1. Volume 130, Issue 3, Pages 855-867 (March 2006)
Loss of the Major Duodenal Papilla Results in Brown Pigment Biliary Stone Formation in Pdx1 Null Mice 
Akihisa Fukuda, Yoshiya Kawaguchi, Kenichiro Furuyama, Sota Kodama, Takeshi Kuhara, Masashi Horiguchi, Masayuki Koizumi, Koji Fujimoto, Ryuichiro Doi, Christopher V.E. Wright, Tsutomu Chiba 
Gastroenterology 
Volume 130, Issue 3, Pages (March 2006)
DOI: /j.gastro
Copyright © 2006 American Gastroenterological Association Terms and Conditions

2. Figure 1
Formation of brown pigment CBD stones in pdx1 null mice. (A and B) Stereomicroscopic views of the common bile duct (CBD) at P7. The CBD is normal in wild-type mice (A), whereas multiple CBD stones whose color is yellowish-brown are formed in pdx1 null mice (B). (C and D) H&E staining. No CBD dilatation is observed in wild-type mice (C), whereas marked CBD dilatation is observed in pdx1 null mice with stones (D) at P7. (E) Hall staining of the stone in pdx1 null mice. Hall staining indicates that the stones are pigmented but not cholesterol stones. Arrows, CBD stones; arrowheads, CBD.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

3. Figure 2
Expression pattern of pdx1 and loss of peribiliary glands and mucin-producing cells in the CBD in pdx1 null mice. (A and B) PAS staining. Peribiliary glands and PAS-positive mucin-producing cells in the CBD are well developed in wild-type mice (A), but they are absent in pdx1 null mice (B) at P7. Pdx1 expression by immunofluorescence (C and D) and PAS staining (E and F) in wild-type mice at P7. Pdx1 is expressed in the CBD epithelium including the mucin-producing cells and peribiliary glands (C and E). It is also expressed in the Brunner’s glands (D and F). (G and H) PAS staining. Peribiliary glands in the CBD (arrowheads) and an evagination of the CBD epithelium (solid arrows) are observed at P1 (H) but not at E18.5 (G) in wild-type mice. Arrows, mucin-producing cells; arrowheads, peribiliary glands; and frames, Brunner’s glands. (I–L) Stereoscopic views of pdx1lacZ/+ embryos at E12.5 (I) and E15.5 (K), showing pdx1 expression as visualized by X-gal staining. J and L are the magnified views of the periampullary region of the same samples shown in I and K, respectively. (M and N) Hematoxylin staining of the sections of I and K, respectively. The developing CBD (arrowheads in I–N), MDP (arrows in I–N), accessory pancreatic duct (asterisks in I–L), and surrounding duodenum are expressing pdx1 during embryogenesis.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

4. Figure 3
Loss of the major duodenal papilla in pdx1 null mice. (A–D) Stereomicroscopic views of the major duodenal papilla (MDP) at P3. In wild-type mice (A and B), the MDP in the descending portion of the duodenum and well-developed duodenal villi are observed. In pdx1 null mice (C and D), the MDP is completely lost and replaced by an irregular-shaped orifice, which directly channels to the CBD. Note that almost no duodenal villi are seen (C). The CBD is cannulated from the MDP (B) and the orifice (D). (E–L) Histologic analysis at P7 (E–H) and at E18.5 (I–L). H&E staining (E, G, I, and K) and expression of α-smooth muscle actin (SMA) by immunohistochemistry (F, H, J, and L). The MDP is well developed in wild-type mice (E and F). The terminal portion of the CBD is covered by the protruded duodenal tissue, the MDP (E). α-SMA (+) muscular structure including the sphincter of Oddi is shown (frames in F). In pdx1 null mice (G and H), the MDP is not formed and is replaced by a large orifice of the CBD without any duodenal covering. α-SMA (+) muscular structure is specified, but the protrusion into the duodenal lumen is far shorter in length (compare H and F). This impaired structure suggests loss of valve function. Earlier in development, in wild-type mice, the MDP is absent in pdx1 null mice (K and L), whereas wild-type mice have already established the basic MDP structure (I and J) at E18.5. The orifice of the CBD in pdx1 null mice is already larger than that of wild-type mice without duodenal covering. Arrowheads indicate the MDP in wild-type mice and the orifice of the CBD in pdx1 null mice.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

5. Figure 4
The precise boundaries between the epithelium of the duodenum and CBD. Immunostaining for Cdx2 shows the precise boundaries between the epithelium of the duodenum and CBD at the duodeno-biliary junction (arrows). Note that the epithelium of the CBD has spread laterally to the duodenal side, resulting in a larger orifice of the CBD in pdx1 null mice at P7 (G and H) compared with that at E18.5 (C and D). B, D, F, and H are high-power images of A, C, E, and G, respectively. Immunostaining for Cdx2/DAPI is also shown in inset in G. St, stomach; Du, duodenum.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

6. Figure 5
Pdx1 null mice had shorter periampullary duodenal villi. (A–H) H&E staining. Pdx1 null mice have shorter duodenal villi in the periampullary region than wild-type mice, but distal duodenal villi are indistinguishable between them at P7 (compare A and B). Also at P3, Pdx1 null mice have shorter duodenal villi in the periampullary region than wild-type mice (compare E and F), but there is no difference in the height of duodenal villi between wild-type mice and pdx1 null mice at E18.5 (compare G and H). Arrow indicates the orifice of the CBD, and arrowheads indicate the dorsal pancreatic ductule.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

7. Figure 6
Reduced cell proliferation of the periampullary duodenal epithelial cells in pdx1 null mice. (A–F) Double immunostaining for pHH3 (red) and DAPI (blue). PHH3-positive cells are less observed in the periampullary duodenal epithelial cells in pdx1 null mice compared with those in wild-type mice at E18.5 (A and B), P3 (C and D), and P7 (E and F). (G) Quantification of proliferative indices (percentage pHH3-positive cells/duodenal epithelial cells) at various stages. Asterisks indicate P < .05.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions

8. Figure 7
Increased apoptosis of the periampullary duodenal epithelial cells in pdx1 null mice. (A–G) Apoptotic nuclei (green) are labeled by TUNEL. All nuclei are simultaneously costained with DAPI (blue). In wild-type, only a small population of TUNEL (+) apoptotic cells are observed at the top of the duodenal villi (A and C), and no TUNEL (+) cells are observed in the CBD (E), whereas great numbers of TUNEL (+) apoptotic cells are observed in the periampullary duodenal villi in pdx1 null mice at E18.5 (B) and P7 (D, F, and G). Note that the epithelium of the CBD is not apoptotic (arrowheads in F and G). Around the duodeno-biliary junction of the mutant, TUNEL (+) cells are detected in the Cdx2-positive duodenal mucosa (G and Figure 4G, serial sections). Arrows, the MDP or the large orifice at the duodeno-biliary junction; arrowheads, CBD. (H) Electron micrographs of the periampullary duodenal epithelial cells in pdx1 null mice at P7. Arrowhead indicates low-density matrix from a nuclear-membranous component, and arrows indicate apoptotic bodies with chromatin condensation and nuclear fragmentation, characteristic features of apoptosis. (I) Apoptotic indices (percentage TUNEL-positive cells/duodenal epithelial cells) at E18.5 and P7. Asterisks indicate P < .05.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Terms and Conditions