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First published online 26 January 2006
doi: 10.1242/dev.02252

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Foxf1 and Foxf2 control murine gut development by limiting mesenchymal Wnt signaling and promoting extracellular matrix production

¹ Department of Cell and Molecular Biology, Göteborg University, Göteborg, Sweden.
² Department of Biochemistry, Hamamatsu University School of Medicine, Hamamatsu, Japan.
³ The Electron Microscopy Unit, Department of Anatomy and Cell Biology, Göteborg University, Göteborg, Sweden.

View larger version (81K): [in a new window]   Fig. 1. Foxf mutants have aganglionic megacolon with smooth muscle hypoplasia and occasional anal atresia. (A,B) Lower gastrointestinal tract of E18.5 wild-type (A) fetus and Foxf1-/+; Foxf2-/+ litter mate (B). Arrowheads indicate the colon, which is thin-walled and distended in the mutant. (C) Vibratome section of the intestine of a wild-type E12.5 embryo, whole-mount in situ hybridized with a Foxf2 probe, showing Foxf2 expression in the mesenchyme next to the endodermal epithelium. (D,E) Higher magnification of the colon in A and B. (F) Anal atresia and megacolon in E18.5 Foxf2-/- mutant. (G-I) Hematoxylin and Eosin stained sagittal sections through the rectum and lower colon of E18.5 wild-type (G), Foxf1-/+; Foxf2-/+ (H) and Foxf2-/- (I); insets show higher magnifications of the distal colon wall, which is thin and flat in the mutants. (J,K) Thin (1 µm) section of wild-type (J) E18.5 distal colon showing the stratification with epithelium (ep), mesenchyme (mc), circular (cm) and longitudinal (lm) musculature, and mesothelium (mt). The mesodermal layer in the Foxf1-/+; Foxf2-/+ (K) is hypoplastic and consists of loosely associated, poorly differentiated SMCs. (L,M) Immunostaining with antiserum against the neuronal marker neurofilament shows the plexus of enteric nerves in the mesodermal layer of E18.5 wild-type distal colon (L), whereas no neurons are detected in the mutant (M; Foxf1-/+; Foxf2-/+). Insets show the distal colon wall at higher magnification and arrowheads indicate the enteric plexus. Staining in the gut lumen is due to the non-specific stickiness of the meconium and goblet cell mucins. (N,O) Immunostaining with anti-SMA reveals SMC hypoplasia in Foxf2-/- distal colon (arrowheads), but normal SMC investment of arteries (Ar).

View larger version (187K): [in a new window]   Fig. 2. Lack of extracellular matrix, weak cell adhesion and poorly developed endoplasmic reticulum in fibroblasts of Foxf mutant gut. TEM images of E18.5 wild-type (A,C,E) and Foxf1-/+; Foxf2-/+ (B,D,F) distal colon. (A) Normal stratified colon with (from top) epithelium, mesenchyme, the two muscle layers and mesothelium (compare Fig. 1J). (B) Foxf1-/+; Foxf2-/+ colon with flat epithelium (top right) and poorly differentiated, dissociating SMCs (compare with Fig. 1K). (C) A basement membrane (white arrowheads) delimits the epithelium (top) from the mesenchymal layer and bundles of fibrillar collagens (black arrowheads) are embedded in the basal laminae surrounding individual fibroblasts and smooth muscle cells. Endoplasmic reticulum in fibroblasts is well developed and filled with electron-dense proteins for secretion (arrow). (D) Hardly any ECM or collagen fibers can be seen in the mutant colon. The basement membrane is indistinct and instead gaps of extracellular space (white arrowheads) separate epithelium (top) from mesenchyme. Gaps are also frequent between mesodermal cells (black arrowhead). (E) Wild-type fibroblasts have well-developed endoplasmic reticulum (white arrowheads), often filled with proteins (arrow). (F) Foxf mutant colon mesodermal cells have a poorly differentiated SMC phenotype with little endoplasmic reticulum (white arrowhead).

