AJP - GI Watch the video to see how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Gastrointest Liver Physiol 291: G163-G170, 2006. First published February 9, 2006; doi:10.1152/ajpgi.00019.2006
0193-1857/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Tables and Figures
Right arrow All Versions of this Article:
291/1/G163    most recent
00019.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Takano-Maruyama, M.
Right arrow Articles by Ohno, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Takano-Maruyama, M.
Right arrow Articles by Ohno, H.

MUCOSAL BIOLOGY

Foxl1-deficient mice exhibit aberrant epithelial cell positioning resulting from dysregulated EphB/EphrinB expression in the small intestine

Masumi Takano-Maruyama,1 Koji Hase,1 Hiroshi Fukamachi,2 Yasutaka Kato,3 Haruhiko Koseki,4 and Hiroshi Ohno1,5

1Laboratory for Epithelial Immunobiology and 4Developmental Genetics, Research Center for Allergy and Immunology, RIKEN, Kanagawa; 2Department of Molecular Oncology, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, Tokyo; 3Second Department of Internal Medicine, School of Medicine, Chiba University, Chiba; and 5International Graduate School of Arts and Sciences, Yokohama City University, Kanagawa, Japan

Submitted 17 January 2006 ; accepted in final form 31 January 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The winged helix transcription factor Foxl1, expressed in the gut mesenchyme, regulates epithelial cell proliferation and differentiation through the Wnt/beta-catenin pathway. To better understand the role of Foxl1 in epithelial morphogenesis, we examined the tissue structure and positioning of epithelial cells in the small intestine of Foxl1-deficient mice. The small intestine of Foxl1-deficient mice manifested aberrant crypt structure, including widely distributed Paneth cells, which coincided with the ectopic and increased expression of EphB2 and EphB3, which are key regulators of epithelial cell positioning. Furthermore, real-time quantitative PCR indicated that a subset of Wnt family genes was highly expressed in the gut mesenchyme of Foxl1-deficient mice compared with that of wild-type mice. Such an increase in Wnt expression was remarkable in the mesenchyme, where the aberrant Paneth cell positioning was observed by in situ hybridization. Foxl1 plays an important role in the maintenance of crypt architecture and epithelial cell positioning through the mesenchymal-epithelial interaction in the small intestine. This interaction is essential for the normal regulation of the Wnt/beta-catenin pathway and the subsequent EphB/EphrinB expression.

Foxl1; EphB/EphrinB; Wnt; mesenchymal-epithelial interaction; gut

THE GUT MESENCHYME plays an essential role in the proliferation and differentiation of epithelial cells. Although the precise molecular machinery regulating the mesenchymal-epithelial interaction has not been fully elucidated, a number of mesenchymal factors have been reported to influence epithelial homeostasis in the gut (17, 20, 29, 39). Among them, the winged helix transcription factor Foxl1 is considered to be one of the key regulators of epithelial proliferation and differentiation. Foxl1 is expressed in the mesenchyme of developing and adult gastrointestinal tract. The deletion of Foxl1 results in the delayed formation of gastric glands and intestinal villi during fetal development in mice (16). Adult Foxl1-deficient (–/–) mice exhibit hyperplasia of the gastric mucosa and the intestinal villi. Expansion of the crypt compartment is also observed in these mice. Such disorders are associated with the abnormal proliferation of epithelial cells in the gastrointestinal tract (26). These findings clearly suggest that Foxl1 works as a negative regulator of the proliferation of gut epithelial cells.

Foxl1 target genes remain to be clarified; however, the genes encoding the molecules involved in the Wnt/beta-catenin signaling pathway could be among the possible targets of Foxl1. Previous studies have shown that 7 of the 19 Wnt family molecules are expressed in the murine gastrointestinal tract during development and in adulthood (22, 33). The binding of Wnt to the Frizzled receptor expressed on epithelium induces the stabilization and translocation of beta-catenin to the nucleus (23, 36). Nuclear beta-catenin serves as a transcriptional coactivator for TCF4, and the beta-catenin/TCF4 complex promotes the expression of c-myc, leading to epithelial cell proliferation (9, 35). It is therefore supposed that the activity of the beta-catenin/TCF4 complex may determine the fate of intestinal epithelial stem cells, namely, whether to proliferate or to differentiate. A previous study has suggested that Foxl1 negatively regulates the beta-catenin/TCF4 signaling pathway, because the nuclear translocation of beta-catenin is apparently enhanced in the stomach and the small intestine of Foxl1–/– mice (26). Furthermore, Foxl1 deficiency leads to a marked increase in tumor multiplicity in gastric and colorectal cancers induced by a mutated APC tumor suppressor gene (27) that is known to promote the degradation of beta-catenin. This indicates that Foxl1 could cooperate with APC in terms of controlling epithelial cell proliferation.

We and others have recently reported that Foxl1–/– mice demonstrate multiple pathological symptoms, including impaired acid secretion by gastric parietal cells, defect in small intestinal uptake of D-glucose, and abnormal formation of gut-associated lymphoid follicles (7, 8, 18, 19, 27). These observations raise the possibility that Foxl1 may function as a multifunctional regulator in the context of the mesenchymal-epithelial interaction. In the present study, we show that Foxl1 deficiency leads to disorder in epithelial cell positioning in the murine small intestine. This phenomenon is mainly explained by the dysregulation of the EphB/EphrinB system, which is critical for epithelial cell positioning (2), because the balanced expression of EphB/EphrinB is dysregulated in the gut of Foxl1–/– mice. Furthermore, the aberrant expression of EphB/EphrinB is coincident with the enhanced expression of Wnt by mesenchymal cells, suggesting that Foxl1 regulates the beta-catenin/TCF4 pathway and the subsequent epithelial EphB expression via mesenchymal Wnt expression. Collectively, Foxl1 is involved in not only the proliferation but also the positioning of epithelial cells. Such a coordinated regulation of epithelial proliferation and positioning may be important for the formation and maintenance of the crypt-villus structure in the gut.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mice. Foxl1–/– mice were backcrossed onto a BALB/c background 10 times (7). The morning of the appearance of a vaginal plug was considered to be 0.5 day postcoitus (dpc). All animal experiments were carried out in accordance with the guidelines for the care and use of laboratory animals of RIKEN.

