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• W2013351311 abstract "Background & Aims: The transcription factor SOX9 has been shown previously to have an essential role in the differentiation of a small number of discrete cell lineages. In the intestine, Sox9 is expressed in the epithelial cells of the crypts and is a target of Wnt signaling. Methods: To examine the function of SOX9 in the intestine, we inactivated the Sox9 gene in intestinal epithelial cells by generating mice that harbored a conditional Sox9 gene and a Villin-Cre transgene. Results: In the absence of SOX9, Paneth cells were not formed, but the differentiation of other intestinal epithelial cell types was unaffected. The lack of SOX9 also lead to crypt enlargement, to a marked increase in cell proliferation throughout the crypts, and to replacement of the Paneth cells by proliferating epithelial cells. Conclusions: We conclude that SOX9 is required for the differentiation of Paneth cells. Our results elucidate an essential step in the differentiation of gut epithelium. Background & Aims: The transcription factor SOX9 has been shown previously to have an essential role in the differentiation of a small number of discrete cell lineages. In the intestine, Sox9 is expressed in the epithelial cells of the crypts and is a target of Wnt signaling. Methods: To examine the function of SOX9 in the intestine, we inactivated the Sox9 gene in intestinal epithelial cells by generating mice that harbored a conditional Sox9 gene and a Villin-Cre transgene. Results: In the absence of SOX9, Paneth cells were not formed, but the differentiation of other intestinal epithelial cell types was unaffected. The lack of SOX9 also lead to crypt enlargement, to a marked increase in cell proliferation throughout the crypts, and to replacement of the Paneth cells by proliferating epithelial cells. Conclusions: We conclude that SOX9 is required for the differentiation of Paneth cells. Our results elucidate an essential step in the differentiation of gut epithelium. See editorial on page 710. See editorial on page 710. The inner surface of the small intestine is composed of crypts and villi and is covered by a single layer of epithelial cells. The upper two thirds of the crypts is occupied by transiently proliferating cells, which upon exiting the cell cycle undergo differentiation into four different cell types, namely enterocytes, which are the absorptive cells, and three secretary cell types, mucin-secreting goblet cells, hormone-secreting enteroendocrine cells, and antimicrobial agents- secreting Paneth cells. Enterocytes, goblet cells and enteroendocrine cells migrate upwards from the crypts onto the villi to be subsequently shed in the lumen of the gut, whereas Paneth cells migrate towards the bottom of the crypts. Each crypt also contains a small number of stem cells located at position +4 above the Paneth cells. These stem cells are needed for the regeneration of the transient proliferative compartment and hence the differentiation of the epithelial cells.1Reya T. Clevers H. Wnt signalling in stem cells and cancer.Nature. 2005; 434: 843-850Crossref PubMed Scopus (2966) Google Scholar, 2Mills J.C. Gordon J.I. The intestinal stem cell niche: there grows the neighborhood.Proc Natl Acad Sci U S A. 2001; 98: 12334-12336Crossref PubMed Scopus (90) Google Scholar, 3Bach S.P. Renehan A.G. Potten C.S. Stem cells: the intestinal stem cell as a paradigm.Carcinogenesis. 2000; 21: 469-476Crossref PubMed Scopus (268) Google Scholar Terminal differentiation of Paneth cells, which occurs beginning around P11 to P14, requires Wnt signaling through the Wnt receptor Frizzled-5.4van Es J.H. Jay P. Gregorieff A. van Gijn M.E. Jonkheer S. Hatzis P. Thiele A. van den Born M. Begthel H. Brabletz T. Taketo M.M. Clevers H. Wnt signalling induces maturation of Paneth cells in intestinal crypts.Nat Cell Biol. 2005; 7: 381-386Crossref PubMed Scopus (487) Google Scholar The essential event in the Wnt signaling cascade is the accumulation and nuclear translocation of β-catenin, which then interacts with members of the Lef/Tcf family of transcription factors to activate target genes.1Reya T. Clevers H. Wnt signalling in stem cells and cancer.Nature. 