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http://dmm.biologists.org/lookup/doi/10.1242/dmm.030163Access the most recent version at DMM Advance Online Articles. Posted 11 May 2017 as doi: 10.1242/dmm.030163http://dmm.biologists.org/lookup/doi/10.1242/dmm.030163Access the most recent version at
First posted online on 11 May 2017 as 10.1242/dmm.030163
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Figures
Figure 1. Microfluidic array for parallel imaging of live embryos. (A) Torso/ERK
signaling antagonizes Cic-dependent gene repression. (B) Schematic of the microfluidic
array with close-up views of a single-trapping unit in both layers. (C) Illustrations depicting a
single embryo (red) within a trapping unit; dorsal-ventral (DV) and anterior-posterior (AP)
cross-sections, and three-dimensional (3D) representations depicted. (D) Superficial fluid
velocity at the mid-plane of the trapping unit predicted by the finite element model. (E)
Representative image of a loaded microfluidic device: black triangles indicate embryos
trapped within the array.
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Figure 2. Image processing of time lapse data of Cic dynamics. (A) Time-lapse images
of a representative Capicua-Venus (CicV) expressing embryo. (B) Time-lapse images of a
single optical cross-section the blastoderm embryo. (C) Representative space-time plot of
Cic dynamics. Vertical axis represents egg length (L) from anterior to posterior (0-1),
horizontal axis represents imaging time, and color axis represents CicV intensity. White
vertical dotted lines indicate nuclear cycle transitions with cycles labeled for stages 4 and 5
of embryogenesis. Time is set to zero at the start of imaging.
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Figure 3. Low dimensional approximation of Cic dynamics. (A-C) Reconstruction of Cic
dynamics using singular value decomposition for WT (A), TorD4021 (B), MEKF53S (C). Single
embryo raw data (left column), associated first spatial mode and projection (middle column),
and reconstructed heat maps (right column). Time is set to zero at the 13th mitotic division.
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Figure 4. Statistical analysis of Cic dynamics in the wild type and mutant embryos. (A-
C) Dynamics of the amplitudes of the first spatial mode for the wild type, TorD4021, and
MEKF53S. (D) Comparison of mean amplitude in cycle 14 for each genotype reveals
significant pairwise differences between genotypes, except for the MEK mutants.
Throughout the figure, the shaded regions indicate one standard deviation. Students t-test,
two-tailed: P value, not significant (n.s.), * <.05, ** < .005, *** < .0005.
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Figure 5. Single-cell analysis of the Cic import rates. (A-C) Mean single cell CicV
intensity traces for the wild type, TorD4021 (A), MEKF53S (B), and MEKE203K (C). The wild type:
black; mutant: red in each plot. (D) Comparison of recovery times reveals significant pairwise
differences between genotypes. Throughout the figure, the shaded regions and error bars
indicate one standard deviation. Students t-test, two-tailed: P value, not significant (n.s.), *
<.05, ** < .005, *** < .0005.
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Figure 6. Statistical comparison of spatial profiles. (A-C) Profiles of the dominant modes
for the wild type (A), TorD4021 (B), and MEKF53S (C) across the embryo length (L). (D) Pairwise
comparison of characteristic length, 𝜆. The gray shaded regions indicate one standard
deviation. Students t-test, two-tailed: P value, not significant (n.s.), * <.05, ** < .005, *** <
.0005.
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Figure S1: Trapping efficiency and fluid dynamics modeling of non-optimized two-layer anterior-posterior array. (A) Representative image of a loaded microfluidic array wherein white * indicate embryos that were successfully trapped within the array and black * indicate empty traps. (B) 3D finite-element modeling results of fluid dynamics across a single trapping unit with superficial velocity at the array mid-plane depicted.
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Disease Models & Mechanisms 10: doi:10.1242/dmm.030163: Supplementary information
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Figure S2: Optical aberrations when imaging at the mid-sagittal plane in a simplified (AP) array. (A) Optical confocal micrographs of a representative CicV expressing embryo imaged within the previously describe AP array at the mid-sagittal plane. CicV fluorescence (top left), brightfield (bottom left), and merged (right). * indicates optical aberrations caused by PDMS sidewall. Specifically, CicV reflection in the PDMS is evident as well as dimming in blastoderm, which is further characterized in panel B. (B) Cic gradient across the egg length (EL) from anterior to posterior (0-1) along the top most epithelium in the embryo depicted in A (black line). The expected curvature of the Cic gradient is depicted (red line). Shaded region highlights the affected portion of the gradient.
Disease Models & Mechanisms 10: doi:10.1242/dmm.030163: Supplementary information
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Crop Segment Filter Skeleton Extend Merge
B C D E F
Figure S3: Image processing pipeline for single-cell data for Cic dynamics. (A) Cropped CicV heat-map for nuclear cycle 14 is segmented (B) followed by filtering (C) which removes unwanted objects and nuclei from outside the region of interest. Image from (C) is skeletonized (D) which reduces nuclei spatial locations to a single point along the AP axis. Skeletonized images are then extended (E) to approximate nuclei spatial locations for all times. (F) Merged image of A and E where white horizontal dotted lines indicate central region of embryo wherein single-cell analysis was performed. (G) Single-cell CicV expression for the cells identified in I. Time is set to zero at thirteenth mitotic division.
Disease Models & Mechanisms 10: doi:10.1242/dmm.030163: Supplementary information
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Figure S4: Calculation of characteristic time constant describing single-cell Cic nucleocytoplasmic shuttling. (A) Single-cell CicV traces for 234 single-cells in WT embryos. (B) Representative single-cell CicV trace (black dots) from A with best fit model (red line). I is intensity at time t, Io is intensity at time to, which represented the onset of nuclear cycle 14. If is the maximum intensity reached at steady-state. 𝜏 is the characteristic time constant used to describe CicV nucleocytoplasmic shuttling. Each single-cell CicV trace was fitted to the inset equation using GraphPad Prism software and the characteristic time constant 𝜏 was averaged among all identified cells from all embryos in each genetic background and compiled in figure 5D.
𝐼 = 𝐼𝑜 + (𝐼𝑓−𝐼𝑜)[1 − 𝑒1
𝜏𝑡𝑜−𝑡 ]
Disease Models & Mechanisms 10: doi:10.1242/dmm.030163: Supplementary information