Whole mount staining. The small intestine was dissected into the small pieces and fixed in methanol for 1 h at –20°C. The pieces were rinsed with PBS and immersed in PBS containing FITC-conjugated Ulex europaeus I (UEA-1, 20 µg/ml; Vector Laboratories, Burlingame, CA) and rhodamine-conjugated wheat germ agglutinin (WGA, 25 µg/ml; Vector Laboratories) for 1 h at room temperature. The specimens were examined with an LSM510 confocal microscope (Carl Zeiss, Deutschland, Germany).

Immunohistochemistry. The small intestine was dissected and fixed overnight in 4% paraformaldehyde in PBS at 4°C, embedded in paraffin wax, and processed into 3-µm sections. The sections were deparaffinized in xylene, rehydrated through a graded series of ethanol, boiled in 0.01 M citrate buffer (pH 6.0) for 20 min, and incubated with 0.5% blocking reagent (TSA Fluorescence System; Perkin-Elmer, Boston, MA) in TBS [0.1 M Tris·HCl (pH 7.5) and 0.15 M sodium chloride] for 30 min. Incubation with primary antibodies against EphB2, EphB3, and EphrinB1 (2.5 µg/ml; R&D Systems, Minneapolis, MN) was performed for 3 h followed by incubation with biotin-conjugated donkey anti-goat IgG antibody (0.5 µg/ml; Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h. The sections were quenched with 3% H2O2 in PBS for 10 min. Signal amplification and detection was performed with an Elite ABC kit (Vector Laboratories) using FITC-conjugated tyramide (Perkin-Elmer) as the substrate according to the manufacturer's instructions. To detect Paneth cells, the sections were incubated with rhodamine-conjugated UEA-1 (20 µg/ml; Vector Laboratories) for 5 min. In some experiments, further incubation was performed after EphB2 staining with rabbit anti-Ki67 antibody (1:1,000; Novocastra Laboratories, Newcastle, UK) for 2 h. For visualization of antibody binding, the sections were incubated with Cy3-conjugated anti-rabbit IgG antibody (1.2 µg/ml; Jackson ImmunoResearch Laboratories) for 1 h.

Separation of epithelial cells and mesenchymal cells. The small pieces of the small intestine were washed with ice-cold calcium- and magnesium-free Hanks' balanced salt solution (HBSS). The pieces were immersed in HBSS containing 30 mM EDTA for 7 min. The epithelial monolayer was peeled off with 29-gauge needles under monitoring with a transillumination stereomicroscope (MZ12.5; Leica Microsystems, Heerbrugg, Switzerland) to separate epithelial cells from mesenchymal cells (11, 15). Hematoxylin and eosin staining of the sections confirmed that these preparations were almost exclusively composed of either epithelial monolayers or the mesenchymal tissue [Supplemental Fig. 1 (Supplemental data for this article may be found at http://ajpgi.physiology.org/cgi/content/full/00019.2006/DC1.)].


Figure 1
View larger version (122K):
[in this window]
[in a new window]
  		Fig. 1. Abnormal architecture of the small intestinal crypts in Foxl1–/– mice. A and B: whole mount specimens of the jejunum from wild-type (WT) mice (A) and Foxl1–/– mice (B) at 12 wk old were stained with lectins Ulex europaeus I (UEA-1) and wheat germ agglutinin (WGA) and analyzed with a confocal microscope. UEA-1 (green) stains the L-fucose epitope on Paneth cells, and WGA (red) stains the N-acetylglucosamine epitope of the extracellular matrix. Confocal images (x-y plane) orthogonal to the villi-crypt axis are shown. The area encircled by a dotted line in B represents an abnormally large single crypt in Foxl1–/– mice. C: the width of crypts was measured at eight different fields of the jejunum. *P < 0.05. D and E: Periodic acid-Schiff and hematoxylin staining of the jejunal tissue sections from WT (D) and Foxl1–/– mice (E). The localization of Paneth cells was restricted to the crypt bottom in WT mice (arrowheads), whereas these cells were often found in the middle of the crypts (arrows) in addition to the crypt bottom (arrowhead) in Foxl1–/– mice. Scale bars = 50 µm.

 
Immunoblot analysis. The separated epithelial cells and mesenchymal cells were homogenized with lysis buffer [50 mM HEPES-KOH (pH 7.4), 1% Triton X-100, 150 mM sodium chloride, 5 mM EDTA, 5 mM EGTA, 20 mM sodium fluoride, and 1 mM dithiothreitol]. After centrifugation, the supernatants were obtained, and the protein contents were quantified with BCA protein assay reagents (Pierce, Rockford, IL) following the manufacturer's instructions. The supernatants were boiled with Laemmli's buffer, separated on 8% SDS-PAGE, and transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore, Billeria, MA). The membranes were incubated with goat anti-mouse EphB2 antibody, followed by horseradish peroxidase-conjugated donkey anti-goat IgG antibody (0.26 µg/ml; Jackson ImmunoResearch Laboratories). Specific binding was detected using the SuperSignal Detection System (Pierce) according to the manufacturer's instructions. The blots were stripped and reprobed with goat anti-mouse EphB3 antibody or mouse anti-glyceraldehyde-3-phosphate dehydrogenase antibody (0.25 µg/ml; Chemicon, Temecula, CA).