2005; 434: 843-850Crossref PubMed Scopus (2966) Google Scholar One of the Wnt/Tcf4 target genes in the intestine is Sox9, which is expressed in the intestinal crypts.5Blache P. van de Wetering M. Duluc I. Domon C. Berta P. Freund J.N. Clevers H. Jay P. SOX9 is an intestine crypt transcription factor, is regulated by the Wnt pathway, and represses the CDX2 and MUC2 genes.J Cell Biol. 2004; 166: 37-47Crossref PubMed Scopus (364) Google Scholar, 6Jay P. Berta P. Blache P. Expression of the carcinoembryonic antigen gene is inhibited by SOX9 in human colon carcinoma cells.Cancer Res. 2005; 65: 2193-2198Crossref PubMed Scopus (57) Google Scholar SOX9 is a member of the SOX family of transcription factors and, similar to other members of this family, it contains a DNA binding domain that shows sequence homology to the testis-determining factor SRY.7Wright E.M. Snopek B. Koopman P. Seven new members of the Sox gene family expressed during mouse development.Nucleic Acids Res. 1993; 21: 744Crossref PubMed Scopus (191) Google Scholar SOX9 is essential for the differentiation of a small number of cell lineages and appears to control a different repertoire of genes in each of these cell types.8Akiyama H. Chaboissier M.C. Behringer R.R. Rowitch D.H. Schedl A. Epstein J.A. de Crombrugghe B. Essential role of Sox9 in the pathway that controls formation of cardiac valves and septa.Proc Natl Acad Sci U S A. 2004; 101: 6502-6507Crossref PubMed Scopus (202) Google Scholar, 9Mori-Akiyama Y. Akiyama H. Rowitch D.H. de Crombrugghe B. Sox9 is required for determination of the chondrogenic cell lineage in the cranial neural crest.Proc Natl Acad Sci U S A. 2003; 100: 9360-9365Crossref PubMed Scopus (321) Google Scholar, 10Chaboissier M.C. Kobayashi A. Vidal V.I. Lutzkendorf S. van de Kant H.J. Wegner M. de Rooij D.G. Behringer R.R. Schedl A. Functional analysis of Sox8 and Sox9 during sex determination in the mouse.Development. 2004; 131: 1891-1901Crossref PubMed Scopus (449) Google Scholar, 11Stolt C.C. Lommes P. Sock E. Chaboissier M.C. Schedl A. Wegner M. The Sox9 transcription factor determines glial fate choice in the developing spinal cord.Genes Dev. 2003; 17: 1677-1689Crossref PubMed Scopus (454) Google Scholar, 12Bi W. Deng J.M. Zhang Z. Behringer R.R. de Crombrugghe B. Sox9 is required for cartilage formation.Nat Genet. 1999; 22: 85-89Crossref PubMed Scopus (1361) Google Scholar, 13Akiyama H. Chaboissier M.C. Martin J.F. Schedl A. de Crombrugghe B. The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6.Genes Dev. 2002; 16: 2813-2828Crossref PubMed Scopus (1329) Google Scholar To determine whether SOX9 has a role in the intestine, we inactivated the Sox9 gene in the intestinal epithelial cells in mice by using the Cre-LoxP system. We report here that in the absence of SOX9, Paneth cells do not form. The lack of SOX9 also leads to increased crypt dimensions and enhanced proliferation in the crypts. Mice carrying Sox9flox, a Sox9 allele in which the DNA segment that includes exons 2 and 3 is flanked by loxP sites, were a generous gift from Andreas Schedl and Marie-Christine Chaboissier (University of Nice, Nice, France).10Chaboissier M.C. Kobayashi A. Vidal V.I. Lutzkendorf S. van de Kant H.J. Wegner M. de Rooij D.G. Behringer R.R. Schedl A. Functional analysis of Sox8 and Sox9 during sex determination in the mouse.Development. 2004; 131: 1891-1901Crossref PubMed Scopus (449) Google Scholar The 12.4-kb VilCre mouse transgenic line14Madison B.B. Dunbar L. Qiao X.T. Braunstein K. Braunstein E. Gumucio D.L. Cis elements of the villin gene control expression in restricted domains of the vertical (crypt) and horizontal (duodenum, cecum) axes of the intestine.J Biol Chem. 2002; 277: 33275-33283Crossref PubMed Scopus (542) Google Scholar was used to produce intestinal epithelium–specific inactivation of Sox9. In the first cross, VilCre transgenic mice were mated with Sox9flox/+ mice. The offspring inheriting VilCre and the Sox9flox allele then were mated with Sox9flox/+ mice to obtain Sox9flox/flox;VilCre mice. Mice were genotyped by polymerase chain reaction on tail DNA by using the following primer pairs for Cre and Sox9flox. VilCre: AAC AGC ATT GCT GTC ACT TGG T, and TGG GCC AGC TAA ACATGC TT. Sox9flox: GAC AGT GCC AAG CAA GCA ACT, TGA CGT CCA GAC ACA GCA TAG G, CAA AAT GGT AAT GAG TCA TAC ACA GT, and ACA TGG AGG ACG ATT GGA GAA. Note that all mice were bred and maintained in a specific pathogen-free facility. Mice were sacrificed and the intestines were removed and flushed gently with cold phosphate buffered saline (PBS) and then 4% formaldehyde. Intestines were fixed overnight in 4% formaldehyde at room temperature, embedded as Swiss rolls in paraffin, and sectioned to 5–6 μm. Sections were deparaffinized in xylene, then rehydrated in ethanol and