Sfrp controls apicobasal polarity and oriented cell division in developing gut epithelium.

Epithelial tubular morphogenesis leading to alteration of organ shape has important physiological consequences. However, little is known regarding the mechanisms that govern epithelial tube morphogenesis. Here, we show that inactivation of Sfrp1 and Sfrp2 leads to reduction in fore-stomach length in mouse embryos, which is enhanced in the presence of the Sfrp5 mutation. In the mono-cell layer of fore-stomach epithelium, cell division is normally oriented along the cephalocaudal axis; in contrast, orientation diverges in the Sfrps-deficient fore-stomach. Cell growth and apoptosis are not affected in the Sfrps-deficient fore-stomach epithelium. Similarly, cell division orientation in fore-stomach epithelium diverges as a result of inactivation of either Stbm/Vangl2, an Fz/PCP component, or Wnt5a. These observations indicate that the oriented cell division, which is controlled by the Fz/PCP pathway, is one of essential components in fore-stomach morphogenesis. Additionally, the small intestine epithelium of Sfrps compound mutants fails to maintain proper apicobasal polarity; the defect was also observed in Wnt5a-inactivated small intestine. In relation to these findings, Sfrp1 physically interacts with Wnt5a and inhibits Wnt5a signaling. We propose that Sfrp regulation of Wnt5a signaling controls oriented cell division and apicobasal polarity in the epithelium of developing gut.


Introduction
Generation of the gastrointestinal (GI) tract is initiated by formation of the primitive gut tube during embryogenesis. Subsequently, this tube differentiates regionally along the cephalocaudal axis, giving rise to the esophagus, stomach, small intestine and colon, as well as acquiring specific morphologies, which are generated through morphogenetic mechanisms. Regional specification of the gut tube involves interactions between splanchnic mesoderm and endoderm epithelium [1]. However, the morphogenetic mechanisms governing gut formation remain poorly understood.
Secreted Frizzled-related protein (Sfrp) is a secreted Wnt antagonist that interacts directly with the Wnt ligand [6]. The Sfrp gene family, which consists of five members in both the human and mouse genomes, is classified into the Sfrp1 (Type 1) and FrzB subfamilies based on amino acid sequence similarity [6]. Sfrp1, Sfrp2 and Sfrp5 comprise the Sfrp1 subfamily (referred to as Type 1 Sfrps) [6]. Type 1 Sfrps inhibit the Wnt/ß-catenin pathway in vitro. Type 1 Sfrps exhibit characteristics of Wnt inhibition that differ from those of FrzB Sfrps (Sfrp3 and Sfrp4), a phenomenon that can probably be attributed to Wnt ligand specificity [7,8]. Genetic analysis has revealed the functional redundancy of Sfrp1, Sfrp2 and Sfrp5; moreover, Sfrp1/2/5 genetically interact with Stbm/Vangl2 (also known as Ltap), an ortholog of Drosophila Strabismus/Van Gogh Fz/PCP core component [9]. These observations suggest a redundant role for Type 1 Sfrps in the regulation of the Wnt/ßcatenin and the Fz/PCP pathways.
The body axis of Sfrp1/2/5 compound mutants is shortened [9]. This observation suggests that a concomitant shortening of the axial visceral organs, i.e., the GI tract, may occur. Therefore, we focused on the forming gut tube and epithelial morphogenesis. Our results suggest that Sfrp-regulation of Wnt5a signaling is required for the regulation of epithelial cell polarity, oriented cell divisions and apicobasal (AB) polarity, and lengthening of the developing gut.

Sfrp1, Sfrp2, and Sfrp5 Are Required for Fore-Stomach Morphogenesis
During mouse embryonic development, the primitive gut tube is generated by embryonic day (E) 9. Subsequently, the gut tube develops the organ buds of the lung, stomach, liver and pancreas, which are apparent at E10.5. In the developing gut, Sfrp1, Sfrp2 and Sfrp5 are regionally expressed along the cephalocaudal axis. At E10.5, Sfrp1 was expressed in the splanchnic mesoderm from the caudal region of the prospective stomach to the midgut. At E12.5, expression was observed in the mesenchyme of the colon, as well as in the caudal region of the fore-stomach and the small intestine ( Figure S1A, D, G, J, N). Sfrp2 was expressed in the splanchnic mesoderm of the prospective esophagus at E10.5. Later, at E12.5, Sfrp2 expression expanded to the rostral region of fore-stomach mesenchyme ( Figure S1B, E, H, K, N). Sfrp2 expression was also detected in colon epithelium at this stage ( Figure S1L). In addition, Sfrp5 expression was present in endoderm cells of the presumptive midgut region at E8.75 [10]. Sfrp5 expression remained in evidence in the midgut endoderm at E9.5, a stage lacking obvious expression of Sfrp1 and Sfrp2 in the gut tube (data not shown). During the later stages of E10.5-12.5, Sfrp5 expression was observed in the duodenum epithelium ( Figure S1C, F, I, M, N). Despite expression in the developing gut, no obvious morphological abnormality was identified in the gut of Sfrp1, Sfrp2 and Sfrp5 single knock-out embryos as far as we examined [9,11,12], possibly because, as in other tissues, the long-range effect of an Sfrp as a secreted factor can compensate for the function of other Sfrps in those mutants [9,12].
In order to establish the redundant role of Sfrp1, Sfrp2 and Sfrp5 in gut formation, the gut tube was examined in Sfrp1 subfamily compound mutant mice. Sfrp1 and Sfrp5 (Sfrp12/2 Sfrp52/2) and Sfrp2 and Sfrp5 (Sfrp22/2 Sfrp52/2) double homozygous mutants appeared to be normal in terms of GI tract formation. In contrast, embryos carrying a double homozygous mutation in both Sfrp1 and Sfrp2 (Sfrp12/2 Sfrp22/2) displayed severe shortening of the gut tube, e.g., smaller stomach and shorter intestine, at E13.5 ( Figure  S2B, E). The reduction in the length/size of the gut in the E13.5 Sfrp12/2 Sfrp22/2 embryos was enhanced in the presence of an Sfrp5 heterozygous mutation (Sfrp12/2 Sfrp22/2 Sfrp5+/2), which is suggestive of a redundant role for Sfrp1, Sfrp2 and Sfrp5 in gut formation ( Figure S2E, F).