To detect the nuclear translocation of beta-catenin, the isolated epithelial cells were homogenized in sucrose solution [10 mM HEPES-KOH (pH 7.4), 0.25 M sucrose, 5 mM EDTA, 1 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride] using 22-gauge needles. The homogenates were centrifuged at 600 g for 10 min. The nuclear fraction and membrane/cytosolic fraction were prepared from the precipitate and supernatant, respectively, as described previously (21). Aliquots of these fractions containing 20 µg of protein were denatured, separated on 8% SDS-PAGE gels, and transferred to PVDF membranes. The membranes were incubated with mouse anti-beta-catenin antibody (1 µg/ml; Zymed Laboratories, San Francisco, CA), followed by goat horseradish peroxidase-conjugated anti-mouse IgG antibody (0.26 µg/ml; Jackson ImmunoResearch Laboratories). Specific binding was detected using the SuperSignal Detection System (Pierce) according to the manufacturer's instructions.

Real-time quantitative RT-PCR analysis. Total RNAs were isolated from the separated epithelial cells and mesenchymal cells with TRI-zol reagent (Invitrogen, Carlsbad, CA), and first-strand cDNAs were synthesized with ReverTra Ace-{alpha} (Toyobo, Osaka, Japan). Real-time quantitative PCR was performed using SYBR Green PCR Master Mix and an ABI PRISM 7900 Analyzer (Applied Biosystems, Foster City, CA). Amplification of the expected single products was confirmed using 2% agarose gel and ethidium bromide staining. All procedures were performed in accordance with the manufacturer's instructions. The amount of actin mRNA in each sample was quantified and used as the internal control for comparison among the different samples. The primer sets are shown in Supplemental Table 1.

In situ hybridization. A 417-bp DNA fragment corresponding to nucleotide positions 685–1101 of mouse Wnt4 cDNA (GenBank accession no.: NM_009523) was subcloned into pGEM-T-Easy vector (Promega, Madison, WI). Digoxigenin-labeled RNA probes were prepared by in vitro transcription with T7 or SP6 RNA polymerase and Digoxigenin RNA Labeling Mix (Roche Diagnostics, Penzberg, Germany). Paraffin-embedded small intestine sections (6 µm) of Foxl1–/– or wild-type (WT) mouse were deparaffinized, treated with protease K, and hybridized with 100 ng/ml digoxigenin-labeled RNA probes at 60°C for 16 h. Specific binding was detected by incubating with alkaline phosphate (AP)-conjugated anti-digoxigenin antibody (Roche Diagnostics) for 2 h at room temperature and visualized with purple AP substrate (Roche Diagnostics) overnight. The sections were counterstained with nuclear fast red.

Statistical analysis. Results are expressed as means ± SD. Statistical analysis of differences between two groups was performed using the unpaired Student's t-test (two-tailed analysis). Differences with P values of <0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Abnormal crypt structure in Foxl1–/– mice. To assess the crypt structure in Foxl1–/– mice, we performed whole mount staining of the small intestine using lectins UEA-1 and WGA. In the crypt region, UEA-1 has been reported to react with mature Paneth cells (4, 6), whereas WGA recognizes heparan sulfate proteoglycans (HSPGs) constituting the extracellular matrix (25). Because HSPGs are abundantly distributed at the mesenchymal-epithelial interface in the small intestine (12, 32), WGA staining is able to outline the whole crypt structure. Confocal microscopic analysis clearly showed that the size and shape of crypts were uniform throughout the small intestine in WT mice (Fig. 1A). In contrast, the crypt structure was irregular in Foxl1–/– mice, where parts of the crypts were enlarged by more than twofold compared with WT crypts (Fig. 1, B and C). Whereas UEA-1+ Paneth cells were restricted to the crypt bottom in WT mice (Figs. 1D and 2E), these cells were often found in the middle of the crypts in Foxl1–/– mice (Figs. 1E and 2F). The abnormalities in the crypt structure were observed throughout the small intestine, particularly in the jejunum.


Figure 2
View larger version (60K):
[in this window]
[in a new window]
  		Fig. 2. Immunohistochemical analysis of EphB/EphrinB expression in the small intestine. The jejunal tissue sections from WT (A, C, E, and G) and Foxl1–/– (B, D, F, and H) mice at 12 wk old were stained with antibodies against EphB2 (A-D), EphB3 (E and F), and EphrinB1 (G and H), respectively. Proliferative cells were visualized by Ki67 antigen staining (red, A-D), and Paneth cells were labeled by UEA-1 lectin (red, E and F). Open arrowheads indicate the crypt-villus junction (A-D). Closed arrowheads denote EphB2 expression extending to the villi (B and D), mispositioned EphB3+ Paneth cells (F), and a decreased EphrinB1 expression (H) in Foxl1–/– mice. Scale bars = 25 µm.