stained. For measurements of crypt widths and depths, samples were obtained 15 cm below the pylorus, fixed as described earlier, embedded vertically, and 6 μm cross-sections were stained with H&E. Lengths were computed from H&E digital images using Metamorph software (Molecular Devices, Sunnyvale, CA). Mice were injected intraperitoneally with BrdU (Zymed) (10mg per kg body weight) and sacrificed 2 hours or 40 hours after BrdU administration. Tissues were fixed in 4% formaldehyde, embedded in paraffin and 5 μm sections were stained with the BrdU staining kit (Zymed) following the manufacturer’s protocol. The primary antibodies and concentrations used were as follows: rabbit anti-SOX9 at 1:5015Lefebvre V. Huang W. Harley V.R. Goodfellow P.N. de Crombrugghe B. SOX9 is a potent activator of the chondrocyte-specific enhancer of the pro alpha1(II) collagen gene.Mol Cell Biol. 1997; 17: 2336-2346Crossref PubMed Google Scholar, mouse anti-Ki67 at 1:200 (Novocastra Laboratories Ltd.), mouse anti-β-catenin at 1:500 (BD Biosciences), rabbit anti-Lysozyme at 1:500 (Dako Cytomation), rabbit anti-synaptophysin at 1:200 (Dako Cytomation), rabbit anti-chromogranin A at 1:2000 (Immunostar). PolyHRP conjugate (Zymed) was used as secondary reagent. Staining was developed with True Blue substrate for anti-SOX9, and DAB for the other antibodies. Sections used for colabeling of SOX9 and β-catenin by immunofluorescence, were first incubated with primary antibodies and after several washes with PBS, the fluorophore-conjugated secondary antibodies (Molecular Probes) were added: donkey-anti-rabbit-555 for SOX9 antibody and goat-anti-mouse-488 for b-catenin antibody. Nuclear counter-stain was performed with TOTO3. Images were generated with a confocal microscope (Zeiss 510 LSM). Colocalization of β-catenin and SOX9 in the nucleus of epithelial cells in the intestinal crypts was analyzed using the colocalization plug-in of Imaris software (Bitplane Inc., St Paul, MN). Apoptotic cells were detected using Apoptag Plus Peroxidase In Situ Apoptosis Detection Kit (Chemicon). Sections were deparaffinized, rehydrated, treated with 0.2 N HCl, digested in proteinase K solution, postfixed, treated in acetic anhydride solution, and hybridized overnight for 24–72 hours at 68°C with digoxigenin (DIG)-labeled probes in 5× standard saline citrate, pH 4.5, 50% formamide, 2% blocking powder (Roche, Almere, The Netherlands), 5 mmol/L ethylenediaminetetraacetic acid, 50 μg/mL yeast transfer RNA, 0.05% 3[3-cholaminopropyl diethylammonio]-1-propane sulfonate, and 50 μg/mL heparin. Sections then were rinsed in 2× standard saline citrate and washed for 3 × 15 minutes at 65°C in 2× standard saline citrate/50% formamide. After rinses in Tris-buffered saline/Tween (Fisher Scientific, Pittsburgh, PA), sections were blocked for 30 minutes in Tris-buffered saline/Tween (Fisher Scientific) containing 0.5% blocking powder.16 Sections then were incubated in blocking solution overnight at 4°C with alkaline phosphatase–conjugated antidigoxigenin (1:2000 dilution; Roche). After washing in Tris-buffered saline/Tween (Fisher Scientific), the color reaction was performed with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate solution. In vitro transcription reactions to generate labeled probe were performed as follows: 1 μg of linearized DNA was incubated at 37°C for more than 2 hours with 4 μL transcription buffer (Promega, Leiden, The Netherlands), 2 μL dithiothreitol 0.1 mol/L (Promega), 2 μL DIG RNA labeling mix,16Jenny M. Uhl C. Roche C. Duluc I. Guillermin V. Guillemot F. Jensen J. Kedinger M. Gradwohl G. Neurogenin3 is differentially required for endocrine cell fate specification in the intestinal and gastric epithelium.EMBO J. 2002; 21: 6338-6347Crossref PubMed Scopus (360) Google Scholar 1 μL ribonuclease inhibitor (Promega), and 1.5 μL T7, T3, and SP6 polymerase (Promega) in a total volume of 20 μL. Six-month-old mice were sacrificed and the entire intestine was removed. The fecal samples were collected directly from the ileum portion of each of Sox9flox/flox;VilCre mice and littermate Sox9flox/+ mice, weighed, resuspended in PBS, and then serial dilutions were performed. In parallel experiments, an equivalent section of the ileum of wild-type and mutant mice were dissected, weighed, homogenized, and then serial dilutions were performed. Serial dilutions of each sample were spread on standard methods agar plates (BD, Franklin Lakes, NJ) and nutrient agar plates (BD) and after incubation the colony