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. 2000 Apr 17;149(2):471-90.
doi: 10.1083/jcb.149.2.471.

Multiple forces contribute to cell sheet morphogenesis for dorsal closure in Drosophila

D P Kiehart  1 , C G Galbraith, K A Edwards, W L Rickoll, R A Montague
Affiliations

Multiple forces contribute to cell sheet morphogenesis for dorsal closure in Drosophila

D P Kiehart et al. J Cell Biol. .

Abstract

The molecular and cellular bases of cell shape change and movement during morphogenesis and wound healing are of intense interest and are only beginning to be understood. Here, we investigate the forces responsible for morphogenesis during dorsal closure with three approaches. First, we use real-time and time-lapsed laser confocal microscopy to follow actin dynamics and document cell shape changes and tissue movements in living, unperturbed embryos. We label cells with a ubiquitously expressed transgene that encodes GFP fused to an autonomously folding actin binding fragment from fly moesin. Second, we use a biomechanical approach to examine the distribution of stiffness/tension during dorsal closure by following the response of the various tissues to cutting by an ultraviolet laser. We tested our previous model (Young, P.E., A.M. Richman, A.S. Ketchum, and D.P. Kiehart. 1993. Genes Dev. 7:29-41) that the leading edge of the lateral epidermis is a contractile purse-string that provides force for dorsal closure. We show that this structure is under tension and behaves as a supracellular purse-string, however, we provide evidence that it alone cannot account for the forces responsible for dorsal closure. In addition, we show that there is isotropic stiffness/tension in the amnioserosa and anisotropic stiffness/tension in the lateral epidermis. Tension in the amnioserosa may contribute force for dorsal closure, but tension in the lateral epidermis opposes it. Third, we examine the role of various tissues in dorsal closure by repeated ablation of cells in the amnioserosa and the leading edge of the lateral epidermis. Our data provide strong evidence that both tissues appear to contribute to normal dorsal closure in living embryos, but surprisingly, neither is absolutely required for dorsal closure. Finally, we establish that the Drosophila epidermis rapidly and reproducibly heals from both mechanical and ultraviolet laser wounds, even those delivered repeatedly. During healing, actin is rapidly recruited to the margins of the wound and a newly formed, supracellular purse-string contracts during wound healing. This result establishes the Drosophila embryo as an excellent system for the investigation of wound healing. Moreover, our observations demonstrate that wound healing in this insect epidermal system parallel wound healing in vertebrate tissues in situ and vertebrate cells in culture (for review see Kiehart, D.P. 1999. Curr. Biol. 9:R602-R605).

