74 Shimada et al. uted in close proximity to endocrine cells from the fetal stage, we hypothesized that the endocrine cells and NC derivatives might use a common cellular adhesion system. Moreover, because the NC derivatives were distributed in closer proximity to alpha cells than to beta cells during islet development, we believed that a cell-cell interaction of these cell types would be important for unique cellular distributions of the endocrine cells and NC derivatives, as observed during islet development. To investigate cell-cell interactions between the NC derivatives and alpha cells during the later stages of pancreatic development, we confirmed the expression of several CAMs. Previous studies have proven the expression of CAMs by endocrine cells [15, 28]. We focused on CADM-1 (SynCAM), a Ca2+-independent CAM, because a previous study showed that SynCAM mediates nerve-islet cell interactions [17]. SynCAM has 3 immunoglobulin-like motifs in its extracellular domain [29, 38] and is expressed by a variety of cell types including epithelial and non- epithelial cells [1, 12–14, 38]. Previous reports have shown that SynCAM can interact with cells by homophilic binding, such as that which occurs between neurons [1] or between mast cells and neurons [8]. Our results suggest that homophilic binding of the SynCAM expressed by endocrine cells and NC derivatives contributes to endocrine cell-NC derivative interactions during islet development. Moreover, during the later stages of pancreatic development, NC derivatives are distributed in closer proximity to alpha cells than to beta cells, and SynCAM is also markedly expressed by NC derivatives and alpha cells at that time point. On the other hand, SynCAM expression was barely confirmed at the cell membranes of beta cells distributed near NC derivatives and alpha cells. Considering the results from previous studies as well as those from our own SynCAM expression analysis, it appears that SynCAM contributes to the aggregation of alpha cells from embryogenesis. In contrast, it is assumed that SynCAM does not contribute to the aggregation of beta cells. We propose that a mechanism regulating homophilic binding of SynCAM is responsible for the tendency of NC derivatives to be distributed in closer proximity to alpha cells than to beta cells. However, the structure of islets is also known to be mediated by several other CAMs. For example, disruption of neural CAM (NCAM) induces alpha cells to become more randomly distributed within the islets [7], and dis- ruption of E-cadherin (ECAD) also disrupts aggregation of beta cells [3]. Although whether NCAM and ECAD disrup- tions mediate NC derivative-endocrine cell interactions remains unclear, we suggest that SynCAM is not the primary mechanism behind endocrine cell-NC derivative interactions; however, it is a mediating factor in NC derivative-endocrine cell interactions during embryogenesis. A recent study reported that sympathetic axon terminals innervate alpha cells rather than beta cells in human islets, which have a more complex structure than rodent islets [27]. It, therefore, appears that the cellular distribution of NC derivatives that is associated with the relationship between alpha cells and nerves is not due to the distinctive structure of the rodent islets in which the beta cells occupy the center and the alpha cells are distributed at the periphery. We suggest that cell-cell interactions associated with specific SynCAM expression in the fetal pancreas are one of the factors by which the axon terminals innervate the alpha cells. In conclusion, we elucidated the temporal and spatial cellular distribution of NC derivatives and endocrine cells during pancreatic development using Wnt1-Cre/Floxed- EGFP mice. Our results strongly suggested that NC derivatives are important for islet development and for the differentiation and the maturation of endocrine cells because NC derivatives tended to gradually approach the endocrine cells during pancreatic development. Moreover, during the later stages of pancreatic development, the NC derivatives were distributed in closer proximity to alpha cells than to beta cells. Analysis of SynCAM expression during islet development indicated that NC derivatives might be distrib- uted in closer proximity to alpha cells due to homophilic binding between the SynCAM expressed by the alpha cells and NC derivatives during islet development. Therefore, we suggest that SynCAM is an important factor in the relationship whereby alpha cells are distributed in the proximity of NC derivatives in adults. It is, therefore, our strong belief that interactions among NC derivatives, alpha cells, and beta cells are all essential for the induction of a physiologically competent islet, particularly from the point of view of the development and regeneration of endocrines. V. Acknowledgments We wish to thank Dr J. Miyazaki for providing the indicator mice (B6;D2-Tg(CAG-CAT-EGFP)39Miya; referred to as “CAG-CATloxP/loxP-EGFP”). We are also grateful to Dr T. Miyake and Dr T. Yoshida for their helpful discussions and comments and to T. Kimura for technical assistance. This study was supported in part by a Grant-in- Aid from the Ministry of Health, Labour and Welfare, Japan (H21-Nanchi-ippan-039 to T.S.), a Grant-in-Aid for Scien- tific Research from the Japan Society for the Promotion of Science (to K.F.), and a Novo Nordisk Pharma Insulin Award (to K.F.). VI. References 1. Biederer, T., Sara, Y., Mozhayeva, M., Atasoy, D., Liu, X., Kavalali, E. T. and Sudhof, T. C. (2002) SynCAM, a synaptic adhesion molecule that drives synapse assembly. Science 297; 1525–1531. 2. Burris, R. E. and Hebrok, M. (2007) Pancreatic innervation in mouse development and beta-cell regeneration. Neuroscience 150; 592–602. 3. Dahl, U., Sjodin, A. and Semb, H. (1996) Cadherins regulate aggregation of pancreatic beta-cells in vivo. Development 122; 2895–2902. 4. Danielian, P. S., Muccino, D., Rowitch, D. H., Michael, S. K. and Mcmahon, A. P. (1998) Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase. Curr. 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