Fig. 2: Time inference from histone images of fixed embryos with image size rescaling.
a Fixation induces embryo shrinkage. Left: confocal images of live (top) and fixed (bottom) his2av-mrfp1 embryos at the mid-sagittal plane. Scale bars, 50 μm. Insets, magnified views of individual nuclei. Scale bars, 5 μm. Top right: size comparison between live (n = 14) and fixed (n = 155) embryos, with two-sided t-test (p = 1.2 × 10−9. ****p < 0.0001). Lines, boxes, whiskers, and points indicate the median, interquartile range (IQR), 1.5 × IQR, and outliers of the distribution, respectively. Bottom right: comparison of nuclear diameter between live (n = 11, 18, and 22 embryos for nc11–13) and fixed embryos (n = 28, 33, and 38 embryos for nc11–13), with two-sided t-test (p = 1.1 × 10−4, 2.8 × 10−5, and 1.2 × 10−12 for nc11–13, respectively. ***p < 0.001; ****p < 0.0001). b Schematic of determining the optimal rescaling magnitude. Live histone images over nc12 (top) were used to extract the trend of nuclear diameter over time (bottom left, mean (line) ± s.d. (shading), data from n = 18 live embryos). This trend was compared iteratively with reconstructed data from rescaled fixed-embryo images (bottom left, red dots, data from n = 33 fixed embryos) to optimize the rescaling magnitude. Scale bar, 5 μm. c Schematic of independent time inferences for anterior, medial, and posterior regions of a fixed his2av-mrfp1 embryo at nc13. Magnified views of representative nuclei from each region are shown below. Right: results from different regions were averaged for an overall time prediction. Scale bars, 50 μm (embryo) and 5 μm (magnified views). d Differences in inferred times between anterior and medial regions, and between posterior and medial regions, during late mitotic interphase for each nuclear cycle (mean ± s.e.m.). Anterior-medial difference: n = 10, 5, and 10 embryos for nc11–13, respectively; posterior-medial difference: n = 7, 10, and 6 embryos for nc11–13, respectively. Source data are provided as a Source Data file.
