cells occur naturally in extrahepatic bile ducts of mice The biliary system in mouse and in humans...
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Introduction In recent years the development of the pancreas has received much attention, largely in response to the challenge of diabetes mellitus as a major problem of human health (Edlund, 1999; Murtaugh and Melton, 2003; Slack, 1995; St-Onge et al., 1999). The pancreas contains five types of endocrine cell, mostly assembled into the islets of Langerhans, of which the insulin-producing � cells are the most numerous. Some of the other endocrine cell types, producing glucagon, somatostatin, pancreatic polypeptide or ghrelin, may be found elsewhere in the body, but until now the � cells were thought to be confined to the pancreas.
Insulin therapy for diabetes has been highly successful and has become very sophisticated, but the disease still produces a considerable burden of distressing complications and represents a substantial cost to health budgets around the world (Zimmet et al., 2001). One possible route forward, especially for type 1 diabetes, is through an induced augmentation of �- cell mass. Some success has been achieved in recent years using islet grafts into the liver (Kendall et al., 2001; Shapiro et al., 2002), but at present the main limitation to the wider application of this technique is the limited supply of islets from human organ donors. This has encouraged clinicians and developmental biologists to think of ways in which the mass of � cells might be augmented either through expansion in vitro, or differentiation from embryonic stem cells, or differentiation from other tissue types (Roche et al., 2003).
One potential source of new � cells is the liver. The pancreas and liver arise from adjacent areas in the anterior endoderm of the developing embryo (Wells and Melton, 1999). This
suggests that the developmental commitment of the liver and pancreas differs by the expression of only a few genes, and so by manipulating the expression of those genes it might also be possible to convert liver into pancreas. A number of labs have shown that it is possible to induce expression of a variety of endocrine and exocrine pancreatic genes in the liver by overexpression of transcription factors such as pancreatic and duodenal homeobox gene 1 (Pdx1, also known as insulin promoter factor 1, IPF1) and neurogenin 3 (Ber et al., 2003; Horb et al., 2003; Sapir et al., 2005; Zalzman et al., 2003; Zalzman et al., 2005). In the course of some experiments attempting to introduce various genes into the liver, we discovered that our control animals already had some � cells in their livers: more specifically they were associated with the large bile ducts found in the hilar region of the liver, which is the region near the gall bladder where the liver lobes come together around the bile ducts and major blood vessels.
The biliary system in mouse and in humans originates in the liver parenchyma, where bile is secreted by hepatocytes into a system of intrahepatic ducts. These drain into larger extrahepatic ducts, which join with the cystic duct from the gall bladder to form the common bile duct. This is joined by the main pancreatic duct shortly before entry into the duodenum. The epithelium of the intrahepatic bile ducts is derived from bipotential precursor cells within the hepatic parenchyma (Lemaigre and Zaret, 2004; Zhao and Duncan, 2005). The extrahepatic biliary tract develops separately from the intrahepatic ducts and becomes anastomosed to them by an unknown mechanism (Lemaigre, 2003; Shiojiri, 1997). A recent study by Sumazaki and colleagues demonstrated the
Insulin-secreting � cells were thought to reside only in the pancreas. Here, we show that � cells are also present in the extra-hepatic bile ducts of mice. They are characterised by insulin and C-peptide content, the presence of secretory granules that are immunoreactive for insulin, and the ducts exhibit glucose-stimulated insulin secretion. Genetic lineage labelling shows that these � cells arise from the liver domain rather than the pancreas and, by histological study, they appear to be formed directly from the bile duct epithelium in late embryogenesis. Other endocrine cell types (producing somatostatin and pancreatic polypeptide) are also found in close association with the bile-duct- derived � cells, but exocrine pancreatic tissue is not
present. This discovery of � cells outside the mammalian pancreas has implications for regenerative medicine, indicating that biliary epithelium might offer a new source of � cells for the treatment of diabetes. The finding also has evolutionary significance, because it is known that certain basal vertebrates usually form all of their � cells from the bile ducts. The mammalian bile-duct-derived � cells might therefore represent an extant trace of the evolutionary origin of the vertebrate � cell.