 
Dysregulated expression of EphB/EphrinB in Foxl1–/– mice. The histochemical analyses presented above and the Ki67 staining shown in Fig. 2, B and D, revealed the abnormal positioning of Paneth cells and proliferative epithelial cells in Foxl1–/– mice. The epithelial cell positioning in the crypt-villus axis is regulated by the repulsive interaction between tyrosine kinase receptors EphB2/EphB3 and their ligand EphrinB1 in the small intestine (2). Because EphB/EphrinB are the target molecules of the beta-catenin/TCF4 signaling pathway and this pathway is activated in Foxl1–/– mice (26, 35), we postulated that the aberrant epithelial cell positioning in Foxl1–/– mice may result from the dysregulation of EphB/EphrinB expression. To this end, we first examined the expression pattern of EphB/EphrinB in the jejunum by immunohistochemistry. Although EphB2 was predominantly expressed in Ki67+ proliferative epithelial cells in both WT and Foxl1–/– mice, its distribution was quite different between them. Whereas EphB2 expression was restricted to the lower half of the crypt in WT mice (Fig. 2, A and C), a broader expression of EphB2 was observed throughout the crypt except its base in Foxl1–/– mice (Fig. 2, B and D). Some EphB2+ cells were observed even in the middle of villi (Fig. 2, B and D). EphB3 was expressed in UEA-1+ Paneth cells that resided at the crypt base in WT mice (Fig. 2E), as described in a previous report (2). However, in Foxl1–/– mice, the EphB3+ Paneth cells were widely distributed throughout crypts (Fig. 2F). EphrinB1 was observed at the crypt-villus junction in WT mice (Fig. 2G) in accordance with a previous observation (2). Although the distribution was almost similar, a weaker EphrinB1 expression was sporadically observed in Foxl1–/– mice (Fig. 2H).

To determine the amount of EphB/EphrinB proteins in the jejunal epithelium, we separated the epithelial cell layer from the mesenchyme (Supplemental Fig. 1 and Ref. 11) and performed Western blot analysis. A marked increase in EphB2 protein expression was observed in Foxl1–/– mice compared with WT mice (Fig. 3, A and B), which was consistent with its broader distribution in Foxl1–/– mice as observed by histochemical analysis (Fig. 2, B and D). EphB3 protein expression also tended to be upregulated in Foxl1–/– mice compared with WT mice, although the difference was not statistically significant. Collectively, these data suggest that the epithelial cell positioning in Foxl1–/– mice could be altered because of the ectopic upregulation of EphB2/EphB3 in conjunction with the downregulation of EphrinB1.


Figure 3
View larger version (16K):
[in this window]
[in a new window]
  		Fig. 3. Immunoblot analysis of epithelial EphB2 and EphB3 expression. A: lysates from isolated epithelial cells of WT mice and Foxl1–/– mice at 12 wk old were subjected to immunoblot analysis. Representative results of 3 independent experiments are shown. B: the band intensity of EphB2 and EphB3 in WT mice (open bar) and Foxl1–/– mice (filled bar) was quantitated with a Lumino image analyzer and normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal control. The expression levels of each molecule are described as the degree of change relative to WT epithelium. The mean values ± SD of 3 samples from different mice are shown. *P < 0.05.

 
Widespread EphB2 expression during crypt formation in Foxl1–/– mice. In the developmental murine small intestine, EphB2/EphB3 and EphrinB1 are expressed in the intervillous region and villi in a mutually exclusive manner. The reciprocal interaction between EphB and EphrinB plays critical roles in the compartmentalization of proliferative cells and differentiated cells and is essential for regular crypt development (2). To examine whether the regulation of EphB2 expression is already disordered during crypt development in Foxl1–/– mice, we analyzed the expression pattern of EphB2 in small intestine at three different perinatal stages: 18.5 dpc, the stage before crypt formation; postnatal day 3 (P3), the early stage of crypt development before significant crypt morphogenesis; and postnatal day 10 (P10), the late stage of crypt formation where the cells constituting crypts settle in their appropriate positions. During these stages, EphB2 expression was limited to the intervillous region (18.5 dpc) and the bottom of crypts (P3 and P10) in control Foxl1+/– littermates (Fig. 4, A, C and E). These EphB2+ cells constitutively expressed the Ki67 proliferative marker (data not shown), consistent with a previous report (2). On the other hand, in Foxl1–/– mice, EphB2 was expressed throughout from the intervillous region to the villi at 18.5 dpc (Fig. 4B). At the later stage in Foxl1–/– mice, EphB2+ cells were not restricted to the crypt bottom but also observed in the middle of the villi (Fig. 4D). At P10, EphB2+ cells occasionally formed clusters in the distal part of the villi, apparently isolated from the EphB2+ cells in the crypts (Fig. 4F). In contrast, EphrinB1, whose expression was observed throughout the villi in control littermates (Fig. 4G), was remarkably downregulated in Foxl1–/– mice (Fig. 4H). These data suggest that the Foxl1 deficiency causes dysregulation of EphB/EphrinB expression, thereby leading to the aberrant positioning of proliferating epithelial cells at the developmental stage of crypt in Foxl1–/– mice. This may disturb the processes necessary for normal crypt formation, eventually resulting in crypt expansion in the adult small intestine.


Figure 4
View larger version (75K):
[in this window]
[in a new window]
  		Fig. 4. Disordered EphB/EphrinB expression in Foxl1–/– mice during crypt development. The sections of small intestines from control littermate (Foxl1+/–) mice (A, C, E, and G) and Foxl1–/– mice (B, D, F, and H) at 18.5 days postcoitus (dpc) (A and B) and postnatal days 3 (C, D, G, and H) and 10 (E and F) were stained with antibodies against EphB2 (A-F) and EphrinB1 (G and H). Widespread EphB2 expression coupled with downregulation of EphrinB1 was observed in Foxl1–/– mice. Open arrowheads indicate the crypt-villus junction (A-H). Closed arrowheads indicate EphB2 expression in the middle of villi (D) and EphB2+ cell clusters in the villi (F). Scale bar = 25 µm.

