Fig. 4.

Frequency distribution of GFP-Dvl puncta. (a) Cells expressing GFP-Dvl were segmented as described above (red line around the cell) and the segmentation used to define a cortical strip of 5 μm (defined by outer red line and inner yellow line), which allows for puncta that are cortical, i.e. in very close proximity to the cell edge, to be counted. The cell domains were defined by the diagonals of a bounding rectangle (marked by white lines) and the length of each domain measured. The puncta identified and counted are marked by a red circle. (b) Puncta counts in each domain normalised to cell cortex length showed no statistically significant bias in their distribution. (c) The puncta count for each domain was normalised to the domain length, to account for different contour shapes between domains. No statistically significant bias was found between the four cell quadrants. Error bars are ±2× Standard error of the mean. ((c)–(e)). Controls for live imaging analysis of GFP-Dvl. (c) Single frame from a live stage 12.5 explant expressing 100 pg/embryo GFP-Dvl, showing a weak, diffuse fluorescence. (d) A stage 12.5 explant expressing 300 pg/embryo GFP-Dvl, fixed for 2 h at room temperature in MEMFA and imaged in 30% glycerol/PBS, showing bright cytoplasmic fluorescence and puncta. (e). Single frame from a live stage 12.5 explant expressing 250 pg/embryo of GFP, showing diffuse cytoplasmic fluorescence with no puncta. All injections were done at the 32-cell stage in blastomere C1.