Instability in the composition of gut bacterial communities (dysbiosis) has been linked to common human intestinal disorders, such as Crohn's disease and colorectal cancer. Here, we show that dysbiosis caused by Nod2 deficiency gives rise to a reversible, communicable risk of colitis and colitis-associated carcinogenesis in mice. Loss of either Nod2 or RIP2 resulted in a proinflammatory microenvironment that enhanced epithelial dysplasia following chemically induced injury. The condition could be improved by treatment with antibiotics or an anti-interleukin-6 receptor-neutralizing antibody. Genotype-dependent disease risk was communicable via maternally transmitted microbiota in both Nod2-deficient and WT hosts. Furthermore, reciprocal microbiota transplantation reduced disease risk in Nod2-deficient mice and led to long-term changes in intestinal microbial communities. Conversely, disease risk was enhanced in WT hosts that were recolonized with dysbiotic fecal microbiota from Nod2-deficient mice. Thus, we demonstrated that licensing of dysbiotic microbiota is a critical component of disease risk. Our results demonstrate that NOD2 has an unexpected role in shaping a protective assembly of gut bacterial communities and suggest that manipulation of dysbiosis is a potential therapeutic approach in the treatment of human intestinal disorders.

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To explore the role of homeobox genes in the intestine, the human colon adenocarcinoma cell line Caco2-TC7 has been stably transfected with plasmids synthesizing Cdx1 and Cdx2 sense and antisense RNAs. Cdx1 overexpression or inhibition by antisense RNA does not markedly modify the cell differentiation markers analyzed in this study. In contrast, Cdx2 overexpression stimulates two typical markers of enterocytic differentiation: sucrase-isomaltase and lactase. Cells in which the endogenous expression of Cdx2 is reduced by antisense RNA attach poorly to the substratum. Conversely, Cdx2 overexpression modifies the expression of molecules involved in cell–cell and cell–substratum interactions and in transduction process: indeed, E-cadherin, integrin-β4 subunit, laminin-γ2 chain, hemidesmosomal protein, APC, and α-actinin are upregulated. Interestingly, most of these molecules are preferentially expressed in vivo in the differentiated villi enterocytes rather than in crypt cells. Cdx2 overexpression also results in the stimulation of HoxA-9 mRNA expression, an homeobox gene selectively expressed in the colon. In contrast, Cdx2-overexpressing cells display a decline of Cdx1 mRNA, which is mostly found in vivo in crypt cells. When implanted in nude mice, Cdx2-overexpressing cells produce larger tumors than control cells, and form glandular and villus-like structures.Laminin-1 is known to stimulate intestinal cell differentiation in vitro. In the present study, we demonstrate that the differentiating effect of laminin-1 coatings on Caco2-TC7 cells is accompanied by an upregulation of Cdx2. To further document this observation, we analyzed a series of Caco2 clones in which the production of laminin-α1 chain is differentially inhibited by antisense RNA. We found a positive correlation between the level of Cdx2 expression, that of endogenous laminin-α1 chain mRNA and that of sucrase-isomaltase expression in these cell lines.Taken together, these results suggest (a) that Cdx1 and Cdx2 homeobox genes play distinct roles in the intestinal epithelium, (b) that Cdx2 provokes pleiotropic effects triggering cells towards the phenotype of differentiated villus enterocytes, and (c) that Cdx2 expression is modulated by basement membrane components. Hence, we conclude that Cdx2 plays a key role in the extracellular matrix–mediated intestinal cell differentiation.