Epithelial specification in the gut is tightly controlled by crosstalk between splanchnic mesoderm and endoderm epithelium [17]. The stomach of compound mutant embryos demonstrated normal expression of Islet1 ( Figure 1D) and Barx1 [18], which are specific markers for stomach mesenchyme ( Figure 1E). The nonglandular stomach of compound mutant embryos was significantly smaller than that of control embryos at E16.5; however, normal characteristic cell types were detected at histological levels in the glandular and non-glandular stomach. The mucosa appeared to be thicker and tightened in Sfrp12/2 Sfrp22/2 Sfrp5+/2 nonglandular stomach in comparison with control non-glandular stomach ( Figure S3).
To determine correlation between reductions of the anteriorposterior (a-p) body axis and abnormal gut formation in Sfrp12/2 Sfrp22/2 and Sfrp12/2 Sfrp22/2 Sfrp5+/2 embryos, we examined the shape of the gut at earlier stages. Reductions in the length of the hindgut and the caudal half of the midgut were already apparent at E10.5. The shortening of the caudal gut tube was closely related to reduction of the a-p body axis in compound mutant embryos at earlier stages [12] ( Figure 1F). In contrast, marker analysis of Shh, Pdx1 [19], Barx1 and Sfrp5 in Sfrp12/2 Sfrp22/2 and Sfrp12/2 Sfrp22/2 Sfrp5+/2 embryos demonstrated that the regions corresponding to the prospective stomach and duodenum were unaffected at E10.5 ( Figure 1F-I). Thus, the region corresponding to the stomach and the duodenum in Sfrpsdeficient embryos is specified and generated in normal length at E10.5; moreover, organ bud formation is initiated in a manner consistent with that in control embryos. Organ bud formation occurs following the establishment of the a-p body axis; consequently, we concluded that deficiency of Type 1 Sfrps leads to a reduction in the size of the fore-stomach in a manner that is independent of the mechanism that shortens the a-p body axis.

Author Summary
The gastrointestinal tract is generated from the primitive gut tube during embryogenesis. The primitive gut differentiates regionally along the cephalocaudal axis. Individual regions simultaneously acquire specific morphologies through morphogenetic mechanisms. The regional specification of the gut tube is controlled by cross-talk between the mesenchyme and epithelium. However, the morphogenetic mechanisms governing gut formation remain poorly understood. Secreted Frizzledrelated protein (Sfrp) is an inhibitor of the Wnt pathway, members of which are expressed in the developing gut. A deficiency of Sfrp genes (Sfrp1, Sfrp2, and Sfrp5) results in reduction of fore-stomach length in mice. During normal fore-stomach formation, cell division is oriented along the cephalocaudal axis; in contrast, reduced fore-stomach length in Sfrps-deficient mice is associated with the divergence of oriented cell division in tubular epithelial cells. Thus, oriented cell division is one of the essential components in fore-stomach morphogenesis. In addition, Sfrps-deficient small intestine epithelium fails to maintain proper apicobasal polarity. We also found that Wnt5ainactivation leads to a phenotype similar to that induced by Sfrps-deficiency in the developing gut, and that Sfrp1 inhibits Wnt5a signaling. We propose that Sfrp regulation of Wnt5a signaling is required for oriented cell division and that it modulates apicobasal polarity in gut epithelium during organ elongation.

Sfrps Are Required for Oriented Cell Division in Fore-Stomach Epithelium
Although the stomach region is enlarged at E11.5, one day after the initiation of organ bud formation, the greater curvature of the fore-stomach is not well expanded as observed at later stages. The expansion of the greater curvature becomes obvious from around E12. We measured the size of E12.5 Sfrps-deficient fore-stomach, in order to gain insight into the character of the size reduction defect in the fore-stomach. The length of the greater curvature was greatly reduced in the fore-stomach of Sfrp12/2 Sfrp22/2 Sfrp5+/2 embryos in comparison with that of control embryos (790675 mm in control and 406653 mm in Sfrp12/2 Sfrp22/2 A broken line depicts Shh-negative epithelium along the greater curvature. Curved arrows, an arrow and double arrowheads denote the fore-stomach, the junction of the esophagus and fore-stomach and the pyloric sphincter, respectively. A white arrowhead defines the caudal end of the fore-stomach. A black arrowhead indicates the rostral end of marker gene expression. Fs, fore-stomach. Scale bar: 500 mm. (F-I) A region of presumptive stomach and duodenum is generated normally in Sfrp12/2 Sfrp22/2 and Sfrp12/2 Sfrp22/2 Sfrp5+/2 embryos at E10.5, which is suggested by the expression patterns of Shh (F), Pdx1 (G), Sfrp5 (H) and Barx1 (I). The area between the arrowheads identifies a Shh-negative region in the gut epithelium (F). Arrows depict the rostral and caudal ends of marker gene expression (G, H). Ce, cecum; Lb, lung bud; Pb, pancreatic bud; St, stomach. Scale bar: 500 mm. doi:10.1371/journal.pgen.1000427.g001 Sfrp5+/2 fore-stomachs, n = 3, P,0.01). In contrast, the width of the Sfrp12/2 Sfrp22/2 Sfrp5+/2 stomach was significantly increased at the junction of the fundus and the body (347633 mm in control and 453644 mm in Sfrp12/2 Sfrp22/2 Sfrp5+/2 fore-stomachs, n = 3, P,0.05; Figure S4A-C). Thus, Sfrps deficiency induces lateral expansion of the fore-stomach, which may be suggestive of a defect in morphogenesis.