View larger version (95K): [in a new window]   Fig. 3. Tissue disintegration due to ECM deficiency in E18.5 Foxf2-/- intestine. (A,B) Hematoxylin and Eosin stained sections of colon from wild type (A) and Foxf2-/- (B). The mutant has a distended distal colon and the mesodermal layers separate from each other and from the epithelium (top in close-up) because of poor cell adhesion. (C-K) Immunostaining with antisera against a sheet forming collagen (type IV), a fibrillar collagen (type I) and SMA. Both collagens are reduced throughout the length of the intestine (from duodenum to rectum) in Foxf2-/-. The mutant intestinal wall (I) is flimsy in appearance compared with the wild type (H), owing to lack of collagen fibers. Both the small intestine (I) and colon (E; here from a proximal, non-distended part) has a smaller diameter in the mutant than in wild-type littermates (C,H). Foxf1-/+; Foxf2-/+ (D; proximal colon) also has reduced amounts of ECM components, but less extreme than in Foxf2-/-. (L,M) Immunostaining with anti-SMA in wild type (L) and Foxf1-/+; Foxf2-/+ (M) E18.5 small intestine shows ectopic expression of SMA in intravillus mesenchyme of the mutant. (N-Q) Cell-autonomous requirement for Foxf proteins for collagen expression in intestinal fibroblasts. Primary fibroblasts were prepared from E18.5 wild-type intestine and transfected with a plasmid expressing GFP (N,O) or a dominant-negative Foxf protein fused to GFP (P,Q). After 24 hours, cells were fixed and stained with an antiserum against collagen I (red). Collagen staining is seen in cytoplasmic vesicles and fibers between the cell and the glass substrate. In cells expressing the dominant-negative Foxf protein, identified by their green nuclear fluorescence, collagen staining is reduced dramatically. Blue, DAPI nuclear staining.

View larger version (114K): [in a new window]   Fig. 4. Overproliferation and failure to induce apoptosis generate excessive epithelial cells in Foxf mutants. (A-C) Low (top) and high (bottom) magnifications. Hematoxylin and Eosin staining of E18.5 small intestine sections from wild-type (A), Foxf1-/+; Foxf2-/+ (B) and Foxf2-/- (C). Normal villi (A) are covered by a smooth monolayer of highly polarized epithelial cells with basal nuclei. Villi in the compound heterozygote (B) are large, club-shaped with multilayered epithelia, whereas in Foxf2 null mutants (C), the epithelial cells detach from the mesenchyme and adhesion between epithelial cells from adjacent villi create inter-villus cross linking and luminal obstruction. (D-F) Altered distribution of E-cadherin immunostaining reveals loss of epithelial polarity. In normal, polarized epithelial cells (D), E-cadherin is confined to the lateral membrane, where adherence junctions connect adjacent cells, and basal and apical membranes lack this cell-adhesion molecule. In the compound Foxf heterozygote (E), internal layers of epithelial cells have ubiquitous membrane-associated staining, but the outer layer has apical membranes free of E-cadherin. In Foxf2-/- small intestine (F) the epithelial cells show many signs of lost polarity, including E-cadherin staining along the entire circumference. (G-I) Dissolution of the proliferation boundary in Foxf mutants. Immunostaining for PCNA – a proliferation marker – identifies actively cycling cells, which in wild-type E18.5 small intestine epithelium (G) are confined to the intervillus pockets (predecessors of crypts of Lieberkühn). In Foxf mutants (H,I), the boundary between the basal proliferative and the distal post-mitotic compartments is missing, and PCNA-positive epithelial cells are found throughout the villi. (J-M) Partial resistance to apoptosis in Foxf2-/- intestinal epithelium. TUNEL assay (red nuclei, apoptotic cells) of E18.5 wild-type (J,K) and Foxf2-/- (L,M) small intestine. (J) Most parts of the normal intestine show no or few apoptotic cells at this stage. (K) Where local slippage between mesenchyme and epithelium causes poor contact between epithelial cells and basement membrane, apoptosis is induced in the affected cells. (L) In the light of the severe ECM deficiency and the beginning separation between epithelium and mesenchyme (arrowheads), the Foxf2-/- intestinal epithelium contains surprisingly few apoptotic cells. (M) Not until the final stages of tissue disintegration in the worst affected areas, where the epithelial cells detach completely from the villus core, does the frequency of apoptotic cells increase significantly.

