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First published online 7 July 2004
doi: 10.1242/dev.01259

Development 131, 3795-3804 (2004)
Published by The Company of Biologists 2004
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SOX9 specifies the pyloric sphincter epithelium through mesenchymal-epithelial signals

Brigitte Moniot1,*, Sandrine Biau1,*, Sandrine Faure2, Corinne M. Nielsen3, Philippe Berta1, Drucilla J. Roberts3 and Pascal de Santa Barbara1,{dagger}

1 Institut de Génétique Humaine, UPR 1142 CNRS, 141 rue de la Cardonille, 34396 Montpellier, Cedex 5, France
2 Centre de Recherche en Biologie Macromoléculaire, CNRS FRE 2593, 1919 Route de Mende, 34293 Montpellier, Cedex 5, France
3 Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Fruit Street, Boston, MA 02114, USA



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  		Fig. 1. Expression of Sox9 mRNA and SOX9 protein in 5-day-old (E5) chick GI tract. (A) Whole-mount in situ hybridization of E5 dissected gut using antisense Sox9 riboprobe. (B1-F2) Paraffin cross-sections along AP axis as indicated in A, stained with anti-SOX9 (B1,C1,D1,E1,F1) or anti-HNK1 (B2,C2,D2,E2,F2) antibodies. Cross-sections of the gut were taken at the following levels: esophagus and lung bud (B1,B2), posterior region of the stomach (C1,C2), midgut anterior to the umbilicus (D1,D2), caeca (E1,E2) and hindgut (F1,F2). Note SOX9 expression in the mesoderm of the posterior region of the stomach (C1). Mesodermal SOX9 expression is also detected in the lung and caeca (red arrows, B1,E1). SOX9 is strongly expressed in the endoderm of the esophagus, lung, pancreas and hindgut (A,B1,E1). Some SOX9-positive cells are present in the midgut epithelium (insert, D1). SOX9 expression is observed in the mesentery (black arrow, E1), but not in the Remark's nerve, which is essentially composed of neural crest-derived cells as shown by anti-HNK1 antibodies (E2). cae, caeca; cl, cloaca; eso, esophagus; gizz, gizzard; hg, hindgut; mg, midgut; panc, pancreas; pyl, pyloric sphincter; RN, Remark's nerve; sto, stomach.

 


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  		Fig. 2. Expression of Sox9 mRNA and SOX9 protein in 9-day-old (E9) chick GI tract. (A) Whole-mount in situ hybridization of E9 dissected gut using antisense Sox9 riboprobe. Paraffin cross-sections along AP axis as indicated in A, stained with anti-SOX9 (B1,C1,D1,E1,F1) or anti-HNK1 (B2,C2,D2,E2,F2) antibodies. Cross-sections of the gut were done at the following levels: esophagus (B1,B2), pyloric sphincter (C1,C2), midgut anterior to the umbilicus (D1,D2), caeca (E1,E2), hindgut (F1,F2) and cloaca chamber (G1,G2). Note that mesodermal SOX9 expression strongly demarcates the pyloric sphincter (A,C1). SOX9 is strongly expressed in the epithelia of the esophagus, the small intestine and the cloaca chamber (B1,D1,G1), but faintly in the hindgut (F1). Abbreviations: an, anus; cae, caeca; cl, cloaca; eso, esophagus; gizz, gizzard; hg, hindgut; liv, liver; mg, midgut; panc, pancreas; pv, proventriculus; pyl, pyloric sphincter.

 


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  		Fig. 3. SOX9 expression in human embryo GI system. (A-E) Immunohistochemistry analyses using anti-SOX9 antibodies on paraffin sections from a 7.5-week-old (7.5 w) human embryo. (A) Lung. (B) Pancreas. (C) Posterior region of the stomach. (D) Small intestine. (E) Rectum. Expression of SOX9 protein is found in human viscera epithelial layers and there is exclusive mesodermal expression of SOX9 protein at the pyloric level in the stomach (red arrow, C). (F) Immunohistochemistry with anti-SOX9 antibodies on paraffin wax sections from prenatal human small intestine (22 w). Note the expression of SOX9 in the proliferative compartment of the villi (red arrow, F). mg, midgut; panc, pancreas; prenat, prenatal; pyl, pyloric structure; rect, rectum; si, small intestine; sto, stomach.

 


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  		Fig. 4. Expression of E subgroup Sox genes, Bmp4 and Nkx2.5 in 7-day-old (E7) chick stomach. (A) Whole-mount in situ hybridization of E7 dissected gut using antisense Sox9 riboprobe. Sox9 expression strongly demarcates the stomach from the duodenum and is only observed in the mesoderm of the pyloric sphincter (red arrow, A). (B,C) Whole-mount in situ hybridization of E7 dissected gut, using antisense Sox8 (B) and Sox10 (C) riboprobes. Sox8 and Sox10 expression in the stomach is restricted to ENS cells (B,C). (D,E) Whole-mount in situ hybridization of E7 dissected gut using antisense Bmp4 (D) and Nkx2.5 (E) riboprobes. Note that Bmp4 is expressed in the mesenchyme of the duodenum and the pylore (red arrow, D), but not in the gizzard. Nkx2.5 expression is observed only in the pyloric mesenchyme (red arrow, E). gizz, gizzard; pv, proventriculus; pyl, pyloric sphincter.

