NC Derivatives during Islet Development 71 suggest that endocrine cells other than beta cells are present between the cells with high PDX1 expression and the NC derivatives. To identify these intervening cells, we examined the cellular distribution of alpha cells, which are PDX1- negative during late-stage pancreatic development. Most of the intervening cells were identified as PDX1-negative alpha cells (Fig. 3C, F, I; arrows). These results indicate that during late-stage pancreatic development, alpha cells are found between NC derivatives and cells with high PDX1 expres- sion; hence, most NC derivatives distributed in close proximity to the cells with high PDX1 expression do not come into contact with beta cells directly (Fig. 3C, F, I; arrows and arrowheads). During late-stage pancreatic development, most NC derivatives tended to be distributed in close proximity to alpha cells To investigate the cellular distribution of the NC derivatives and of each endocrine cell type during pancreatic development, we confirmed the cellular distribution of these cells during late-stage pancreatic development. Immuno- staining experiments at the early stage of pancreatic development indicated that some NC derivatives tended to be distributed in close proximity to endocrine cells (data not shown). At E16.5, most NC derivatives tended to be distributed in closer proximity to alpha cells (Fig. 4B; arrows) than to beta cells (Fig. 4C; arrowheads). Moreover, Fig. 5. SynCAM expression in the mouse pancreas. (A–F) Immunohistochemical staining of pancreatic regions of Wnt1-Cre/Floxed-EGFP mouse embryos. Staining was performed with SynCAM antibody (green), glucagon antibody (blue), and PDX1 antibody (red). (G–L) Immuno- histochemical staining of embryos at E12.5, E16.5 and 3W; staining was performed with GFP antibody (green), SynCAM antibody (RED), and glucagon antibody (blue). At E12.5 and E14.0, all pancreatic epithelial cells and glucagon-positive cells were positive for SynCAM (A, B, D, and E), but at E16.5, SynCAM expression was limited to the membranes of glucagon-positive cells and cells with high PDX1 expression (C and F). In adult pancreas, SynCAM was mainly expressed by NC derivatives and alpha cells (G and H). At E16.5, SynCAM was expressed by membranes of NC derivatives and glucagon positive cells (L). A similar SynCAM expression pattern was confirmed at E12.5 (I, J and K). Bars=20 µm (A–C), 10 µm (D–F and J–L), 50 µm (I), 100 µm (G), 20 µm (H).