View larger version (136K): [in a new window]   Fig. 5. Accumulation of ß-catenin in Foxf mutant intestinal epithelium. ß-Catenin immunostaining (A-D,F-K) and TUNEL assay (E; red nuclei indicate apoptotic cells) of E18.5 small intestine of wild type (A,F,H,J), Foxf1-/+; Foxf2-/+ (B-E) and Foxf2-/- (G,I,K). (A) In normal E18.5 small intestine, ß-catenin is most abundant in the basal epithelial cells of the intervillus pits and decreases along the villus axis (non-specific staining in the gut lumen is due to the stickiness of the meconium). (B-D) In Foxf mutants, ß-catenin concentration stays high all the way to the villus tip. Foxf1-/+; Foxf2-/+ differs from Foxf2-/- in having a mosaic pattern with sharp boundaries between regions of the epithelium with high and low ß-catenin (C,D; arrowheads in B). (E) Patches of TUNEL-positive (apoptotic) cells match the low ß-catenin mosaic pattern. (F,G) Higher magnifications show nuclear localization of ß-catenin at base of villi in wild type (F), but ubiquitous nuclear ß-catenin in Foxf mutants (G). (H) Only membrane-associated ß-catenin is found only in cells from upper half of wild-type villus; nuclei (white arrowhead) are negative. (I-K) Both nuclear (white arrowheads) and membrane-associated ß-catenin are found in cells from the upper half of Foxf2-/- villus (I; circular organelle in adjacent cell is a goblet cell mucus vesicle) and from the base of wild-type (J) and Foxf2-/- villus (K). Epithelial depolarization is beginning in K, in spite of the seemingly intact contact with the mesenchyme: the membrane associated ß-catenin has begun to expand from the lateral and into the apical surface (black arrowhead) and nuclei are centrally located in the cells. (L-Q). Normal or reduced amounts of the major glycosaminoglycan proteoglycans in Foxf mutant intestine. Immunostaining with antisera against nidogen (Entactin; L,O), perlecan (M,P) and syndecan 1 (N,Q) on cryosections of E18.5 small intestine from wild type (L-N) and Foxf2-/- (O-Q). Nidogen and perlecan are constituents of the mesenchymal ECM and present in normal amounts in Foxf mutants. Syndecan 1 is membrane anchored, present on the basolateral face of epithelial cells and reduced in Foxf mutant intestine.

View larger version (75K): [in a new window]   Fig. 6. Reduced Foxf gene dosage has pleiotropic effects on paracrine signaling in the intestinal mesenchyme: more Wnt and less Bmp. (A) Whole-mount in situ hybridization of E14.5 embryos with a Wnt5a probe. Expression is significantly higher in Foxf2-/- mutant (right) than in wild-type (left) small intestine. (B-E) X-gal staining of E15.5 Bmp4lacZ/+ embryos. Bmp4-driven ß-galactosidase activity is significantly reduced in the gastrointestinal tract of Foxf2-/- (C), compared with wild type (B). The same embryos have comparable levels of staining in organs where Foxf2 is not expressed, such as eyelids and hair follicles (D,E). (F-J) Bmp signaling inhibits Wnt5a expression in gastrointestinal mesenchyme. Wnt5a whole-mount in situ hybridization of gastric (G,I) and intestinal (F,H,J) explants from Foxf2-/- (F-I) or wild-type (J) E12.5 embryos cultured for 24 hours with beads (arrowheads) implanted into the mesenchyme. Beads contained BSA (control; F,G), Bmp4 (H,I) or noggin (J). The uneven white outline of the explant in J is from remnants of the filter on which the explant was cultured. (K-P) Inhibition of hedgehog signaling by cyclopamine (20 µM; n-p) reduces the expression of Foxf genes (L,M,O,P) to the same extent as of a known hedgehog target, Ptch1 (K,N). E12.5 intestinal explants were cultured for 24 hours in the presence or absence of cyclopamine and analyzed by whole-mount in situ hybridization.

View larger version (44K): [in a new window]   Fig. 7. Summary of paracrine interactions and a model for a degenerative cycle that could explain the local variation of phenotype in Foxf mutant intestine. In wild type, both Foxf genes are activated in mesenchymal cells by hedgehog produced by the epithelium. Cell-autonomous stimulation of ECM production is more dependent on Foxf2 than on Foxf1, whereas both proteins activate Bmp4. Bmp4 inhibits Wnt5a expression in the mesenchyme and there may also be other mechanisms (`?') through which Foxf proteins restrict Wnt5a. The ECM provides tight contact between epithelium and mesenchyme, induces epithelial polarity and ensures efficient signaling of both Hh and Wnt ligands. The loss of both Foxf2 alleles will result in reduced Bmp4 and increased Wnt5a expression, but foremost in a radical decrease of several ECM components. The ECM deficiency will lead to loss of epithelial polarity, but not to apoptosis at this stage because of the stabilized ß-catenin that results from increased Wnt signaling. However, the poor adhesion, particularly between epithelium and the weakened basement membrane, creates an unstable situation where even moderate physical strain (e.g. from peristalsis) will separate the epithelium from the mesenchyme. Once parted, mesenchymal cells will experience a reduced Hh signaling, expression from the remaining Foxf alleles (in this example the two Foxf1 alleles) will drop and the phenotype deteriorates further. When ECM production falls below a certain level, the tissue disintegrates and epithelial cells end up too far from the source of Wnt5a to be rescued from apoptosis. Foxf1-/+; Foxf2-/+ compound heterozygotes initially have a less dramatic reduction in ECM, which sustains tissue integrity longer and supports more efficient epithelio-mesenchymal signaling, allowing the epithelium to overgrow. Given the reduced Foxf gene dose and weakened basement membranes, the tissue is, however, vulnerable to the same degenerative cycle, which may be the cause of the observed low-ß-catenin/apoptosis mosaic pattern.