66 Shimada et al. the mouse embryo, with the appearance of ventral pancreatic and dorsal buds in a specific portion of the intestinal tract [10]. The pancreatic bud can be detected by the expression of pancreatic and duodenal homeobox 1 (PDX1), which is localized in endocrine cells as development proceeds. These pancreatic buds grow, branch, differentiate, and eventually merge to form the pancreas. As development proceeds, the endocrine cells also differentiate and mature, and gradually form aggregations that eventually merge to form pancreatic islets. The development of the pancreas thus requires not only pancreatic mesoderm and endoderm, but also innerva- tion by ectodermally derived tissues, which occurs at E12.5 [2]. In a recent study, ectoderm-derived neural crest (NC) derivatives were shown to accumulate in close proximity to pancreatic epithelial cells at E10.0 [24]. NC cells are pluripotent and mobile, and they differentiate into many cell types, including peripheral and enteric neurons, glia, melanocytes, and smooth muscle cells [9, 19, 20, 33, 40]. Signals from NC derivatives are also known to be essential for the maturation of beta cells [24]. However, despite the importance of NC derivatives in the development of endocrine cells, little is known about the cellular distribution of both NC derivatives and endocrine cells, or the process whereby NC derivatives enclose the latter during the development of the pancreas. Here, we report our examina- tion of the temporal and spatial distribution of NC derivatives and endocrine cells during the development of the pancreas and our observations of the behavior of the NC derivatives during the formation of pancreatic islets. primary and secondary antibody working dilution green fluorescent protein (GFP) green fluorescent protein (GFP) Insulin Glucagon Glucagon Glial fibrillary acidic protein (GFAP) S100 neurofilament 160 kDa (NF-M) tyrosine hydroxylase (TH) calcitonin gene-related peptide (CGRP) choline acetyltransferase (ChAT) neuronal protein HuC/HuD Pancreatic and duodenal homeobox 1 (PDX1) SynCAM Alexa Fluor 488 Alexa Fluor 555 Alexa Fluor 633 DyLight549 1:1000 1:1000 1:500 1:2000 1:200 1:500 1:1000 1:1000 1:1000 1:2000 1:30 1:50 1:400 1:500 1:1000 1:1000 1:1000 1:500 II. Materials and Methods Animals All experiments were performed in accordance with the guidelines of the local ethics committee of the Jikei University School of Medicine. Transgenic Wnt1-Cre mice expressing Cre recombinase under control of the Wnt1 promoter/enhancer [B6.Cg-Tg (Wnt1-Cre) 11Rth Tg (Wnt1-GAL4) 11Rth/J] were mated with indicator mice (CAG-CATloxP/loxP-EGFP) [4, 16] to obtain Wnt1- Cre/Floxed-EGFP mice. Wnt1-Cre/Floxed-EGFP mice express enhanced green fluorescent protein (EGFP) in all NC cells and their derivatives. When a vaginal plug ap- peared, the day was designated 0.5 days post-coitum. Wnt1- Cre/Floxed-EGFP embryos were selected on the basis of EGFP expression. Adult Wnt1-Cre/Floxed-EGFP mice were selected for PCR-based genotyping with primers for the Cre and CAT genes. The embryos used for the experiments were collected at different developmental stages, which were defined as follows: early stage pancreatic development, E10.5–E12.0; mid-stage pancreatic development, E12.5– E15.5; and late stage pancreatic development, E16.5–E20. Immunohistochemistry For histological analyses, samples were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4°C overnight, soaked in different concentrations of sucrose/PBS buffer (10, 20, and 30%) at 4°C for 1 hr, embedded in Tissue-Tek OCT compound, and frozen. Frozen sections type chick polyclonal rabbit polyclonal guinea pig polyclonal mouse monoclonal rabbit polyclonal rabbit polyclonal rabbit polyclonal chick polyclonal rabbit polyclonal rabbit polyclonal rabbit polyclonal mouse monoclonal rabbit polyclonal chick polyclonal goat anti-rabbit, goat anti-chick, goat anti-guinea pig goat anti-rabbit, streptoavidin conjugated goat anti-rabbit, goat anti-guinea pig, streptoavidin conjugated goat anti-chick source Aves Abcam Dako Sigma Cell Signaling Technology Dako Dako Aves Millipore Calbiochem Abcam Molecular Probes Trans Genic MBL Molecular Probes Molecular Probes Molecular Probes Jackson ImmunoResearch Table 1. Primary and secondary antibodies for immunohistchemistry Abcam: Cambridge, UK; Aves: Oregon, USA; Calbiochem: Darmstadt, Germany; Cell Signaling Technology: Danvers, USA; Dako: Copenhaven, Denmark; Jackson ImmunoResearch: West Grove, PA, USA MBL: Nagoya, Japan; Millipore: Billerica, USA; Molecular Probes: Merelbeke, Belgium; Sigma-Aldrich; St. Louis, MO, USA; Trans genic: Kumamoto, Japan.