Complete canthi removal reveals that forces from the amnioserosa alone are sufficient to drive dorsal closure in Drosophila.
Canthi maintain purse string curvature (necessary for their dorsalward forces), and zipping at the canthi shortens leading edges, ensuring a continuous epithelium at closure completion.Dissection of one or both canthi resulted in tissue recoil and flattening of each purse string.How the embryo coordinates multiple, large forces (each of which is orders of magnitude greater than the net force) during native closure and is also resilient to multiple perturbations are key extant questions.
Affiliation: Department of Biology, Duke University, Durham, NC 27708.
- Animal Structures/embryology*/metabolism/ultrastructure
- Drosophila melanogaster/cytology/embryology*/metabolism
- Embryonic Development*
- Mechanotransduction, Cellular*
- Serous Membrane/metabolism/ultrastructure*
- Biomechanical Phenomena
- Embryo, Nonmammalian
- Epithelial Cells/cytology/metabolism
- Laser Capture Microdissection
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Figure 4: Leading edges recoil and flatten after canthus removal. Fiducials at cell–cell boundaries along each leading edge were tracked through time, including periods before, during, and after canthus removal procedures. (A, A′) Schematics illustrating Euclidean and ventralward distance measurements plotted over time (B, C and B′, C′, respectively. (B, B′) Plots report distance measurements between a reference fiducial (orange bar in inset) and three distinct fiducials (ovals in inset, in increasing distance from cut edge: yellow, red, blue) for the anterior-top, freed edge. Free edges refer to the leading edges detached from the canthus due to canthus removal. Yellow arrowheads show rapid shortening of purse strings. (C, C′) Plots compare distance measurements between each fiducial near freed edges (yellow ovals in inset) and its corresponding reference fiducial (bars in inset) in a single embryo. Colors in each plot correspond to distance measurements between the same-colored reference fiducial and the yellow fiducial proximal to the cut. The orange bar corresponds to the anterior-top free edge, pink to posterior-top, purple to anterior-bottom, and light blue to posterior-bottom. Vertical dashed lines in B, B′, C, and C′ indicate the start of the posterior (maroon) and anterior (green) canthus removal cuts. (D) Inverse images of a confocal fluorescence micrograph of an embryo ubiquitously expressing DE-cadherin-GFP before canthus removal (left) and after (right). Image overlay illustrates the procedure used to find leading edge curvature. A snakes (active contour) algorithm locates leading edges via optimally positioned snake points (blue dots). The snake points are fit to a polynomial (red lines), from which the curvature of the curve furthest from the midline is computed and plotted (E, F). (E) Plot of curvature over time for a double canthus removal embryo (green) and a native control embryo (gray). (F). Plot of curvature over time for four double canthus removal embryos (green, purple, orange, green) and control (gray). Scale bar, 10 μm (D).
We measured the extent to which the leading edge contracts after canthus removal by following the movements of fiducials on or near the purse strings before, during, and after laser dissection. Four sets of fiducials were followed (each embryo has two purse strings, and the double canthus cuts free two ends on each purse string) on GFP-moe-ABD embryos. For each free end, three fiducials were tracked, and their distance to a fourth, reference fiducial was measured and plotted as a function of time (Figure 4, A–C). The reference fiducial was the one located farthest from the cut edge, closest to the symmetry point, and did not move much, if at all. The plots indicate that the purse strings rapidly shorten (arrows in Figure 4B confirming the observations in Figures 7 and 8 of Kiehart et al., 2000). Moreover, the extent to which segments of the purse strings shorten is inversely proportional to the proximity of the segment to the cut end of the purse string (compare the movement of the fiducials and the extent of segment shortening in Figure 4B). These data are consistent with the embryo's response to other, noncanthi laser surgical cuts whereby there is little tissue movement >∼50 μm from the cut. The shortening of the purse strings decreases the tension, T.