 
Increased Wnt4 and Wnt11 expression in the mesenchyme of Foxl1–/– mice. A previous study has demonstrated that Foxl1 is involved in the regulation of the beta-catenin signaling pathway (26). We also confirmed that the nuclear beta-catenin translocation was enhanced in the small intestinal epithelium of Foxl1–/– mice by Western blot analysis (Fig. 5). Because EphB/EphrinB are the target molecules of the beta-catenin/TCF4 complex and the activity of the beta-catenin pathway is controlled by Wnt signaling, we hypothesized that the aberrant expression of Wnt family members and/or their coreceptor HSPGs are involved in the ectopic expression of EphB2 and EphB3 in Foxl1–/– mice. To test this hypothesis, we separately prepared the epithelium and mesenchyme from the jejunum of WT and Foxl1–/– mice and analyzed the expression of mRNAs of seven Wnt molecules (Wnt2, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt11, and Wnt14; see Refs. 28 and 33) and 12 HSPGs, the expressions of which have been documented in the developing and adult gastrointestinal tract (1, 34). More than one-half of the Wnt molecules tested were predominantly expressed in the mesenchyme in both WT and Foxl1–/– mice (Supplemental Table 2). There were, however, marked differences in Wnt4 and Wnt11 expression between WT and Foxl1–/– mice, where the expression levels of these molecules were fourfold higher in Foxl1–/– mice than in WT mice (Fig. 6, A and B). On the other hand, the mRNA expression levels of most HSPGs showed no apparent differences between WT and Foxl1–/– mice, with the exception of syndecan2 and glypican3, which were slightly increased in the epithelium and mesenchyme, respectively, of Foxl1–/– mice (Supplemental Table 2).


Figure 5
View larger version (36K):
[in this window]
[in a new window]
  		Fig. 5. Enhanced nuclear translocation of beta-catenin in the small intestinal epithelium of Foxl1–/– mice. The membrane/cytosolic fraction (lanes 1 and 2) and the nuclear fraction (lanes 3 and 4) were prepared from the jejunal epithelium of WT (lanes 1 and 3) and Foxl1–/– (lanes 2 and 4) mice at 12 wk old. These samples were subjected to immunoblot analysis to examine the intracellular localization of beta-catenin.

 

Figure 6
View larger version (10K):
[in this window]
[in a new window]
  		Fig. 6. Increase in mesenchymal Wnt expression in Foxl1–/– mice. Epithelial and mesenchymal tissues were separated from the jejunum of WT (open bars) and Foxl1–/– (filled bars) mice at 12 wk old and subjected to quantitative real-time PCR analysis. The mRNA expression levels of Wnt4 (A) and Wnt11 (B) were normalized to that of beta-actin as the internal control, and were described as the degree of change relative to WT epithelium. Mean values ± SD of 3 (WT) or 4 (Foxl1–/–) samples from different mice are shown. *P < 0.05.

 
To further examine whether the overexpression of Wnts in Foxl1–/– mice is correlated with the aberrant epithelial cell positioning, we analyzed the tissue distribution of Wnt4 mRNA by in situ hybridization. Wnt4 mRNA was expressed in the mesenchymal cells adjacent to the crypt in WT mice (Fig. 7, A and B). This observation further confirmed that the mesenchyme is the primary site of Wnt4 production in the small intestine. Wnt4 mRNA was expressed at a relatively higher level in Foxl1–/– mice than in WT mice, and the expression was widely distributed in the mesenchyme from the villi to the crypt-villus junction (Fig. 7, D and E). Of note is that the intense expression of Wnt4 was observed in the mesenchyme close to Paneth cells ectopically localized at the upper crypts in Foxl1–/– mice (Fig. 7E). Taken together, these data provide evidence that the disruption of the positioning of Paneth cells may result from the upregulated expression of Wnts in mesenchymal cells in Foxl1–/– mice.


Figure 7
View larger version (108K):
[in this window]
[in a new window]
  		Fig. 7. In situ hybridization analysis of Wnt4 expression in the jejunum. The jejunal tissue sections from WT (A-C) or Foxl1–/– (D-F) mice were hybridized with the antisense probe to Wnt4 mRNA (A, B, D, and E) or the control sense probe (C and F). Arrows indicate the enhanced mesenchymal Wnt4 expression frequently observed near the mispositioned Paneth cells (arrowheads) in the upper crypts, which are easily distinguished from other epithelial cells by their characteristic granule-enriched cytoplasm. Scale bars = 25 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Foxl1 in the gut mesenchyme has been considered to control the growth and differentiation of gastrointestinal epithelial cells. Additionally, we described here that Foxl1 could contribute to the sorting of epithelial cells, because Foxl1–/– mice displayed mispositioning of proliferative epithelial cells and Paneth cells in the small intestine. In the adult small intestine, epithelial stem cells are located near the crypt base and produce transit amplifying cells. These cells give rise to terminally differentiated epithelial cell lineages as they migrate upward with the exception of Paneth cells, which downmigrate to the bottom of the crypts (5, 30). Such epithelial cell allocation is most likely controlled by the complementary expression of EphB2/B3 and its ligand EphrinB1 (2). EphB2 and EphB3 are expressed by proliferative epithelial cells and Paneth cells, respectively, whereas EphrinB1 is strongly induced in the differentiated epithelial cells at the crypt-villus junction, and its expression is gradually decreased toward the lower region of crypts. The interaction between EphB and EphrinB generates repulsive cues (14, 38) that allow EphB-expressing cells to keep away from the EphrinB1-expressing domain. Consequently, the localization of proliferative epithelial cells and Paneth cells is compartmentalized to the bottom of crypts in the normal small intestine (2). However, the expression balance between EphB and EphrinB is disturbed in Foxl1–/– mice, where EphB2 and EphB3 expression is upregulated and, inversely, EphrinB1 expression is sporadically decreased. This observation suggests that Foxl1 plays an important role in the expression and regulation of these molecules.

