Early depletion of Smad1 results in disruption of epiblast derivatives and commitment to visceral endoderm. (A) Smad1 knockdown was induced on day 2 of EB differentiation, and EBs were analyzed for morphologic changes and GFP expression over the following several days. Induction was maintained by addition of doxycycline at day 5; day 7 EBs were transferred to gelatinized tissue-culture plates and examined both for their morphology and their ability to adhere to the plate surface. (B) Day 2–induced EBs were harvested at day 3.75 of differentiation and cultured in methylcellulose medium with cytokines permissive for differentiation into BL-CFC colonies. Colonies were identified and counted by their distinctive morphology 4 days later. For each sample, n = 3 and the graph is a representative example taken from a pool of at least 4 experiments. Error bars indicate SEM; *P < .01 compared with uninduced controls. Transcription of genes associated with ectoderm (C), mesoderm (D), endoderm (E), and, more selectively, visceral or definitive endoderm (F) were analyzed by qPCR in day 4.5 EBs that had been induced at day 2. Data were analyzed using the 2^−ΔΔCT method¹⁹  and reported as fold change in mRNA transcript level, and were normalized to control cells that were not treated with doxycycline. Gapdh transcript levels were used as a reference control, and shown are representative results from an experiment that was repeated at least 3 times. Error bars indicate SEM; *P < .01 compared with uninduced controls. (G) Visceral endoderm commitment in day 2–induced EBs was corroborated by flow cytometric analysis after staining for epCAM (pan-endoderm) and DPP4 (visceral endoderm) -specific fluorophore-conjugated Abs using cells from dissociated EBs over a 4-day time course from day 3 to day 6 of EB cultures. (H) FLK1 and c-KIT staining was analyzed from day 3 to day 6 by flow cytometry to measure the potential population of hemato-vascular precursors in EBs induced at day 2, compared with control uninduced samples.