Since this defect might be associated with an abnormality in epithelium, the histology of Sfrps-deficient fore-stomach epithelium was examined. The cell number per area of epithelium (2000 mm 2 ) was slightly increased in the greater curvature of Sfrpsdeficient fore-stomachs; however, this observation was statistically insignificant ( Figure S4E). Due to the low frequency of multinuclei along the apicobasal (AB) axis, the greater curvature epithelium at E12.5 is considered a mono-cell layer in both Sfrp12/2 Sfrp22/2 Sfrp5+/2 and control fore-stomachs ( Figure  S4D, F).
Since cell density in the epithelium of Sfrps-deficient forestomachs appeared to be increased in comparison with the controls, we examined AB polarity. Sub-cellular distribution of marker proteins (e.g., atypical Protein Kinase C (aPKC), ß1integrin, E-cadherin and F-actin) [21][22][23] in Sfrp12/2 Sfrp22/2 Sfrp5+/2 fore-stomachs was identical to localization in control fore-stomachs (data not shown). However, the distribution patterns did not suggest a strong establishment of AB polarity even in the controls. Thus, these observations suggest that the defect of cell division orientation is associated with fore-stomach morphogenesis phenotype.

Fz/PCP Pathway Modulates Epithelial Oriented Cell Division in the Fore-Stomach
We next examined which Wnt pathway is regulated by Sfrps in the fore-stomach, since Sfrps regulate the Wnt/ß-catenin and the Fz/PCP pathways [9]. The Wnt/ß-catenin pathway is highly activated in fore-stomach epithelium at E13.5, as evidenced by TOPGAL reporter activity [24] ( Figure S6A, B). The activity levels were not altered in fore-stomach epithelium of Sfrp12/2 Sfrp22/2 Sfrp5+/2 embryos in comparison with that in control embryos. In contrast, TOPGAL activity, which was markedly diminished at the boundary of the control fore-and hindstomachs, extended into the hind-stomach region in Sfrp12/2 Sfrp22/2 Sfrp5+/2 embryos ( Figure S6C, D). However, as shown by FoxA2 [25] and Sox2 [26] protein expression, no significant patterning defect was observed at the junction of fore-and hindstomach epithelium ( Figure S6E-J). Moreover, the TOPGAL activation pattern in Sfrp12/2 Sfrp22/2 Sfrp5+/2 stomach was not correlated with the defect observed in fore-stomach morphogenesis.
We also examined fore-stomach morphogenesis in Lp/Lp embryos carrying mutations in Stbm/Vangl2, a component of the Fz/PCP pathway, because Type 1 Sfrps genetically interact with Stbm/Vangl2 [9]. Stbm/Vangl2 is expressed in stomach epithelium [27]. Marker analysis revealed that fore-stomach size/length was greatly reduced in E13.5 Lp/Lp embryos ( Figure 3A). In addition, a morphological defect was associated with divergence of cell division orientation in the epithelium of the greater curvature epithelium ( Figure 3B, C, also see Figure 2B). These observations suggest that the Fz/PCP pathway modulates lengthening of the fore-stomach during oriented cell division.
These data present the possibility that dys-regulation of the Fz/ PCP pathway perturbs oriented cell division in Sfrps-deficient forestomach epithelium. To investigate whether the Fz/PCP pathway is affected in Sfrps-deficient fore-stomach, we examined the subcellular distribution of Frizzled3 (Fz3) and Dishevelled-2 (Dvl-2) [28,29]. Proper sub-cellular distribution of Fz/PCP pathway components is essential for the pathway activity [30]. Fz3 and Fz6 are mammalian homologues of the Drosophila Fz receptor in the Fz/PCP pathway. Fz3 localized to the apical surface of epithelial cells in control greater curvature at E13.5; in addition, local enrichment of the protein at the site of cell-cell adhesion was not observed. Dvl-2 co-localized with Fz3 in the apical region of the Figure 2. Divergence of cell division orientation in the epithelium of Sfrps-deficient fore-stomach. (A) Cell division orientation was visualized with anti-ß1-integrin (red), anti-acetylated a-tubulin (green) and DAPI (blue) staining in the greater curvature epithelium of the forestomach of control and Sfrp12/2 Sfrp22/2 Sfrp5+/2 embryos at E13.5. The top of the confocal image is oriented in the direction of the fundus. Scale bar: 40 mm. An inset shows higher magnification of the mitotic cells indicated by an arrowhead. The cell division axis is indicated by a bar, whereas the vertical axis is denoted by an asterisk. (B) Arrows indicate the direction of the fundus (f), pylorus (p) as well as right (r) and left (l) in the schematic diagram of the stomach at E12.5,E13.5. (C-H) Cell division orientation converged within 645u of the cephalocaudal axis in control forestomach epithelium, whereas it diverged in Sfrp12/2 Sfrp22/2 Sfrp5+/2 fore-stomach epithelium at E12.5 (C, D) and E13.5 (F, G). Statistical analysis revealed a significant difference in convergence of cell division orientation along the cephalocaudal axis between control and Sfrps-deficient forestomachs at E12.5 (D) and E13.5 (G). In contrast, no significant difference in the frequency of cell division along the AB axis is evident between control and Sfrp12/2 Sfrp22/2 Sfrp5+/2 fore-stomachs at E12.5 (E) and E13.5 (H). (I, J) Oriented cell division is not observed in hind-stomach epithelium of controls or Sfrp12/2 Sfrp22/2 Sfrp5+/2 mutants at E13.5. (K) Cell division along the AB axis occurs at a similar frequency in control (35.164.93%, n = 3) and Sfrp12/2 Sfrp22/2 Sfrp5+/2 (34.365.5%, n = 3) hind-stomachs at E13.5. doi:10.1371/journal.pgen.1000427.g002 fore-stomach epithelium (arrowheads in Figure 3Da-c). Colocalization of Fz3 and Dvl-2 was less apparent in the apical surface of hind-stomach epithelium (data not shown). Dvl-2 expression, which was also observed in the basal side of the epithelium, overlapped with that of ß1-integrin (Figure 3Db; data not shown). Significantly, Fz3 and Dvl-2 displayed diffuse distribution in the middle of the greater curvature of the Sfrpsdeficient fore-stomachs (n = 3; Figure 3Dd-f). Thus, this finding indicates that the Fz/PCP pathway is affected in Sfrps-deficient fore-stomach. Moreover, Sfrp regulation of the Fz/PCP pathway appears to be correlated with the defect observed in fore-stomach morphogenesis.