The increase in EphB2 expression and the decrease in EphrinB1 expression are also observed at the developmental stage of Foxl1–/– mice. In the embryonic intestine, proliferative epithelial cells segregate in the flat intervillous region that eventually forms the nascent crypt. The newly generated epithelial cells in the nascent crypt begin to migrate upward, and thereby the mature crypt structure is gradually developed in 2 wk (31, 37). The EphB/EphrinB system most likely contributes to the crypt development as well, because EphB2/B3 double-deficient mice display atrophy of the nascent crypt as well as mispositioned proliferative epithelial cells that intermingle with differentiated epithelial cells in the villi. In contrast, Foxl1–/– mice display the expansion of the nascent crypt, which consequently gives rise to an enlarged crypt compartment in the adult small intestine, as shown here and in a previous report (26). Not only is the crypt elongated but it shows multiple branching as well, which may result from the fusion of nascent crypts and/or incomplete crypt fission during crypt formation (37, 39). Such hyperplastic lesions of the intestinal crypt in Foxl1–/– mice may be caused not only by the increase in the number of proliferative cells as reported (16, 26) but also by the extension of the EphB-expressing region during crypt development.

The mode of action by which Foxl1 promotes the mesenchymal-epithelial interaction has been a subject of research interest. The intestinal epithelium of Foxl1–/– mice tends to accumulate more nuclear beta-catenin than that of WT mice, which, in turn, forms a complex with TCF4 and acquires transcriptional activity (3, 24, 26, 35). The target genes of the beta-catenin/TCF4 complex are mainly the regulators of cellular proliferation and survival (9). Interestingly, however, they also include Paneth cell-specific antimicrobial peptides and matrix metalloproteinase-7/matrilysin (36), implying a role of beta-catenin signaling in the maturation of this cell lineage. Furthermore, EphB/EphrinB expression is inversely controlled by beta-catenin signaling, because the inhibition of beta-catenin/TCF4 with the dominant negative form of TCF4 results in the downregulation of EphB2 and EphB3 and the upregulation of EphrinB1 in a colorectal cancer cell line (2). Taking into account these reports and our observations, the physiological activity of Foxl1 on epithelial cell proliferation and positioning is probably mediated by the modulation of the beta-catenin activity. Consistent with this notion, several of the Wnts tested here are upregulated in the intestinal mesenchyme of Foxl1–/– mice. The enhanced expression of Wnt4 is particularly remarkable in the mesenchyme, around which mispositioned Paneth cells are observed. On the other hand, Perreault et al. (26) reported that there were no differences in the Wnt mRNA expression between Foxl1–/– and WT mice using whole intestinal tissues as samples. This discrepancy is likely because of the technical differences in sample preparation. In the present study, we utilized a mesenchymal purification technique that involved removal of the epithelial monolayer from the intestinal tissue, which was expected to improve the sensitivity for detecting mesenchymal Wnt expression. Another possibility that may account for the discrepancy is the distinct genetic background of the mice used in the two studies, i.e., F1 hybrids of 129SvEv and C57BL6 (26) versus BALB/c (the present study). Recent reports have indicated that certain Wnt molecules (Wnt3, -6, and -9a) are specifically expressed in the crypt epithelium (9, 36), suggesting their autocrine/paracrine role in promoting the beta-catenin signaling pathway. Consistent with that notion, we also detected Wnt6 mRNA expression in the epithelium but not in the mesenchyme of the small intestine; however, the expression of Wnt6 is unlikely controlled by Foxl1, because there were no significant differences in the Wnt6 expression between WT and Foxl1–/– mice (Supplemental Table 2). It remains to be clarified whether Foxl1 directly regulates mesenchymal Wnt expression.

There might be yet other mechanisms that link Foxl1 and the Wnt/beta-catenin signaling pathway. Kaestner et al. (16) showed the downregulation of Bmp2 and Bmp4 in the gastrointestinal tract of Foxl1–/– mice. Bmp4, which is expressed in the intestinal mesenchyme, functions by binding to its receptor Bmpr1a on epithelial cells. According to a recent model, Bmp signaling inhibits beta-catenin through phosphatase and tensin homolog deleted on chromosome 10 (PTEN) activity regulation, thereby suppressing the self-renewal of stem cells in the intestinal epithelium (10, 13). These observations suggest that Foxl1 may regulate the beta-catenin activity also by controlling Bmp4 expression, although further evidence is required to confirm this suggestion.

In conclusion, the present study indicates that Foxl1 contributes to the regulation of epithelial cell positioning through the regulation of Wnt/beta-catenin signaling and the subsequent expression of EphB/EphrinB. These observations reinforce the importance of Foxl1 in promoting the mesenchymal-epithelial interaction that is essential to maintaining intestinal homeostasis.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by Grants-in-Aid for Scientific Research, Scientific Research in Priority Areas, and Protein 3000 Project from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Uehara Memorial Foundation, and the Naito Foundation.


