1 Department of Histopathology, Imperial College School of Medicine, Hammersmith Campus, DuCane Road, London, W12 0NN, UK
2 Histopathology Unit, Imperial Cancer Research Fund, Lincolns Inn Fields, London, WC2A 3PX, UK
*Author for correspondence (e-mail: s.Kirkland{at}ic.ac.uk)
Accepted March 14, 2001
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SUMMARY |
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We have developed an in vitro model system of intestinal epithelial stem cells to facilitate the direct analysis of stem cells undergoing lineage commitment and differentiation. Using this culture system, we can now directly investigate the role of cell-matrix signalling in stem-cell decisions. In this study, collagen-IV synthesis has been followed in monolayers of multipotent cells that have been induced to differentiate into absorptive, goblet and enteroendocrine cells. Our experiments demonstrate that commitment to the enteroendocrine lineage is specifically accompanied by the expression of type-IV collagen that remains enteroendocrine-cell associated. Undifferentiated cells, absorptive cells and goblet cells do not express collagen IV. To confirm that the differential lineage-specific expression of collagen IV observed in the model system was representative of the in vivo situation, collagen-IV synthesis was analysed in isolated human colorectal crypts and tissue sections using immunocytochemistry and in situ hybridisation. These studies confirmed the in vitro findings, in that implementation of the enteroendocrine differentiation program involves synthesis and accumulation of a collagen-IV matrix. Thus, human colorectal enteroendocrine cells are unique in the colorectal crypt in that they assemble a cell-associated collagen-IV-rich matrix not observed on other colorectal epithelial cells.
This study provides the first evidence for differential matrix synthesis between colorectal epithelial lineages in human colorectal epithelium. The specialised pericellular environment of the enteroendocrine cells might explain some of the unique phenotypic characteristics of this cell lineage. Furthermore, these findings suggest a potential mechanism whereby individual epithelial cells could modulate their cell-matrix signalling even while rapidly migrating in heterogeneous sheets over a shared basement membrane.
Key words: Intestinal epithelium, Extracellular matrix, Collagen IV, Stem cell, Enteroendocrine, Lineage commitment
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INTRODUCTION |
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Cell-matrix interactions, mediated via integrin receptors, are known regulators of epithelial differentiation (Adams and Watt, 1993), epithelial stem cell fate (Zhu et al., 1999) and cell migration (Lauffenburger and Horwitz, 1996; Palecek et al., 1997). In the intestine, extracellular matrix (ECM) proteins are present at the epithelial-mesenchymal interface early in development (Simon-Assmann et al., 1995) and changes in spatial distribution are associated with morphogenetic events (Simo et al., 1991). In the adult small intestine, broad differential expression of laminin chains between the crypt (proliferative) and villous (differentiated) basement membranes (Beaulieu and Vachon, 1994; Simon-Assmann et al., 1994) have indicated a role for laminin in intestinal differentiation. This has been confirmed using in vitro studies with the Caco-2 cell line, where enterocytic differentiation requires laminin synthesis (DeArcangelis et al., 1996). Therefore, epithelial matrix protein synthesis regulates epithelial phenotype. In vitro studies have further shown that intestinal epithelial cells bind preferentially to collagen IV (Moore et al., 1994) and that collagen synthesis promotes intestinal epithelial cell migration (Moore et al., 1992; Goke et al., 1996; Wilson and Gibson, 1997).
