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Development. Author manuscript; available in PMC Jul 24, 2008.
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PMCID: PMC2442161
HALMS: HALMS287635
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SOX9 specifies the pyloric sphincter epithelium through mesenchymal-epithelial signals

SUMMARY

Gastrointestinal (GI) development is highly conserved across the vertebrates. Although several transcription factors and morphogenic proteins are involved in the molecular controls of GI development, the interplay between these factors is not fully understood. We report herein the expression pattern of Sox9 during GI development and provide evidence that it functions in part to define the pyloric sphincter epithelium. SOX9 is expressed in the endoderm of the GI tract (with the exclusion of the stomach) and its derivate organs, lung and pancreas. Moreover, SOX9 is also expressed in the mesoderm of the pyloric sphincter, structure that demarcates the stomach from the duodenum. Using viral misexpression technique, we show that Sox9 expression in the pyloric sphincter is under the control of the BMP signaling pathway, which plays key role in the development of this structure. By misexpressing SOX9 in the mesenchymal stomach, we show that SOX9 is able to transdifferentiate the adjacent gizzard epithelium into pyloric sphincter-like epithelium through the control of mesodermal-epithelial signals mediated in part by Gremlin, a modulator of the BMP pathway. Our results suggest that SOX9 acts in the regionalization of the foregut/midgut boundary and is necessary and sufficient to specify the pyloric sphincter epithelial properties.

Keywords: Animals, Bone Morphogenetic Proteins, metabolism, Cell Differentiation, Chick Embryo, Epithelium, anatomy & histology, embryology, metabolism, Gene Expression Regulation, Developmental, Gizzard, cytology, embryology, metabolism, High Mobility Group Proteins, genetics, metabolism, Humans, In Situ Hybridization, Intercellular Signaling Peptides and Proteins, metabolism, Mesoderm, cytology, physiology, Morphogenesis, Muscle, Smooth, embryology, Oligonucleotides, Antisense, genetics, metabolism, Pylorus, anatomy & histology, embryology, metabolism, Signal Transduction, physiology, Stomach, anatomy & histology, embryology, metabolism, Transcription Factors, genetics, metabolism
Keywords: Pyloric sphincter, SOX, SOX9, stomach, chick

INTRODUCTION

The vertebrate gastrointestinal (GI) tract is a remarkably complex, three dimensional, specialized and vital organ system derived from a simple tubular structure. The GI tract includes the lumenal digestive system of the esophagus, stomach, intestines, and colon (which we will refer as gut) and the derivate organs, thyroid, lungs, liver, and pancreas (Roberts et al., 2000). The gut is composed of the three germ layers, endoderm (which forms the epithelial layer), mesoderm (which forms the smooth muscle layers and myofibroblast), and ectoderm (which includes the enteric nervous system, and most anterior and posterior luminal epithelia). Originally, the gut develops from two invaginations at the anterior (anterior intestinal portal, AIP) and posterior (caudal intestinal portal, CIP) ends of the embryo, which elongate and fuse to form a straight tube. During development the gut becomes patterned along the anterior-posterior (AP), the dorsoventral (DV), the left-right (LR), and the radial (cross-sectional, crypt-villous) axis (de Santa Barbara et al., 2002a). Specific regional differentiation along AP axis will give rise to the foregut (pharynx, esophagus, and stomach), midgut (small intestines), and hindgut (colon, and rectum). During adult life, the gut epithelium is constantly regenerating and its epithelial pattern must be maintained throughout life (de Santa Barbara et al., 2003). In addition, the gut needs reciprocal signals between the mesoderm and endoderm during the patterning process during development (Roberts et al., 1998) up to adulthood for epithelial regeneration (Clatworthy and Subramanian, 2001).

Over the past years, several molecular factors involved during GI tract development have been identified (for review, see (Roberts, 2000; de Santa Barbara et al., 2002a)). We and others have shown that Hox genes play important and active roles in patterning the gut along AP axis and controls normal gut epithelial differentiation (Roberts et al., 1998; Zakany and Duboule, 1999; Aubin et al., 2002; de Santa Barbara and Roberts, 2002b). Morphogenic factors also play key roles during gut development and differentiation. Endodermal Shh expression was described to regulate morphogenesis and mesenchymal differentiation through the induction of Bmp4 in the adjacent mesoderm (Roberts et al., 1995; Sukegawa et al., 2000). Ramalho-Santos and colleagues showed more generally that Hedgehog genes play important roles during GI organogenesis, enteric nervous system (ENS) development, epithelial proliferation and differentiation (Ramalho-Santos et al., 2000). Bmp4, which is expressed in the gut mesoderm, is involved in controlling growth and differentiation of the GI musculature (Roberts et al., 1998; Smith et al., 2000a; Nielsen et al., 2001). The importance of all these factors in gut patterning is highlighted by their remarkable conservation across vertebrate species (Smith et al., 2000b).

