Fibroblast growth factor 10 alters the balance between goblet and Paneth cells in the adult mouse small intestine

Abstract

Intestinal epithelial cell renewal relies on the right balance of epithelial cell migration, proliferation, differentiation, and apoptosis. Intestinal epithelial cells consist of absorptive and secretory lineage. The latter is comprised of goblet, Paneth, and enteroendocrine cells. Fibroblast growth factor 10 (FGF10) plays a central role in epithelial cell proliferation, survival, and differentiation in several organs. The expression pattern of FGF10 and its receptors in both human and mouse intestine and their role in small intestine have yet to be investigated. First, we analyzed the expression of FGF10, FGFR1, and FGFR2, in the human ileum and throughout the adult mouse small intestine. We found that FGF10, FGFR1b, and FGFR2b are expressed in the human ileum as well as in the mouse small intestine. We then used transgenic mouse models to overexpress Fgf10 and a soluble form of Fgfr2b, to study the impact of gain or loss of Fgf signaling in the adult small intestine. We demonstrated that overexpression of Fgf10 in vivo and in vitro induces goblet cell differentiation while decreasing Paneth cells. Moreover, FGF10 decreases stem cell markers such as Lgr5, Lrig1, Hopx, Ascl2, and Sox9. FGF10 inhibited Hes1 expression in vitro, suggesting that FGF10 induces goblet cell differentiation likely through the inhibition of Notch signaling. Interestingly, Fgf10 overexpression for 3 days in vivo and in vitro increased the number of Mmp7/Muc2 double-positive cells, suggesting that goblet cells replace Paneth cells. Further studies are needed to determine the mechanism by which Fgf10 alters cell differentiation in the small intestine.

Keywords: Fgf10; Fgfr2b; differentiation; small intestine.

Fig. 1.

Expression of FGFR1, FGFR2, and their ligands in human ileum. A and B: hematoxylin and eosin (H&E) staining of normal human ileum showing crypt and villus structures (A) and higher magnification of A (B). C and D: immunohistochemistry (IHC) using anti-FGFR1 antibody from Santa Cruz (Flg) (C) and anti-FGFR1 from Abcam (D) on human ileum. E: negative control (Cont.) using matching anti-IgG. E′: negative control using the competition peptide (Pep) for Flg antibody. F and G: IHC using anti-FGFR2 antibody from Santa Cruz (Bek) (F) and anti-FGFR2 antibody from Abcam (G) on human ileum. H: negative control using matching anti-IgG. H′: negative control using the competition peptide for Bek antibody. I and J: qRT-PCR for FGFs ligands (I, n = 5 at least), FGFR1b, and FGFR2b (J, n = 9) on human ileum. Scale bars are 100 μm.

Fig. 2.

Expression of FGFR1, FGFR2, and FGFR2b ligands in the small intestine. A–C: LacZ (blue) and nuclear red (pink) staining on tissue sections from duodenum (A), jejunum, (B) and ileum (C) of adult Fgf10-LacZ mice. D: RT-PCR for Fgf10 and β-actin on separated epithelial and mesenchymal fractions from adult mouse duodenum (n = 3). E–G: IHC staining for Fgfr1 on mouse adult duodenum (E), jejunum, (F) and ileum (G). E′–G“: higher magnifications of crypts from panels E, F and G. H–J: IHC staining for Fgfr1 on mouse adult duodenum (H), jejunum, (I) and ileum (J). H′–J′: higher magnifications of crypts from H, I and J. K: qRT-PCR for Fgf10 family members on adult mouse duodenum (Duo), ileum (Ile), and jejunum (Jej) and embryonic day 14.5 whole mouse embryo (positive control). Negative controls did not show any bands; n = 3. To show the entire villus height, the duodenum sections are shown at lower magnifications than the jejunum and ileum panels. Scale bars are 100 μm in A–C, F, G, I, and J; 200 μm in E–H; and 20 μm in E′–G′ and H′–J′.

Fig. 3.

Increased villus height in the duodenum and jejunum after Fgf10 overexpression. A–D: hematoxylin and eosin staining of jejunum sections of control not treated with doxycycline (Dox; A, A′, A″), Dox-treated controls (B, B′, B″), Rosa26^rtTA/−; tet(o)sFgfr2b/+ (C, C′, C″), and Rosa26^rtTA/−; tet(o)Fgf10/+ animals (D, D′, D″). A′–A″, B′–B″, C′–C″, D′–D″ are higher magnifications of A, B, C, and D, respectively. E: qRT-PCR validating the expression of Fgf10 in the controls vs. Rosa26^rtTA/−; tet(o)Fgf10/+ animals (n = 6). E: qRT-PCR showing the expression of exogenous sFgfr2b transcripts in the controls vs. Rosa26^rtTA/−; tet(o)sFgfr2b/+ animals (n = 6). G: quantification of the crypt depths. H: quantification of the villus height. I: quantification of the ratio villus/(villus + crypt). N = 6 for each group, *P ≤ 0.05. Scale bars are 500 μm in A–D, 200 μm in A′–D′, and 20 μm in A″–D″.

Fig. 4.