forming units were counted. At E13.5 all epithelial cells in the developing intestine were positive for SOX9 (Figure 1A). At E15.5 SOX9 was found in proliferative pockets between the nascent villi (Figure 1B), indicating that SOX9 was expressed well before the differentiation of Paneth cells. We also confirmed that in adult mice SOX9 was expressed in all epithelial cells in the lower two thirds of the crypts, namely in the domains of transiently proliferating cells and of Paneth cells (Figure 1G).5Blache P. van de Wetering M. Duluc I. Domon C. Berta P. Freund J.N. Clevers H. Jay P. SOX9 is an intestine crypt transcription factor, is regulated by the Wnt pathway, and represses the CDX2 and MUC2 genes.J Cell Biol. 2004; 166: 37-47Crossref PubMed Scopus (364) Google Scholar As expected, SOX9 protein was found in the nucleus. The overlap of SOX9 and β-catenin immunofluorescence in the bottom of the crypts strongly suggested that Paneth cells express SOX9 (Figure 1C–F). This was shown further by the presence of typical Paneth cell granules in SOX9-positive cells in the bottom of the crypts (Figure 1I). The Villin gene is first activated at E9.0 in the presumptive intestinal hindgut endoderm and its expression extends to the small and large intestinal endoderm at E10.0. After the crypt-villus axis is formed, villin is expressed in all epithelial cells of the intestine.4van Es J.H. Jay P. Gregorieff A. van Gijn M.E. Jonkheer S. Hatzis P. Thiele A. van den Born M. Begthel H. Brabletz T. Taketo M.M. Clevers H. Wnt signalling induces maturation of Paneth cells in intestinal crypts.Nat Cell Biol. 2005; 7: 381-386Crossref PubMed Scopus (487) Google Scholar, 14Madison B.B. Dunbar L. Qiao X.T. Braunstein K. Braunstein E. Gumucio D.L. Cis elements of the villin gene control expression in restricted domains of the vertical (crypt) and horizontal (duodenum, cecum) axes of the intestine.J Biol Chem. 2002; 277: 33275-33283Crossref PubMed Scopus (542) Google Scholar In transgenic mice, which harbor a 12.4-kb Villin promoter driving Cre, expression of Cre after E12.5 mimics that of the endogenous Villin gene.14Madison B.B. Dunbar L. Qiao X.T. Braunstein K. Braunstein E. Gumucio D.L. Cis elements of the villin gene control expression in restricted domains of the vertical (crypt) and horizontal (duodenum, cecum) axes of the intestine.J Biol Chem. 2002; 277: 33275-33283Crossref PubMed Scopus (542) Google Scholar To investigate the role of the transcription factor SOX9 in the intestinal epithelium, we used mice containing a conditional allele of Sox913Akiyama H. Chaboissier M.C. Martin J.F. Schedl A. de Crombrugghe B. The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6.Genes Dev. 2002; 16: 2813-2828Crossref PubMed Scopus (1329) Google Scholar and generated Sox9flox/flox;VilCre mice by using the mating scheme described in the Materials and Methods section. Cre-mediated deletion of Sox9 creates a null allele.17Kist R. Schrewe H. Balling R. Scherer G. Conditional inactivation of Sox9: a mouse model for campomelic dysplasia.Genesis. 2002; 32: 121-123Crossref PubMed Scopus (87) Google Scholar Sox9 immunohistochemistry staining of Sox9flox/flox;VilCre mice showed a nearly complete absence of SOX9 in the crypt compartment of the intestine (Figure 1H and J), indicating that the Sox9 gene was excised effectively in the intestinal epithelial cells. The absence of Sox9 in mutant mice was confirmed between the ages of 2 weeks and 1 year. Because VilCre is not expressed in epithelial cells of the pancreas, the cells that stained for SOX9 in the pancreas in Sox9flox/flox;VilCre mice provided a positive control for SOX9 in immunohistochemical staining (data not shown). We also noted that the nuclei of a small number of epithelial cells in the villi of wild-type mice but not of Sox9flox/flox;VilCre mice stained positive for SOX9 (Figure 1G, arrowhead). These cells did not show morphologic features of either goblet cells or Paneth cells and did not stain for synaptophysin, a marker of enteroendocrine cells (data not shown). Mutant mice appeared overtly normal, were fertile, and the litters were of normal size. Their weight between 2 weeks and 1 year was indistinguishable from that of littermate controls. Interestingly, histologic examination showed that the width as well as the depth of the crypts were increased significantly in the Sox9 mutant mice (Figure 2), implying that there could be a considerable increase in proliferative cell numbers in the crypts of Sox9 mutant mice. The overall