator and grows as the ring approaches the pole (Fig. 3A and supplementary material). Consequently, when the ring is near the equator, tension in the animal-vegetal (AV) direction within the ring is expected to be much smaller than circumferential tension (supplementary material). A detailed calculation that considers the force balance of a constricting ring positioned on a sphere and connected to the EVL at one end shows that AV tension scales as h/R cot θ, with θ being the polar angle, h the width of the ring, and R the radius of the yolk cell. As a result, AV tension is expected to increase during epiboly progression and reach at most ~20% of circumferential tension at 80% epiboly (supplementary material). We tested this assumption by cutting the actomyosin network along a 20-μm-long line 20 μm away from the EVL/YSL border in an orientation parallel to the EVL margin (Fig. 2A and movie S5). Contrary to our expectations, we found that the initial recoil velocity of the actomyosin network for parallel cuts was approximately half of the initial recoil velocity for perpendicular cuts at 60 to 70% epiboly (vcut = 7.5 ± 1.0 μm/s, N = 40) and remained constant during epiboly progression (fig. S2D and Fig. 2B). Notably, characteristic decay times were similar for both parallel and perpendicular cuts and at different stages during epiboly (fig. S2, B to E), suggesting that material properties of the actomyosin network are constant and isotropic. We conclude that AV tension is much larger than expected if the actomyosin ring were acting as a constricting ring only.

Fig. 3

Theoretical description of EVL epiboly. (A) The EVL and actomyosin ring are represented by two thin and compressible viscous layers with an internal active tension, lying on a sphere and mechanically connected at the EVL margin. (i) Circumferential tension within the ring coupled to the embryo curvature moves the EVL toward the closest pole. (ii) Myosin-dependent retrograde flow coupled to friction against an underlying substrate generates a pulling force on the EVL. (B) Global fit of the model predictions for flows (blue curves, left), ring advancement velocity (blue curve, middle), and tensions (green and red dots, right) compared to the experimentally measured EVL and actomyosin ring flow profiles, and actomyosin ring relative tensions obtained from laser ablation (gray curves and dots in the respective panels) at successive epiboly stages. Experimental data are fitted to Eqs. 42 to 46 in the supplementary material. Tensions are normalized to the average ring tension 〈tEmbedded Image The fitting procedure yields the hydrodynamic length l = 76 ± 10 μm indicative of nonzero friction exerted on the actomyosin ring.

To elucidate where the additional tension in the AV direction comes from, we analyzed the dynamic distribution of actin and myosin-2 within the YSL at high spatial and temporal resolution using spinning disk confocal microscopy. Formation of the actomyosin ring was accompanied by flows of actin and myosin-2 from vegetal parts of the YSL toward the margin of the EVL (Fig. 2C and movie S8). These retrograde actomyosin flows were slow (0.3 μm/min) at the onset of EVL epiboly (40% epiboly), but increased their retrograde velocity as epiboly proceeded, reaching maximal flow velocities of 1.2 μm/min around 70% epiboly stage (Fig. 2D and fig. S3).

To investigate if retrograde flows within the YSL could give rise to additional tension along the AV axis of the YSL actomyosin network, we developed a theoretical description of YSL actomyosin network dynamics within the framework of thin films of active fluids (1214) (supplementary material). Cortical flows have previously been associated with gradients of actomyosin contractility along the direction of flow (12, 15). We considered the actomyosin ring to be an isotropic thin-film fluid with myosin-2–dependent active tension (Fig. 3 and supplementary material). The animal side of the ring is mechanically connected to the EVL (4), and tension and velocity are continuous at the interface between the two layers (Fig. 3A). On the vegetal side of the ring, myosin-2 density, and consequently active tension, gradually decreases toward zero, consistent with tension measurements at the vegetal pole (fig. S2F and movie S9). This active tension gradient can give rise to contractile flow (Fig. 3 and supplementary material). The ring here is in a “stationary state,” where flow within the ring is compensated and balanced by continuous actomyosin turnover and regrowth (fig. S5), reminiscent of situations in crawling cells where retrograde actin flow is compensated by continuous polymerization at the leading edge (16). Turnover also endows the material with fluid character (12, 17).

