Evidence that tissue recoil in the early Drosophila embryo is a passive not active process

Figures

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  FIGURE 1:. The kinetics of myosin depletion using the AID2 degron system. (a) The dynamics of myosin depletion quantified as the time-course of fluorescence intensity within a region of interest. (b and b’) Myosin fluorescence at the cellularization front in a control embryo and in a 5-Ph-IAA-AM injected embryo at the same time-point in cellularization. Confocal slices were taken 26 microns beneath the surface of the embryo. The main images were taken with the same microscope settings and processed in identical ways. The insets are magnified images, adjusted separately for brightness to allow for easier visualization in the 5-Ph-IAA-AM sample. Scale bars are 25 μm in the main figures and 10 μm in the insets. (c and c’) Representative snapshots showing the time-course of sqhGFP-AID depletion. Scalebar: 25 μm.

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  FIGURE 2:. Ferrofluid-based assay used to quantify tissue material properties in different conditions. (a–a’’) Snapshots of the ferrofluid pulling assay performed on untreated (control) embryos. Scalebar: 50 μm. (b and c) Zoomed in regions of interest illustrating the dynamics of the droplet being pulled by the magnet in two experiments. (b) is an uninjected control embryo, while (c) is a 5-Ph-IAA-AM injected sqhGFP-AID embryo. Note that the cellularization front (red arrowhead) is much more apparent in the untreated control case: this is a representative effect of the treatment, making the cellularization front appear “floppy” and uneven. Scalebar: 20 μm.

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  FIGURE 3:. Quantification of tissue material properties after myosin depletion. Different color traces are repetitions of the same experiment in different embryos. Top panels show the measurements in the untreated (control) embryos. Middle panels correspond to embryos in which sqhGFP-AID was depleted using the AID2 technique. Bottom panels correspond to embryos in which sqhFRB-GFP was depleted using the nanobody-GFP aggregation-based technique. Left-most figures show the displacement of the ferrofluid droplet as a function of time. Middle figures represent the same data on a log–log plot. Right-most figures show plots of the recoil dynamics with logarithmic y-axis. Average values ± SEM are given in the main text. Single-factor ANOVA failed to show a statistical difference among all three samples for the calculated relaxation time-constant (p = 0.11) and percent recoil (p = 0.92). Control n = 9, AID2 n = 6, Aggregation assay n = 9.

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  FIGURE 4:. Aggregation-based assay for depletion of abundant proteins in the early Drosophila embryo. (a) Schematic illustrating the basis of the method (see text). (b–b’’’) A representative sqhFRB-GFP embryo that has been injected with biotinylated anti-GFP nanobody and streptavidin. Yellow signal (b’’) is coinjected dextran indicating distribution of the injected material. Gastrulation fails in the vicinity of the injection site. Red arrow indicates region with properly forming ventral furrow. White arrowhead indicates region where sqhFRB-GFP forms aggregates; the ventral furrow fails to form in this region. Image was taken 5–10 min after the injection. Scalebar: 30 μm. (c–c’’). The same aggregation technique as in (b) performed at an earlier timepoint, during cellularization. Image was taken 5–10 min after the injection. Furrow canals lack the usual rounded shape, indicating loss of tension. (c’) is a zoomed in image of the box indicated in (c). (c’’) is from the same box, except 4 μm closer to the surface; this image was included to show the aggregates, which are more obvious in this plane of section. (d). Uninjected control sqhFRB-GFP embryo at the same stage as in (c) showing the…