Dubuis et al. - Source_Data_Files

Complete source data for Dubuis et al. contains two folders: 1. Folder "FC_Calibration" contains the source data used for calibration of the time-dependent furrow canal depth. It displays the chi_2 minimized furrow canal depth (delta_FC) for the 8 live imaged Drosophila embryos as function of time during n.c. 14 (see Materials and Methods). It also shows the mean furrow canal depth averaged over the 8 embryos (mean_delta_FC) and adjusted mean in fixed tissues (mean_delta_FC_fixed) that we used to estimate the age of the 163 fixed embryos. Column 1 contains the time in minutes after the onset of n.c. 14. Columns 2 to 9 contain the chi_2 minimized furrow canal depths (delta_FC_x, x being the embryo number) in for the 8 live imaged embryos. NaN means that we couldn't detect the furrow canal at that particular time. Column 10 contains the mean furrow canal depth averaged over the 8 embryos (mean_delta_FC) measured in micrometers. Column 11 contains the the furrow canal depth, measured in micrometers, which we used to estimate the age of fixed embryos. It was obtained by a 5% shrinkage of the previous column (see Materials and Methods). 2. Folder "Images" contains the original images of 201 Drosophila embryos at blastoderm stage immunostained against the four main gap genes (Kni, Kr, Gt, Hb). For each embryo with provide 5 12-bit images, each corresponding to a different optical channel (see Materials and methods). Channel 0 -- rat @ Kni (488nm); Channel 1 -- bright field (delta_FC); Channel 2 -- guinea pig @ Gt (568nm); Channel 3 -- rabbit @ Kr (594nm); Channel 4 -- mouse @ Hb (647nm). Images were taken with a Leica 20x HC PL APO NA 0.7 oil immersion objective, and with sequential excitation wavelengths of 488, 546, 594 and 633 nm. The bandwidth of the detection filters were set up as shown in Figure 1A to minimize fluorophore cross-talk while still allowing good detection in each optical channel. For each embryo, three high-resolution images (1024x1024 pixels, with 12 bits and at 100 Hz) were taken along the anterior-posterior axis (focused at the midsagittal plane) at 1.7 magnified zoom and averaged together. With these settings, the linear pixel dimension corresponds to 0.44 um

Dubuis et al. - Processed_Profiles

Each file contains the source data for gap gene (Hb, Kr, Gt, Kni) processed dorsal gene expression levels measured using immunofluorescence techniques in 23 Drosophila embryos with FC depth comprised between 10 and 20 microns. We only selected embryos that were imaged close to their midsagittal plane (Orientation '1'). Column 1 contains the embryo number referring to its position on the slide (see Images.zip). Column 2 contains an information about the azimuthal orientation of the embryo on the slide ('1' if the confocal plane is closer to midsagittal plane, '2' if it is closer to the coronal plane). Column 3 contains the furrow canal depth (delta_FC) measured in micrometers. Column 4 contains the corresponding estimated age in minutes. Columns 5 to 1004 contain a 1x1000 vector representing the dorsal time-corrected gene expression level of the gap gene (Hb, Kr, Gt, Kni) in the embryo. The 1000 points are equally spaced along the AP axis. Thus, g345 represents the gene expression level at 34.5%EL. NaN means that we coudn't reliably detect the profile intensity at that position (usually near the edges). Columns 1-4 are identical in the four files

Dubuis et al. - Raw_Profiles

Each file contains the source data for raw gap gene (Hb, Kr, Gt, Kni) dorsal intensity profiles measured using immunofluorescence techniques in 163 Drosophila embryos during n.c. 14. Column 1 contains the embryo number referring to its position on the slide (see Images.zip). Column 2 contains an information about the azimuthal orientation of the embryo on the slide ('1' if the confocal plane is closer to midsagittal plane, '2' if it is closer to the coronal plane). Column 3 contains the furrow canal depth (delta_FC) measured in micrometers. Column 4 contains the corresponding estimated age in minutes. Columns 5 to 1004 contain a 1x1000 vector representing the dorsal intensity profile of the gap gene (Hb, Kr, Gt, Kni) in the embryo. The 1000 points are equally spaced along the AP axis. Thus, Intensity345 represents the intensity at 34.5%EL. NaN means that we couldn't reliably detect the profile intensity at that position (usually near the edges). Columns 1-4 are identical in the four files

Experimental data averaged over the data sets.

<p>Experimental data averaged over the data sets.</p

Propagation of wavefronts by chemical and mechanical signaling.

