Quantifying interface co-alignment

Interface orientations, relative to the embryonic axes, were calculated for PSB, -1 and +1 interfaces at all time points in movies from eve-EGFP, GAP43-mCherry or wg^CX4; eve-EGFP, GAP43-mCherry and for PSB, S1/2B, S2/3B, +1 and -1 interfaces at all times points in movies from sqh^AX3; sqh-GFP, GAP43-mCherry embryos. All distributions of interface orientations (from 0, parallel to the AP embryonic axis, to 180°) were reflected around 90°, producing distributions from 0°, AP-aligned, to 90°, DV-aligned. As a measure of co-alignment, the proportion of interfaces oriented between 60 and 90° relative to the AP axis was plotted over time, from -20 to 60 min. Cumulative frequencies were calculated for each reflected distribution of interface orientations at 40 min (corresponding to when Myosin II levels were significantly different). Two-sample Kolmogorov-Smirnov tests on the cumulative frequency distributions of interface orientation were used to compare treatments (Prism, GraphPad).

We repeated the analysis, treating the distribution of interface angles from 0° to 180° as a circular distribution, and calculating the parameter of concentration (κ) of the von Mises probability density function. Both the plots of κ versus time and the comparison of distributions at 40 min gave very similar results to the above methods (data not shown).