Figure 3: The data needed for Bayesian network equations can be easily acquired. Here, confocal analysis and qRT-PCR are examples of data acquisition that can be used to generate plots such as those found in Figures 4 and 6. (a) Immunofluorescence shows expression of Nanog in the nuclei of undifferentiated cells. This expression decreases drastically after 2 days of differentiation. HNF3β expression is first visible at d2 in suspension. White arrowhead points to nuclear localization of this transcription factor. By d10, expression is localized to the right half of the EB. Noggin expression is first seen at day 5 and, as expected of a secreted factor, localizes to vesicles in the cytoplasm (white arrows). At day 10 Noggin is confined to the left half of the EB. Ap2-α is first detected at d5 in suspension. Like the other markers, by d10 Ap2-α shows specific staining in one portion of the EB. 5 μm scale applies to all images in upper panel. 10 μm scale bar applies to all images in lower panel. (b) qRT-PCR of EBs at specified days of differentiation illustrates one method for understanding timing of germ layer development using germ layer-specific markers Fgf5 for ectoderm, Hnf-4 for endoderm, and Brachyury for mesoderm.