<p>a) Color plot showing the chemical wavefront in two dimensions. The wave starts in the center (red dot) with a single Dirac delta peak release. The color coding indicates when a nucleus releases its chemical to the bulk, going from red through the different hues of the rainbow to violet. b) Plot showing the best fits (blue and purple lines) of the diffusion model with time delay to the to the two sets of experimental data (black and gray dots with error bars). Although the time delay manages to balance the trend that the wavefront speed increases in the region of interest (but not before), the model fails to describe the observed data. Here . c) Color plot showing the mechanical wavefront in two dimensions for totally anisotropic dipoles, including their orientations, which are picked at random, and free boundary conditions. The color coding is the same as in Figure a. d) Color plot showing the mechanical wavefront in two dimensions for totally isotropic dipoles and semi-periodic boundary conditions (periodic in vertical direction, free in horizontal direction). Wavefronts are initialized at both free ends simultaneously and travel to the center, as in the experimental system. e) Plot showing fit (purple) of the displacements calculated from the model to the experimentally obtained displacements (blue) following the onset of metaphase. Fit parameters same as in Figure e (set 1). Error bars obtained by averaging over a slice of , as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077216#pone-0077216-g001" target="_blank">Figure 1a</a>. f) Plot showing fits (blue and purple lines) of the mechanical model for isotropic force dipoles and semi-periodic boundary conditions to the two sets of experimental data (black and gray dots with error bars). Fit parameters: , where is the dipole strength and the spacing in cycle 10, , and (blue line/black datapoints), (purple line/gray datapoints).</p

Wavefront propagation and speeds.

<p>a) -coordinate of nuclei at the onset of metaphase (blue diamonds) and anaphase (red pluses) vs. time for the wavefront shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077216#pone-0077216-g001" target="_blank">Figure 1</a>. Both events show two clear wavefronts moving in from near the embryo poles (solid lines). b) Ratio of the speeds of the wavefronts as measured by the onset of anaphase () and metaphase (), for different embryos and cycles. Each embryo is indicated by a different symbol and color, with the closed and open symbols representing two different measurement sets. Ratios for a given cycle and different embryos are slightly separated horizontally. c) Wavefront speed vs. cycle. Two of the embryos contribute two waves per cycle (coming in from opposite poles, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077216#pone-0077216-g001" target="_blank">Figure 1a</a>; blue squares and green diamonds). Although the actual propagation speeds vary significantly from one embryo to the next, they all follow the same trend, decreasing with successive cycles. d) Time interval between the onset of metaphase and anaphase vs. cycle. e) Log-linear plot of wavefront speeds vs. cycle, normalized by the speed of its first observed wavefront (if the first observed wave front is in cycle 10) or 0.71 times its first observed wavefront (if the first observed wavefront is in cycle 11). The black open circles connected by a dashed line corresponds to a scaling of 0.71 per cycle, showing that all embryos follow the same exponentially decaying trend. f) Average distance between nearest neighbors on a logarithmic plot. The dashed line corresponds to a dependence , where is the cycle number and . In Figures b–f, the same symbol/color corresponds to the same embryo.</p

Observation of wavefronts and mechanical response.

<p>a) Image of a <i>Drosophila</i> embryo during mitosis at the end of cycle 11, with the detected chromosomal contours overlaid. Anaphasic wavefronts (orange dashed curved lines), the long axis (green dashed straight line) and a typical slice perpendicular to the long axis (green parallel straight lines) are indicated. b) Sketch of the three main states in image analysis: interphase (circular contours), metaphase (compressed elliptical contours), and anaphase (highly extended elliptical contours, perpendicular to metaphase contour). See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077216#pone.0077216.s001" target="_blank">Figure S1</a>. c) Ratio of the two elliptical axes of the detected shape of the nuclear DNA/chromosomes vs. time in cycle 11, averaged over an -slice (as shown in a); error bars indicate variation within the slice. The transitions between interphase and metaphase, as well as the onset of anaphase, are sharp and indicated respectively by dotted (blue) and dashed (orange) vertical lines. The slice shown was taken at . d) Kymograph showing the elliptical axes ratio, (where white indicates values larger than 1 and black indicates values smaller than 1), as a function of position and time. The dotted and dashed lines indicate the onsets of metaphase and anaphase, as in Figure c. e) Average -displacement of the nuclei within one slice vs. time. After a nucleus has divided, we use the average position of its two daughters. The slice shown is identical to the one in Figure c. f) Kymograph showing the collective motion of nuclei in slices taken at different positions along the long axis of the embryo. White indicates motion in the positive direction, black in the negative direction. Dotted and dashed lines again indicate the onsets of metaphase and anaphase. Note that the displacements occur sometime after these onsets, but follow the same wavefront pattern.</p