As intestinal epithelial phenotype is regulated by interactions with ECM proteins, this has led to speculation that specific regional differences in the biochemical composition of the ECM and/or matrix receptor expression might specify epithelial cell behaviour in intestinal epithelium. However, the intestinal basement membrane has a slower turnover than the epithelium (Trier et al., 1990) and a relatively uniform composition. The few reports of differential expression of matrix molecules or receptors have described broad regional differences between crypt and villus in the small intestine (Beaulieu, 1992; Beaulieu and Vachon, 1994; Simon-Assmann et al., 1994) and crypt and lumenal surface in the colon (Koretz et al., 1991; Koukoulis et al., 1993). Although such reports strongly indicate a role for matrix molecules in intestinal epithelial biology, the patterns of expression do not reflect the complexity of the multilineage differentiation program. For example, there have been no reports of microheterogeneity in matrix protein or receptor expression that would be compatible with stem-cell- or lineage-specific cell-matrix signalling (Potten et al., 1997). Consequently, regions of the colorectal crypt that appear to have a uniform basement membrane composition and epithelial matrix receptor expression contain a panoply of colorectal epithelial phenotypes (including anchored or migratory and undifferentiated or lineage committed). Therefore, it is not clear how cell-matrix signalling is specified at the single-cell level to generate the observed phenotypic diversity.
To elucidate the molecular mechanisms regulating intestinal stem-cell lineage commitment and differentiation, we have developed an in vitro stem-cell model. This model uses a cloned human rectal epithelial cell line, HRA-19, that has tripotential progenitor cell characteristics (Henderson and Kirkland, 1996) and can be induced to differentiate into all colorectal epithelial lineages (i.e. absorptive, goblet and enteroendocrine) (Henderson and Kirkland, 1996). Differentiation requires transfer of HRA-19 cells to serum-free medium (Henderson and Kirkland, 1996) but does not need specialised ECM-coated substrates. The model facilitates the direct analysis of multipotent colorectal epithelial cells undergoing commitment in the absence of basement-membrane proteins or mesenchymal cells. Thus, epithelial matrix synthesis, a regulator of epithelial phenotype, can be investigated as cells execute their differentiation program.
In this study, we have chosen to investigate type-IV-collagen synthesis during HRA-19 differentiation as collagen-IV substrates promote enteroendocrine lineage commitment in the HRA-19 model (S.C.K. and K.H., unpublished). In addition, ascorbic acid, an essential cofactor for collagen biosynthesis, promotes enteroendocrine- and goblet-lineage commitment of HRA-19 cells (Henderson and Kirkland, 1996). Furthermore, several studies have indicated that collagen-IV synthesis is required for intestinal-cell migration (Moore et al., 1992). This study describes the analysis of type-IV-collagen synthesis in human colorectal epithelial cells as they differentiate along enteroendocrine, goblet and absorptive lineage pathways.
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MATERIALS AND METHODS |
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Differentiating monolayers were obtained by trypsinising HRA-19 cells from T25 flasks and seeding into eight-chamber Permanox multislides in serum-free medium. The serum-free medium used in all experiments was Dulbeccos Eagles medium (Gibco, Paisley, UK) containing sodium pyruvate (110 µg ml-1), kanamycin (100 µg ml-1), insulin (2 µg ml-1), transferrin (2 µg ml-1) and ascorbic acid (10 µg ml-1). Monolayers were fixed at day 3 or 4 for immunocytochemistry.
Isolation of normal human colonic crypts
Normal colonic mucosa was obtained from resections for colorectal carcinoma at a site distant from the tumour. Colonic crypts were liberated from the mucosa by a modification of the method described by Whitehead et al. (1987). Mucosa was incubated for 1.5 hours in the following solution: NaCl (8 g l-1), KCl (0.2 g l-1), Na2HPO4·12H2O (2.9 g l-1), KH2PO4 (0.2 g l-1) EDTA (0.2 g l-1), 0.5% phenol red (1.5 ml), followed by vigorous shaking to liberate mainly intact crypts. Extended incubations in this solution reduced collagen staining. Isolated crypts were dried onto microscope slides overnight then immunostained.
