Fig. 1. Notch pathway activation assessed by Hes1 expression in developing mouse pancreas. (A,D,G) Single channel and merged confocal images demonstrating co-expression of Hes1 (red) and Pdx1 protein (green) in E13.5 mouse pancreas. (B,E,H) Single channel and merged confocal images demonstrating co-expression of Hes1 (red) and Ptf1-p48 protein (green) in E13.5 mouse pancreas. (C,F,I) Double fluorescent in situ hybridization demonstrating absence of Hes1 expression in Ngn3-positive endocrine precursors. Cells with cytoplasmic Hes1 transcripts (red) do not contain transcripts for Ngn3 (green). (J) Immunostaining for Hes1 and E-cadherin on E14.5 pancreas reveals nuclear Hes1 protein in central undifferentiated epithelium as well as in epithelial branches and centroacinar cells (arrow). (K) Magnified view of area outlined in J, demonstrating nuclear Hes1 protein in cells comprising peripheral epithelial branches (arrow), but exclusion of Hes1 from acinar cluster, indicated by broken line. (L) Immunostaining for Hes1 and amylase, demonstrating Hes1 protein in nuclei of amylase-negative terminal ductal/centroacinar cells, but not in differentiating amylase-positive acinar cells. Dashed lines indicate contour of representative Hes1-negative acinar cell, identified by basal nuclei and apical collection of amylase-positive zymogen granules. Dotted lines indicate contour of representative Hes1-positive, amylase-negative terminal ductal/centroacinar cell. Images in C,F,I and J-L generated by 3D image deconvolution. Scale bars: 10 µm.