Steps in obtaining a quantified gene expression profile from a Nematostella in situ hybridization. A) An unaltered embryo geometry (chosen from a list of interpolated geometries) is projected on an in situ hybridization of Nvnos2 at the end of gastrulation (original image from ). In the original image, the arrowhead highlights gene expression in the aboral ectoderm and the asterisk denotes the future mouth. Endoderm and ectoderm are labeled ‘en’ and ‘ec’, respectively. B) The points in the graphical geometry are manually dragged over the cell layer boundaries of the observed Nematostella embryo. C) After applying a color inversion, the embryo is decomposed into parallel sections along the cell layer. D) For each section in figure 4C, the average red, green, blue and greyscale intensities are plotted in an expression profile as a function of its position along the cell layer. The segment on the aboral end of the decomposition corresponds to 0 on the horizontal axis; from here, the decomposition proceeds counterclockwise along the cell layer. After crossing the endoderm-ectoderm boundaries (which correspond to the vertical black lines in the plot), the decomposition again reaches the aboral end, corresponding to the right side of the horizontal axis in the intensity plot. Arrows highlight artefacts (see example 1 in the main text). The main profile is plotted as a solid graph. E) After vertically and horizontally shifting the main profile from panel D, artefacts are removed, noisy regions are smoothed and the profile is symmetrized. On the horizontal axis, the endoderm center lies at normalized cell layer position 0 and the ectoderm center is normalized at −50 and +50, while the maximum intensity is normalized to 100 on the vertical axis.