Cell Division Orientation in Wnt5a2/2 Fore-Stomach Epithelium Wnt signaling inhibition by Sfrp usually involves an associating Wnt ligand [6]. The following observations suggest that Wnt5a is inhibited by Sfrps during fore-stomach morphogenesis: Wnt5a, a typical non-canonical Wnt ligand gene, is expressed in forestomach mesenchyme, where defects of the Sfrps-deficient stomach were found; Wnt5a and Type1 Sfrps genetically interact with Stbm/Vangl2 to regulate the Fz/PCP pathway [9,31].
To address the possibility that Wnt5a is an inhibitory target of Sfrps in the fore-stomach, fore-stomach phenotype in Wnt5a homozygous (Wnt5a2/2) mutant embryos was surveyed. Significantly, the Wnt5a2/2gut displayed similarities to the Sfrpsdeficient gut, with the exception of ectopic branching of the small intestine ( Figure 4A, double arrows in the panel depicting Shh expression). First, fore-stomach formation was defective in the Wnt5a2/2 embryos at E13.5 ( Figure 4A). Hind-stomach formation was less affected in the E13.5 Wnt5a2/2 embryos, although the hind-stomach appeared to be affected and was smaller at later stages, such as E16.5 (data not shown). Second, canonical Wnt/ß-catenin signaling was not altered in that region as evidenced by TOPGAL activity, a reporter of the Wnt pathway [24] ( Figure 4A). Third, fore-stomach malformation was associat- ed with divergence of cell division orientation in the greater curvature epithelium ( Figure 4B, C, also see Figure 2B). Oriented cell division in the horizontal plane of the greater curvature of the fore-stomach, which was evident in the controls, was not obvious in the Wnt5a2/2 mutants ( Figure 4B).
Statistical analyses revealed a significant difference between convergence of cell division orientation in the control (83.264.14% of 232 cells, n = 3) and Wnt5a2/2 fore-stomach epithelia (37.064.48% of 257 cells, n = 3, P,0.0001; Figure 4C, left). The cell proliferation ratio determined with anti-phospho- Wnt5a inactivation results in defective fore-stomach formation at E13.5. TOPGAL activity is unaltered in Wnt5a2/2 fore-stomach epithelium; however, the size of the forestomach is significantly reduced in Wnt5a 2/2 embryos, as evidenced by Shh expression and the higher expression domain of Pitx1. Islet1 and Barx1 are expressed in the mesenchyme of the hind-and whole stomachs. A broken line defines Shh-negative epithelium along the greater curvature. Curved arrows, an arrow and double arrowheads denote the fore-stomach, the junction of the esophagus and fore-stomach and the pyloric sphincter, respectively. The white arrowhead identifies the caudal end of the fore-stomach. The black arrowhead indicates the rostral end of marker gene expression. Scale bar: 500 mm. (B) Cell division orientation is diverged in Wnt5a-inactivated fore-stomach epithelium at E13.5. (C, D) Statistical analyses revealed a difference between convergence of cell division orientation in control and Wnt5a2/2 fore-stomach epithelia (C, left). Oriented cell division is absent in control and Wnt5a2/2 hind-stomach epithelia (D, left). A significant difference in the frequency of cell division along the AB axis is not evident in either the fore-stomach of the control and Wnt5a2/2 or the corresponding hind-stomachs (right in C and D). doi:10.1371/journal.pgen.1000427.g004 Histone H3 staining was not altered in Wnt5a2/2 fore-stomach epithelium (18.360.2 positive cells in1610 5 mm 3 , n=3) in comparison with control fore-stomach epithelium (18.761.0 positive cells in1610 5 mm 3 , n = 3). Oriented cell division was not observed in either control (54.063.59% of 137 cells, n = 3) or Wnt5a2/2 hind-stomach epithelium (53.461.22% of 131 cells, n = 3; Figure 4D). No significant difference in the frequency of cell division along the AB axis was detected between control and Wnt5a2/2 fore-stomachs (35.463.91% and 34.062.65% of 359 and 389 cells, respectively, n = 3); a similar situation was apparent with respect to corresponding hind-stomachs (34.161.54% and 36.764.76%, respectively, n = 3; Figure 4C, D, right). Thus, oriented cell division is disrupted in the epithelium of Wnt5a2/2 fore-stomachs as well as in the Sfrps-deficient fore-stomach.