 


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  		Fig. 5. Modulation of the BMP signaling pathway in the stomach affects Sox9 expression. Whole-mount in situ hybridization using antisense Sox9 riboprobe on GFP- (A), Bmp4- (B), Nkx2.5- (C) and Noggin- (D,E) misexpressing E8 stomachs. The morphological change of Bmp4-misexpressing stomach is characterized by a reduced musculature of the gizzard (compare B with A). Noggin misexpression in the stomach gives rise to a range of phenotypes (D,E). Moderate phenotype present proventriculus fate change with gland formation inhibition and size increase (D). Severe Noggin phenotype is mainly characterized by stomach/duodenum connection defect and gizzard-like phenotype found in the whole stomach (E). Neither GFP (A) as control nor Nkx2.5 (C) misexpression affects stomach morphology. Sox9 expression is upregulated in Bmp4-misexpressing stomach (B), strongly downregulated in Noggin-misexpressing stomachs (D,E) and not affected in Nkx2.5- (C) or GFP- (A) misexpressing stomachs. Red and black arrows indicate pyloric sphincter area. (F) Bmp4-misexpressing stomach was sectioned and probed with anti-SOX9 antibodies and revealed SOX9 ectopic expression in the gizzard (red arrowheads, F).

 


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  		Fig. 6. Misexpression of SOX9 in the gizzard mesoderm specifies the gizzard epithelium into pyloric epithelium. Histological sections of control E9 stomach (A,B), SOX9- (C) and SOX9{Delta}Cter- (D) misexpressing E9 stomachs. Immunohistochemistry analyses with {alpha}3C2 antibodies confirmed that the infection was restricted to the mesoderm of the stomach (inset in C and D) and clearly excluded from the endoderm. Control gizzard epithelium cells present keratin long cilia (A). Control pyloric sphincter epithelium cells are characterized by bulbous cilia (B). Upon mesodermal SOX9 misexpression, the gizzard epithelial cells present bleb-like cilia (C), while after mesodermal SOX9{Delta}Cter misexpression, the pyloric epithelial cells present keratin long like cilia (D). (E-G) Analyses of Pdx1 expression by in situ hybridization on section of GFP- (E), SOX9- (F) and SOX9{Delta}Cter- (G) misexpressing stomachs using antisense Pdx1 riboprobe. There is normal expression of Pdx1 in the pyloric endoderm. Ectopic Pdx1 expression is detected in SOX9-misexpressing gizzard epithelium (red arrowheads, F). In SOX9{Delta}Cter-misexpressing stomach, no Pdx1 expression is detected in the gizzard epithelium and downregulation of Pdx1 is observed in the pyloric epithelium (G). Note the expression of Pdx1 in the pancreas in these SOX9{Delta}Cter-misexpressing stomach (inset, G). duo, duodenum; e, endoderm; gizz, gizzard; m, mesoderm; panc, pancreas; pyl, pyloric structure.

 


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  		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 antisense Gremlin riboprobe. Gremlin expression strongly demarcates the stomach from the duodenum and is present in the mesoderm of the pyloric sphincter (red arrow, A). (B) Histological section of Gremlin-misexpressing E9 gizzard. After mesodermal Gremlin misexpression, the gizzard epithelial cells present bleb like cilia (B). Immunohistochemistry with {alpha}3C2 antibodies show retrovirus infection restricted to the mesoderm of the gizzard (upper inset B). GFP- (C), SOX9- (D) and SOX9{Delta}Cter- (E) misexpressing E7 stomach sections followed by detection of Gremlin expression by in situ hybridization using antisense Gremlin riboprobe. Gremlin is expressed in the mesoderm and adventitia of the pyloric sphincter (C). Ectopic expression of Gremlin in the mesodermal stomach is observed after retroviral SOX9 misexpression in the gizzard (red arrowheads, D). Downregulation of Gremlin expression is correlated with retroviral SOX9{Delta}Cter misexpression in the pyloric sphincter (black arrowheads, E), while Gremlin expression is normal in the adventitia. duo, duodenum; e, endoderm; gizz, gizzard; m, mesoderm; pv, proventriculus; pyl, pyloric structure.

 


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  		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 be divided in proventriculus (glandular stomach) and gizzard (muscular stomach). The pyloric sphincter is a highly conserved structure present in all vertebrates, which (in avians) anatomically separates the gizzard from the duodenum. Shh from epithelium induces Bmp4 expression in the adjacent mesenchyme, except in the gizzard where Bapx1 prevents Bmp4 expression. In the small intestine, Bmp4 activates the BMP signaling pathway in the mesoderm and endoderm (S.F. and P.d.S.B., unpublished). In the pyloric sphincter, Bmp4 is able to activate the expressions of Nkx2.5 and Sox9, which are both sufficient to induce pyloric epithelial phenotype through mesenchymal-epithelial signal modulation. Importantly, our data show that there is no cross-regulation between Sox9 and Nkx2.5 at the transcriptional level. SOX9 is able to control Gremlin expression in the pyloric sphincter mesenchyme. Gremlin, a diffusible factor, could modulate endodermal BMP pathway activation, in order to induce specific pyloric epithelium differentiation.

 

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© The Company of Biologists Ltd 2004