SOX genes, encoding high-mobility group (HMG) domain-containing transcription factors, have been identified as key players in numerous developmental processes including sex determination, neurogenesis, muscle differentiation, chondrogenesis, and endoderm specification (Wegner, 1999). Recently, Sox17 was shown to be necessary for the gut endoderm development (Kanai-Azuma et al., 2002). Among then, SOX9 was initially identified as the gene responsible for campomelic dysplasia (CD) syndrome, an autosomic dominant disease characterized by skeletal malformations associated with sex reversal (Cameron and Sinclair, 1997). Studies on SOX9 mainly focused on its role in skeletal and gonadal development. However, CD patients often display abnormalities in visceral organs and brain, suggesting a role of SOX9 in some aspects of the GI and central nervous system development. Indeed, dysmorphogenesis have been described in CD affected individuals in their GI tract, tracheopulmonary system, urinary tract, and heart (Maroteaux et al., 1971; Houston et al., 1983). GI tract anomalies are characterized by megacolon and intestinal malrotation (Houston et al., 1983). Due to the high mortality, only few studies have addressed GI diseases or malformations in CD patients (Piper et al., 2002).

In this study, we have investigated the expression and function of SOX9 in visceral pattern formation using the chick embryo as a model system. We found that SOX9 was expressed throughout the gut endoderm sparing the gizzard during both avian and human GI development. Mesodermal expression is mainly restricted to the pyloric sphincter mesoderm. We provide evidence that Sox9 expression in the pyloric sphincter structure is under control of the BMP signaling pathway. Ectopic expression of SOX9 through retroviral misexpression technique in the stomach mesoderm induces the transdifferentiation of the adjacent gizzard epithelium into a pyloric sphincter-like epithelium, whereas SOX9 loss-of-function expression in the pyloric mesoderm affects the differentiation of the pyloric epithelium. Taken together, our results show that SOX9 patterns the foregut/midgut boundary and is necessary and sufficient to induce the differentiation of the pyloric epithelium.

MATERIALS AND METHODS

Chick and Human embryos

Timed fertilized white Leghorn eggs (Haas Farm, France) were incubated at 38°C in a humidified incubator (Coudelou, France) until used experimentally. Staged-embryos (Hamburger and Hamilton, 1951) were harvested, washed in fresh PBS and then fixed in freshly made 4% paraformaldehyde in PBS for 2 hours. Fixed-embryos were washed in PBS and processed further either through a graded series of methanol-PBS to 100% methanol and kept at −20°C until used for whole-mount in situ hybridization studies, or frozen in cryomount (Fisher Scientific) for cryosectioning and immunostaining. Human embryonic tissues were obtained from surgical abortions as part of a program approved by both ethics committees from CNRS and French National Ethic Committee. Embryos were staged according to the Carnegie stages (O’Rahilly, 1983).

Immunohistochemistry and in situ hybridization on gastrointestinal system

Immunohistochemical stainings were performed on cryostat sections mainly as previously described (de Santa Barbara and Roberts, 2002b) using standard techniques and the Vectastain ABC detection system (Vector Laboratories, Inc, CA) following the manufacturer’s directions. Used anti-SOX9 (αSOX9) antibodies were raised against the transactivation (TA) domain of human SOX9 protein and used for immunohistochemistry analyses diluted 1:100 in TBST (de Santa Barbara et al., 1998; de Santa Barbara et al., 2000). A full characterization in chick both by Western blot and immunohistochemistry is available in Supplemental data. Anti-avian retroviral GAG protein (α3C2) antibodies were used as previously described (de Santa Barbara and Roberts, 2002b). Anti-HNK-1 (αHNK-1) antibodies were purchased from NeoMarkers and used diluted 1:400. These antibodies specifically recognize migrating neural crest-derived cells.

