<i>Fgf10</i> overexpression during homeostasis changes gastric gland histology but not epithelial proliferation in glandular stomach.

<p>(A–B) Hematoxylin and eosin staining of sections of adult glandular stomach in control littermate (<i>R26^rtTA/+; tet(O)Fgf10/+</i> without dox) (A) and mutant <i>Fgf10</i> overexpressing mice (<i>R26^rtTA/+; tet(O)Fgf10/+</i> with dox) (B). There is a visible decrease in the number of parietal cells and an obvious shift of the mucous neck cells from the luminal side towards the gastric gland base (black arrowheads). (C) qRT-PCR showing a significant increase in Fgf10 expression in the mutants vs controls (*p<0.05). (D-E) PCNA immunostaining of sections of adult glandular stomach in control littermate (<i>R26^rtTA/+; tet(O)Fgf10/+</i> without dox) (D) and mutant <i>Fgf10</i> overexpressing mice (<i>R26^rtTA/+; tet(O)Fgf10/+</i> with dox) (E). (F) Quantification of the percentage of PCNA-positive epithelial cells showed a statistically significant increase in the percentage of PCNA positive epithelial cells indicating increased gastric epithelial proliferation in mutant <i>Fgf10</i> overexpressing mice (<i>R26^rtTA/+; tet(O)Fgf10/+</i> with dox) compared to control littermates (<i>R26^rtTA/+; tet(O)Fgf10/+</i> without dox). Dox was given for 10 days. The scale bar is 50 µm. M = mesenchyme, GE = gastric epithelium, L = lumen.</p

Gastric epithelial differentiation is significantly altered by <i>Fgf10</i> overexpression during homeostasis.

<p>Immunostaining for markers of terminally differentiated gastric epithelial cells of sections of adult glandular stomach in control littermate (<i>R26^rtTA/+; tet(O)Fgf10/+</i> without dox) (A,D,G,J) and mutant <i>Fgf10</i> overexpressing mice (<i>R26^rtTA/+; tet(O)Fgf10/+</i> with dox) (B,E,H,K). (A-B) Mucous neck cells are identified by GSII and (C) quantification reveals a significant increase in mutant <i>Fgf10</i> overexpressing mice compared to control littermates (*p<0.05). (D,E) Chief cells are marked by intrinsic factor and (F) quantification shows a significant decrease in chief cell number (*p<0.05). (G,H) Endocrine cells are identified by chromogranin A and (I) quantification demonstrates no significant change in endocrine cell number. (J,K) Parietal cells are visualized with H/K ATPase immunostaining and (L) quantification demonstrates a significant reduction in mutant <i>Fgf10</i> overexpressing mice compared to control littermates (*p<0.05). Dox was given for 10 days. The scale bar is 50 µm. GE = gastric epithelium, L = lumen.</p

<i>Fgf10</i> overexpression during homeostasis does not cause metaplasia of the gastric epithelium.

<p>(A–C) CDX2, a marker of intestinal metaplasia (IM), immunostaining in adult colon (positive control) (A), control littermate (<i>R26^rtTA/+; tet(O)Fgf10/+</i> without dox) (B) and mutant <i>Fgf10</i> overexpressing mice (<i>R26^rtTA/+; tet(O)Fgf10/+</i> with dox) (C). (D-F) HE4, a marker of spasmolytic polypeptide expressing metaplasia (SPEM), immunostaining in human epididymis (positive control) (D), control littermate (<i>R26^rtTA/+; tet(O)Fgf10/+</i> without dox) (E) and mutant <i>Fgf10</i> overexpressing mice (<i>R26^rtTA/+; tet(O)Fgf10/+</i>) (F). Dox was given for 10 days. The scale bar is 50 µm.</p

FGF10-FGFR2b mediated signaling is not required for normal gastric histology and epithelial proliferation during homeostasis.

<p>Hematoxylin and eosin staining of sections of adult glandular stomach in control littermate (<i>R26^+/+; tet(O)sFgfr2b/+</i> with dox) (A) and mutant mice (<i>R26^rtTA/+; tet(O)sFgfr2b/+</i> with dox) (B) showing normal stomach histology. (C) qRT-PCR confirming robust expression of <i>sFgfr2b</i> in the mutants as compared to controls where the level of <i>sFgfr2b</i> is nearly undetectable. PCNA immunostaining of sections of adult glandular stomach in control littermate (<i>R26^+/+; tet(O)sFgfr2b/+</i> with dox) (D) and mutant mice (<i>R26^rtTA/+; tet(O)sFgfr2b/+</i> with dox) (E) and (F) quantification of the percentage of PCNA-positive epithelial cells showing no significant change in gastric epithelial proliferation in mutant mice compared to control littermates. Dox was given for 1 month. The scale bar is 50 µm. M = mesenchyme, GE = gastric epithelium, L = lumen.</p

Normal expression pattern of <i>Fgf10,</i> its receptors, all other FGFR2b ligands in adult glandular stomach.

<p>(A) PCR of adult glandular stomach and E14.5 wildtype whole embryo (positive control). RT negative controls for both are all negative. (B,E) β-galactosidase staining of adult glandular stomach in <i>Fgf10^LacZ/+</i> (B) and wildtype <i>Fgf10^+/+</i> (E) mice. <i>Fgf10</i> expression occurs only in the mesenchyme. Immunohistochemical analysis of wildtype adult glandular stomach sections stained with anti-FGFR1 (C) or anti-FGFR2 (D). (F,G) Negative control stainings, in which the primary antibody was absent, show minimal to no signal. FGFR1 and FGFR2 staining is strong throughout the gastric epithelium whereas the mesenchymal staining is much weaker. The scale bar is 50 µm. M = mesenchyme, GE = gastric epithelium, L = lumen.</p

FGF10-FGFR2b mediated signaling is not required for gastric epithelial differentiation during homeostasis.