Sfrps Are Involved in AB Polarity in Small Intestine Epithelium
In addition to a defect in fore-stomach morphogenesis, the intestine was shortened substantially in the Sfrps-deficient embryos in association with shortening of the anterior-posterior (a-p) body axis ( Figure S7). Moreover, the observed reduction in the length of the intestine in E13.5 Sfrp12/2 Sfrp22/2 embryos was enhanced in the presence of an Sfrp5 heterozygous mutation (Sfrp12/2 Sfrp22/2 Sfrp5+/2) ( Figure S7F). Thus, cell migration associated with a-p axis elongation may be involved in gut morphogenesis. The small intestine was remarkably shortened in Sfrp12/2 Sfrp22/2 and Sfrp12/2 Sfrp22/2 Sfrp5+/2 embryos at E13.5; therefore, Sfrps-deficient gut tubes were examined to determine the effect of these molecules on regionalization of the small intestine. Cdx2 is expressed in the epithelium of the small and large intestines [32]. In the gut derived from Sfrp12/2 Sfrp22/2, Sfrp12/2 Sfrp22/2 Sfrp5+/2 and control embryos, the rostral boundary of Cdx2 expression was observed at the pyloric sphincter, a junction between the stomach and the duodenum ( Figure S7A, arrow). Hoxa4 is expressed in the mesenchyme from the prospective duodenum to a portion of the ileum (rostral small intestine) in control embryos [33], whereas Sfrp5 is expressed in the epithelium ( Figure S7B, C). Wnt5a and Hoxc6, which are marker genes for the caudal small intestine, are expressed in the mesenchyme ( Figure  S7D, data not shown) [34,35]. The expression of these markers was indicative of the regionalization of the small intestine in Sfrp12/2 Sfrp22/2 and Sfrp12/2 Sfrp22/2 Sfrp5+/2 embryos. Furthermore, Hoxd13 expression was observed in the caudal large intestine of Sfrps-deficient and control embryos ( Figure S7E). Thus, the expression of these markers indicates that (Type 1) Sfrps do not affect regional specification of the gut tube at E13.5.
Interestingly, the small intestine of Sfrp12/2 Sfrp22/2 and Sfrp12/2 Sfrp22/2 Sfrp5+/2 embryos at E13.5 displayed cell clumps, which disrupted the internal surface of the epithelial tube ( Figure 5A, arrowhead). The clump of epithelial cells occurred continuously from the jejunum to the ileum. In control embryos at E13.5, the region corresponding to the prospective jejunum and ileum within the small intestine exhibited a smooth apical surface ( Figure 5A). In contrast, cell clumps were not obvious in the Sfrpsdeficient small intestine at E16.5. It is possible that an increase in cell proliferation may contribute to the generation of cell clumps within the epithelium. However, a BrdU incorporation assay failed to detect an increase in cell proliferation rates in the clumps as well as the entire gut epithelium (36.163.38% of total 1030 control epithelium cells, n = 3; 33.664.63% of total 1265 Sfrp12/2 Sfrp22/2 Sfrp5+/2epithelium cells, n = 3) ( Figure S8A-C). In addition, cell density was not significantly increased in the epithelium of Sfrp12/2 Sfrp22/2 Sfrp5+/2 small intestine ( Figure S8D). Based on these observations, we hypothesize that this histological abnormality appears to be related to a defect in epithelial morphogenesis.
To address this possibility in greater detail, we analyzed the localization of AB polarity markers. Activated aPKC (phospho-aPKC) has been implicated in the establishment of AB polarity in mammalian cells [23]. We observed proper localization of aPKC to the apical region of control small intestine epithelium. In contrast, specific sub-cellular localization of aPKC disappeared in a clump of epithelial cells in Sfrp12/2 Sfrp22/2 Sfrp5+/2 small intestine ( Figure 5B-G). Antibody staining against ß1-integrin revealed a round cell shape in the clump of epithelium ( Figure 5D, E; Video S1, S2). In addition, E-cadherin was concentrated at the apicolateral cytoplasmic membrane in control epithelium ( Figure 5F). However, it was widely distributed in the cytoplasmic membrane of the epithelial cell clump ( Figure 5G, asterisk; Video S3, S4). These observations indicate the involvement of Type 1 Sfrps in the regulation of AB polarity in the small intestine epithelium.
Since a relationship between AB polarity and the PCP pathway was suggested previously [30,36], we assessed sub-cellular distributions of Fz3 and Dvl-2 in the small intestine. In control epithelium derived from small intestine corresponding to the jejunum and the ileum at E13.5, Fz3 occupied the apical region and co-localized with Dvl-2 ( Figure 6F). However, Fz3 and Dvl-2 were not concentrated in the apical region of the Sfrps-deficient small intestine epithelium, especially in the clump of epithelial cells ( Figure 6G). No difference in sub-cellular distributions of Fz3 and Dvl-2 was detected in other regions of the Sfrp12/2 Sfrp22/2 Sfrp5+/2 gut tube relative to that of the control gut tube. Thus, Type 1 Sfrps affect AB cell polarity in conjunction with the regulation of the sub-cellular distribution of core Fz/PCP factors in the small intestine epithelium.

AB Polarity in Wnt5a2/2 Small Intestine Epithelium
TOPGAL reporter activity [24] indicated that up-regulation did not occur in the canonical pathway within the Sfrp12/2 Sfrp22/ 2 Sfrp5+/2 small intestine epithelium ( Figure S9A, B). Since similar defects in terms of oriented cell division were observed in the stomachs of Sfrp12/2 Sfrp22/2 Sfrp5+/2 and Wnt5a inactivated embryos, we examined AB polarity in the small intestine of Wnt5a2/2 embryos at E13.5. The epithelial structure was disrupted by epithelial cell clumps in the region corresponding to the jejunum and the ileum of Wnt5a2/2 embryos ( Figure 6A). In addition, the apical distribution of aPKC was disturbed in Wnt5a2/2 small intestine epithelium ( Figure 6B-E), suggesting a defect in AB polarity. Moreover, this epithelial abnormality was associated with defective Fz3 and Dvl-2 sub-cellular distributions in the Wnt5a2/2 small intestine ( Figure 6H). The gut phenotypes observed in Sfrp12/2 Sfrp22/2 Sfrp5+/2 embryos appeared to be more severe than those in Wnt5a2/2 embryos. However, Wnt5a2/2 embryos displayed significant phenotypic similarities to Sfrps-deficient embryos.