Antisense RNA probes were previously described for cBmp4 (Nielsen et al., 2001), cGremlin (Capdevila et al., 1999), cNkx2.5, cShh (Smith et al., 2000a), cSox8 (Bell et al., 2000), cSox9 (Healy et al., 1999), cSox10 (Cheng et al., 2000), cWnt11 (Theodosiou and Tabin, 2003), cPdx1 and cSox2 (Grapin-Botton et al., 2001). DIG labeled ribroprobes were made following manufacturer’s instructions (Roche). Whole-mount in situ hybridization experiments were performed using a standard protocol (Roberts et al., 1998). Cryosections at 10 μm onto Superfrost Plus slides (Fisher Scientific) were air dried for 4–18 hours and kept at −20°C until used. Fixed and stained embryos were embedded in paraffin and sectioned at 8 μm for histologic analysis. Haematoxylin and eosin staining was performed using standard techniques. In situ hybridization and immunohistochemistry on paraffin sections were performed as previously described (de Santa Barbara and Roberts, 2002b).

Constructs and viral infection in the chick gastrointestinal tract

The viral constructs that we used were previously described, including vectors transducing Bmp4 (Roberts et al., 1998), Noggin (Smith and Tabin, 1999), Nkx2.5 (Smith et al., 2000a), Gremlin (Capdevila et al., 1999) and GFP (de Santa Barbara and Roberts, 2002b).

New constructs were produced and characterized in this study (see Supplemental data). The full-length and C-terminal deleted (ΔCter) human SOX9 cDNAs were cloned into the shuttle vector Slax13 and then subcloned into RCAS(A) vector. Full-length and ΔCter SOX9 RCAS vectors were transfected into chick embryonic fibroblasts, and virus harvested and tittered using standard techniques (Morgan and Fekete, 1996). Only high infectious viruses were used in the experiments described. In order to target the presumptive stomach mesoderm, misexpression experiments were performed on stage 10 embryos according to the published fate map (Matsushita, 1995). Approximately 1–5 μl of freshly thawed virus, dyed with 1% fast green, were injected per embryo. Eggs were then placed at 38°C until harvested.

Photography

Images were collected in whole-mount under a Nikon SMZ1000 scope and in section under Zeiss Axiophot microscope, both using Nikon DXM1200 camera.

RESULTS

Spatial and temporal expression pattern of SOX9 in the developing GI system

To examine the expression pattern of Sox9 during chick GI development, we first performed whole-mount in situ hybridization on 5 days old (E5) dissected gut (stage 26–27). At this stage, Sox9 expression was detected at different sites of the GI system, in the stomach, the hindgut and derivate organs such as lung, and pancreas (Fig. 1A).

Fig. 1
Expression of Sox9 mRNA and SOX9 protein in 5 days old chick GI tract. (A) Whole-mount in situ hybridization of E5 dissected gut using antisens Sox9 riboprobe. (B1-F2) Paraffin cross-sections along AP axis as indicated in (A), stained with anti-SOX9 (B1,C1,D1,E1,F1) ...

To define more in details this complex expression pattern, we took advantage of anti-SOX9 antibodies that we previously developed and used on human and mouse embryo tissues (de Santa Barbara et al., 1998; de Santa Barbara et al., 2000; Gasca et al., 2002). We first confirmed the specificity of these antibodies on chick embryo extracts and tissues (see Supplementary data) and then examined the expression pattern of SOX9 during GI development. At E5, SOX9 expression was detected in different epithelia of the gut, at high levels in the esophagus (Fig. 1B1), hindgut (Fig. 1F1) and cloaca (data not shown), and at lower levels in the midgut epithelium where its expression is detected in isolated positive epithelial cells (insert, Fig. 1D1). In addition, SOX9 expression was strong in derivate organ epithelia: lung (Fig. 1B1), pancreas, and liver (data not shown). We also found discrete areas of SOX9 expression in the lung mesoderm that we were unable to detect by in situ hybridization (red arrow, Fig. 1B1). At E5, the development of the long bilateral caeca has just begun and SOX9 protein was detected in the endoderm, but also in the mesoderm of the caecal tips (red arrows, Fig. 1E1) and in the mesenteric structures (black arrow, Fig. 1E1). In addition, SOX9 expression was detected in the mesoderm of the posterior region of the stomach (Fig. 1A, C1). Since ENS cells colonize the mesodermal layer to assure gut innervation and at this stage of development are part of this layer, we analyzed whether or not mesodermal SOX9 expression co-localized with ENS cells by immunohistochemical analyses using anti-HNK-1 antibodies (Fig. 1B2–F2). The anti-HNK-1 antibodies specifically recognize migrating neural crest-derived cells. At E5, ENS cells are migrating along AP axis and are not yet organized into plexi (Fig. 1D2). SOX9 stainings detected in the lung, stomach and caeca mesoderm did not co-localize with HNK-1 (compare respectively Fig. 1B1, C1, E1 with B2, C2, E2). These data demonstrate that SOX9 is expressed at different level of the GI system, in the endoderm and in the mesoderm and that SOX9 expressing mesodermal cells are not ENS cells.

