70 Shimada et al. O). Therefore, in the late stage of pancreatic development, some of the NC derivatives tended to be distributed in close proximity to beta cells; this is different from the cellular distribution of NC derivatives and endocrine cells in adults. To investigate this difference, detailed distribution of both NC derivatives and PDX1-positive cells in the later stages of pancreatic development was examined further. With development, most NC derivatives tended not to be in close proximity to PDX1-expressing cells Immunostaining experiments to detect endocrine cells and EGFP at the early stage of pancreatic development indicated that a few NC derivatives tended to be distributed in close proximity to endocrine cells (data not shown). Moreover, from E14.5, some NC derivatives displayed a tendency to approach cells with high PDX1 expression (Fig. 2F, J–H; arrows). As pancreatic development ad- vanced, only beta cells showed high PDX1 expression. Our results, therefore, showed that the majority of cells with high PDX1 expression have a beta-cell lineage. This supports previous findings that signals from NC derivatives regulate the mass, proliferation, and maturation of beta cells. However, we also confirmed that NC derivatives were distributed in closer proximity to alpha cells rather than beta cells during the postnatal stages. During late-stage pancreatic development, although NC derivatives were distributed in close proximity to cells with high PDX1 expression (Fig. 3B and H; arrowheads), several other cell types remained present between the cells with high PDX1 expression and the NC derivatives (Fig. 3B, E, H; arrows). Therefore, we Fig. 4. Distribution of NC derivatives and endocrine cells during pancreatic development. (A–I) Immunohistochemical staining of pancreatic regions of Wnt1-Cre/Floxed-EGFP mouse embryos during late embryonic stage. Staining was performed with insulin antibody (blue) (A, D, and G) (magenta) (C, F, and I), glucagon antibody (RED) (A, D, and G) (magenta) (B, E, and H), and GFP antibody (green). (J) Pancreatic region of an E18.5 Wnt1-Cre/Floxed-EGFP mouse embryo stained with insulin antibody (blue), glucagon antibody (red), and GFP antibody (green) and the location of the 20-µm grid. From E16.5 on, NC derivatives tended to be distributed in closer proximity to alpha cells (arrows) than to beta cells (arrowheads). To confirm the gradual process by which NC derivatives migrated into closer proximity to endocrine cells, the characteristics of the cells that appeared in the 20-µm grid were examined based on the images obtained from the immunostaining experiments in embryos from stages later than E14.5. The results were as follows: (1) as development of the pancreas advanced, the frequency of the appearance of endocrine cells and NC derivatives in the same grid also increased (K); and (2) the frequency of the appearance of glucagon-expressing cells and NC derivatives in the same grid also increased, indicating that alpha cells were in closer proximity to NC derivatives than to beta cells (L). Therefore, as development advanced, the tendency for NC derivatives to be distributed in closer proximity to alpha cells increased. Bar=100 µm (A–I).