Sfrps Modulate Wn5a Signaling
Although the morphological abnormalities detected in the gut of Wnt5a2/2 embryos resembled those in Sfrps-deficient gut, this observation did not necessarily equate to a similarity in signaling regulation. In fact, the loss or gain of Wnt5a function results in dys-regulated convergent extension (CE) movements in vertebrates [37,38]. Additionally, previous reports imply that Sfrp2 antagonizes Wnt5a signaling [39].
To establish a molecular relationship between Sfrps function and Wnt5a signaling, the signaling activity of the Wnt5a pathway in the gut was assayed in terms of phospho c-Jun levels. It is well established that Wnt5a activates c-Jun N-terminal kinase (JNK). In turn, JNK phosphorylates c-Jun [40,41]. Currently, phospho c-Jun is the only available marker in the pathway detectable with antibody staining (Figure 7A). Phospho c-Jun-positive cells were observed in small intestine epithelium at E13.5; however, it was scarcely detected in fore-stomach epithelium. In Sfrps-deficient small intestine, phospho c-Jun levels were elevated significantly in the epithelium in comparison with the control small intestine epithelium ( Figure 7A, B). Moreover, phospho c-Jun-positive cells were frequently observed in the mesenchyme of Sfrp12/2 Sfrp22/2 Sfrp5+/2 embryos ( Figure 7A). In contrast, phosphoc-Jun levels were decreased in Wnt5a2/2 epithelium ( Figure 7A, B). Immunofluorescence staining was repeated three times, followed by statistical analysis of the staining intensity. The results were statistically significant (i.e., Control, 10068.16%; Sfrpsdeficient, 13969.24%; Wnt5a2/2, 52.5611.4%; P,0.01) upon comparison between control and Sfrps-deficient or Wnt5a2/2 small intestines (Figure 7 B). These observations suggest that Type 1 Sfrps may inhibit Wnt5a signaling. Therefore, we tested this possibility. In a co-culture immunoprecipitation assay, Wnt5a coprecipitated with Sfrp1 from the lysate containing Sfrp1 and Wnt5a; this observation indicated that Sfrp1 interacts with Wnt5a ( Figure 7C). We also examined the effect of Sfrp on JNK activation induced by Wnt5a. In conditioned medium, Wnt5a elevated the levels of phospho-JNK, an active form, in HEK293T cells. However, Sfrp1, in the presence of Wnt5a, reduced the levels  of active JNK ( Figure 7D). Thus, Type 1 Sfrps are capable of inhibiting Wnt5a signaling.

Oriented Cell Division in Epithelium Is Associated with Fore-Stomach Morphogenesis
In Zebrafish, oriented cell division is a driving force of a-p axis elongation during gastrulation and neural tube morphogenesis during neurulation [42][43][44]. Moreover, in Drosophila, oriented cell division in the imaginal disc affects adult organ shape [45]. In both processes, oriented cell division is disrupted by a defect in the Fz/ PCP pathway [42,43,45]. Thus, the Fz/PCP pathway regulates cell division orientation with respect to tissue elongation during embryonic development. However, this involvement was unknown in mammals.
We observed divergence of cell division orientation in the greater curvature of the fore-stomach in Sfrps-deficient and Wnt5a2/2 embryos. Both Wnt5a and Type1 Sfrps genetically interact with Stbm/Vangl2 [9,31]. Moreover, oriented cell division was disrupted in the fore-stomach epithelium of Lp/Lp embryos. Thus, these observations are indicative of Fz/PCP pathway regulation of epithelial oriented cell division in the fore-stomach. Significantly, the components of the Fz/PCP pathway, i.e., Fz3, Dvl2 and Vangl2, are expressed in the epithelium [27]; in contrast, co-expression is not evident in the mesenchyme. Furthermore, sub-cellular distribution of Fz3 and Dvl-2 was affected in Sfrpsdeficient fore-stomach epithelium. Therefore, the Fz/PCP pathway components in the epithelium appear to be involved in the regulation of oriented cell division.
Following the initiation of organ bud formation at around E10.5, the stomach is dramatically enlarged over several days. Previous data of Nyeng et al. [26] suggest that the epithelium of the fore-stomach initiates terminal differentiation at E15.5. Hence, most of the cells in fore-stomach epithelium at E12.5 and E13.5 could be immature cells continuing cell division during these stages. When we observed cell division in fixed samples, approximately 3% of epithelial cells demonstrated division in the greater curvature of the fore-stomach at E12.5. However, cell proliferation occurs rapidly in the epithelium; additionally, an increment of epithelial cell number generates the largest number of dividing cells in the entire fore-stomach epithelium during organ development. Therefore, cell division orientation could be one of essential components contributing to fore-stomach morphogenesis.
The phenotype described by fore-stomach shortening and mitotic orientation defect in Sfrp12/2 Sfrp22/2 Sfrp5+/2 stomachs might be most severe in all mutants; additionally, the phenotype in Lp/Lp stomachs might be relatively milder in comparison to that found in other mutants. Although the difference between the mitotic orientation defects of the mutants was statistically insignificant, a weak tendency in the severity of mitotic orientation defect, which could be correlated with the severity of fore-stomach shortening defects, may occur. The weak tendency might be due to our observation of cell division involving an instantaneous event of an individual cell, whereas the morphological features of the fore-stomach shortening defect was a result of accumulated cellular events.
A defect in the cell rearrangement process (e.g., increased radial intercalation) [46] might be a possible element in the induction of fore-stomach shortening. However, epithelial cell division along the AB axis occurred at low frequencies and the mono-cell layer was maintained in Sfrps-deficient fore-stomach epithelium at E12.5. Therefore, oriented cell division and allocation of the divided cells along the cell division axis could contribute to organ lengthening as one of the earlier events in fore-stomach morphogenesis. The fore-stomach is the most remarkable structure in the developing gut tube, which is generated during mid-gestation. Most internal organs are generated from an epithelial tubule structure, with the tube altering its shape depending on the function of the organ. It is possible that oriented cell division is a common mechanism that is essential for tubular morphogenesis of the internal organs.