The function of SOX9 was recently shown to be dependent and regulated by its nuclear-cytoplasmic translocation in the developing gonad (de Santa Barbara et al., 2000; Gasca et al., 2002). Only nuclear staining of SOX9 protein was observed in the developing GI tract (Fig. 1B1–F1), whereas we observed SOX9 cytoplasmic expression in the developing chick gonad before sexual differentiation, suggesting that SOX9 regulation might be context-dependent (data not shown).

At E9 (stage 35), Sox9 expression was strongly detected in GI derivate organs (such as lung, pancreas and liver), in the hindgut and in the midgut (Fig. 2A). At this stage, SOX9 expression in the esophageal epithelium is high at the base of the villi and lower in the apex (Fig. 2B1). Strong nuclear expression of SOX9 protein was detected in the midgut and caeca epithelia (Fig. 2D1, E1), as well as in the hindgut and the cloaca epithelia (Fig. 2F1, G1) and the Fabricius bursa epithelium (data not shown). Mesodermal expression of SOX9 was observed at the caecal tips (Fig. 2E1). In chick, the stomach consists of two portions, the proventriculus (avian glandular stomach) and the gizzard (avian muscular stomach), which are distinct both morphologically and physiologically (Romanoff, 1960). In addition, the connection between the gizzard and the duodenum is demarcated by the pyloric sphincter. This structure is composed of mesodermal restriction that allows to maintaining food in the stomach and later to flowing gastric contents into the duodenum lumen. Sox9 expression was also detected in the stomach in a ring form demarcating the boundary between the stomach and the duodenum (Fig. 2A). SOX9 protein is present in the pyloric sphincter mesenchyme (Fig. 2C1). We also noticed that no co-localization between SOX9 and HNK-1 positives cells was observed (compare respectively Fig. 2C1 with C2).

Fig. 2
Expression of Sox9 mRNA and SOX9 protein in 9 days old chick GI tract. (A) Whole-mount in situ hybridization of E9 dissected gut using antisens Sox9 riboprobe. Paraffin cross-sections along AP axis as indicated in (A), stained with anti-SOX9 (B1,C1,D1,E1,F1) ...

In order to determine whether SOX9 expression in the GI system is conserved in human, we examined the expression of SOX9 protein in human embryonic tissues. At 7.5 weeks gestational age, SOX9 expression was similar to that seen in the chick. Epithelial SOX9 expression was detected in the lung, pancreas, small intestine, and rectum (respectively, Fig. 3A,B,D,E) and mesodermal SOX9 expression in the posterior region of the stomach at the pyloric area (red arrow, Fig. 3C). Our results also indicate that SOX9 expression changes during the differentiation state of the small intestine epithelium; its expression is restricted at the proliferative compartment of the villi, suggesting potential function for SOX9 during the epithelial differentiation process (red arrow, Fig. 3F).

Fig. 3
SOX9 expression in human embryo GI system. (A–E) Immunohistochemistry with anti-SOX9 antibodies on paraffin sections from a 7.5 weeks old human embryo. (A) Lung. (B) Pancreas. (C) Posterior region of the stomach. (D) Small intestine. (E) Rectum. ...

Taken together, our data indicate that SOX9 exhibits a restricted expression pattern in the GI system. SOX9 is expressed in different GI epithelia and in the mesoderm of the pyloric sphincter in vertebrates.

E subgroup Sox gene expression in the chick stomach

SOX proteins constitute a large family of transcription factors characterized by the presence of a HMG domain. Sequence homology outside of HMG domains allowed distinguishing 8 different groups (Schepers et al., 2002). Sox9 belongs to the E subgroup and shares strong homology with Sox8 and Sox10, the other members of this subgroup. In addition, overlapping functions and expressions of these E subgroup genes have been previously described (Montero et al., 2002; Schmidt et al., 2003).

