68 Shimada et al. nerve-related cells surrounding the islets were assumed to originate from the NC cells. We confirmed the types of cells the NC derivatives differentiated into in the pancreas using a transgenic mouse line that expresses EGFP in all NC cells and their derivatives (Fig. 1C). NC derivatives were widely distributed throughout the pancreas and were located particularly close to blood vessels and the pancreatic islets. Most of the NC derivatives enclosed the islets (Fig. 1D). NC derivatives surrounding the islets primarily differentiated into the nervous system (Figs. 1E–J). Moreover, most NC derivatives were distributed in close proximity to alpha cells located at the margins of the islets (Fig. 1A, B, and D; arrows). Therefore, our data suggest that cell-cell inter- actions may occur between the NC derivatives and alpha cells. To investigate their physical relationship, the cellular distribution of both NC derivatives and endocrine progenitor cells during pancreatic development was examined further. Relationship between endocrine progenitor cells and NC derivatives during pancreatic development The localization of NC derivatives that migrated to the pancreas was examined using Wnt1-Cre/Floxed-EGFP mice at each developmental stage. The prospective area of the pancreas was identified by the pancreatic epithelial marker Fig. 2. Distribution of NC derivatives during pancreatic development. (A–L) Immunohistochemical staining of pancreatic regions of Wnt1-Cre/ Floxed-EGFP mouse embryos at each embryonic stage. Staining was performed with pancreatic and duodenal homeobox 1 (PDX1) antibody (magenta) and GFP antibody (green). (A) At embryonic day 9.5 (E9.5), Wnt1-Cre/Floxed-EGFP embryos did not have any NC derivatives in close proximity to the ventral and dorsal pancreatic buds (arrow). (B) At E10.5, NC derivatives were observed in a close proximity to both pancreatic buds. They were closer to the dorsal pancreatic bud than to the ventral pancreatic bud (arrowheads). (C and D) From E11.0, NC derivatives surrounded PDX1-positive cells. (E) At E12.5, as branching of buds progressed, NC derivatives were distributed along the pancreatic epithelial cells. (F) At E14.5, several NC derivatives tended to be located close to cells with high PDX1 expression (arrows). (J–L) Higher magnification image of the region in G–I, where NC derivatives were distributed in close proximity to PDX1-positive cells (arrows). (M–O) Immunohistochemical staining of pancreatic regions of mouse embryos is shown; staining was performed with insulin antibody (blue), glucagon antibody (red), and PDX1 antibody (green). During later stages of pancreatic development, PDX1-positive cells were mainly localized in insulin-positive rather than glucagon-positive cells. Bar=100 µm (B–O). VB=ventral pancreatic bud, DB=dorsal pancreatic bud.