Fig. 4. Dorsal endoderm in Flk1^-/- embryos does not express Ptf1a, insulin and glucagon. (A-C) RT-PCR analysis of the dorsal endoderm in the region of the pancreatic bud. (A) The expression levels of factors involved in early pancreatic development were lower in the dorsal endoderm of Flk1^-/- embryos than in that of control embryo. (B) The expression of Ptf1a, insulin, and glucagon was not observed in the dorsal endoderm of Flk1^-/- embryos even with three or six additional PCR cycles. PCR cycle ranges of embryo tissues: Pdx1, 35-41; Ptf1a, 34-40; Prox1, 33-39; Ngn3, 34-40; Neurod1, 34-40; insulin, 33-39; glucagon, 27-33; actin, 23-29. (C) The expression of Ptf1a starts by 15S in wild-type embryos, when the aorta has extensive contact with Pdx1-positive cells. Short exposure, 12 hours; long, 5 days. In some experiments, Ptf1a expression was observed in the 12-15 S range (data not shown). PCR cycles for Ptf1a, 39, 42, 45; actin 28, 31, 34. (D,E) Glucagon immunostaining confirms the absence of glucagon expression in the dorsal endoderm of Flk1^-/- embryos. Somite stages are indicated in the each panel.