1. Sonic hedgehog regulates gastric gland morphogenesis in man and mouse
Gijs R. Van Den Brink, James C.H. Hardwick, Maikel P. Peppelenbosch, Sander J.H. Van Deventer, Guido N.J. Tytgat, Menno A. Brink 
Gastroenterology 
Volume 121, Issue 2, Pages (August 2001)
DOI: /gast
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2. Fig.1
The different cell types of the gastric tubular unit.Based on Karam and Leblond.2
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

3. Fig.2
Shh is expressed in murine stomach.Immunoblot, demonstrating that both (A) Shh precursor (Santa Cruz antibody) and (B) cleaved amino-terminal Shh signal protein (R&D antibody) are found in the murine stomach but not in the duodenum.(C) Ihh is expressed in both kidney and liver, but not in stomach or duodenum.Both liver and kidney were Shh negative (R&D antibody).
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

4. Fig.3
Compartmentalized Shh expression in the stomach.The Santa Cruz anti-Shh precursor antibody was used for all immunohistochemistry.(A, B, E, F, G, I, J) Human stomach.(C, D, H) Murine stomach.Shh precursor protein is expressed in both human and murine stomach (brown precipitate, A, C), but no expression was found in the small intestine of either species (B, D).An Shh blocking peptide competed efficiently with binding of the anti-Shh antibody (anti-Shh without blocking peptide in E versus anti-Shh plus a 50-fold excess blocking peptide in F).Use of a periodic acid–Schiff stain that stains the pit cells dark purple combined with an Shh stain shows that Shh is exclusively expressed in the gland compartment in both human (Shh stained blue, G) and murine (Shh stained brown, H) gastric units.The expression of the pit cell marker MUC5AC (brown in I and J) shows that Shh expression (blue in I and J) shows no overlap with MUC5AC and is expressed exclusively in the gland region.Note the graded Shh expression in the human stomach (A, J) from high at the pit–gland transition (arrows) to low deeper in the gland (asterisks).Original magnifications: A, ×25; B–D, ×50; E, F, ×40; G, H, ×200; I, ×25; J, ×100.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

5. Fig.4
Identification of Shh expressing cell type.(A–C) Human stomach.Double stain of Shh (blue) and parietal cell marker H+,K+-ATPase (brown), performed as described in Materials and Methods, showing complete overlap.Again a clear expression gradient is observed, parietal cells close to the pit express high Shh (B, arrows, blow-up of area boxed with continuous line in A), whereas this expression gradually diminishes toward the gland base (C, asterisks, blow-up of area boxed with broken line in A).(D–F) Murine stomach.Serial sections showing expression of Shh (D, brown) and the H+,K+-ATPase (E, brown).In the murine gland, Shh is expressed by parietal cells (arrows) and zymogenic cells (asterisks).As can be seen in the combined Shh (brown)/periodic acid–Schiff (pink) stain in F, Shh is also expressed in mucous neck cells.Arrows in F denote cells positive for both Shh and the periodic acid–Schiff reaction.Original magnifications: A, ×25; B, C, ×100; D, E, ×200; F, ×1000.
Gastroenterology  , DOI: ( /gast )
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6. Fig.5
Expression of Shh transcriptional targets.(A–F) Human stomach.(A, B) Expression of HNF3β in parietal cells is evident in a double stain of HNF3β (brown) and parietal cells (blue) performed as described in Materials and Methods (arrows in B denote positive cells).(C) BMP2 expression and (E) Ptc expression (antibody from Dr.Töftgard) are both confined to the gland region as shown by parietal cell staining in an adjacent section (D, F), which helps indicate the gland region.(G–L) Murine stomach.(G, J) Expression of HNF3β (arrows denote positive cells) in parietal cells is identical to that in the human stomach using the same double staining method as in A and B.(H, I) Expression of (H) Ptc and (I) the parietal cell marker H+,K+-ATPase.Ptc is expressed mainly in parietal cells (arrows) and zymogenic cells (asterisk).The expression pattern of (K) BMP4 and (L) BMP2 shows localization of BMP4 to stromal cells (arrows in K), whereas BMP2 is expressed in the epithelial cells (arrows in L).Original magnifications: A, ×200; B, ×1000; C–I, ×200; J, ×1000; K, L, ×200.
Gastroenterology  , DOI: ( /gast )
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7. Fig.6
Reduced expression of HNF3β, Isl-1, and BMP4 in cyclopamine-treated mice.Immunoblots showing HNF3β, Isl-1, BMP2, BMP4, and actin expression (A) in whole gastric lysates from 7 control (lanes 1–7) and 7 cyclopamine-treated (lanes 8–14) mice.Signals were quantified with a PhosphorImager, corrected for the β-actin signal, and expressed in relative units in B.Values are means + SE.*Statistically significant differences between control and cyclopamine-treated animals (P = for HNF3β; P = for Isl-1; P = for BMP4; Student t test).
Gastroenterology  , DOI: ( /gast )
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8. Fig.7
Inhibition of Shh increases gland cell proliferation in the murine stomach.Panel 1. (A, B) BrdU stain, arrows denote BrdU-positive cells.Compared with controls (A), BrdU incorporation is substantially increased in the precursor cells in the isthmus in cyclopamine-treated mice (B).(C–H) PCNA expression.Whereas PCNA staining in the pit cells remained unaltered in treated animals (E) compared with control (C), gland cell expression increased considerably (D, control; F, treated).(G, H) As can be seen at higher magnification, expression increased especially in zymogenic cells (compare H with control in G).Panel 2. Graphs depicting the mean number of BrdU- and PCNA-positive nuclei per crypt/gland in the stomach and duodenum of cyclopamine-treated mice (□, n = 8) and controls (■, n = 8).Error bars indicate SE.Values in treated mice were significantly different from controls (stomach: P = 0.2 for BrdU, P < for PCNA; duodenum: P = 0.02 for BrdU, P = 0.03 for PCNA, Student t test).Original magnifications: A,B, ×200; C, D, ×250; E, F, ×400.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

9. Fig.7
Inhibition of Shh increases gland cell proliferation in the murine stomach.Panel 1. (A, B) BrdU stain, arrows denote BrdU-positive cells.Compared with controls (A), BrdU incorporation is substantially increased in the precursor cells in the isthmus in cyclopamine-treated mice (B).(C–H) PCNA expression.Whereas PCNA staining in the pit cells remained unaltered in treated animals (E) compared with control (C), gland cell expression increased considerably (D, control; F, treated).(G, H) As can be seen at higher magnification, expression increased especially in zymogenic cells (compare H with control in G).Panel 2. Graphs depicting the mean number of BrdU- and PCNA-positive nuclei per crypt/gland in the stomach and duodenum of cyclopamine-treated mice (□, n = 8) and controls (■, n = 8).Error bars indicate SE.Values in treated mice were significantly different from controls (stomach: P = 0.2 for BrdU, P < for PCNA; duodenum: P = 0.02 for BrdU, P = 0.03 for PCNA, Student t test).Original magnifications: A,B, ×200; C, D, ×250; E, F, ×400.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions