Reciprocal epithelial-mesenchymal FGF signaling is required for cecal development - PubMed (original) (raw)

Comparative Study

. 2006 Jan;133(1):173-80.

doi: 10.1242/dev.02175. Epub 2005 Nov 24.

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Comparative Study

Reciprocal epithelial-mesenchymal FGF signaling is required for cecal development

Xiuqin Zhang et al. Development. 2006 Jan.

Abstract

Fibroblast growth factor (FGF) signaling mediates reciprocal mesenchymal-epithelial cell interactions in the developing mouse lung and limb. In the gastrointestinal (GI) tract, FGF10 is expressed in the cecal mesenchyme and signals to an epithelial splice form of FGF receptor (FGFR) 2 to regulate epithelial budding. Here, we identify FGF9 as a reciprocal epithelial-mesenchymal signal required for cecal morphogenesis. Fgf9 null (Fgf9(-/-)) mouse embryos have agenesis of the embryonic cecum, lacking both mesenchymal expansion and an epithelial bud. In the cecal region of Fgf9(-/-) embryos, mesenchymal expression of Fgf10 and Bmp4 is notably absent, whereas the expression of epithelial markers, such as sonic hedgehog, is not affected. Using epithelial and whole explant cultures, we show that FGF9 signals to mesenchymal FGFRs and that FGF10 signals to epithelial FGFRs. Taken together, these data show that an epithelial FGF9 signal is necessary for the expansion of cecal mesenchyme and the expression of mesenchymal genes that are required for epithelial budding. Thus, these data add to our understanding of FGF-mediated reciprocal epithelial-mesenchymal signaling.

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Figures

Fig. 1

Fig. 1. Fgf9, FGFR1 and FGFR2 expression in the embryonic cecum

(A,B) Whole-mount in situ hybridization was performed on isolated E11.5 (A) and E13.5 (B) wild-type GI tracts to detect Fgf9 expression. Insets show small intestine and colon. Fgf9 expression was uniformly expressed in the epithelium of the cecum, small intestine and colon. (C) Frozen section of an E13.5 whole-mount in situ hybridization showing Fgf9 expression in the cecal epithelium (arrows). (D–F) Immunohistochemical analysis of E12.5 cecal sections stained with an anti-FGFR1 (E) or anti-FGFR2 (F) polyclonal antibody. Control (D) staining, in which the primary antibody was absent, shows no signal. D′, E′ and F′ are higher magnification views of the boxes in D, E and F, respectively. FGFR1 (E′) and FGFR2 (F′) are expressed in both cecal epithelium (black arrows) and mesenchyme (blue arrows). Co, colon; I, Ileum; SI, small intestine; Ce, cecum.

Fig. 2

Fig. 2. The development of the cecum is abnormal in both _Fgf9_−/− and _Fgf10_−/− embryos

(A–F) Morphologic comparison of the development of the cecum in WT, _Fgf9_−/− and _Fgf10_−/− embryos at E12.5, E14.5 and E18.5. (G–I) Hematoxylin and Eosin (H&E) stained sections of E14.5 WT, _Fgf9_−/− and _Fgf10_−/− cecum. In WT embryos (A–G), the cecum is located at the ileo-colonic junction. (A–C,H) _Fgf9_−/− embryos show a normal appearing epithelial tube throughout the small intestine and colon, but no identifiable cecal bud. (D–F,I) _Fgf10_−/− embryos develop a mesenchymal bud at the ileo-colonic junction, but no epithelial budding or expansion into cecal mesenchyme was observed. (F) By E18.5, the cecal mesenchymal bud appeared to degenerate in _Fgf10_−/− tissue (arrow). Dashed lines indicate the border between the epithelium and mesenchyme in the cecal region. Arrows indicate the position of the cecal tip or the position of the ileo-colonic junction in the absence of a cecum. M, mesenchyme.

Fig. 3

Fig. 3. Reduced cell proliferation rates in _Fgf9_−/− cecal mesenchyme and epithelium

(A,B) H&E stained paraffin sections from E12.5 wild-type (A) and _Fgf9_−/− (B) cecum. The amount of mesenchyme (M) in _Fgf9_−/− cecal tissue is reduced compared with in wild-type tissue. The insets show a low-power view of the entire cecal section; the boxed region indicates the enlarged area shown. (C,D) BrdU labeling of adjacent cecal sections demonstrating reduced cell proliferation in both the mesenchyme and epithelium (arrow) in _Fgf9_−/− (D) tissue compared with in wild-type tissue (C). Dashed line indicates the border of mesenchyme and epithelium; the solid line indicates the apical cap of the cecal epithelium. (E) Quantification of percentage of BrdU-positive nuclei showing significantly decreased cell proliferation in both cecal mesenchyme (*P<0.001) and epithelium (**P<0.01) in _Fgf9_−/− tissue.

Fig. 4

Fig. 4. FGF9 signaling is required for Fgf10 expression

Whole-mount in situ hybridization was performed on E11.5 and E12.5 embryonic GI tracts from wild-type (WT), _Fgf9_−/− and _Fgf10_−/− embryos. Fgf10 was expressed in the cecum mesenchyme in WT tissue (A,C), but was completely absent in _Fgf9_−/− tissue (B,D). By contrast, Fgf9 was expressed in cecum epithelium from WT and _Fgf10_−/− tissue (E,F). Co, colon; I, terminal ileum.

Fig. 5

Fig. 5. Effect of FGF9 and FGF10 on growth of cecal explants

E12.5 embryonic wild-type cecums were removed and embedded in Matrigel. BSA-, FGF9- or FGF10-soaked heparin beads were placed one bead diameter from the cecal explant. (A–C) During the first 12 hours, the cecal epithelium and mesenchyme did not significantly extend toward either the FGF9- or FGF10-soaked bead compared with the BSA-soaked control bead. (D–F) After 84 hours, the cecal epithelium had expanded and extended toward the FGF10 bead (E,E′,H) compared to the BSA bead (D,D′,G); however, no mesenchymal growth was observed (red solid line in D′,E′,G,H). By contrast, cecal mesenchyme showed significant growth accompanied by epithelial expansion when placed adjacent to an FGF9 bead (F,F′,I). Note the significantly thickened mesenchymal regions in F′ and I (line). D′, E′ and F′ show higher magnification brightfield views of the explants in D, E and F, respectively. (G–I) H&E stained frozen sections showing the histology of cultured cecal explants at 84 hours. Red dashed lines indicate the border of mesenchyme and epithelium. The solid lines in D′–F′, G–I indicate the thickness of the cecal mesenchymal layer.

Fig. 6

Fig. 6. Distal small intestinal epithelium extends toward an FGF10, but not an FGF9, source

BSA-, FGF9- or FGF10-soaked beads were placed in Matrigel two-bead diameters from a wild-type distal intestinal epithelial explant in which mesenchymal tissue had been removed. (A–D) Intestinal epithelium did not grow when exposed to a BSA-soaked control bead over an 84-hour period. (E–H) In response to an FGF9 bead, intestinal epithelium showed a modest dilation but no directional extension towards the bead. (I–L) In response to an FGF10 bead, intestinal epithelium showed a significant dilation and extension towards the bead.

Fig. 7

Fig. 7. Loss of Bmp4 but not Shh expression in the developing cecum of _Fgf9_−/− mice

Whole-mount in situ hybridization was performed on E12.5 and E13.5 embryonic GI tracts from wild-type (WT), _Fgf9_−/− and _Fgf10_−/− embryos. (A–D) At E12.5 and E13.5, Bmp4 was expressed strongly in the cecal mesenchyme in WT tissue (A,B) and _Fgf10_−/− tissue (C,D). (E,F) At these same stages, Bmp4 expression was absent in _Fgf9_−/− cecum. (G,H) At E12.5, Shh was expressed in cecal epithelium in WT tissue, and its expression was not significantly affected by the absence of expression of either Fgf9 or Fgf10.

Fig. 8

Fig. 8. Model of reciprocal FGF signaling in cecum development

During early cecal development, epithelial expression of FGF9 signals to mesenchymal FGFRs (FGFR1c and FGFR2c) to stimulate local mesenchymal proliferation and gene expression (Fgf10, Bmp4). Mesenchymal FGF10 signals to the epithelial FGFR2b to stimulate epithelial proliferation and extension of the epithelial bud into the expanded cecal mesenchyme. FGF9 is only able to induce mesenchymal proliferation and Fgf10 expression in the permissive junctional mesenchyme.

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