designed as a translation-blocking morpholino, whereas cFlamingo1-MO (5'- ... embryos was electroporated with either 2mM control morpholino or cFlamingo1 ...
doi: 10.1038/nature06211
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Supplementary Figure S5. Expression patterns of Wnt-PCP genes at the stages indicated at the top of each column. a-e: cFlamingo1; f-j: cPrickle; k-o: cVanGoghlike2.
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doi: 10.1038/nature06211
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Supplementary Figure S6 Electroporation of Dsh-∆DEP construct led in the majority of cases to an ingression domain in the shape of a posterior crescent (see Fig. 3 c). Occasionally however (when electroporation was less even), multiple small streaks appeared to arise within the electroporated domain, as shown in the figure below in whole mount (left) and sections (upper and lower right). Brown reveals anti-GFP staining, brachyury in blue.
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doi: 10.1038/nature06211
Supplementary methods Embryo imaging. A round hole was drilled into the bottom of a 35mm Petri dish and its edges elevated by 1mm using a glass ring. A round coverslip (φ = 32mm, 100µm thick, VWR International 631-0162) was sealed to the bottom of the dish with high vacuum silicone grease (BDH 33135 3N). In the central well, liquid agar-albumen (0.33% agar, Sigma A-7002, 83% egg albumen) was poured and allowed to set. Electroporated embryos on their glass rings with vitelline membrane were carefully lifted and placed into the imaging dish. The glass ring was then secured in place with a circular plastic support wedged between it and the sides of the dish. The whole assembly was then inverted onto the lid of the dish, on which a well filled with egg albumen was placed. The edges of the lid were then sealed with Parafilm and the assembly placed under the objective (x20, N.A. = 0.5 or x40, N.A. = 0.8) of a Leica SP2 microscope equipped with a Tsunami XI infrared laser and a thermo-regulated hood. To excite both EGPF and DsRed-Express, the infrared laser was tuned to 910nm.
Cell
tracking.
Movie
processing
was
done
with
the
ImageJ
software
(http://rsb.info.nih.gov/ij/). To outline the tracks of fluorescent cells in the low-power movie S1, the “Kalman filter” (http://rsb.info.nih.gov/ij/plugins/kalman.html, contributed by C. P. Maurer) was applied. Individual cells were tracked using the “Manual Tracking” plug-in by F. Cordelières (http://rsb.info.nih.gov/ij/plugins/track/track.html). For the analyses described here, only the cells in the epiblast were considered. Cell divisions. In movie S2, 73 cell divisions can be observed in 193 histone 2B-EGFPlabelled cells that pass through the imaged region (predicting an overall population doubling time of 12.2 hours; however, some cells divide twice and others not at all during the filming period). Analysis of protrusive activity. In the maximum intensity projections of cytoplasmic or membrane labeling of cells, the area of selected cells was measured, and a circle was drawn centered on their center of gravity which extended up to two sides of the cell. The area of regions of the cells not covered by the core circle was considered as a measure of
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doi: 10.1038/nature06211
their protrusive activity. The orientation of the cells was assessed by fitting an ellipse and measuring its inclination with ImageJ’s measurement tool. Cells in the intercalation domain put out significantly larger protrusions (31.0 +/- 15.6% of total cell area) than the ones in the lateral domain (22.1 +/- 6.8%; p
