72 Shimada et al. Fig. 6. SynCAM expression between each endocrine cell type during late-stage of pancreatic development. (A–C) Immunohistochemical staining of pancreatic regions of Wnt1-Cre/Floxed-EGFP mouse embryos during late development stage. Staining was performed with SynCAM antibody (green), glucagon antibody (red), and insulin antibody (blue). (D–H) Immunoreactivity of SynCAM expression between alpha cells and beta cells is shown. During later stages of pancreatic development, SynCAM was expressed at the cell boundaries between alpha cells (1, 2, and 3 in A, B, and C) and between alpha cells and beta cells (7, 8, and 9 in A, B, and C). SynCAM expression was also confirmed between the cell boundaries of a few beta cells (4, 5, and 6 in A, B, and C, respectively). Bar=10 µm for all Figures. as development advanced, this tendency was remarkably increased (Fig. 4D–I). Therefore, we showed that NC derivatives tended to approach alpha cells rather than beta cells in the later stages of pancreatic development. To confirm the gradual process whereby NC derivatives moved closer to both endocrine cell types, the characteristics of cells that appeared in a 20-µm grid were examined using images obtained from the immunostaining experiments performed during stages after E14.5 (Fig. 4J). The rate at which the NC derivatives and endocrine cells were found to co-exist in the same grid increased as the development advanced. These results suggest that NC derivatives tended to approach the endocrine cell region rather than the exocrine cell region (Fig. 4K). Moreover, the NC derivatives enclos- ing the endocrine cells were distributed in closer proximity to alpha cells than to beta cells. At E16.5, approximately 60% of the NC derivatives enclosing endocrine cells were distributed in close proximity to alpha cells. This ratio increased to approximately 70% at E20. On the other hand, the rate at which NC derivatives were distributed in close proximity to beta cells was substantially decreased (Fig. 4L). During late-stage pancreatic development, both alpha cells and NC derivatives expressed a common cell adhesion molecule (CAM) Most NC derivatives were distributed in closer prox- imity to alpha cells than to beta cells during islet develop- ment. To investigate the cell-cell interactions between NC derivatives and alpha cells, we confirmed the expression of several CAMs. In adult mice, CADM-1, also referred to as “SynCAM”, a Ca2+-independent homophilic trans-CAM, was expressed by NC derivatives and the membranes of alpha cells (Fig. 5G, H). During embryogenesis, we observed the temporal and spatial expression of SynCAM in the developing pancreas and confirmed that SynCAM was expressed by the cell membranes of all pancreatic epithelial cells by E14.0 (Fig. 5A, B, D, E). At E12.5, SynCAM ex- pression was confirmed in not only the cell membranes of the pancreatic epithelial cells but also in the NC deriva- tives (Fig. 5I–K). As development advanced, SynCAM expression was limited to the NC derivatives, the cell membranes of endocrine cells, PDX1-positive cells, and a few exocrine cells (Fig. 5C, F, L). SynCAM expression by both NC derivatives and alpha cells was confirmed from E16.5 (Fig. 5L). Among the endocrine cells, the cell mem- branes of most alpha cells expressed SynCAM, but almost none of the cell membranes of beta cells did (Fig. 6A–C; arrows). SynCAM expression in the pancreas was mainly observed in the cell membranes of alpha cells and NC derivatives (Fig. 7A–C, G–I, M–O; arrowheads). It was also confirmed that the endocrine cells came into contact with the NC derivatives. SynCAM expression by the cell membranes of beta cells was barely detectable, and, when present, was at the cell boundaries of interacting NC derivatives and beta cells (Fig. 7D–F, J–L, P–R; arrowheads) or alpha cells and beta cells (Fig. 6A–C; arrowheads). SynCAM expression by the cell membranes of beta cells that were not distributed in close proximity to NC derivatives or alpha cells was either very weak or absent (Fig. 7F, L, R; the section enclosed with dotted lines). Therefore, SynCAM expression was confirmed from E16.5 on in areas where NC derivatives came into contact with endocrine cells (Fig. 7S). These results show that during the later stages of pancreatic development, SynCAM expression is detected at the cell boundaries between NC derivatives and alpha cells as well as between NC derivatives and some beta cells.