In order to determine whether stomachal SOX9 expression is a common feature of all E subgroup Sox members or whether this expression pattern is specific to SOX9, we performed in situ hybridization with Sox8, Sox9 and Sox10 riboprobes on chick E7 stomach. Expression of all 3 genes was detected in the stomach, with specific expression patterns (Fig. 4A–C). Sox8 expression was detected in the pancreas and weak expression was also observed in ENS cells (Fig. 4B). Sox10 expression was restricted to ENS cells (Fig. 4C) and Sox9 expression was exclusively observed in the pyloric sphincter mesoderm (Fig. 4A). All together, our results demonstrate that SOX9 is the only E subgroup SOX member expressed in the pyloric sphincter mesoderm in vertebrates.

Fig. 4
Expression of E subgroup Sox genes, Bmp4 and Nkx.2.5 in 7 days old chick stomach. (A) Whole-mount in situ hybridization of E7 dissected gut using antisens Sox9 riboprobe. Note that Sox9 expression strongly demarcates the stomach from the duodenum and ...

BMP signaling regulates Sox9 expression in the stomach

Previous studies showed that BMP signaling pathway is necessary for normal stomach development (Smith et al., 1999). Bmp4 is expressed in the mesenchyme of the whole gut with the exception of the gizzard (Fig. 4D). Bmp4 regulates both muscular thickness and pyloric sphincter specification (Roberts et al., 1998; Smith et al., 1999). Bmp4 function is mediated in part by the homeobox-containing gene Nkx2.5, which is expressed in the pyloric mesoderm (Fig. 4E) and is necessary and sufficient to control epithelial pyloric differentiation through mesenchymal-epithelial interactions (Smith et al., 2000a).

Then overlapping expression patterns in the stomach suggest a potential connection between BMP signaling pathway and SOX9. We thus investigated whether BMP pathway could regulate Sox9 expression. We used the avian retroviral system to specifically misexpress Bmp4, Nkx2.5, and the BMP-antagonist Noggin in the stomachal mesoderm (Roberts et al., 1998; Nielsen et al., 2001) and monitored Sox9 expression by in situ hybridization experiments (Fig. 5). GFP misexpression was used as control. Retrovirus presence in the analyzed stomach was confirmed by immunohistochemistry using antibodies directed against the avian retroviral GAG protein (α3C2) (data not shown). As previously reported, Bmp4 misexpression in the stomach induced a gross phenotype characterized by a small gizzard with thin musculature (Roberts et al., 1998), whereas the morphology of the pyloric sphincter was normal (Fig. 5B). However, we observed a broad domain of Sox9 expression that extends from the pyloric sphincter to the right part of the gizzard (double red arrows, Fig. 5B), suggesting that activation of BMP signaling pathway in the stomachal mesoderm upregulates Sox9 expression. Examination of cryostat sections also indicated that not all stomachal cells are expressing SOX9 in response to Bmp4 misexpression, suggesting that only few competent cells are able to respond to BMP4 activation (red arrowheads, Figs. 5F). In similar experiments, Smith and Tabin (1999) reported an extension of the expression domain of Nkx.2.5 in the stomach in response to Bmp4 misexpression. As previously described (Smith and Tabin, 1999), we observed that Nkx2.5 misexpression in the stomachal region did not affect the morphology of the stomach (Fig. 5C) and whole-mount in situ hybridization analyses revealed that Sox9 expression pattern was not affected in Nkx2.5-misexpressing stomach (Fig. 5C). Misexpression of Noggin, a specific secreted antagonist of the BMP signaling pathway, in the stomach induced the predicted phenotype of muscular hypertrophy (compare Fig. 5D, E with B). E8 Noggin-misexpressing stomachs develop a range of phenotypes from moderate to severe (Fig. 5D, E). Moderate phenotype was associated with a mild foregut/midgut boundary perturbation associated with down-regulation of Sox9 expression (compare Fig. 5D with A). Severe phenotype was marked by an increase of the muscular mass associated with pyloric morphologic defect, and correlated with the inhibition of Sox9 expression in the malformed pyloric structure (compare, Fig. 5E with A).

Fig. 5
Modulation of the BMP signaling pathway in the stomachal mesoderm affects Sox9 expression. Whole-mount in situ hybridization using antisens Sox9 riboprobe upon GFP (A), Bmp4 (B), Nkx.2.5 (C), and Noggin (D,E) misexpressing E8 stomach. Note morphological ...

Taken together, these results demonstrate that the BMP pathway regulates Sox9 expression in the pyloric sphincter.

