FGF9-Pitx2-FGF10 signaling controls cecal formation in mice

Abstract

Fibroblast growth factor (FGF) signaling to the epithelium and mesenchyme mediated by FGF10 and FGF9, respectively, controls cecal formation during embryonic development. In particular, mesenchymal FGF10 signals to the epithelium via FGFR2b to induce epithelial cecal progenitor cell proliferation. Yet the precise upstream mechanisms controlling mesenchymal FGF10 signaling are unknown. Complete deletion of Fgf9 as well as of Pitx2, a gene encoding a homeobox transcription factor, both lead to cecal agenesis. Herein, we used mouse genetic approaches to determine the precise contribution of the epithelium and/or mesenchyme tissue compartments in this process. Using tissue compartment specific Fgf9 versus Pitx2 loss of function approaches in the gut epithelium and/or mesenchyme, we determined that FGF9 signals to the mesenchyme via Pitx2 to induce mesenchymal Fgf10 expression, which in turn leads to epithelial cecal bud formation.

Figure 1. Pitx2 expression in the embryonic cecum

(A-C) Whole mount in situ hybridization was performed on isolated E12.5 (A), E13.5 (B) and E14.5 (C) GI tracts to detect Pitx2 expression. Pitx2 was strongly expressed in both epithelium and mesenchyme of the cecum, while Pitx2 expression was restricted to the epithelium of the small intestine and absent in the colon. (D-F) Vibratome sections of the ceca were performed at 20 μm to show the localization of Pitx2 at E12.5 (D), E13.5 (E) and E14.5 (F). The sections showed expression of Pitx2 in both epithelium and mesenchyme of the cecum. (G,H) Vibratome sections of the small intestine to show the localization of Pitx2 at E12.5 (G) and E14.5 (H). The sections showed an epithelial expression of Pitx2 at E12.5 (G) and both mesenchymal and epithelial expression at E14.5 (H). Ile, ileum; Col, colon; cec, cecum; epi, epithelium; mes, mesenchyme. Scale bars in A-C are 500 microns, scale bars in D-I are 100 microns.

Figure 2. Deletion of mesenchymal Pitx2 leads to cecal agenesis

(A) Pitx2 whole mount in situ hybridization (WMISH) confirms the deletion of mesenchymal Pitx2 in the mutants (B) as compared to the controls at E11.5 (A). Comparison of the cecum development in E12.5 (C-D), E14.5 (E-F) and E16.5 (G-H) WT gut (C, E, G) and Pitx2^Dermo1-Cre gut (D, F, H). Pitx2^Dermo1-Cre mutants did not show any cecum formation (D, F, H) whereas wild type littermates showed a normal developing cecum (C, E, G). (I,J) Comparison of control (I) versus Pitx2^Shh-Cre (J) ceca at E14.5. No difference is noticeable in the development of the mutant cecum compared to the control. (K) WISH using sense probe for Pitx2 did not show any staining in E12.5 wild type cecum. (L) Whole mount view of representative E14.5 GI tracts of Pitx2^Dermo1-Cre and littermate controls. White arrows show the location of the cecum on each GI tract. The black arrow shows the diverticulum (M) Quantification of the small intestine (SI) and colon lengths of Pitx2^Dermo1-Cre mutants normalized to control littermates (100%) indicates that mutant SI are shorter when compared to controls while no difference is observed for the colons. The bars represent means ± s.e.m. Asterisk indicates a statistical difference (p=0.0214; n=7). Scale bars are 500 microns.

Figure 3. Decrease in cell proliferation in Pitx2^Dermo1-Cre mutants

(A, B) Paraffin sections from E12.5 control (A) and Pitx2^Dermo1-Cre (B) stained with H&E. Note the absence of cecal bud in the mutant along with a decreased mesenchyme when compared to the control. (C-F) BrdU labeling of control (C, E) and mutant (D, F) cecal sections showed a decreased proliferation in both epithelium and mesenchyme of the mutants. (G) Quantification of the number of BrdU positive cells in the epithelium and the mesenchyme (n=6). Scale bars are 50 microns.

Figure 4. Gene expression in E11.5 Pitx2^Dermo1-Cre cecum

Comparison of Fgf10, Bmp4 (C-D) and Fgf9 (E-F) expression by WMISH in ceca isolated at E12.5. Fgf10 and Bmp4 are expressed in the mesenchyme of wild type E12.5 cecum (A and C respectively). Fgf10 expression is absent from the cecum of the mutants (B) while Bmp4 expression is maintained in the mutant ceca (D). Fgf9 is expressed in both epithelium and mesenchyme of E12.5 WT cecum (E) and its expression is restricted to the epithelium of the mutant ceca (F). Insets in E and F show a high magnification of the cecal bud. (G-I) Negative controls using the sense probes for Fgf10 (G), Bmp4 (H) and Fgf9 (I) did not show any staining. (J) Quantification of gene expression by RT-qPCR (n=12). (K) Schematic illustration of the parts of the ceca from controls and mutants that were used for the analyses. Scale bars are 500 microns.

Figure 5. FGF9 controls the expression of Pitx2 and Fgf10

(A) qPCR for Pitx2, Fgf10, Fgfr2b and Fgfr2c after in vitro culture of ceca in the absence or presence of 250 ng/mL rh FGF9 for 12 hours (n=4 in duplicates). (B-D) Comparison of the cecum development at E14.5 in wild type (B), Fgf9^Dermo1-Cre (C) and Fgf9^CMV-Cre (D) mutants. The wild type cecum has a well developed cecal bud surrounded by a thick layer of mesenchyme (B); deletion of Fgf9 in the mesenchyme resulted in a thinning of the mesenchymal layer surrounding the epithelial bud that forms properly (C), whereas the complete deletion of Fgf9 resulted in impaired cecal bud formation (D). Pitx2 (E-G) and Shh (H-J) expression in mutants compared to the control. Note the complete lack of Pitx2 expression in the mesenchyme of Fgf9^CMV-Cre gut (G- inset shows a high magnification of the cecal bud). No detectable change in Shh expression was detected in either mutants (I, J) compared to control (H). Scale bars are 250 microns.

Figure 6. Pitx2 overexpression induced the expression of Fgf10 in the absence of Fgf9

Relative Fgf10 mRNA expression showed significant increase of Fgf10 expression after transfection of NIH 3T3 cells (negative for Fgf9) with Pitx2a vector (n=4, p=0.03).

Figure 7. Model of FGF/Pitx2/FGF10 signaling in the developing cecum

We propose that epithelial FGF9 acts via FGFRc receptor isoforms in the mesenchyme to control cecal mesenchyme expansion and Fgf10 expression. Both activities are mediated by Pitx2. In turn FGF10 acts on the epithelium to trigger the invasion of the epithelial bud into the cecal mesenchyme.