architecture of villi appeared similar to that in the wild-type intestine. Strikingly the characteristic cytosolic granules typical of Paneth cells, as visualized by H&E staining, were completely absent in the cells of the crypts of Sox9flox/flox;VilCre mice throughout the small intestine (Figure 2A and B). The absence of lysozyme immunostaining (Figure 3A and B) and of Matrix metalloproteinase 7 (mmp7) mRNA (Figure 3C and D) further supported the view that Paneth cells were missing. In addition, in situ hybridizations revealed that Cryptdin-1 and Cryptdin-6 mRNA were readily detectable in the small intestine of control mice, but were absent in the Sox9flox/flox;VilCre mice (Figure 3E and F, and data not shown). In contrast, the average number of Goblet cells per villus as detected by periodic acid–Schiff (PAS) staining did not show any significant difference between mutant and control (Figure 4A–C). Likewise, the average number of enteroendocrine cells per villus detected by synaptophysin immunostaining was similar in mutant and control mice (Figure 4D–F). In the colon the average number of goblet cells and enteroendocrine cells per crypt also was unchanged (Figure 4C and F). Moreover, transmission electron microscopy of the villi of mutant mice showed the presence of abundant enterocytes with typical microvilli, which were indistinguishable from those in control mice (data not shown).Figure 4Goblet cell and enteroendocrine cell numbers are unchanged in the absence of Sox9. (A) PAS–positive cells of Sox9flox/flox, and (B) Sox9flox/flox;VilCre mice. (D) Anti-synaptophysin immunostaining–positive cells in jejunum of Sox9flox/flox. (E) Sox9flox/flox;VilCre. (C) Quantification of goblet cells and (F) enteroendocrine cells per villus in different segments of the small intestine and per crypt of the colon in Sox9flox/flox mice and Sox9flox/flox;VilCre mice by counting periodic acid–Schiff–positive cells and anti-synaptophysin-positive cells, respectively. Open bars represent Sox9flox/flox, and closed bars represent Sox9flox/flox;VilCre mice. Error bars represent standard errors (N = 3 animals per genotype).View Large Image Figure ViewerDownload Hi-res image Download (PPT) In wild-type mice, the cells above Paneth cells in the lower two thirds of the crypts are proliferating. Some rare proliferating cells also are found intercalated among Paneth cells in the crypts. In contrast, in Sox9flox/flox;VilCre mice, proliferating cells occupied the entire crypts, including the crypt bottoms where Paneth cells reside in wild-type mice. This was visualized by the presence of Ki67-positive cells throughout the crypts (Figure 5A and B). Furthermore, when mice were injected with bromodeoxyuridine (BrdU) and sacrificed 2 hours later, BrdU-positive cells were detected in the bottom of the crypts in Sox9flox/flox;VilCre mice more frequently than in wild-type mice (Figure 5C–G). Importantly, the average number of BrdU-positive cells per crypt in Sox9 mutant mice was significantly larger than in control mice in all segments of the intestine (Figure 5E). Because SOX9 is expressed in the crypts of the colon, we examined the number of BrdU-positive epithelial cells in the colon: BrdU-positive epithelial cells were twice as frequent in the colon of Sox9 mutant than in wild-type mice. In a different experiment in which mice were sacrificed 40 hours after BrdU injection, the leading and trailing edges of the BrdU-positive cells in the villi were comparable in mutant and control mice (data not shown), strongly suggesting that loss of Sox9 did not affect cell migration from crypts to villi. In addition, very few apoptotic cells, measured by the terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay, were seen in either Sox9 mutant or control crypts, and no obvious difference was seen between mutant and control (data not shown). To verify whether the absence of Paneth cells leads to a larger bacterial load in the crypts that might account for the increased crypt cell proliferation, we computed the number of colony forming units from feces samples collected in the ileum portion of the intestine and also from the homogenized tissue of a segment of the ileum. No difference was observed in total colony forming units between 6-month-old Sox9 mutant and wild-type littermate controls. We conclude that it was unlikely that the increased proliferation was the consequence of a larger bacterial load resulting from a lack of Paneth cells, but was the consequence of the absence of SOX9. Our