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First published online 19 December 2006
doi: 10.1242/jcs.03330

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cells occur naturally in extrahepatic bile ducts of mice

James R. Dutton^*, Naomi L. Chillingworth^*, Daniel Eberhard^*, Claire R. Brannon, Mark A. Hornsey, David Tosh and Jonathan M. W. Slack

Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Bath, BA2 7AY, UK

View larger version (97K): [in this window] [in a new window]   Fig. 1. Insulin-positive cells in the hilar region. (A) Bile duct system of an adult mouse made visible by injection of the gall bladder with 0.5% Trypan Blue. The boxed area is the hilar region where the ectopic cells are located. gb, gall bladder; panc, pancreas; duo, duodenum. (B) Whole-mount stain of the ducts in the hilar region of an adult mouse using Dolichos biflora lectin (red), and antibody against insulin (green). The insulin-positive cells are in clusters in the connective tissue surrounding the ducts. Bar, 500 µm. (C-F) Insulin-positive cells (green), associated with bile ducts: (C) 18.5-day embryo, (D) 1 day postnatal, (E) 7 days postnatal, (F) 6 months. (G) Two insulin-positive cells just within the liver parenchyma of a 3-week-old mouse. Bars, 20 µm. (H) Analysis of the hilar duct system in 30 mice of different ages (E18.5 to age 6 months). Each vertical line represents a series of serial sections from one mouse of which every fifth slide (five sections) was examined. The horizontal bars indicate those sections containing one or more insulin-positive cells.

View larger version (12K): [in this window] [in a new window]   Fig. 2. Quantitative data. (A) Average number of foci per animal. (B) Average number of cells per focus. (C) Percentage of insulin-positive cells on the sections found within 10 µm of the ductal epithelium. n=5 mice per group, except data for 2-month-old mice, where n=10. For the postnatal mice insulin-positive cells were counted on every fifth slide (where one slide contains 4x4 µm or 5x4 µm sections) of serial sections through the hilar region. For embryos, every other slide was examined (approximately 8-µm-intervals). The number of animals with insulin-positive cells was five of five for 1-day- and 6-month-old animals, four of five for 7-day- and 3-week-old animals and for embryos at E18.5, and nine of ten for 2-month-old animals. Error bars represent standard errors.

View larger version (33K): [in this window] [in a new window]   Fig. 3. Formation and migration of endocrine cells. (A) Single somatostatin-positive cell in the duct epithelium. (B) Typical cluster showing SS- and insulin-positive cells. (C,D) Triple-stained large endocrine cell clusters: (C) insulin (green), SS (red), glucagon (blue); (D) insulin (green), PP (red), glucagon (blue). Bars, 20 µm. (E) Hypothetical mechanism for endocrine cell formation from duct epithelium.

View larger version (45K): [in this window] [in a new window]   Fig. 4. Characterisation of cells. (A,A') C-peptide (red) and insulin (green). Bar, 20 µm. (B) TEM showing cells with -cell-like secretory granules (arrowheads). Bar, 2 µm. (C) Immuno-EM showing a positive signal for insulin in the lower right cell. The upper right cell has secretory granules that are not insulin-positive. Bar, 0.5 µm.

View larger version (92K): [in this window] [in a new window]   Fig. 5. Alb-Cre lineage labelling. (A) Newborn Alb-Cre/R26R lacZ mouse stained with X-gal. The gall bladder (g) and bile duct system are labelled, as is a piece of liver left attached in the hilar region (h). The pancreas (p) is virtually unlabelled, the visible blue being surface background stain and a small part of the pancreatic duct; d, duodenum. (B,B',B") Section showing an insulin-positive cell labelled with X-gal in the duct epithelium. Bar, 20 µm.