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Figures

Figure 1
Figure 1
Low magnification micrographs of two embryos provide an overview of morphogenesis from extended germ band through dorsal closure. Two embryos with slightly different orientations give an overall view of the movements examined in this study (a–d, nearly sagittal view; and e–h, dorsal view). The posterior end of the germ band is indicated by arrows in a–c. (a) Germ band–extended embryo. (b) Germ band retraction has begun. The amnioserosa is indicated (AS; time, 54 min since a). (c) Germ band retraction, 80–90% complete (time, 1 h 21 min since a). The leading edge of the lateral epidermis has not yet accumulated much actin and is irregularly shaped (c, arrowheads). (d) Germ band retraction is essentially complete (time, 1 h 51 min since a). The leading edge of the lateral epidermis has begun to accumulate more actin in local regions (d, arrows). (e) End of germ band retraction, beginning of dorsal closure. The stage here is approximately the same as shown in d. (f) Dorsal closure is 50–75% complete. The leading edge of the lateral epidermis is resolved into a smoothly arcing front and appears as a bright line, indicating that it has begun to accumulate more actin (time, 2 h 33 min since e). (g) Dorsal closure 80–90% done. Even at low magnification, the change in cell shape from polygonal, at the end of germ band retraction, to elongate at this stage of dorsal closure is apparent. This is seen in the lateral epidermis (elongated perpendicular to the long axis of the embryo) and in the amnioserosa (elongated parallel to the long axis of the embryo; time, 3 h 9 min from e). (h) Dorsal closure complete. The bright bar indicates that the embryo just finished closing. In later panels (not shown), no scar is observed. Bright spots are denticles. A 25×, 0.8 NA lens was used to collect these images. Bar, 100 μm.
Figure 2
Figure 2
Schematic of tissue organization and simplified analysis of intrinsic tension/stiffness. (a) Schematic of embryo at early dorsal closure (traced from Campos-Ortega, 1985 Fig. 3.8A). Tissues and structures referred to in the text are appropriately labeled. Large arrows depict the beam of the irradiating laser for ablation of cells in the lateral epidermis, the leading edge of the lateral epidermis and the amnioserosa. Note that, at these early stages, the leading edge of the lateral epidermis and the amnioserosa overlie yolk (see text). (b–d) Schematic diagrams depict idealized, Hookean behavior (i.e., conforms to Hooke's Law) of a tissue. b shows an idealized tissue that varies in stiffness. Thus, the spring constant (or stiffness) of the spring to the left is half that of the one on the right. Shown are the springs at rest length, i.e., there is no intrinsic tension. c shows the ideal tissue with tension applied across it. Note that the stiffer tissue (spring) on the right extends half as much as the tissue (spring) on the left. This is because the tension across these two tissues in series is identical and, because F = kx and x = F/k, the amount stretched (x) will be half for the stiffer spring. d shows what happens when the tissue is cut (here between the two springs) and the tissue on either side of the cut regains its equilibrium length. Note that the end of the spring on the left moves half as far as the one on the right (d, arrows, dotted circle indicates position of linkage before tissue cutting).
Figure 3
Figure 3
A dorsal view of cell shape change in dorsal closure. Overall changes in the shape of the leading edge of the lateral epidermis are shown in a–e and in schematic form in a′–e′. f plots the change in the length of the leading edge as it changes at time 0 (a) through to when it remains a bright bar of tightly apposed cells in e. Note that the length of the leading edge in e can also be interpreted as 0. The graph shows that a change in length is essentially linear with time. Analysis of the change in area (g) indicates that individual clusters of cells (open symbols) change area at the same rate as the amnioserosa disappears (solid triangles), implicating changes in the area of the amnioserosa with progress of the leading edge of the lateral epidermis (see text).
Figure 4
Figure 4
The amnioserosa loses cells during dorsal closure. A high magnification view of the amnioserosa shows a cell that accumulates actin at its apical end and drops out of the plane of the tissue (arrow). Time in minutes (from the start of the time-lapsed sequence) is shown in the lower left. a′–c′ are identical to a–c, but have a set of cells outlined in white to show the overall change in shape.
Figure 5
Figure 5
Changes in the lateral epidermal cell shape during dorsal closure stages show that the cells start off polygonal and become elongate. (a) Germ band (GB) retraction in progress (time is 5 min after the start of the time-lapsed sequence). Cells of the lateral epidermis (labeled GB) are polygonal in shape and the interface between the amnioserosa (AS) is scalloped (arrowhead). (b) Germ band retraction is complete. Arrowheads again depict scalloped edge (time is 61 min after the start of the time-lapsed sequence). (b) Graph of the length of the leading edge of the lateral epidermis marked by double-headed arrows in d–f. The abscissa of c represents the time for d minus 70 min. d–f are micrographs showing the elongation of the cells of the lateral epidermis (times are 104, 164, and 202 min after the start of the time-lapsed sequence). Double-headed arrows show the set of 10 cells whose overall length was reduced to ∼68% of their original length during the course of the time-lapsed sequence shown. Points for each panel are labeled in c.