Increased cell proliferation in the duodenum of mutants overexpressing Fgf10. A–D: IHC for PCNA on duodenum sections from controls not treated with Dox (A), Dox-treated controls (B), Rosa26^rtTA/−; tet(o)sFgfr2b/+ (C), and Rosa26^rtTA/−; tet(o)Fgf10/+ animals (D). A′, B′, C′, and D′ are higher magnifications of the inset in A, B, C, and D, respectively. E–H: immunofluorescence (IF) staining for phospho-histone H3 (Phh3) on duodenum sections from controls not treated with Dox (E), Dox-treated controls (F), Rosa26^rtTA/−; tet(o)sFgfr2b/+ (G), and Rosa26^rtTA/−; tet(o)Fgf10/+ animals (H). I: quantification of cell proliferation in the crypts as the percentage of the number of PCNA+ positive to the number of total cells per crypt. J: quantification of the number of Phh3+ cells per 100 crypts. K: quantification of cell death in the villi as the number of active caspase 3 + cells per 100 villi. The results are expressed as means ± SE of at least 5 independent animals per group. *P ≤ 0.05; **P ≤ 0.01. Scale bars are 200 μm in A–D, 20 μm in A′–D′, and E–H.

Fig. 5.

Fgf10 overexpression results in increased goblet cells and decreased Paneth cells. A–C: chromogranin A IF staining in the jejunum of Dox-treated control (A), Rosa26^rtTA/−; tet(o)sFgfr2b/+ (B), and Rosa26^rtTA/−; tet(o)Fgf10/+ animals (C). D: quantification of the number of chromogranin A-positive cells per total number of epithelial cells. E–G: lysozyme staining (red) a marker of Paneth cells in the jejunum of Dox-treated control (E), Rosa26^rtTA/−; tet(o)sFgfr2b/+ (F), and Rosa26^rtTA/−; tet(o)Fgf10/+ (G). H: quantification of the number of lysozyme-positive cells per total number of epithelial cells per crypt. I–K: Alcian blue staining to label goblet cells in the jejunum of Dox-treated control (I), Rosa26^rtTA/−; tet(o)sFgfr2b/+ (J), and Rosa26^rtTA/−; tet(o)Fgf10/+ animals (K). L: quantification of the number of goblet cells per total number of epithelial cells. M: quantification of the number of goblet cells per total number of epithelial cells in the crypts of control Dox and Rosa26^rtTA/−; tet(o)Fgf10/+. N: quantification of the number of goblet cells per total number of epithelial cells in the villi of Dox-treated control and Rosa26^rtTA/−; tet(o)Fgf10/+. The results in D, H, L, M, and N are expressed as means ± SE of 6 independent animals per group. *P ≤ 0.05; **P ≤ 0.01; ***P < 0.0001. Scale bars are 100 μm.

Fig. 6.

FGF10 acts directly on the epithelium to induce goblet cells and decrease Paneth cells and stem cell markers. A and B: 3D images of whole mount IF staining of Phh3 and Hoechst on enteroid cultures in absence (A) or presence of rhFGF10 (200 ng/ml). C: quantification of the number of Phh3-positive cells per mm³. Results are expressed as means ± SE of 5 independent experiments; 3 enteroids were analyzed in each experiment. D: LacZ staining of ileal enteroids from Axin2-LacZ reporter mouse in absence (control) or presence (+FGF10) of rhFGF10 and qRT-PCR for Axin2 transcripts in wild-type enteroids treated or not with rhFGF10. E: qRT-PCR on RNA from wild-type enteroids untreated (black bars) or treated (gray bars) with 200 ng/ml of rhFGF10 for Muc-2, lysozyme, Atoh1, Klf4, Spdef, Ascl2, Hes1, Hopx, Lgr5, Lrig1, and Sox9. F and G: IF staining for Phh3 (red) and ECadherin (green) on enteroids isolated from Rosa26^rtTA/−; tet(o)Fgf10/+ treated (G) or not (F) with Dox. H: quantification of the percent of Phh3+ cells per total number of DAPI. I and J: IF staining for Mmp7 (green) and Muc2 (red) on enteroids isolated from Rosa26^rtTA/−; tet(O)Fgf10/+ treated (J) or not (I) with Dox. K: quantification of the percent of Muc2+ and Mmp7+ cells per total number of DAPI. L and M: IF staining for lysozyme (red) on enteroids isolated from Rosa26^rtTA/−; tet(o)Fgf10/+ treated (M) or not (L) with Dox. N: quantification of the percent of lysozyme+ cells per total number of DAPI. O: quantification of the percent of double-positive cells for Mmp7 and Muc2 compared with the total number of Mmp7+ cells. P and Q: Mmp7 and Muc2 IF staining on ileum from control animals (P) and Rosa26-RtTA; tet(o)Fgf10 animals treated with Dox for 3 days. Arrows show double-positive cells. R: qRT-PCR confirming Fgf10 overexpression in the enteroids treated with Dox compared with the untreated enteroids. S: qRT-PCR to assess gene expression of Lgr5 and Lrig1 following Fgf10 overexpression. Scale bars are 100 μm in A and B, 50 μm in F and G, and 25 μm in I, J, L, and M. *P ≤ 0.05; **P ≤ 0.01.