Key words: � cell, Insulin, Bile duct, Pancreas, Liver, Diabetes, Cre- lox
� 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 *These authors contributed equally to this work †Author for correspondence (e-mail: firstname.lastname@example.org)
Accepted 7 November 2006 Journal of Cell Science 120, 239-245 Published by The Company of Biologists 2007 doi:10.1242/jcs.03330
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formation of foci of ectopic pancreatic tissue in the extrahepatic bile ducts of mice homozygous for a null allele of Hes1 (hairy and enhancer of split), a mediator of Notch signalling (Sumazaki et al., 2004). This suggests that within the liver-domain-derived tissues, the extrahepatic bile ducts may be particularly closely related to pancreas in terms of the combination of transcription factors whose expression controls differentiation.
Here, we show that there is a small population of extra- pancreatic endocrine cells within or adjacent to bile ducts in the hilar region of the adult liver. At least four of the normal pancreatic endocrine types are present, and the insulin-positive cells appear to be genuine � cells rather than some other cell population that is making insulin. We have used albumin-Cre mice to label the liver domain during development and show that the ectopic � cells originate from this region rather than migrating from the pancreas. On the basis of the morphological analysis the cells appear to be formed directly from the bile duct epithelium in late embryogenesis and to multiply slowly thereafter to form small clusters in the duct mesenchyme. Other endocrine cell types are also found in close association with the bile-duct-derived � cells but exocrine pancreatic tissue is not present.
Although the extrapancreatic endocrine cells are not numerous, their existence is highly significant for two distinct reasons. First, the fact that they arise spontaneously suggests that the extrahepatic bile ducts must have a similar transcription factor code to the pancreatic endocrine cells, and this means that the biliary epithelium may offer a new source from which to make � cells for the treatment of diabetes. Second, they provide a clue concerning the evolutionary origin of the vertebrate � cell. The agnatha, which are the most basal living vertebrates, have no exocrine pancreas and appear to normally form all their � cells from the bile ducts (Youson, 2000). The bile-duct-derived � cells of mice may therefore represent an extant trace of this primordial method of �-cell development in modern mammals.
Results Endocrine cells are found in bile ducts of the hilar region The hilar region is the location in the body where the liver lobes meet with the principal blood vessels, bile ducts and the gall bladder (Fig. 1A). During an analysis of the livers of some experimental mice we found insulin-positive cells in this region and it soon became clear that these cells existed also in normal control mice. They are found where the extra-hepatic bile ducts branch to individual liver lobes (Fig. 1A,B). Insulin-positive cells or cell clusters were found in bile ducts of most mice with those examined ranging in age from embryonic day 18.5 (E18.5) up to 6 months.
Sections showing insulin-positive cells associated with large bile ducts are shown in Fig. 1C-F, and the locations are further documented in an analysis of serial transverse sections from 30 mice shown in Fig. 1H. The cells are not seen in mid- gestation embryos but first become visible in the late embryo, from E17.5. At E17.5 most of the cells are isolated individuals and about 50% lie within the ductal epithelium or touch it (Fig. 1C). At postnatal stages they are mostly found in the duct connective tissue that surrounds the epithelium and very few are in the epithelium itself, consistent with the view that, once formed, they migrate from the epithelium (Fig. 1D-F). A few
cells are actually found within the parenchyma of the liver (Fig. 1G) so they can presumably migrate this far from the duct. With advancing age, the total number of foci remains constant and the proportion of isolated cells falls while the proportion of small clusters increases (Fig. 2A-C). This morphological study suggests that the endocrine cells arise from the duct epithelium in late embryonic life, migrate into the neighbouring connective tissue and then divide slowly to yield small cell clusters.
Hilar region insulin-positive bile duct cells are not a peculiarity of one individual mouse strain because they were found in both CD1 and C57BL/6 mice. The cells were found in both sexes and in most but not every individual mouse (31 of 35 strain CD1s and 5 of