<p>Mucous neck cells are marked by GSII immunostaining of sections of adult glandular stomach in control littermate (<i>R26^+/+; tet(O)sFgfr2b/+</i>with dox) (A) and mutant mice (<i>R26^rtTA/+; tet(O)sFgfr2b/+</i> with dox) (B) and (C) quantification reveals no difference in mucous neck cell number. Chief cells are identified by intrinsic factor immunostaining of sections of adult glandular stomach in control littermate (<i>R26^+/+; tet(O)sFgfr2b/+</i> with dox) (D) and mutant mice (<i>R26^rtTA/+; tet(O)sFgfr2b/+</i> with dox) (E) and (F) quantification reveals no significant change in chief cell number. (G,H) Endocrine cells are identified by chromogranin A and (I) quantification shows no significant change in mutant <i>sFgfr2b</i> overexpressing mice compared to control littermates. Parietal cells are identified by H/K ATPase immunostaining of sections of adult glandular stomach in control littermate (<i>R26^+/+; tet(O)sFgfr2b/+</i>with dox) (J) and mutant mice (<i>R26^rtTA/+; tet(O)sFgfr2b/+</i> with dox) (K) and (L) quantification reveals no significant change in parietal cell number. Dox was given for 1 month. The scale bar is 50 µm. GE = gastric epithelium, L = lumen.</p

FGF10-FGFR2b mediated signaling is not required for parietal cell differentiation during homeostasis.

<p>Hematoxylin and eosin staining of sections of adult glandular stomach in control littermate (<i>R26^+/+; tet(O)sFgfr2b/+</i> with dox) (A) and mutant mice (<i>R26^rtTA/+; tet(O)sFgfr2b/+</i> with dox) (B) showing normal stomach histology. (C) qRT-PCR confirming a significant increase in the expression of <i>sFgfr2b</i> in the mutants as compared to controls (*p<0.05). Parietal cells are identified by H/K ATPase immunostaining of sections of adult glandular stomach in control littermate (<i>R26^+/+; tet(O)sFgfr2b/+</i> with dox) (D) and mutant mice (<i>R26^rtTA/+; tet(O)sFgfr2b/+</i> with dox) (E) and (F) quantification reveals no significant change in parietal cell number. Dox was given for 3 months. The scale bar is 50 µm. M = mesenchyme, GE = gastric epithelium, L = lumen.</p

CC10 and LacZ staining in the distal lung compartment after naphthalene injury.

<p>CC10 and LacZ co-staining in <i>TOPGAL</i> control lungs at low (<b>A</b>) and high (<b>B</b>) magnification show strong expression of <i>TOPGAL</i> in the CC10-positive cells. A decrease in the Clara cells after naphthalene injury is shown in the distal airways (<b>C, D</b>). <i>BATGAL</i> expression is not detected in the distal airways (<b>E, F</b>) of adult control lungs, and still absent from the epithelium (<b>G, H</b>) in the distal compartment of the naphthalene-treated lungs. <i>Axin2^LacZ</i> sections showed very low level staining in the distal airways of the control adult lungs (<b>I, J</b>), and an increased expression after injury (<b>K, L</b>). Scale bars are 100 µm.</p

LacZ expression in whole embryos of <i>TOPGAL</i>, <i>BATGAL</i> and <i>Axin2^LacZ</i> mice.

<p>(<b>A</b>) E11.5 <i>TOPGAL</i> embryo shows staining in the forebrain, the nasal process, the inner ear, the apical ectodermal ridge (AER) of the limb, the epithelium of the mammary placode, the somites and the tip of the tail. (<b>B</b>) E12.5 <i>TOPGAL</i> embryo shows LacZ expression in ectodermal appendages, the whisker placodes in the nasal region as well as the AER, the mammary buds in between the limbs and discrete mesenchymal condensation within the limbs. (<b>C</b>) E11.5 <i>BATGAL</i> embryo shows expression throughout the embryo with a “salt and pepper” pattern with higher signals in ectodermal domains such as the nasal process, the forebrain, the AER and the tip of the tail. (<b>D</b>) At E12.5, the <i>BATGAL</i> embryo still shows “salt and pepper” expression throughout the embryo. LacZ expression is found in the mammary buds but significant staining was also found in the surrounding tissue. (<b>E</b>) E11.5 <i>Axin2^LacZ</i> embryo shows homogenous LacZ expression throughout the embryo with higher expression in the AER and the developing mammary placode. (<b>F</b>) At E12.5, individualized whisker placodes as well as mammary buds and the AER are clearly positive for <i>Axin2^LacZ</i>. In addition, strong LacZ expression is found in the developing ear and in the somites.</p

Vibratome sections of E13.5 <i>TOPGAL</i>, <i>BATGAL</i> and <i>Axin2^LacZ</i> lungs.

<p><i>TOPGAL</i> is expressed in both epithelium and mesenchyme at the level of the bronchi (<b>A, B</b>) and restricted to the epithelium in the distal lung (<b>C</b>). <i>BATGAL</i> “salt and pepper” expression is found in a heterogeneous fashion in the lung (<b>D</b>). The expression is restricted to the mesenchyme adjacent to the bronchial epithelium (<b>E</b>). In the distal lung, <i>BATGAL</i> is sporadically expressed in the epithelium (<b>F</b>). <i>Axin2^LacZ</i> is found exclusively in the mesenchyme adjacent to the epithelium at the bronchial level (<b>G, H</b>). While in the distal lung, LacZ expression is found mainly in the epithelium and at lower level in the mesenchyme (<b>I</b>).</p