Fig. 5. NGN3+ cells form islets. Correct expression of the Ngn3 promoter-driven transgene is assessed by in situ hybridization on adjacent sections. (A,B) Adjacent sections of an E13.5 transgenic pancreas probed with Ngn3 (A) or Cre-ER^TM (B) cRNA. To study the lineage of the Ngn3-expressing cells, TM is given to embryos at different stages and the pancreata of double transgenic animals are examined for HPAP expression or co-expression of HPAP with various hormones at 3-8 weeks of age. (C,D) HPAP staining in islet cells when TM was injected at E8.5 or E12.5, respectively (8-week-old pancreata are shown). (E) A confocal image that shows hormone (Ins, Glu, Som, PP) and HPAP double-positive cells (arrows) when TM was injected at E8.5. Each picture is a single scan. Because HPAP is a membrane-bound protein while the hormones are cytoplasmic proteins, we do not always see colocalization of HPAP and hormone, as in HPAP/PP staining (i, islets, broken red lines). Scale bars: in B, 25 µm for A,B; in D, 25 µm for C,D; in E, 5 µm.