Figure 6
Figure 6
Mechanical wounds to the leading edge of the lateral epidermis cause hemocytes to accumulate and can disrupt the actin-rich supracellular purse-string. (a–c) Early dorsal closure–staged embryo with a micropipette inserted into its flank. Time is shown in minutes after insertion of the micropipette. With time, hemocytes begin to accumulate (b and c, arrowheads) as a response to wounding. (d–f) Before, right after, and after healing of a mechanically induced lesion in the leading edge of the lateral epidermis. Time is shown in minutes with respect to insertion of the micropipette. In d, the intact leading edge is shown, with the fluorescently filled micropipette out of focus and coming in horizontally from the right, just above the center of the panel. (e) After wounding, the leading edge is disrupted. (f) With time, the wound heals and dorsal closure is completed.
Figure 7
Figure 7
Rapid changes in the leading edge after laser ablation demonstrate that actin and myosin at the leading edge of the lateral epidermis form a contractile purse-string that is under tension. (a) Kalman-filtered image of the embryo just before laser ablation. (b–d) Frames from videotape of the ablation taken 10, 20, and 40 s after a single 3-ns pulse of UV irradiation. White lines show the position of landmark points in the image as the tissue spreads in response to the wound. Note that the longest traces are those that track the position of points in the leading edge. There is minimal change in the architecture of the amnioserosa. The lateral epidermis moves an intermediate amount. e is a Kalman-filtered image taken after the wound stabilized.
Figure 8
Figure 8
Reproducibility of the wounding process is demonstrated by this panel of three different embryos wounded at the leading edge of the lateral epidermis. Arrowheads indicate part of the leading edge that recoiled away from the site of laser irradiation.
Figure 9
Figure 9
Wounding the amnioserosa and the lateral epidermis. (a and b) The amnioserosa shown before (a) and after (b) laser wounding. Note that the tissues surrounding the wound site spread outward, away from the wound, indicating that the tissue is under isotropic tension. (c and d) The lateral epidermis before (c) and after (d) wounding. Here, the spread of the tissue is anisotropic. In each case, white lines indicate the trajectory of points in the tissue after the irradiation. White dots mark the starting point and black dots mark the end point of the formation of a mature wound before the initiation of healing. Arrowheads indicate the location of part of the leading edge. (e and f) Multiple wounds to the flank of the lateral epidermis cause the leading edge to advance toward the dorsal midline. Arrowheads mark the lateral extent of the wounds, and are positioned to indicate the middle of the wound measured along the dorsal-ventral axis. As a consequence of the ablations, the leading edge of the lateral epidermis has prematurely advanced toward the dorsal midline. Bar for a–d is shown in d; bar for e and f is shown in f.
Figure 10
Figure 10
Recovery of the embryo after two and four wounds to the leading edge. a–e show a time-lapsed sequence of healing following two wounds to the leading edge. f–j show a similar sequence following 4 wounds to the leading edge. Arrowheads in a and f indicate the edges of the wounded lateral epidermis. Arrows in a–c, f, and g point to the accumulated actin at the site of the wound, indicating the formation of a purse-string response to wounding.
Figure 11
Figure 11
Repeated wounding of the leading edge does not stop dorsal closure. The leading edge was wounded as soon as healing was apparent (as observed by a through-focus analysis). Time is shown from the first panel in hours/minutes. The left column of panels shows the embryo just before wounding. The right panel shows the embryo after the first wound (b) and after subsequent wounds designed to disrupt the mechanical integrity of the leading edge.
Figure 12
Figure 12
Repeated wounding of the amnioserosa does not stop dorsal closure. The amnioserosa was wounded as soon as healing was apparent (as observed by a through-focus analysis). The time is shown from the first panel in hours/minutes. The left column shows the embryo just before wounding. The right column shows the embryo after the first wound (b) and after subsequent wounds designed to disrupt the mechanical integrity of the leading edge.
Figure 13
Figure 13
A schematic diagram of dorsal epithelial tissues in a midstage 14 embryo indicates the forces that contribute to cell movement during dorsal closure. (a) Tracing of the shape of the leading edge (LE) of the lateral epidermis and of cell shape in the amnioserosa (AS, traced from images of a wild-type embryo before wounding). b–d show wounds to different epithelial tissues. In each case, the ablating spot is shown as a gray circle, and the outline of the leading edge of the lateral epidermis before wounding is shown as a heavy dotted line (in some cases it superimposes on the outline of the leading edge after wounding). Arrows depict the direction of tissue movement after ablation. (b) A wound to the leading edge of the lateral epidermis causes recoil of the leading edge away from the site of ablation and away from the dorsal midline. (c) A wound to the amnioserosa causes nearly isotropic spreading away from the site of ablation. (d) A series of wounds to the flank of the lateral epidermis releases tension on the leading edge and allows it to advance prematurely towards the dorsal midline.
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