Our results implicate the Fz/PCP pathway, in association with Sfrp regulation of Wnt5a, in the regulation of oriented cell division. The reason that mutations in opposing regulatory components lead to similar defects in oriented cell division may be due to the loss of planer cell polarity in both defects. Animals with mutations in opposing Fz/PCP pathway regulatory components frequently exhibit similar phenotypes [47].
The current in vitro data and data representing the response of downstream effectors of Wnt5a signaling suggested that Sfrp inhibits Wnt5a signaling. Genetic analysis was also conducted via the generation of Wnt5a, Sfrp1 and Sfrp2 compound mutant embryos. A survey of the morphology and internal organs of these embryos was suggestive of no rescue and no enhanced phenotype in Wnt5a+/2 Sfrp12/2 Sfrp22/2 embryos in comparison with Sfrp12/2 Sfrp22/2 embryos at E10.5, E12.5 and E13.5. Thus, the genetic analysis was not beneficial in terms of evaluation of an interaction between Sfrps function and Wnt5a signaling. This observation is likely attributable to the insufficient capacity of the heterozygous mutation to reduce Wnt protein expression under an effective dosage.
The Wnt5a transcript is highly expressed in fore-stomach mesenchyme; in contrast, the expression is weaker in hindstomach mesenchyme at E13.5. Based on transcriptional expression patterns, an active Wnt5a protein gradient may be generated in conjunction with Sfrps along the cephalocaudal axis in the stomach. Further investigation is necessary in order to understand the mechanism by which the protein gradient is involved in epithelial oriented cell division through the Fz/PCP pathway.

Wnt Regulation of AB Polarity in Small Intestine Epithelium
Previous studies have identified the role of various molecules including the conserved PAR-aPKC complex in the regulation of epithelial AB polarity [23]. Additionally, the involvement of PCP pathway components in AB polarity of epithelial cells has been suggested [36,48]. However, little is known regarding intracellular signaling regulation. The intercellular signaling regulation could coordinate AB polarity in developing organs. We observed a similar AB polarity defect in both Sfrps-deficient and Wnt5a inactivated small intestines. In contrast, we could not identify clear evidence suggesting abnormality of AB polarity in the forestomach; AB polarity was not well established even in wild-type fore-stomach epithelium throughout the assessment of sub-cellular distribution of protein markers.
Although Sfrps are capable of inhibiting Wnt5a signaling, it is possible that mutations in opposite regulatory components result in similar defects as both defects lead to a loss of AB polarity. Previously published data indicate that Wnt5a activates JNK [40,41], which is an essential effecter of CE movement [49]. JNK phoshorylates paxillin, a component of the focal adhesion complex [50]. In addition, Wnt5a is able to activate focal adhesion kinase (FAK) [39]. Paxillin and FAK are known to play a role in cell migration [39,50]; thus, the possibility exists that Wnt5a promotes cell migration. However, FAK and paxillin are also required for the maintenance of adherence junctions via N-cadherin-based cell-cell adhesion. Regarding this function, FAK and paxillin are involved in a mechanism of the down-regulation of the activity of the small GTP-binding protein Rac1 [51]. In contrast, Wnt5a signaling is also involved in the up-regulation of Rac1 [39]. Interestingly, the constitutive active and dominant negative forms of Rac1 lead to a phenotype similar to that observed upon the loss of E-cadherin at sites of cell-cell contact [52,53]. Furthermore, E-cadherin provides a clue with respect to the development of AB polarity leading to the recruitment of the PAR-aPKC complex to immature adherence junctions [23]. In this respect, appropriate levels of Wnt5a signaling activity may be essential for the modulation of AB polarity. Hence, we propose that Sfrps and Wnt5a are putative components of intracellular signaling regulation in order to coordinate AB polarity in the developing small intestine.
Control of AB polarity via Wnt signaling has been suggested. In Xenopus and Drosophila, Dvl is necessary for basolateral membrane localization of Lgl (Lethal giant larvae), which encodes a protein with multiple WD-40 motifs that regulates AB polarity [48]. Dvl interacts with Lgl. Moreover, Fz8, but not Fz3 and Fz7, regulate Lgl sub-cellular localization [48], which suggests that the basolateral localization of Fz8, Dvl and Lgl is required for the establishment of AB polarity [48]. In contrast, we demonstrated that Dvl-2 is concentrated at the apical surface of gut epithelium and that this apical localization overlaps with Fz3 localization. Thus, these observations suggest that AB polarity regulation in gut epithelium is distinct from that previously described in Xenopus and Drosophila.
In summary, our results indicate a link between Sfrps function and Wnt5a signaling in the regulation of epithelial cell polarity including oriented cell division and AB cell polarity in developing gut. Sfrps are known as tumor suppressor genes, which are epigenetically silenced in many types of cancer, especially in colorectal cancer [7]. In contrast, up-regulation and downregulation of Wnt5a are observed in gastric and colon cancer, as well as in cancer progression [39,54]. Sfrps-regulation of Wnt5a signaling may provide novel insight into the progression and aggressiveness of GI tract cancer.

