Fig. 4.

Patterning with activin and Wnt3a is required for optimal pancreatic specification. (A,B) Quantitative PCR (QPCR) analysis evaluating PDX1 and INS expression at different stages of differentiation in cultures generated from populations treated without activin (No ACT) at day 5, with activin for 2 days (d5-d7; 2dACT) and with activin for three days (d5-d8; 3dACT). Cultures were treated with SB431542 (SB) and noggin (NOG) following NCR treatment and were terminated at day 15, 17 and 18 for No Act, 2dAct and 3dAct, respectively. Expression levels were normalized to TBP and are relative to the 2dACT sample at d17 (set at 100). Bars represent mean±s.d. Asterisks indicate statistical significance as determined by t-test. P=0.032 (PDX1) and P=0.027 (INS). n=3. (C,D) Day 17 cultures were analyzed by qPCR for the expression of intestinal (CDX2) and pancreatic (INS) genes following treatment with DKK1 or Wnt3a. Bars represent mean±s.d. Cultures were treated between days 7 and 9 with FGF10 (F) or with a combination of FGF10 and one of the following: DKK1 at 150 ng/ml (F+DKK), Wnt3a at 1 ng/ml (F+W1), Wnt3a at 3 ng/ml (F+W3), Wnt3a at 9 ng/ml (F+W9). CDX2 expression was significantly downregulated in F+DKK, INS expression was significantly upregulated in F+W3. *P<0.05, **P<0.01 determined by ANOVA with Tukey's HSD test. n=4.