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First published online 24 July 2008
doi: 10.1242/dev.020453

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Fgf9 signaling regulates small intestinal elongation and mesenchymal development

Michael J. Geske¹, Xiuqin Zhang^2,*, Khushbu K. Patel¹, David M. Ornitz² and Thaddeus S. Stappenbeck^1,

¹ Departments of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA.
² Department of Developmental Biology, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA.

View larger version (84K): [in this window] [in a new window]   Fig. 1. Late stage Fgf9-/- embryos were disproportionately shortened when compared with littermate controls. (A) Whole-mount photograph of dissected gastrointestinal tracts of E18.5 Fgf9-/- and Fgf9+/- littermates. The small intestines are indicated by the broken yellow boxes and the site of cecal development by the yellow arrows. The colons are towards the right of the small intestines. (B) Quantification of the mean small intestinal lengths (±s.d.) of E12.5, E14.5, E16.5 and E18.5 Fgf9-/- embryos normalized to control littermates (100%). Controls are wild-type and Fgf9+/- littermates; n=5-7 embryos/group. An asterisk indicates the difference of the means is statistically significant (when compared with controls at the same time point) by Student's t-test (P<0.01). (C) Quantification of the mean small intestinal calibers measured at the mid-point of the proximal third, middle third and distal third (±s.d.) of E18.5 control and Fgf9-/- embryos. No statistically significant differences were observed between these two groups. (D) Hematoxylin and Eosin stained cross-section of control and Fgf9-/- E18.5 proximal small intestines showing premature formation of crypt-like structures in Fgf9-/- mice that were not observed in controls (shown at higher magnification on the right). (E) No other morphological differences were noted, including restriction of BrdU-positive cells to the intervillus epithelium. (F) Quantification of mean number of goblet and enteroendocrine cells per 100 epithelial cells (±s.d.) in proximal, middle and distal small intestines of E18.5 control and Fgf9-/- mice. No statistically significant differences were observed between these two groups. (G) Immunohistochemically stained sections of E18.5 control (top) and Fgf9-/- (bottom) littermates showing that I-fabp (left panels) in the proximal small intestines and Ilbp (right panels) in the distal small intestines were expressed at detectable levels in villus enterocytes of controls but not Fgf9-/- embryos. Scale bars: 100 µm in D; 30 µm in D (higher magnification), E,F.

View larger version (58K): [in this window] [in a new window]   Fig. 2. Fgf9 regulates mesenchymal fibroblast proliferation. (A) Hematoxylin and Eosin stained section (left) and color-coded cartoon (right) showing micro-anatomical regions of a control E18.5 small intestine. (B) Immunohistochemistry of BrdU stained sections of control (left) and Fgf9-/- (right) E18.5 proximal small intestines. The broken red lines indicate the boundaries between the epithelium, mesenchyme and muscle layers. (C) Quantification of the means (±s.d.) of the proportion of S-phase cells (as indicated by BrdU incorporation) in the epithelium, mesenchyme and muscularis propria for E14.5, E16.5 and E18.5 for control and Fgf9-/- embryos. Statistically significant differences between the two groups (n=4 embryos/group) were determined by a Student's t-test (**P<0.01; *P<0.05). (D) Quantification of the mean percent BrdU incorporation (±s.d.) in fibroblasts (prolyl 4-hydroxylase positive) and endothelial cells (Pecam1 positive) from the mesenchyme of E18.5 control and Fgf9-/- small intestines. Statistically significant differences between the two groups were determined by comparison of SEMs using Student's t-test; **P<0.01; n=4 mice/group). Scale bars: 30 µm.

View larger version (43K): [in this window] [in a new window]   Fig. 3. Fgf9 signals through mesenchymal receptors to drive small intestine development/elongation. (A) Section of an E14.5 Dermo1-cre; Rosa26R small intestine stained with X-Gal. The epithelium, mesenchyme and muscularis propria are indicated. Blue staining showed regions of Dermo1-cre activity in all cells in the mesenchyme and muscularis propria. The epithelial compartment showed no detectable staining. (B) Section of an E18.5 Dermo1-cre; Rosa26R small intestine stained with anti-β-galactosidase and anti-Pecam (red, top) or CD45 (red, bottom). Both cells show colocalization of punctuate β-galactosidase with the cell lineage markers. No detectable β-galactosidase staining was detected in the epithelium. (C) PCR products of DNA that was procured from the epithelium and mesenchyme of control and DCR1R2 E18.5 small intestines. The recombined alleles of Fgfr1 and Fgfr2 were detected in the mesenchyme of DCR1R2 mice but not in the epithelium. PCR of vimentin is shown as a control for the isolation of the DNA. (D) Quantification of the means of the small intestinal lengths (±s.d. normalized to wild-type length; n=4-5 mice/group) in mice with loss of mesenchymal Fgfr1 (DCR1), Fgfr2 (DCR2), and both Fgfr1 and Fgfr2 (DCR1R2). Loss of both Fgfrs was additive. Two asterisks indicate statistically significant differences when comparing any two groups (Student's t-test, P<0.01). (E) Quantification of S-phase cells in three cellular compartments (intervillus epithelium, mesenchyme and muscularis propria) of E18.5 control and DCR1R2 small intestines. Mean±s.d. for each group is plotted. Statistically significant differences in the means are denoted by an asterisk (P<0.05 by Student's t-test). Scale bars: 10 µm in A; 5 µm in B.