experiments indicate that in the absence of SOX9 in the intestinal epithelial cells no differentiated Paneth cells were present in the bottom of the crypts. This view is supported by the absence of the characteristic Paneth cell vesicles in the cells of crypt bottoms and by the lack of expression in these cells of typical Paneth cell markers including lysozyme, MMP7, and Cryptdin-1 and Cryptdin-6. The same phenotype was seen throughout the small intestine. These results strongly suggest that SOX9 is needed for the differentiation of Paneth cells including the establishment of the typical secretory machinery of these cells. Because the number of goblet cells and of enteroendocrine cells remain unchanged in the Sox9 mutants compared with wild-type mice, SOX9 appears to have no role in the differentiation and localization of these cells. Several factors have been shown to be required for discrete steps in the differentiation of intestinal epithelial cells.18Sancho E. Batlle E. Clevers H. Signaling pathways in intestinal development and cancer.Annu Rev Cell Dev Biol. 2004; 20: 695-723Crossref PubMed Scopus (425) Google Scholar Cells first adopt either an absorptive or a secretory cell fate based on the balance between Notch and Wnt signaling. In proliferative crypt cells, both signaling pathways are active.19van Es J.H. van Gijn M.E. Riccio O. van den Born M. Vooijs M. Begthel H. Cozijnsen M. Robine S. Winton D.J. Radtke F. Clevers H. Notch/gamma-secretase inhibition turns proliferative cells in intestinal crypts and adenomas into goblet cells.Nature. 2005; 435: 959-963Crossref PubMed Scopus (1224) Google Scholar On exiting the crypts, Notch signaling induces absorptive enterocyte differentiation.19van Es J.H. van Gijn M.E. Riccio O. van den Born M. Vooijs M. Begthel H. Cozijnsen M. Robine S. Winton D.J. Radtke F. Clevers H. Notch/gamma-secretase inhibition turns proliferative cells in intestinal crypts and adenomas into goblet cells.Nature. 2005; 435: 959-963Crossref PubMed Scopus (1224) Google Scholar, 20Fre S. Huyghe M. Mourikis P. Robine S. Louvard D. Artavanis-Tsakonas S. Notch signals control the fate of immature progenitor cells in the intestine.Nature. 2005; 435: 964-968Crossref PubMed Scopus (697) Google Scholar Notch signaling activates expression of the transcriptional repressor Hes1, which in turn represses expression of the Math1 transcription factor. Indeed, loss of Hes1 leads to increased numbers of goblet cells,21Jensen J. Pedersen E.E. Galante P. Hald J. Heller R.S. Ishibashi M. Kageyama R. Guillemot F. Serup P. Madsen O.D. Control of endodermal endocrine development by Hes-1.Nat Genet. 2000; 24: 36-44Crossref PubMed Scopus (929) Google Scholar whereas Math1 knockout mice lack all secretory lineages.22Yang Q. Bermingham N.A. Finegold M.J. Zoghbi H.Y. Requirement of Math1 for secretory cell lineage commitment in the mouse intestine.Science. 2001; 294: 2155-2158Crossref PubMed Scopus (715) Google Scholar The secretory lineage is specified further into either a goblet cell, or an enteroendocrine or a Paneth cell fate. Enteroendocrine cell differentiation is controlled positively by the transcription factor Ngn3,16Jenny M. Uhl C. Roche C. Duluc I. Guillermin V. Guillemot F. Jensen J. Kedinger M. Gradwohl G. Neurogenin3 is differentially required for endocrine cell fate specification in the intestinal and gastric epithelium.EMBO J. 2002; 21: 6338-6347Crossref PubMed Scopus (360) Google Scholar, 23Pinto D. Clevers H. Wnt control of stem cells and differentiation in the intestinal epithelium.Exp Cell Res. 2005; 306: 357-363Crossref PubMed Scopus (219) Google Scholar, 24Schonhoff S.E. Giel-Moloney M. Leiter A.B. Minireview: development and differentiation of gut endocrine cells.Endocrinology. 2004; 145: 2639-2644Crossref PubMed Scopus (247) Google Scholar and negatively by the transcription factor Gfi1.25Shroyer N.F. Wallis D. Venken K.J. Bellen H.J. Zoghbi H.Y. Gfi1 functions downstream of Math1 to control intestinal secretory cell subtype allocation and differentiation.Genes Dev. 2005; 19: 2412-2417Crossref PubMed Scopus (231) Google Scholar Our results elucidate an important and unique step in the cascade of intestinal epithelial cell differentiation by showing that Sox9 specifically is required for the differentiation of Paneth cells. Sox9 has remarkable properties in that it is required for the differentiation of a small number of cell lineages, during which SOX9 has an essential role in decisions of cell fate. The diverse lineages include chondrocytes in the skeleton,12Bi W. Deng J.M. Zhang Z. Behringer R.R. de Crombrugghe B. Sox9 is required for cartilage formation.Nat Genet. 