Antibodies
Type-IV collagen and prolyl-4-hydroxylase antibodies
PCO: rabbit polyclonal to type-IV collagen (Eurodiagnostica, Euro-Path, Cornwall, UK). p1: rabbit polyclonal raised against monomeric collagen
1 (IV) globular domain (Butlowski et al., 1989) (a kind gift from J. Wieslander and T. Hellmark, Lund, Sweden). ZIB7214: rabbit polyclonal against human type-IV collagen (TCS Biologicals, Botolph Claydon, UK). 00-401-106: rabbit polyclonal against type-IV collagen (Rockland, Lorne Lab., Reading, UK). CIV 22: mouse monoclonal to human collagen IV (DAKO, Denmark). Monoclonal antibody to human prolyl-4-hydroxylase ß subunit (3-2B12, TCS Biologicals, Botolph Claydon, UK).
Cell lineage markers
Mouse monoclonal to human chromogranin (LK2H10, Boehringer). Rabbit polyclonal to PYY (Cambridge Research Biochemicals, Cambridge, UK). PR4D4 is a monoclonal antibody that specifically reacts with goblet-cell mucus (Richman and Bodmer, 1987). PR4D4 does not stain absorptive or enteroendocrine cells (Richman and Bodmer, 1987). PR2D3 is a monoclonal antibody that reacts with pericryptal mesenchymal cells (Richman et al., 1987).
Secondary antibodies
Peroxidase-linked secondary antibodies (DAKO, Denmark). Rhodol-Green-conjugated goat anti-mouse IgG (H+L) (Molecular Probes, Cambridge Bioscience, Cambridge, UK). Cy3-conjugated affinity purified donkey anti-rabbit IgG (H+L) and Cy3-conjugated Goat anti-mouse IgG (H+L) (Jackson Immunoresearch, Stratech Scientific Ltd, Luton, UK).
Immunocytochemistry
Cell monolayers
Immunocytochemistry was performed essentially as described previously (Henderson and Kirkland,1996) using primary antibodies at the following concentrations: PCO, 1:50; p1, 1:1000; ZIB 7214, 1:300; 600-410-106, 1:500; CIV22, 1:50; prolyl-4-hydroxylase, 1:50. Rhodol-Green-conjugated goat anti-mouse IgG and Cy3-conjugated donkey anti-rabbit IgG were used as secondary antibodies at 1:200.
Colonic sections
Normal colonic mucosa was obtained from resections for colorectal carcinoma at a site distant from the tumour and fixed in neutral buffered formalin overnight before routine processing and sectioning. Colon sections for type-IV collagen staining required prior digestion for 30 minutes at 37°C with 0.1% protease (Sigma P-4789) in PBS. Sections were stained with primary antibodies at the following concentrations: PCO, 1:10; p1, 1:100; ZIB 7214, 1:50; 600-410-106, 1:100; CIV22, 1:100; prolyl-4-hydroxylase, 1:400. Serial sections were stained alternately with p
1 (1:100) and either rabbit polyclonal antibody to PYY (1:400) or a monoclonal antibody to human chromogranin (1:200) (LK2H10).
Frozen sections were allowed to dry overnight and fixed for 10 minutes in acetone at room temperature before immunostaining. Peroxidase-linked secondary antibodies (Dako) and development in a DAB/Nickel solution (Henderson and Kirkland, 1996) were used to detect primary antibody binding.
Isolated crypts
Isolated crypts were fixed in ethanol for 10 minutes at room temperature using the same antibody concentration used for monolayers except 600-401-106 (1:150), which was used to detect type-IV collagen in double-stained preparations. On whole-crypt preparations, Rhodol-Green-conjugated goat anti-mouse IgG secondary antibody was used at 1:300.
