Apical constriction facilitates epithelial sheet bending and invagination during morphogenesis1,2. Apical constriction is conventionally thought to be driven by the continuous purse-string-like contraction of a circumferential actin and non-muscle myosin-II (myosin) belt underlying adherens junctions3,4,5,6,7. However, it is unclear whether other force-generating mechanisms can drive this process. Here we show, with the use of real-time imaging and quantitative image analysis of Drosophila gastrulation, that the apical constriction of ventral furrow cells is pulsed. Repeated constrictions, which are asynchronous between neighbouring cells, are interrupted by pauses in which the constricted state of the cell apex is maintained. In contrast to the purse-string model, constriction pulses are powered by actin–myosin network contractions that occur at the medial apical cortex and pull discrete adherens junction sites inwards. The transcription factors Twist and Snail differentially regulate pulsed constriction. Expression of snail initiates actin–myosin network contractions, whereas expression of twist stabilizes the constricted state of the cell apex. Our results suggest a new model for apical constriction in which a cortical actin–myosin cytoskeleton functions as a developmentally controlled subcellular ratchet to reduce apical area incrementally.
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We thank D. Kiehart and R. Karess for providing flies; J. Goodhouse for assisting with microscopy; and S. De Renzis, X. Lu, T. Schupbach, A. Sokac, F. Ulrich and Y.-C. Wang for helpful comments on the manuscript. This work is supported by grant PF-06-143-01-DDC from the American Cancer Society to A.C.M., National Institutes of Health/National Institute of General Medical Sciences grant P50 GM071508 to M.K., and by National Institute of Child Health and Human Development grant 5R37HD15587 to E.F.W. E.F.W. is an investigator of the Howard Hughes Medical Institute.
Author Contributions Biological reagents and fly stocks were made by A.C.M. and E.F.W., and experiments were performed by A.C.M. Image analysis methods were developed by M.K. and the live-imaging data were analysed by A.C.M. and M.K. The first draft of the manuscript was written by A.C.M. All authors participated in discussion of the data and in producing the final version of the manuscript.
This file contains Supplementary Figures S1- S4 with Legends (PDF 3390 kb)
Supplementary Video 1 shows a ventral furrow apical constriction visualized with Spider-GFP. The focal plane was adjusted to stay 2 µm below the apical surface along the ventral midline. Time interval between acquired frames was 6 s and the acquisition length was 8 minutes. Video is 36x faster than real-time. (MOV 7419 kb)
Supplementary Video 2 shows a pulsed constriction of ventral furrow cells. Cell outlines are labelled with Spider-GFP. Time interval between acquired frames was 6 s and the acquisition length was 5 minutes. Video is 72x faster than real-time. (MOV 354 kb)
Supplementary Video 3 shows that the pulsed constriction is asynchronous. The colour of the cell indicates the constriction rate (as illustrated by the colour bar in Fig. 1h) for a given time point. Time interval between acquired frames was 6s and the acquisition length was 6 minutes. Video is 18x faster than real-time. (MOV 2533 kb)
Supplementary Video 4 shows apical myosin dynamics during ventral furrow cell apical constriction. Z-projections of Myosin-mCherry (green, 5 µm depth) are merged with a single Spider-GFP z-slice located 2 µm below the apical cortex (red). Time interval between acquired frames was 5 s and the acquisition length was 7.5 minutes. Video is 25x faster than real-time. (MOV 11170 kb)
Supplementary Video 5 shows that myosin coalescence accompanies apical constriction. Z-projections of Myosin-mCherry (green, 5 µm depth) are merged with a single Spider-GFP z-slice located 2 µm below the apical cortex (red and bottom). Time interval between acquired frames was 6 s and the acquisition length was 2.6 minutes. Video is 50x faster than real-time. (MOV 770 kb)
Supplementary Video 6 shows that .Twist and Snail differentially regulate myosin dynamics. Z-projections of Myosin-GFP (5 µm depth) for the indicated genetic backgrounds. Note that myosin coalescence is more prominent in twist mutant embryos than snail mutant embryos. This results in more movement of myosin structures since they are being pulled. Time interval between acquired frames was 10 s and the acquisition length was 16.7 minutes. Video is 100x faster than real-time. (MOV 18122 kb)
Supplementary Video 7 shows that. twistRNAi and snailRNAi mimic the mutant phenotypes. Z-projections of Myosin-GFP for the indicated knock-downs. Time interval between acquired frames was 10 s and the acquisition length was 16.7 minutes. Video is 100x faster than real-time. Note that the phenotypes are not as severe as the null mutants, probably due to remaining Twist and Snail activity. (MOV 10269 kb)
Supplementary Video 8 shows that the Constriction pulses in twistRNAi embryos are not stabilized. Cell outlines 2 µm below the apical cortex are labelled using SpiderGFP. Cell shape is relatively constant in snailRNAi embryos, while fluctuations in cell size and shape occur in twistRNAi embryos. Time interval between acquired frames was 6 s and the acquisition length was 7 minutes. Video is 36x faster than real-time. (MOV 10159 kb)
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Martin, A., Kaschube, M. & Wieschaus, E. Pulsed contractions of an actin–myosin network drive apical constriction. Nature 457, 495–499 (2009). https://doi.org/10.1038/nature07522
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