Immunofluorescence Staining and Immunoprecipitation
The GI tract was isolated from control, Sfrp12/2 Sfrp22/2, Sfrp12/2 Sfrp22/2 Sfrp5+/2, Lp/Lp and Wnt5a2/2 embryos in phosphate buffered saline (PBS) containing 10% fetal calf serum. Whole mount immunofluorescence staining of stomach epithelium was performed as described previously [12] employing anti-acetylated a-tubulin antibody (Sigma, mouse monoclonal clone 6-11B-1) and anti-ß1-integrin antibody (Chemicon, rat monoclonal MAB1997). Chromosomes were visualized by DAPI staining. Images were captured on a BioRad Radiance 2100 Laser Scanning Confocal Microscope System equipped with a Zeiss Axiovert and processed using Adobe Photoshop. Immunofluorescence staining of sectioned tissue was conducted utilizing the following primary antibodies: anti-ß-galactosidase antibody (from Rabbit, Cappel), anti-E-cadherin antibody (Sigma, mouse monoclonal anti-Uvomorulin clone DECMA-1), anti-phospho-aPKC antibody (from rabbit, Cell Signaling), anti-Fz3 antibody (from rabbit, MBL), anti-Dvl-2 antibody (from goat, Santa Cruz, N-19), anti-FoxA2 antibody [58] (from Rabbit) and anti-Sox2 antibody (from goat, Santa Cruz, Y-17), anti-phospho-c-Jun (from Rabbit, Cell Signaling) and anti-BrdU (mouse monoclonal, BD). F-actin was visualized with Rhodamine-conjugated phalloidin. Images, which were captured on a BioRad Radiance 2100 Laser Scanning Confocal Microscope System equipped with a Zeiss Axiovert, were processed using Adobe Photoshop. Images of anti-phospho-c-Jun staining were analyzed by imaging software MultiGauge (Fujifilm) to calculate averages of signaling intensity per area in the epithelium. With respect to this calculation process, staining background intensity in epithelium and mesenchyme was measured, followed by subtraction from the specific signal intensity. For the BrdU incorporation assay, BrdU/PBS solution was injected into a pregnant mouse intraperitoneally (100 mg/g body weight) 1 h before embryo collection.
The immunoprecipitation assay was performed by co-culture immunoprecipitation of L cells expressing Wnt5a (L Wnt-5A, ATCC) and L cells expressing Sfrp1-FLAG (c-terminal tagged with FLAG, L Sfrp1) (1:1 ratio). Sfrp1-FLAG was precipitated with anti-FLAG M2 affinity gel (Sigma) according to the manufacturer's protocol. Goat anti-Wnt5a antibody (R&D Systems, Inc.) was used to detect Wnt5a protein. JNK activity was evaluated based on the levels of phospho-JNK determined by an anti-phospho-JNK antibody (Cell Signaling). Total JNK was detected by anti-JNK antibody (Cell Signaling). Lysates for Western blotting were derived from HEK293T cells incubated for 3 h in L cell conditioned medium. L Sfrp1 was established as a stable transformant cell line. Conditioned media from L cell, L Wnt5a and L Sfrp1 were obtained following a 4-day incubation of the culture medium.

Epithelial Cell Counting
Histological sections (5 mm) stained with Hematoxylin and Eosin (H&E) were prepared from Bouin-fixed and paraffinembedded specimens. Cell number per 2000 mm 2 in fore-stomach epithelium was calculated by counting the nuclei in a 400-mm wide area of a single section. Total cell number in an epithelial cell suspension was determined with a hemocytometer. Cell suspensions were prepared from stomach epithelium separated from mesenchyme following incubation of the sample in PBS containing 0.5 unit dispase and 1.25% pancreatin at room temperature for 30 minutes. Statistical significance, which was evaluated using Welch's t-test, was defined as P,0.05. Error bars indicate SD.

Ethics Statement
All animals were handled in strict accordance with good animal practice as defined by the relevant national and/or local animal welfare bodies, and all animal works were approved by the appropriate committee. Reporter activity indicates slightly enhanced canonical Wnt/bcatenin signaling in Sfrp12/2 Sfrp22/2 Sfrp5+/2 hind-stomach epithelium (D) in comparison with the control (C). Scale bar: 50 mm. Note that b-galactosidase derived from the TOPGAL reporter exhibits cytoplasmic localization in the epithelium (Ep), whereas b-galactosidase derived from the Sfrp1 knock-in locus displays nuclear localization in the mesenchyme (Me). The arrowhead identifies a boundary between fore-and hind-stomach epithelium as determined by epithelial morphology. (E-J) Protein distribution of FoxA2 and Sox2 is not significantly altered in the junction between the fore-and hind-stomach in Sfrp12/2 Sfrp22/2 Sfrp5+/2 embryos in comparison to control embryos at E13.5. Higher FoxA2 expression is found in fore-stomach epithelium; in contrast, the expression weakens at the junction of the fore-and hind-stomach epithelium (arrow). Sox2 expression in fore-stomach epithelium is gradually reduced in the hind-stomach epithelium (bracket). Scale bar: 50 mm. The intestines of Sfrp12/2 Sfrp22/2 and Sfrp12/2 Sfrp22/2 Sfrp5+/2 embryos are regionalized along the cephalocaudal axis, as suggested by the expressions of Cdx2 (A), Sfrp5 (B), Hoxa4 (C), Wnt5a (D) and Hoxd13 (E). Ce, cecum; Co, colon; St, stomach. Scale bar: 500 mm. (F) The length of the small intestine was reduced in Sfrp12/2 Sfrp22/2 embryos (5.560.39 mm; n = 9; P,0.0001) relative to control small intestine at E13.5 (10.161.32 mm; n = 9). The reduction in length was enhanced upon introduction of an Sfrp5 mutant allele (3.760.37 mm; n = 9; P,0.0001). (G) The length of the rostral large intestine coinciding with the Hoxd13-negative region is shortened in Sfrp12/2 Sfrp22/2 (1.3060.076 mm; n = 8; P,0.0001) and Sfrp12/2 Sfrp22/2 Sfrp5+/2 (1.3160.11 mm; n = 8; P,0.0001) embryos in comparison with controls (2.7360.18 mm; n = 9) at E13.5.