SOX9 is necessary and sufficient to specify the pyloric sphincter epithelium

In order to investigate more directly the role of SOX9 in pyloric sphincter development, we used the avian retroviral system to specifically misexpress full-length SOX9. Anterior misexpression of SOX9 into the gizzard mesoderm did not lead to morphological change, but histological analyses demonstrated an effect of SOX9 on gizzard epithelium (Fig. 6C). In addition to morphological and mesodermal differences, the gizzard and the pyloric sphincter present different epithelial specificities. Indeed the gizzard epithelium harbors cells with keratin long cilia allowing resistance to the abrasive grinding (Fig. 6A), and the pyloric sphincter presents epithelial cells with bulbous cilia (Fig. 6B). SOX9 misexpression in the mesoderm of the gizzard modified the adjacent epithelium properties, as this epithelium had pyloric sphincter-like features showing bleb like cilia at the epithelial level (compare Fig. 6C with A). This epithelial transformation was not due to the expression of ectopic SOX9 in the gizzard epithelium, since α3C2 detection demonstrated that retroviral infection was restricted to the gizzard mesodermal (insert, Fig. 6C). In order to characterize the epithelial transformation observed upon SOX9 misexpression, we analyzed by in situ hybridization the expression of different endodermal markers (such as Shh, Sox2, Gata4 and Pdx1). Shh is a general marker of the gut endoderm (Roberts et al., 1995), Sox2 and Gata4 are expressed in the stomach endoderm (Ishii et al., 1998, unpublished data). Pdx1 is a marker of the duodenum epithelium, the pancreas epithelium and the pyloric gland, but Pdx1 expression is not present in the stomach epithelium (Grapin-Botton and Melton, 2000; Fig. 6E). PDX1 expression is also associated to the abnormal presence of pseudopyloric glands in human patient stomach (Sakai et al., 2004). SOX9 misexpression in the stomach did not modify the expression of Shh, Sox2 and Gata4 markers (data not shown). However, in SOX9 misexpressing stomach, we observed an ectopic expression of Pdx1 in the gizzard epithelium, indicating that the transformed gizzard epithelium exhibited characteristics of pyloric epithelium (red arrowheads, Fig. 6F).

Fig. 6
Misexpression of SOX9 in the stomachal mesoderm specifies the stomachal epithelium into pyloric epithelium. Histological sections of control E9 stomach (A,B), SOX9 (C) and SOX9ΔCter (D) misexpressing E9 stomachs. Immunohistochemistry with α3C2 ...

We also used the avian retroviral system to specifically a mutant form of SOX9 deleted of this C-terminal domain (SOX9ΔCter). This deleted mutant form is the one found in human campomelic patients (Sudbeck et al., 1996). We and others showed previously that this mutated form was present in the nuclear compartment of the cell, able to bind DNA, yet unable to activate transcription and finally able to form heterodimer complex with endogenous SOX9 protein (de Santa Barbara et al., 1998; Bernard et al., 2003; data not shown). This suggests that this deleted form might behave as a dominant negative of SOX9 action. Misexpression of SOX9ΔCter in the mesodermal stomach showed no phenotypic modification of mesoderm, but clear perturbation of the pyloric epithelium (Fig. 6D). SOX9ΔCter misexpressing stomach showed gizzard-like features with keratin long cilia at the pyloric epithelial level (compare Fig. 6D with B). In addition and by contrast with the full-length SOX9 misexpression, no obvious epithelial phenotype was present in the gizzard epithelium, suggesting that the transactivation domain of SOX9 is required to transdifferentiate the stomachal epithelium. SOX9ΔCter misexpression in the stomach did not modify the expression of Shh, Sox2 and Gata4 markers (data not shown). However, in SOX9ΔCter misexpressing stomach, we observed a decrease of epithelial Pdx1 expression in the infected pyloric sphincter, indicating that the transformed pyloric epithelium lost pyloric epithelium features (Fig. 6G).

All together, our data show that the transcription factor SOX9 is necessary and sufficient to specify the pyloric sphincter epithelium through mesenchymal-epithelial signals.