1999; 22: 85-89Crossref PubMed Scopus (1361) Google Scholar, 13Akiyama H. Chaboissier M.C. Martin J.F. Schedl A. de Crombrugghe B. The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6.Genes Dev. 2002; 16: 2813-2828Crossref PubMed Scopus (1329) Google Scholar glial cells in the central nervous system,11Stolt C.C. Lommes P. Sock E. Chaboissier M.C. Schedl A. Wegner M. The Sox9 transcription factor determines glial fate choice in the developing spinal cord.Genes Dev. 2003; 17: 1677-1689Crossref PubMed Scopus (454) Google Scholar Sertoli cells,10Chaboissier M.C. Kobayashi A. Vidal V.I. Lutzkendorf S. van de Kant H.J. Wegner M. de Rooij D.G. Behringer R.R. Schedl A. Functional analysis of Sox8 and Sox9 during sex determination in the mouse.Development. 2004; 131: 1891-1901Crossref PubMed Scopus (449) Google Scholar and endocardial cushion cells.8Akiyama H. Chaboissier M.C. Behringer R.R. Rowitch D.H. Schedl A. Epstein J.A. de Crombrugghe B. Essential role of Sox9 in the pathway that controls formation of cardiac valves and septa.Proc Natl Acad Sci U S A. 2004; 101: 6502-6507Crossref PubMed Scopus (202) Google Scholar A recent study has indicated that ablation of one of the Wnt receptors, Fz5, with a K19Cre transgene, caused mislocalization of Paneth cells in the villi; these cells were positive for lysozyme but lacked expression of other Paneth cell markers such as EphB3 and cryptdin-1.4van Es J.H. Jay P. Gregorieff A. van Gijn M.E. Jonkheer S. Hatzis P. Thiele A. van den Born M. Begthel H. Brabletz T. Taketo M.M. Clevers H. Wnt signalling induces maturation of Paneth cells in intestinal crypts.Nat Cell Biol. 2005; 7: 381-386Crossref PubMed Scopus (487) Google Scholar This suggested that in these experiments there was a block at a late stage of differentiation of Paneth cells in the intestines of mice in which Fz5 was inactivated because the early marker lysozyme still was expressed. In contrast, in Sox9 mutants both lysozyme and the late markers were absent, suggesting that Sox9 is needed at an early step in the pathway of Paneth cell differentiation. The difference in phenotype between Fz5 and Sox9 mutants might be the result of the timing of inactivation of the respective genes by either VilCre or K19Cre during Paneth cell differentiation. Embryos lacking the canonical Wnt signaling nuclear effector, Tcf4, had a severe phenotype and died in the perinatal period; they were characterized by a complete inhibition of proliferation of intestinal epithelial cells, showed the presence of enterocytes and Goblet cells, but, in contrast to control embryos, lacked expression of cryptdin-1 and cryptdin-6 as well as Sox9.5Blache P. van de Wetering M. Duluc I. Domon C. Berta P. Freund J.N. Clevers H. Jay P. SOX9 is an intestine crypt transcription factor, is regulated by the Wnt pathway, and represses the CDX2 and MUC2 genes.J Cell Biol. 2004; 166: 37-47Crossref PubMed Scopus (364) Google Scholar, 26Korinek V. Barker N. Moerer P. van Donselaar E. Huls G. Peters P.J. Clevers H. Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4.Nat Genet. 1998; 19: 379-383Crossref PubMed Scopus (1302) Google Scholar We propose that SOX9 controls an early step of Paneth cell differentiation, specifying the commitment of these cells toward the Paneth cell phenotype. In addition, it also is possible that Paneth cell markers might be direct targets of Sox9. In the chondrocyte differentiation pathway Sox9 is needed at several steps throughout the pathway and chondrocyte marker genes also are direct targets of Sox9.13Akiyama H. Chaboissier M.C. Martin J.F. Schedl A. de Crombrugghe B. The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6.Genes Dev. 2002; 16: 2813-2828Crossref PubMed Scopus (1329) Google Scholar A previous study based on DNA transfection experiments of colon cancer cells postulated that SOX9 might contribute to the maintenance of progenitor cells. This hypothesis is compatible with an essential role of SOX9 in the differentiation of Paneth cells. Although the number of goblet cells and enteroendocrine cells were similar in wild-type and mutant mice, we have not excluded that the size of the pool of progenitor cells might be different in the mutant crypts. The lack of Sox9 also leads to an enlargement of crypts throughout the small intestine. A likely explanation for this increase in the depth and the width of the crypts is the