NC Derivatives during Islet Development 73 Fig. 7. SynCAM expression at the cell boundaries between NC derivatives and both endocrine cell types. (A–R) Immunohistochemical staining of pancreatic regions of Wnt1-Cre/Floxed-EGFP mouse embryos during later stages of pancreatic development. Staining was performed with SynCAM antibody (red), glucagon antibody (blue) (A, B, G, H, M, and N), insulin antibody (blue) (D, E, J, K, P, and Q), and GFP antibody. During later stages of pancreatic development, most NC derivatives came into contact with endocrine cells (B, E, H, K, N, and Q; arrows). SynCAM expression occurred at the cell boundaries between NC derivatives and endocrine cells (C, F, I, L, O, and R; arrowheads). The majority of endocrine cells in which SynCAM was expressed were alpha cells. Beta cells that were in contact with NC derivatives were also positive for SynCAM (F, L, R; arrowheads), but SynCAM expression was very weak or not expressed in beta cells distributed far away from NC derivatives (F, L, R; the section enclosed with dotted lines). A schematic diagram of the cellular distribution of NC derivatives and endocrine cells based on SynCAM expression pattern during later stages of pancreatic development (S). This schematic diagram shows that SynCAM was expressed during interactions among alpha cells, NC derivative cells and endocrine cells, and between alpha cells and beta cells. Bars=20 µm (A–R). IV. Discussion Previous studies have shown that signals from NC derivatives regulate the mass, proliferation, and maturation of beta cells [21, 24]. In addition, it has previously been shown that co-culturing pancreatic islets with NC stem cells in vitro promotes the regeneration of functional beta cells [22]. Therefore, it is believed that NC derivatives that migrate to the pancreatic region are important for mainte- nance of the function, development, and maturation of beta cells. Our results relating to the cellular distribution of NC derivatives at each stage of pancreatic development support NC derivative signaling of beta cells. A previous study indicated that such signals are likely to be juxtacrine signals, because neural fibers derived from NC cells are located close to insulin-positive cells at E15.5, and they contact with 98% of the insulin cell clusters by the first postnatal day [24]. However, our results show that the NC derivatives enclosing endocrine cells are located in closer proximity to alpha cells than to beta cells from the later stages of pancreatic development. Moreover, we showed that as development advances, the ratio of NC derivatives distrib- uted in close proximity to alpha cells increases remarkably. Therefore, we suggest that the NC derivatives probably signal beta cells using juxtacrine as well as paracrine signals or a combination of both. This tendency of NC derivatives to be distributed in close proximity to a specific cell type is of interest as it indicates that the NC derivatives that migrate to the pancreatic region are not distributed at random during pancreatic development. The structure of the islet cells might be related to deviations in the cellular distribution of the NC derivatives and each endocrine cell type at the embry- onic stage. In rodents, the general structure of the islets is for beta cells to occupy the center and alpha cells to be distributed in the peripheral regions [23, 32]. We confirmed that in adult mice, nerve fibers and nerve-related cells tended to enclose the majority of islets. Therefore, we believe that NC derivatives and endocrine cells might have unique distributions during embryogenesis as a form of in preparation for the highly-regimented organization of the mature islets. Because NC derivatives tended to be distrib-