NC Derivatives during Islet Development 69 PDX1, whereas NC derivatives were identified by EGFP expression. At E9.5, NC derivatives were not observed in proximity to the epithelia of the pancreatic buds (Fig. 2A; arrow). However, at E10.5, NC derivatives were observed in proximity to the epithelia of the ventral and dorsal pancreatic buds (Fig. 2B). Most NC derivatives observed in close proximity to the pancreatic buds were distributed more on the dorsal side than on the ventral side (Fig. 2B; arrowheads). Therefore, NC derivatives migrated to the prospective region of the pancreas between E9.5–E10.5, and were mostly distributed on the dorsal side of the pancreatic bud. NC derivatives surrounded PDX1-positive cells at E11.0 (Fig. 2C). Many of the NC derivatives in the developing pancreas were scattered along branched pancreatic epithelial cells from E11.5 (Fig. 2D, E). As development advanced, NC derivatives were widely distrib- uted throughout the developing pancreas (Fig. 2F–I). Some of the NC derivatives were distributed in proximity to the cells with a high PDX1 expression from the stage where differentiation into endocrine cells began (Fig. 2F, J–L; arrows). As development advanced, PDX1 expression became localized in endocrine rather than in exocrine cells. From E16.5, PDX1 expression was mainly localized in beta cells, and most alpha cells were PDX1-negative (Fig. 2M– Fig. 3. Glucagon-positive cells tended to be located between PDX1-positive cells and NC derivatives. (A–H) Immunohistochemical staining of pancreatic regions of Wnt1-Cre/Floxed-EGFP mouse embryos during later stages of pancreatic development. Staining was performed with PDX1 antibody (red) and GFP antibody (green), and nuclei were stained with DAPI solution (blue). (C, F, and I) Immunohistochemical staining results with PDX1 antibody (red), GFP antibody (green), and glucagon antibody (white). Figures B, E, and H are higher magnification images of A, D, and G, respectively. NC derivatives were distributed in closer proximity to PDX1-negative cells (arrowheads) than to PDX1-positive cells (arrows). Figures C, F, and I represent the same sections as B, E, and H, respectively, but were stained with glucagon antibody (white). PDX1-negative cells located in close proximity to NC derivative cells were glucagon-positive (arrows). Most NC derivatives tended to be distributed in closer proximity to glucagon-positive cells than to PDX1-positive cells (arrowheads) during later stages of pancreatic development. Bar=50 µm (A, D, G, C, F, and I).