Radial glial cells have been shown to act as neuronal precursors in the developing cortex and to maintain their radial processes attached to the basement membrane (BM) during cell division. Here, we examined a potential role of direct signalling from the BM to radial glial cells in three mouse mutants where radial glia attachment to the BM is disrupted. This is the case if the nidogenbinding site of the laminin ␥1 chain is mutated, in the absence of ␣6 integrin or of perlecan, an essential BM component. Surprisingly, cortical radial glial cells lacking contact to the BM were not affected in their proliferation, interkinetic nuclear migration, orientation of cell division and neurogenesis. Only a small subset of precursors was located ectopically within the cortical parenchyma. Notably, however, neuronal subtype composition was severely disturbed at late developmental stages (E18) in the cortex of the laminin ␥1III4 -/-mice. Thus, although BM attachment seems dispensable for precursor cells, an intact BM is required for adequate neuronal composition of the cerebral cortex.KEY WORDS: Mouse, Basement membrane, Laminin, Cerebral cortex, Lamc1, Itaga6, Hspg2 Development 133, 3245-3254 (2006) DEVELOPMENT 3246 1995), defects may also arise within the cortical parenchyma of the ␣6 integrin -/-mice. We therefore examined a further mouse mutant with deletion of a molecule restricted to the BM, perlecan -/- (Costell et al., 1999) (see also Arikawa-Hirasawa et al., 1999). MATERIALS AND METHODS AnimalsLaminin ␥1III4 heterozygous mice were kept on a SV129 background, ␣6 integrin heterozygous mice (Georges-Labouesse et al., 1998) on C57Bl/6/SV129 background and perlecan heterozygous mice (Costell et al., 1999) on C57Bl/6 background. Crossing of heterozygous mice [the day of the vaginal plug is considered embryonic day (E) 0] allowed to examine wild-type, heterozygous ( +/-) and homozygous mutant embryo ( -/-) littermates. Immunohistochemistry and in-situ hybridizationEmbryonic brains were fixed in 4% paraformaldehyde in phosphatebuffered-saline (PBS) and 12 m frontal sections were cut with a cryostat after cryoprotection. Sections were immunostained using the primary antibodies against the phosphorylated form of Histone H3 (PH3) (rabbit (rbt); Biomol; 1:200), BrdU (mouse IgG1, 1:10, Bioscience Products), calbindin (rbt, 1:2000, SWANT), calretinin (rbt, 1:2000, SWANT), Ki67 (rat Tec-3, 1:50, Dako), pan-Laminin (rbt, 1:50, BD), ␤III-Tubulin (mouse IgG2a, 1:100, Sigma), O4 (mouse IgM, 1:1000, kindly provided by Jack Price), GFAP (mouse IgG1, 1:200, Sigma), RC2 (mouse IgM, 1:500, kindly provided by P. Leprince), BLBP (rbt, 1:1500, kindly provided by Nathaniel Heintz), nestin (mouse IgG1, 1:4, Dev. Hybridoma Bank) and reelin (E4, mouse IgG1, 1:500, kindly provided by André Goffinet). The respective secondary antibodies were from Southern Biotechnology Associates and Jackson ImmunoResearch. Specimens were mounted in Aqua Poly/Mount (Polysciences, Northampton, UK) and analysed with a Confocal Microscope (Leica TCS 4NT; Olympus ...

Intestinal morphogenesis and differentiation are dependent on heterotypic cell interactions between embryonic epithelial cells (endoderm) and stromal cells (mesenchyme). Extracellular matrix molecules represent attractive candidates for regulators of these interactions. The structural and functional diversity of the extracellular matrix as intestinal development proceeds is demonstrated by 1) spatio-temporal specific expression of the classically described constituents, 2) the finding of laminin and collagen IV variants, 3) changes in the ratio of individual constituent chains, and 4) a stage-specific regulation of basement membrane molecule production, in particular by glucocorticoids. The orientation/assembly of these extracellular matrix molecules could direct precise cellular functions through interactions via integrin molecules. The involvement of extracellular matrix, and in particular basement membrane molecules in heterotypic cell interactions leading to epithelial cell differentiation, has been highlighted by the use of experimental models such as cocultures, hybrid intestines and antisense approaches. These models allowed us to conclude that a correct elaboration and assembly of the basement membrane, following close contacts between epithelial and fibroblastic cells, is necessary for the expression of differentiation markers such as digestive enzymes.

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Using several models, we provide the first evidence that the kinase NIK and integrins interact in vitro and in vivo. This interaction is required for proper axonal navigation in C. elegans.

Background-Laminins are major components of basement membranes, well located to interact with platelets upon vascular injury. Laminin-111 (α 1 β 1 γ 1 ) is known to support platelet adhesion but is absent from most blood vessels, which contain isoforms with the α 2 , α 4 , or α 5 chain. Whether vascular laminins support platelet adhesion and activation and the significance of these interactions in hemostasis and thrombosis remain unknown. Methods and Results-Using an in vitro flow assay, we show that laminin-411 (α 4 β 1 γ 1 ), laminin-511 (α 5 β 1 γ 1 ), and laminin-521 (α 5 β 2 γ 1 ), but not laminin-211 (α 2 β 1 γ 1 ), allow efficient platelet adhesion and activation across a wide range of arterial wall shear rates. Adhesion was critically dependent on integrin α 6 β 1 and the glycoprotein Ib-IX complex, which binds to plasmatic von Willebrand factor adsorbed on laminins. Glycoprotein VI did not participate in the adhesive process but mediated platelet activation induced by α 5 -containing laminins. To address the significance of platelet/laminin interactions in vivo, we developed a platelet-specific knockout of integrin α 6 . Platelets from these mice failed to adhere to laminin-411, laminin-511, and laminin-521 but responded normally to a series of agonists. α 6 β 1 -Deficient mice presented a marked decrease in arterial thrombosis in 3 models of injury of the carotid, aorta, and mesenteric arterioles. The tail bleeding time and blood loss remained unaltered, indicating normal hemostasis. Conclusions-This study reveals an unsuspected important contribution of laminins to thrombus formation in vivo andsuggests that targeting their main receptor, integrin α 6 β 1 , could represent an alternative antithrombotic strategy with a potentially low bleeding risk. (Circulation. 2013;128:541-552.)Key Words: blood platelets ◼ integrin α 6 β 1 ◼ laminin ◼ thrombosis