1. Ductal Malformation and Pancreatitis in Mice Caused by Conditional Jag1 Deletion 
Maria L. Golson, Kathleen M. Loomes, Rebecca Oakey, Klaus H. Kaestner 
Gastroenterology 
Volume 136, Issue 5, Pages e1 (May 2009)
DOI: /j.gastro
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2. Figure 1
Progressive loss and fatty replacement of acinar tissue in Jag1loxP/loxP;Pdx1-Creearly mice. (A and B) H&E staining indicates that both control (A) and mutant (B) mice have acinar, islet (arrows), and ductal tissue on postnatal day 3 (original magnification, 120×). (C and D) Wild-type animals have acinar, duct, and islet tissue (arrows) at 6 weeks (C); but, in Jag1 mutants, although part of the pancreas seems normal, large areas are replaced by what appears to be adipose tissue (D; original magnification, 24×). (E and F) Oil-Red-O staining confirms that the tissue replacing the acinar tissue of mutants (F) contains abundant lipids, whereas none is observed in the interior of control pancreata (E; original magnification, 60×).
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3. Figure 2
Pancreas defects in Jag1 mutants. (A and B) Compared with control mice (A), some Jag1-deficient mice (B) display dramatically enlarged pancreatic ducts with very little other pancreas tissue (pancreata are outlined in black). (C) In unweaned P14 mutant mice with the engorged duct phenotype, the intestinal chyme and stool are pale yellow, suggesting that milk is not completely digested.
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4. Figure 3
Pancreatic ducts are malformed in Jag1 mutants. (A) At P14, CK19 staining outlines normal ducts in the control pancreas but enlarged ducts in the mutant (original magnification, 60× with insets at 120×). (B) Ductal casts in 6-week-old control pancreata reveal that only a short segment of the ductal tree is connected to the common bile duct in Jag1-deficient mice (original magnification, 1.6×). (C) Staining on postnatal day 3 pancreata with Ki67 reveals that CK19-positive duct cells proliferate less in mutants than in controls (original magnification, 240×). (D) Acetylated tubulin staining at postnatal day 3 indicates fewer cilia in mutants than in controls (original magnification, 1000×). (E) Quantification of duct cells, Ki67-positive duct cells, and ductal cilia at postnatal day 3. (*P < .05, **P < .01.)
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5. Figure 4
Epithelial Jag1 is limited to the ducts postnatally. (A–C) Jag1 expression at postnatal day 3. Jag1 is expressed in endothelial cells (A) and ducts (B) in control animals. Jag1 is absent from some mutant ducts at P3 (C; arrow; original magnification, 120×). (D–F) Jag1 expression at 6 weeks. Jag1 is expressed in control ducts at 6 weeks (D). Jag1 expression is absent from ducts in the normal-looking pancreatic head (arrow) but can still be observed in endothelial cells (E; arrowhead) and is present in all of the ducts in the abnormal pancreatic tail (F) (original magnification, 200×).
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6. Figure 5
Jag1-deficient pancreata are fibrotic and display thickened, enlarged ducts. Trichrome staining on control (A and C) and mutant (B and D) pancreata at P3 (A and B; original magnification, 120×). P14 (C and D; original magnification, 60×) and Sirius Red staining at 6 weeks (E and F; original magnification, 24×) reveals that mutants at each age have fibrotic areas within the pancreatic parenchyma, as determined by collagen staining (blue, A–D and red, E and F). In addition, at P14 and 6 weeks, ducts are surrounded by collagen deposits in Jag1-deficient animals (D and F).
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7. Figure 6
Increased cell death and inflammation in Jag1loxP/loxP;Pdx1-Creearly pancreata. (A and B) TUNEL staining on P3 control (A) and mutant (B) pancreata. Many more brown, TUNEL-positive cells are apparent in Jag1-deficient pancreas (original magnification, 240×). (C) Quantification of TUNEL-positive acinar cells. (D) Quantification of total acinar cell area reveals that Jag1 mutants have lost approximately half of their acinar mass by P3. (E and F) Immunostaining for the T-lymphocyte marker CD3 in control (E) and Jag1 mutant (F) mice at 6 weeks (original magnification, 120×). Arrow in (E) indicates a rare CD3-positive cell in a control mouse. Inset in mutant shows an H&E of the material found inside the mutant ducts, which is a mixture of immune cells and cell debris (original magnification, 1000×). (*P < .05.)
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8. Figure 7
Lipid-containing cells observed in Jag1 mutants are adipocytes from an extrapancreatic or mesenchymal pancreatic lineage. (A and B) Immunostaining with the adipocyte marker FABP4 on 6-week control (A) and mutant (B) pancreata. Arrows indicate FABP4-postitive cells (original magnification, 240×). (C and D) Lineage tracing using the R26R allele. (C) Cre activation, indicated with blue staining, is observed throughout the acinar, duct (labeled D), and islet tissue of Jag1+/loxP;Pdx1-Creearly;R26R+/− mice. (D) In Jag1loxP/loxP;Pdx1-Creearly; R26R+/− mice with fatty infiltration, Cre activation is observed in ducts and islets but not in adipocytes (outlined) or endothelial cells. Collagen deposits surrounding ducts in controls and increased collagen deposits in mutants are also negative for LacZ (original magnification, 120×).
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9. Figure 8
Aberrant duct structure in Jag1-deficient mice leads to chronic pancreatitis and exocrine pancreas insufficiency. (A) The chronic pancreatitis marker CTGF is up-regulated in Jag1 mutant pancreas (*P > .05). (B and C) Oil-Red-O staining on stool smears from P15 control (B) and mutant (C) mice reveals abundant fat in the stool of unweaned Jag1 mutant mice with severely affected pancreata (original magnification, 40×). (D) Model of progression of pancreatitis in Jag1-deficient mice. (i) Depiction of pancreatic ductal tree in wild-type mice. (ii) Depiction of ductal tree in Jag1-deficient mice before secondary effects distort it. The tail of the ductal tree is not connected to the common bile duct. (iii) Because the tail of the pancreatic ductal tree is not connected to the bile duct, the fluid in the ducts cannot drain out, causing engorgement of the duct. Eventually, this fluid breaches the duct and leaks into the pancreatic parenchyma, killing acinar tissue. (iv) Fibroblasts infiltrate the pancreas, causing fibrosis. These fibroblasts may later transform into adipocytes.
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10. Supplementary Figure 1
Amylase expression is reduced in Jag1 mutant acini. (A–E) Insulin and amylase expression in control (A and B) and mutant (C–E) animals. Amylase granules appear reduced in number in remaining mutant acinar cells (C and D) compared with controls (A and B). Amylase expression is not observed in ducts or islets of controls (A and B) or mutant animals (C and D) nor is it observed in fat-containing cells in the mutant pancreas (E). (A, C, and E: original magnification, 60×; B and D: original magnification, 120×).
Gastroenterology  , e1DOI: ( /j.gastro )
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11. Supplementary Figure 2
Hes1 staining in control (A) and mutant (B) reveals no overt changes in centroacinar cell position frequency.
Gastroenterology  , e1DOI: ( /j.gastro )
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