Applying this model to EVL epiboly, we revealed two contributions to the total force exerted by the ring upon the EVL—one from circumferential tension coupling to geometry, and one from flow. In the absence of friction between the actomyosin ring and adjacent components of the yolk cell, total force on the EVL arises solely through a ring-contraction mechanism that couples to the spherical geometry of the yolk cell (termed “cable-constriction motor”; Fig. 3A). Notably, this cable-constriction motor can drive epiboly only once the actomyosin ring has passed the equator (Fig. 3A and fig. S11). By contrast, when friction is present, total force on the EVL also has a geometry-independent contribution, where retrograde flow of actin and myosin-2 is resisted by friction (termed “flow-friction motor”; Fig. 3A). In the embryo, friction will arise when the flow velocity in the ring is different from the velocity of the adjacent material, such as the yolk cell plasma membrane and the yolk cytoplasm. Consistent with this, we observed differential flow velocities between the actomyosin in the ring and adjacent microtubules within the YSL (fig. S6). This flow-friction motor pulls the EVL in a direction opposite to the actomyosin flow, operates at any stage, and can drive epiboly before passing the equator (Fig. 3A and fig. S11). Notably, friction-resisted flow provides additional tension in the AV direction, consistent with the small degree of tension anisotropy observed in the laser ablation experiments (Fig. 2B). Furthermore, experimentally measured flow profiles within the EVL and the actomyosin ring, as well as the relative tensions obtained from laser ablation, are accurately predicted by our theoretical description at all stages when friction against the substrate is taken into account (Fig. 3B). To conclude, we identified two distinct modes of ring propulsion: a cable-constriction motor due to circumferential contraction of the YSL actomyosin network, and a flow-friction motor due to contraction along the AV axis of the network.

We next asked if the flow-friction motor is sufficient to drive EVL epiboly. To this end, we took advantage of the predicted geometry dependence of the cable-constriction motor. Because the cable-constriction motor cannot exert a net force on the EVL when positioned right at the equator, propulsion by this motor would be hindered when the yolk cell is deformed from its original spherical geometry into a cylindrical shape. We thus deformed the yolk cell into a cylindrical shape by aspirating pre–gastrula-stage embryos (2.5 hpf) into agarose tubes of a diameter smaller than that of the embryo and analyzed resulting changes in EVL movements. To verify that the actomyosin ring is unperturbed in cylindrical embryos, we analyzed the distribution and flow of actin and myosin-2 within the YSL of cylindrical embryos. We found that both the accumulation of actin and myosin-2 in a ring-like structure adjacent to the EVL/YSL border and their retrograde flows from the vegetal pole toward the EVL/YSL border were largely unaffected in cylindrical embryos as compared to normal-shaped control embryos (Fig. 4 and movie S10). This suggests that the actomyosin ring remains intact in cylindrical embryos. We observed that EVL movements were largely unaffected in cylindrical embryos and proceeded with velocities similar to those of spherical control embryos (2.0 ± 0.2 μm/min compared to 1.9 ± 0.1 μm/min at 60 to 70% epiboly; compare Figs. 4D and 2D). This shows that the cable-constriction motor is not essential for EVL epiboly movements and indicates that the flow-friction motor is sufficient to drive this process.

Fig. 4

EVL epiboly without a cable-constriction motor. (A and B) Tg(actb1:GFP-utrCH) embryos labeling F-actin aspirated into cylindrical agarose tubes (diameter = 500 μm) at 2.5 hpf. Brightfield images of cylindrical embryos when control embryos were at 30% epiboly (A) and 100% epiboly (B). Scale bar, 100 μm. (C) EVL margin (blue line) and the 2D vector field of cortical flow (yellow arrows) in cylindrical Tg(actb1:GFP-utrCH) embryos at 60% epiboly. Scale bar, 25 μm. (D) Average actomyosin flow profiles of cylindrical embryos at 60 to 70% epiboly within the EVL and YSL. Negative velocities correspond to flow toward the animal pole.

Our findings have major implications for the function of actomyosin rings in morphogenesis. Whereas the prevalent model of actomyosin ring function assumes circumferential contraction as the main force-generating process, we present evidence that friction-resisted actomyosin flows can represent an equally important process mediating ring function. This raises the possibility of a more general role of cortical flows in morphogenetic pattern formation processes (18).

Supplementary Materials

www.sciencemag.org/cgi/content/full/338/6104/257/DC1

Supplementary Text

Figs. S1 to S16

Materials and Methods

References (1936)

Movies S1 to S10

References and Notes

  1. Acknowledgments: We are grateful to M. Sixt, T. Bollenbach, and E. Martin-Blanco for advice and the service facilities of the IST Austria and MPI-CBG for continuous help. M.B., G.S., S.W.G., and C.-P.H. synergistically and equally developed the presented ideas and the experimental and theoretical approaches. M.B. and P.C. performed the experiments; G.S. developed the theory; and R.H., F.O., and J.R. contributed to the experimental work. This work was supported by a grant from the Fonds zur Förderung der wissenschaftlichen Forschung (FWF) and the Deutsche Forschungsgemeinschaft (DFG) (I930-B20) to C.-P.H., S.W.G., and G.S.
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