observed marked increase in the proliferation of crypt cells. Note that we observed a small increase in the length of the intestine in Sox9 mutant mice, but it is unclear whether this increased length was related to the increased dimension of the crypts. The increased dimensions of the crypts of mutant mice alternatively might be caused by alterations of morphogenic signals resulting from the absence of Sox9 in the crypts. The expansion of proliferating cells into the bottom of the crypts could be a consequence of the absence of differentiated Paneth cells, because proliferating cells would simply migrate to the bottom of the crypts to replace the missing Paneth cells. This interpretation could account for an experiment in which Paneth cells were poisoned using a defensin promoter-driven diphtheria toxin transgene.27Garabedian E.M. Roberts L.J. McNevin M.S. Gordon J.I. Examining the role of Paneth cells in the small intestine by lineage ablation in transgenic mice.J Biol Chem. 1997; 272: 23729-23740Crossref PubMed Scopus (210) Google Scholar In Gfi1-null mice, proliferating cells also occupied the bottom of the crypts.25Shroyer N.F. Wallis D. Venken K.J. Bellen H.J. Zoghbi H.Y. Gfi1 functions downstream of Math1 to control intestinal secretory cell subtype allocation and differentiation.Genes Dev. 2005; 19: 2412-2417Crossref PubMed Scopus (231) Google Scholar In these mice the number of enteroendocrine cells were increased significantly, whereas the numbers of goblet cells and Paneth cells were reduced markedly. In none of these models, however, was crypt enlargement observed. Sox9 has been shown previously to inhibit cell proliferation both after transfection of tissue culture cells and in vivo when expressed at increased levels in chondrocytes of the growth plate.28Panda D.K. Miao D. Lefebvre V. Hendy G.N. Goltzman D. The transcription factor SOX9 regulates cell cycle and differentiation genes in chondrocytic CFK2 cells.J Biol Chem. 2001; 276: 41229-41236Crossref PubMed Scopus (81) Google Scholar, 29Akiyama H. Lyons J.P. Mori-Akiyama Y. Yang X. Zhang R. Zhang Z. Deng J.M. Taketo M.M. Nakamura T. Behringer R.R. McCrea P.D. de Crombrugghe B. Interactions between Sox9 and beta-catenin control chondrocyte differentiation.Genes Dev. 2004; 18: 1072-1087Crossref PubMed Scopus (618) Google Scholar Although we do not know what the mechanism is for the increased cell proliferation in Sox9 mutant intestinal crypts, one possibility might be that Sox9 has an inhibitory function in the activity of the Wnt–β-catenin pathway in crypt epithelial cells acting as a feedback control. We previously provided evidence that Sox9 inhibits the transcriptional activity of β-catenin both in transfection and in Xenopus oocyte experiments.29Akiyama H. Lyons J.P. Mori-Akiyama Y. Yang X. Zhang R. Zhang Z. Deng J.M. Taketo M.M. Nakamura T. Behringer R.R. McCrea P.D. de Crombrugghe B. Interactions between Sox9 and beta-catenin control chondrocyte differentiation.Genes Dev. 2004; 18: 1072-1087Crossref PubMed Scopus (618) Google Scholar Our results thus provide evidence for an essential and specific role of Sox9 in the differentiation of Paneth cells. In addition, Sox9 has an important function in controlling the proliferation of epithelial cells in the crypts. The authors thank Andreas Schedl and Marie-Christine Chaboissier (University of Nice, Nice, France) for the gift of mice with a conditional Sox9 allele; Deborah L. Gumucio (University of Michigan Medical School, Ann Arbor, Michigan) for VilCre mice; Susan J. Henning, Chandra Iyer, Christopher Dekaney (Baylor College of Medicine, Houston, TX), Noah F. Shroyer (Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio), and members of the Henning laboratory (Baylor College of Medicine, Houston, TX) for helpful discussions and guidance. Intestinal Development: The Many Faces of Wnt SignalingGastroenterologyVol. 133Issue 2PreviewThe mammalian intestinal epithelium perpetually renews itself every 3–5 days from a large reservoir of pluripotent stem cells in the lower crypt.1 Intestinal stem cells divide and produce an actively proliferating population of transit amplifying cells that give rise to each of the 4 principal epithelial cell types in the small intestine. As immature cells migrate up the crypt–villus axis, they exit the cell cycle in the villus compartment and differentiate into either absorptive enterocytes, goblet cells, or enteroendocrine cells. Full-Text PDF" @default.
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