Fluorescence intensity quantification and statistical analysis

The fluorescence intensity pertaining to OPP incorporation (Fig. 4b) was quantified for the minimum complete confocal z-series of each embryo as a whole using Fiji (ImageJ)⁹⁸. The confocal images from the channel pertaining to the 488 nm wavelength were analyzed as follows: Image > Stacks > Z Project. The projection-type “Sum Slices” was used to obtain a Z-projected image for the entire embryo. The levels of H4K16ac (Fig. 4g) and RNA pol II S2p (Fig. 4h) were quantified as the fluorescence intensity per cell nuclei, and the cells were defined as inner or outer cells based on the absence or presence of CDX2 immunofluorescence, respectively. The measurements for all of the above were set with the command Analyze > Set Measurements, and the following options were chosen: Area, Mean gray value, and Integrated density. Using the “Polygon selections” tool, an area encompassing the Z-projected embryo image (for OPP) or individual cell nuclei (for H4K16ac and RNA pol II S2p) was demarcated, and the aforementioned measurements were recorded. The selected area was then moved to encompass an area excluding the embryo or cell nucleus, and background measurements were recorded. This process was continued for all the embryos analyzed under both control and p38-MAPKi conditions, and the results were transferred to a spreadsheet for further calculations. The corrected total cell fluorescence (CTCF), in arbitrary units, was measured for each embryo as follows: CTCF = integrated density – (area of selected cell × mean fluorescence of background readings)^99,100 (Supplementary Data 1 tabs marked Fig. 4b, g, h). The calculated CTCFs are plotted in scatterplots, with the means and standard deviations marked. The CTCF differences were statistically tested using the Mann–Whitney test, and the results, unless otherwise stated within individual graphs with specific P values (if statistically nonsignificant), are marked according to the following significance intervals: P < 0.0001 (****), 0.0001–0.001 (***), 0.001–0.01 (**) and 0.01–0.05 (*).