   ACKNOWLEDGMENTS
 
We thank Drs. T. Murakami, H. Takatsu, and F. Nakatsu for critical reading of the manuscript and Y. Takase for expert assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. Ohno, Laboratory for Epithelial Immunobiology, Research Center for Allergy and Immunology, RIKEN, 1-7-22 Suehiro, Tsurumi-ku, Yokohama 230-0045, Japan (e-mail: ohno{at}rcai.riken.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Baeg GH, Lin X, Khare N, Baumgartner S, and Perrimon N. Heparan sulfate proteoglycans are critical for the organization of the extracellular distribution of Wingless. Development 128: 87–94, 2001.[Abstract]
  2. Batlle E, Henderson JT, Beghtel H, van den Born MM, Sancho E, Huls G, Meeldijk J, Robertson J, van de Wetering M, Pawson T, and Clevers H. beta-Catenin and TCF mediate cell positioning in the intestinal epithelium by controlling the expression of EphB/ephrinB. Cell 111: 251–263, 2002.[CrossRef][Web of Science][Medline]
  3. Behrens J, von Kries JP, Kuhl M, Bruhn L, Wedlich D, Grosschedl R, and Birchmeier W. Functional interaction of beta-catenin with the transcription factor LEF-1. Nature 382: 638–642, 1996.[CrossRef][Medline]
  4. Bry L, Falk P, Huttner K, Ouellette A, Midtvedt T, and Gordon JI. Paneth cell differentiation in the developing intestine of normal and transgenic mice. Proc Natl Acad Sci USA 91: 10335–10339, 1994.[Abstract/Free Full Text]
  5. Calvert R and Pothier P. Migration of fetal intestinal intervillous cells in neonatal mice. Anat Rec 227: 199–206, 1990.[CrossRef][Medline]
  6. Falk P, Roth KA, and Gordon JI. Lectins are sensitive tools for defining the differentiation programs of mouse gut epithelial cell lineages. Am J Physiol Gastrointest Liver Physiol 266: G987–G1003, 1994.[Abstract/Free Full Text]
  7. Fukamachi H, Fukuda K, Suzuki M, Furumoto T, Ichinose M, Shimizu S, Tsuchiya S, Horie S, Suzuki Y, Saito Y, Watanabe K, Taniguchi M, and Koseki H. Mesenchymal transcription factor Fkh6 is essential for the development and differentiation of parietal cells. Biochem Biophys Res Commun 280: 1069–1076, 2001.[Medline]
  8. Fukuda K, Yoshida H, Sato T, Furumoto TA, Mizutani-Koseki Y, Suzuki Y, Saito Y, Takemori T, Kimura M, Sato H, Taniguchi M, Nishikawa S, Nakayama T, and Koseki H. Mesenchymal expression of Foxl1, a winged helix transcriptional factor, regulates generation and maintenance of gut-associated lymphoid organs. Dev Biol 255: 278–289, 2003.[CrossRef][Medline]
  9. Gregorieff A and Clevers H. Wnt signaling in the intestinal epithelium: from endoderm to cancer. Genes Dev 19: 877–890, 2005.[Abstract/Free Full Text]
  10. Haramis AP, Begthel H, van den Born M, van Es J, Jonkheer S, Offerhaus GJ, and Clevers H. De novo crypt formation and juvenile polyposis on BMP inhibition in mouse intestine. Science 303: 1684–1686, 2004.[Abstract/Free Full Text]
  11. Hase K, Ohshima S, Kawano K, Hashimoto N, Matsumoto K, Saito H, and Ohno H. Distinct gene expression profiles characterize cellular phenotypes of follicle-associated epithelium and M cells. DNA Res 12: 127–137, 2005.[Abstract]
  12. Hayashi K, Hayashi M, Jalkanen M, Firestone JH, Trelstad RL, and Bernfield M. Immunocytochemistry of cell surface heparan sulfate proteoglycan in mouse tissues. A light and electron microscopic study. J Histochem Cytochem 35: 1079–1088, 1987.[Abstract]
  13. He XC, Zhang J, Tong WG, Tawfik O, Ross J, Scoville DH, Tian Q, Zeng X, He X, Wiedemann LM, Mishina Y, and Li L. BMP signaling inhibits intestinal stem cell self-renewal through suppression of Wnt-beta-catenin signaling. Nat Genet 36: 1117–1121, 2004.[CrossRef][Web of Science][Medline]
  14. Holder N and Klein R. Eph receptors and ephrins: effectors of morphogenesis. Development 126: 2033–2044, 1999.[Abstract]
  15. Iimura M, Nakamura T, Shinozaki S, Iizuka B, Inoue Y, Suzuki S, and Hayashi N. Bax is downregulated in inflamed colonic mucosa of ulcerative colitis. Gut 47: 228–235, 2000.[Abstract/Free Full Text]
  16. Kaestner KH, Silberg DG, Traber PG, and Schutz G. The mesenchymal winged helix transcription factor Fkh6 is required for the control of gastrointestinal proliferation and differentiation. Genes Dev 11: 1583–1595, 1997.[Abstract/Free Full Text]
  17. Karlsson L, Lindahl P, Heath JK, and Betsholtz C. Abnormal gastrointestinal development in PDGF-A and PDGFR-(alpha) deficient mice implicates a novel mesenchymal structure with putative instructive properties in villus morphogenesis. Development 127: 3457–3466, 2000.[Abstract]
  18. Kato Y, Fukamachi H, Takano-Maruyama M, Aoe T, Murahashi Y, Horie S, Suzuki Y, Saito Y, Koseki H, and Ohno H. Reduction of SNAP25 in acid secretion defect of Foxl1-/- gastric parietal cells. Biochem Biophys Res Commun 320: 766–772, 2004.[Medline]