Gremlin mediated SOX9 mesenchymal-epithelial function in the pyloric sphincter

Our results show that SOX9, which is expressed in the mesoderm of the pyloric sphincter, specifies the adjacent epithelium. As SOX9 is a transcription factor, we hypothesized that it may regulate the expression of diffusible ligands, such as Wnts, BMPs or their related inhibitors, which were described to be essential to establish pyloric epithelium phenotype through mesenchymal-epithelial interaction (Theadosiou and Tabin, 2003; Smith et al., 2000a). Wnt11 expression, which is restricted to the mesenchymal pyloric sphincter, was not affected in SOX9 and SOX9ΔCter misexpressing stomach (data not shown). We observed that Gremlin, a modulator of the BMP pathway, was expressed in the mesenchymal pyloric sphincter (Fig. 7A, C). Anterior misexpression of Gremlin in the mesenchymal stomach induced no obvious morphological phenotype, but, as SOX9, an epithelial phenotype characterized by the presence of pyloric bleb like cells in the gizzard epithelium (compare Figs. 7B with with6A).6A). These data suggested that Gremlin might be a potential candidate for a downstream gene regulated by SOX9 in the pyloric sphincter. To test this hypothesis, SOX9 and SOX9ΔCter were misexpressed in the stomach, and Gremlin expression was monitored by in situ hybridization (Fig 7C, D, E). We observed Gremlin ectopic expression in the mesenchymal gizzard with full-length SOX9 (red arrowheads, Fig. 7D), which was not detected in SOX9ΔCter misexpressing stomach (Fig. 7E). Nevertheless, we observed a decrease of mesodermal Gremlin expression in the pyloric structure upon SOX9ΔCter misexpression (black arrowheads, Fig. 7E). Finally, we found that Sox9 expression was not affected in Gremlin misexpressing stomach (data not shown).

Fig. 7
Gremlin modulation of mesenchymal-epithelial interaction in the pyloric sphincter is under SOX9 control. (A) Whole-mount in situ hybridization of E7 dissected gut using antisens Gremlin riboprobe. Note that Gremlin expression strongly demarcates the stomach ...

All these data point to a function of SOX9 in the stomach in specifying the pyloric sphincter epithelium through its expression in the adjacent mesenchyme, and suggest that SOX9 might function in part by modulating Gremlin expression.

DISCUSSION

SOX9 expression in the gastrointestinal system is conserved during evolution

In early studies, SOX9 expression has been mainly observed chondrocytes, neural crest cells and genital ridges (Healy et al., 1999; Spokony et al., 2002; de Santa Barbara et al., 2000). Here, we found that SOX9 was specifically expressed in some restricted areas during GI tract development in chick (summarized in Table 1). SOX9 is present throughout the gut endoderm with the exception of the gizzard endoderm (Fig. 1). We also observed expression of SOX9 in the endoderm of organs derived from the gut tube, the pancreas, liver, and the lung (Figs. 1,,2).2). SOX9 expression is present in the gut endoderm from early stage of development and is maintained until adulthood (data not shown). Since SOX9 is later expressed in the proliferative compartment of the villi (Fig. 3), we might hypothesize a role for SOX9 in stem cell maintenance. In our study, we also observed mesodermal expression of SOX9 in the pyloric sphincter structure (Figs. 1, ,2).2). The pyloric sphincter is an anatomical sphincter that allows food to be grinded in the stomach before flowing in the small intestine. SOX9 strongly demarcates the nascent boundary between foregut/midgut (Fig. 1) and its early expression pattern let us hypothesize a function of SOX9 in the establishment of this boundary (Fig. 2). We also demonstrated that SOX9 expression in the pyloric sphincter is a specific feature of SOX9 and not a common propriety of the E subgroup SOX factors (Fig. 4). This pattern of SOX9 expression in chick GI system is very similar in human and mouse (compare Figs. 1,,22 with with3,3, and data not shown). In addition, Sox100B, the Drosophila gene related to vertebrate SOX9, is expressed in the early hindgut, late midgut endodermal cells, and the anal plates (Hui Yong Loh and Russell, 2000). Thus, SOX9 expression pattern in the developing gut was conserved during evolution, suggesting that SOX9 might exhibit similar functions in this tissue in vertebrates and invertebrates.

TABLE 1
Summary of SOX9 sites of expression in the developing chick GI tract

SOX9 specifies the pyloric sphincter epithelium

These last years, chick embryo has been widely used as a model to study the function of transcription factors mainly expressed in the mesoderm layer during GI tract development (Roberts et al., 1998; Nielsen et al., 2001). In order to investigate the function of SOX9 during the formation and the development of the pyloric sphincter, we decided to use the specific avian virus mediated gene expression technique which we previously showed to be useful in targeting different part of the GI tract and expressing a transgene into the stomach mesoderm (Smith et al., 2000b; Nielsen et al., 2001). We showed that mesodermal gizzard SOX9 misexpression was sufficient to induce the transformation of the gizzard epithelium into pyloric sphincter like epithelium (Fig. 6). When we used virus expressing a mutant form of SOX9, SOX9 deleted of the C-terminal domain (SOX9ΔCter), we observed perturbations in the differentiation of the pyloric epithelium and gizzard like epithelium phenotype (Fig. 6). All these epithelial changes were associated with perturbations of an epithelial pyloric Pdx1 marker expression, strongly suggesting that SOX9 specifies the pyloric sphincter epithelium (Fig. 6). Furthermore, we proved that this effect was mediated by an indirect mechanism using transcriptional regulation of diffusible factors expressed specifically into the pyloric sphincter mesoderm, acting by diffusion to target the pyloric sphincter epithelium (see below).