View larger version (98K): [in this window] [in a new window]   Fig. 4. Fgf9 prevents the mesenchymal fibroblast-myofibroblast transition by inhibition of Tgfβ signaling. Immunofluorescence-stained sections of E14.5, E16.5 and E18.5 small intestines from (A-C,G-I) control and (D-F,J-L) Fgf9-/- embryos. Sections were stained with antisera directed against -SMA (A-F; green) and p-Smad2/3 (G-L; red). At all three time points, the mesenchyme from control embryos showed no reactivity to -SMA and only scattered p-Smad2/3 positive cells were present in this region. By contrast, -SMA in Fgf9-/- embryos displayed numerous positive staining cells in the mesenchyme. Nearly all mesenchymal cells in Fgf9-/- embryos were positive for pSmad2/3. The broken white lines indicate the epithelial-mesenchymal borders. The broken yellow lines indicate the mesenchyme-muscularis propria borders. Scale bar: 30 µm.

View larger version (131K): [in this window] [in a new window]   Fig. 5. A subset of small intestinal mesenchymal cells showed a robust response to Fgf9 and expressed Tgfβ inhibitors. Sections of E18.5 small intestines from (A,D-H) control and (B) Fgf9-/- embryos stained with antisera directed against p-Erk1/2 (green) and co-stained with antisera against (D) Pecam-1, (E) CD45, (F) BrdU, (G) Fst and (H) Fstl1 (in D-H, co-staining is in red). In all panels, the mesenchyme at the base of a single villus is shown. The broken white lines indicate the epithelial-mesenchymal boundaries. The green arrows indicate high p-Erk1/2 positive mesenchymal cells near the base of villi. The red arrows indicate mesenchymal cells that do not colocalize with p-Erk1/2 cells for a given marker (e.g. CD45, Pecam1 and BrdU). The yellow arrows denote mesenchymal cells that colocalize with a given marker (e.g. Fst and Fstl1). (C) Quantification of the mean percentage (±s.d.) of mesenchymal cells with detectable p-Erk in control and Fgf9-/- embryos. The double asterisks indicate a statistically significant difference when comparing Fgf9-/- embryos with controls, as determined by Student's t-test (P<0.01). Scale bars: 10 µm.

View larger version (68K): [in this window] [in a new window]   Fig. 6. The p-Erk1, 2/Fst/Fstl1-positive mesenchymal cells were embryonic small intestinal (esi)MSCs. (A) Fluorescence profiles (x-axis) for E18.5 WT iMSCs (passage 5) stained with antigen specific antibodies (red) and isotype-matched negative controls (black) that showed the majority of these cells (y-axis=cell counts) were CD29, CD44, CD54, CD105 and CD106 positive and CD45 negative. (B) Cultured iMSCs expressed genes encoding secreted Tgfβ inhibitors including follistatin (Fst) and follistatin-like 1 (Fstl1). Quantification of relative mRNA amounts of Fst and Fstl1 in multiple cell lines, as determined by qRT-PCR. Expression of Fst and Fstl1 in iMSCs was compared with myofibroblasts (Mic216), macrophages (RAW 264.7) and gut epithelial cells (AGS). (C) Expression of mRNAs encoding Fst (blue), Fstl1 (orange) and Spry2 (a known transcriptional target of Fgf9, red) in iMSCs increased with Fgf9 treatment. A single asterisk indicates that the mean values for the 8 hour time points were statistically significantly different from 0 hours (P<0.05 by Student's t-test). (D) FACS analysis of intracellular -SMA of MIC-216 (intestinal myofibroblast line) cells either cultured alone or in a transwell co-culture with iMSCs. The x-axis is fluorescence intensity for -SMA and the y-axis is forward scatter (an indication of cell size). The box indicates cell population defined as -SMA high. (E) Quantification of the percentage of -SMA low Mic216 cells cultured alone, in the presence of iMSCs, Fst of Fgf9 (n=3 experiments/condition). *P<0.05 (F,G) Sections of control E18.5 small intestines stained with p-Erk (green) and (F) CD105 (red) and (G) CD54 (red). The latter two markers colocalize with p-Erk1/2 positive mesenchymal cells (yellow arrows). (H) Section of an E14.5 Dermo1-cre; Rosa26R small intestine stained with anti-β-galactosidase and anti-CD54. Scale bars: 10 µm.

View larger version (10K): [in this window] [in a new window]   Fig. 7. Proposed cellular mechanism for the control of small intestinal fibroblast differentiation by Fgf9. Epithelially expressed Fgf9 interacts with its receptors (Fgfr1 and Fgfr2) on mesenchymal stem cells. These cells in turn express inhibitors of Tgfβ signaling that interact with mesenchymal fibroblasts to inhibit their expression of -SMA.

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