  19. Katz JP, Perreault N, Goldstein BG, Chao HH, Ferraris RP, and Kaestner KH. Foxl1 null mice have abnormal intestinal epithelia, postnatal growth retardation, and defective intestinal glucose uptake. Am J Physiol Gastrointest Liver Physiol 287: G856–G864, 2004.[Abstract/Free Full Text]
  20. Kedinger M, Duluc I, Fritsch C, Lorentz O, Plateroti M, and Freund JN. Intestinal epithelial-mesenchymal cell interactions. Ann NY Acad Sci 859: 1–17, 1998.[CrossRef][Web of Science][Medline]
  21. Lee KA and Green MR. Small-scale preparation of extracts from radiolabeled cells efficient in pre-mRNA splicing. Methods Enzymol 181: 20–30, 1990.[Medline]
  22. Lickert H, Kispert A, Kutsch S, and Kemler R. Expression patterns of Wnt genes in mouse gut development. Mech Dev 105: 181–184, 2001.[CrossRef][Web of Science][Medline]
  23. Miller JR and Moon RT. Signal transduction through beta-catenin and specification of cell fate during embryogenesis. Genes Dev 10: 2527–2539, 1996.[Free Full Text]
  24. Molenaar M, van de Wetering M, Oosterwegel M, Peterson-Maduro J, Godsave S, Korinek V, Roose J, Destree O, and Clevers H. XTcf-3 transcription factor mediates beta-catenin-induced axis formation in Xenopus embryos. Cell 86: 391–399, 1996.[CrossRef][Web of Science][Medline]
  25. Ohno J, Tajima Y, and Utsumi N. Binding of wheat germ agglutinin in the matrix of rat tracheal cartilage. Histochem J 18: 537–540, 1986.[Medline]
  26. Perreault N, Katz JP, Sackett SD, and Kaestner KH. Foxl1 controls the Wnt/beta-catenin pathway by modulating the expression of proteoglycans in the gut. J Biol Chem 276: 43328–43333, 2001.[Abstract/Free Full Text]
  27. Perreault N, Sackett SD, Katz JP, Furth EE, and Kaestner KH. Foxl1 is a mesenchymal modifer of Min in carcinogenesis of stomach and colon. Genes Dev 19: 311–315, 2005.[Abstract/Free Full Text]
  28. Pinto D, Gregorieff A, Begthel H, and Clevers H. Canonical Wnt signals are essential for homeostasis of the intestinal epithelium. Genes Dev 17: 1709–1713, 2003.[Abstract/Free Full Text]
  29. Ramalho-Santos M, Melton DA, and McMahon AP. Hedgehog signals regulate multiple aspects of gastrointestinal development. Development 127: 2763–2772, 2000.[Abstract]
  30. Schmidt GH, Wilkinson MM, and Ponder BA. Cell migration pathway in the intestinal epithelium: an in situ marker system using mouse aggregation chimeras. Cell 40: 425–429, 1985.[CrossRef][Web of Science][Medline]
  31. Schmidt GH, Winton DJ, and Ponder BA. Development of the pattern of cell renewal in the crypt-villus unit of chimaeric mouse small intestine. Development 103: 785–790, 1988.[Abstract/Free Full Text]
  32. Simon-Assmann P, Bouziges F, Vigny M, and Kedinger M. Origin and deposition of basement membrane heparan sulfate proteoglycan in the developing intestine. J Cell Biol 109: 1837–1848, 1989.[Abstract/Free Full Text]
  33. Theodosiou NA and Tabin CJ. Wnt signaling during development of the gastrointestinal tract. Dev Biol 259: 258–271, 2003.[CrossRef][Web of Science][Medline]
  34. Tsuda M, Kamimura K, Nakato H, Archer M, Staatz W, Fox B, Humphrey M, Olson S, Futch T, Kaluza V, Siegfried E, Stam L, and Selleck SB. The cell-surface proteoglycan Dally regulates Wingless signalling in Drosophila. Nature 400: 276–280, 1999.[CrossRef][Medline]
  35. van de Wetering M, Sancho E, Verweij C, de Lau W, Oving I, Hurlstone A, van der Horn K, Batlle E, Coudreuse D, Haramis AP, Tjon-Pon-Fong M, Moerer P, van den Born M, Soete G, Pals S, Eilers M, Medema R, and Clevers H. The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell 111: 241–250, 2002.[CrossRef][Web of Science][Medline]
  36. van Es JH, Jay P, Gregorieff A, van Gijn ME, Jonkheer S, Hatzis P, Thiele A, van den Born M, Begthel H, Brabletz T, Taketo MM, and Clevers H. Wnt signalling induces maturation of Paneth cells in intestinal crypts. Nat Cell Biol 7: 381–386, 2005.[CrossRef][Web of Science][Medline]
  37. Wasan HS, Park HS, Liu KC, Mandir NK, Winnett A, Sasieni P, Bodmer WF, Goodlad RA, and Wright NA. APC in the regulation of intestinal crypt fission. J Pathol 185: 246–255, 1998.[CrossRef][Web of Science][Medline]
  38. Wilkinson DG. Multiple roles of EPH receptors and ephrins in neural development. Nat Rev Neurosci 2: 155–164, 2001.[Web of Science][Medline]
  39. Wong WM, Mandir N, Goodlad RA, Wong BC, Garcia SB, Lam SK, and Wright NA. Histogenesis of human colorectal adenomas and hyperplastic polyps: the role of cell proliferaton and crypt fission. Gut 50: 212–217, 2002.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Tables and Figures
Right arrow All Versions of this Article:
291/1/G163    most recent
00019.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Takano-Maruyama, M.
Right arrow Articles by Ohno, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Takano-Maruyama, M.
Right arrow Articles by Ohno, H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.