Relationship between SOX9 and BMP signaling pathway during the specification of the pyloric sphincter epithelium

Establishment and differentiation of the foregut/midgut boundary involved a highly molecular regulated process as Bmp4 (Smith and Tabin, 1999) and Nkx2.5 (Smith and Tabin, 1999; Smith et al., 2000a). Bmp4 activates Nkx2.5 and alter the epithelial differentiation to a pyloric sphincter type (Smith and Tabin, 1999; Smith et al., 2000a). We showed herein that activation and inhibition of BMP signaling pathway by viral misexpression modulated Sox9 expression in the stomach (Fig. 5). Stomachal viral misexpression of Nkx2.5 leads to epithelial phenotype very similar to SOX9 misexpression (compare (Smith et al., 2000b) and Fig. 6). Interestingly, misexpression of SOX9 and SOX9ΔCter in the stomach had no effect on Bmp4 or Nkx2.5 expression (data not shown), suggesting that SOX9 does not regulate the expression of these 2 genes. We propose the following model, in which BMP signaling pathway activates both expressions of Sox9 and Nkx2.5 in the pyloric sphincter (Fig. 8). These two transcription factors could act independently or could interact to specify the pyloric sphincter structure. In support of this, genetic interactions between SOX and NK2 family genes was reported in Drosophila to promote neuroblat formation (Zhao and Skeath, 2002).,

Fig. 8
Model of the molecular pathways and their potential interactions involved during the development of the pyloric sphincter. Schematic representations of avian stomach (left panel) and the molecular pathways involved (right panel). The avian stomach can ...

SOX9 action in this mesenchymal-epithelial mechanism was investigated deeper in order to identify the signal activated by SOX9 in the pyloric mesenchyme and responsible to the pyloric epithelium differentiation. We identified Gremlin as a target gene of SOX9 in this process (Fig. 7). During chondrogenesis, the BMP pathway induces Gremlin expression and Gremlin modulates the BMP activation (Capdevilla et al., 1999). In addition, BMP activation is tightly controlled in the pyloric sphincter epithelium (our unpublished observations). Gremlin could act in this structure to diminish and modulate the BMP activity induces by BMP4; it is noteworthy that pyloric epithelium patterning needs low levels of BMP activity whereas the gizzard is associated with the absence of BMP activity (Fig. 8). Cross-regulatory interactions between BMP signaling pathway and SOX factors might be a reiterated process during development, since it was also demonstrated in vertebrate limb (Chimal-Monroy et al., 2003) and in Drosophila central nervous system development (Cremazy et al., 2000).

In summary, our work revealed new functions of the transcription factor SOX9 during the GI tract development. SOX9 patterns the pyloric sphincter and specifies the pyloric sphincter epithelium through regulation of Gremlin, a diffusible factor modulating BMP pathway. As we previously commented, molecular controls of GI development patterning events are remarkably conserved across species. We might hypothesize that the described molecular controls of pyloric sphincter formation are conserved in human and that alterations in the normal molecular controls of this region are associated with malformations such as hypertrophic pyloric stenosis (Ohshiro and Puri, 1998). Expressional analyzes of these factors on human fetal normal and malformed pyloric sphincter samples will be performed in order to address this hypothesis.

Supplementary Material

Acknowledgments

The authors thank the members of Roberts Laboratory and Franck Girard (IGH, France) for fruitful discussions. The authors are grateful to Patrick Atger for artwork. Thanks to Alexandre and Hugo de Santa Barbara for constant support. D.J.R. is supported by NIH HD39942 and HD34448. P.d.S.B. is supported in part by a postdoctoral fellowship from the Association pour la Recherche sur le Cancer (ARC). We acknowledge the financial support of CNRS, ARC (grant N°4643) and the Fondation pour la Recherche Médicale, Languedoc-Roussillon-Rouergue.

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