In situ hybridisation
Normal human colonic epithelium was fixed in 4% paraformaldehyde in DEPC-treated PBS for 6 hours at 4°C before routine processing and sectioning onto silanised slides. Sections were dewaxed in xylene (three times for 2 minutes each), rehydrated in distilled water for 5 minutes and air dried for 5 minutes. In situ hybridisation was performed using reagents supplied in a custom-made kit from Biognostik (TCS Biologicals, Botolph Claydon, UK). Sections were prehybridised in 50 µl Hybribuffer for 3 hours at 30°C and then incubated overnight at 30°C with 50 µl Hybribuffer containing 50 pmol of double-FITC-labelled oligonucleotide probe. The collagen-IV probe was a 29-base oligonucleotide (ATG GCC AAG TAT CTC ACC TGG ATC ACC CT) representing the reverse complement of bases 589-617 of the total sequence of the 1 chain of human collagen-IV gene (Brazel et al., 1987). Following hybridisation, sections were washed in DEPC-treated SSC (twice for 30 seconds each and once for 5 minutes) and then 0.1% SSC (twice for 7 minutes each). Sections were then incubated in 1:20 normal rabbit serum for 10 minutes at room temperature, then in 1:50 peroxidase-linked rabbit anti-FITC (DAKO) for 1 hour at room temperature. Sections were washed in PBS (three times for 5 minutes each). Peroxidase was visualised by development in Dab/Ni solution for 10 minutes (Henderson and Kirkland, 1996).
Controls included omission of probe and a reverse-control double-FITC probe of 29 bases supplied by Biognostik.
Combined in situ hybridisation/immunocytochemistry
For in situ hybridisation and immunocytochemistry on a single section, chromogranin antibody (1:200) was added to the probe mixture. Following the SCC washes, sections were incubated in PBS containing 1:20 rabbit serum and 1:20 goat serum. Cy3-conjugated goat anti-mouse IgG (Jackson Immunoresearch, Stratech Scientific Ltd, Luton, UK) at 1:300 was included in the anti-FITC solution.
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RESULTS |
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DISCUSSION |
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Although these techniques have demonstrated the novel synthesis of collagen IV by enteroendocrine cells, they have also confirmed previous studies in that collagen IV synthesis could not be demonstrated in most colorectal epithelial cells with undifferentiated, goblet or absorptive phenotypes (Simon-Assmann et al., 1988; Weiser et al., 1990; Perreault et al., 1998). Therefore, whereas previous studies have described epithelial versus mesenchymal matrix protein contributions, our data provide the first evidence of a new principle in intestinal cell-matrix interactions: that synthesis of matrix proteins can be epithelial-lineage specific rather than simply epithelium specific. Both in vivo and in vitro data show that collagen IV remains tightly associated with enteroendocrine cells: neighbouring cells are type-IV-collagen negative, suggesting that the assembly and retention of a type-IV-collagen-rich microenvironment is part of the enteroendocrine differentiation program. Cell-associated type-IV collagen is observed on enteroendocrine cells in vitro and is therefore an inherent feature of the colorectal enteroendocrine lineage and not dependent on the presence of a native basement membrane or mesenchymal cells.
To define the role of type-IV collagen in enteroendocrine lineage commitment further, type-IV collagen expression was followed as cells differentiated along the enteroendocrine lineage pathway. Our data indicate that progression along the enteroendocrine pathway is characterised by a sequential expression of chromogranin, prolyl-4-hydroxylase and, finally, type-IV collagen. Therefore, collagen IV appears to be synthesised only when cells have already committed to the endocrine lineage, indicating that collagen IV is involved in endocrine cell function rather than lineage commitment decisions. The implementation of the enteroendocrine differentiation program must thus involve specific synthesis of matrix proteins that appears to result in the elaboration of a type-IV-collagen-rich pericellular matrix by enteroendocrine cells. The purpose of the collagen-IV-rich matrix in specifying enteroendocrine cell phenotype has yet to be elucidated but the unique migratory behaviour of this lineage suggests one potential role. Several studies have demonstrated that enteroendocrine cells have a much longer lifespan than other intestinal epithelial cells (Tsubouchi and Leblond, 1979; Thompson et al., 1990; DeBruine et al., 1992), strongly indicating that they have a lower migration rate. Therefore, enteroendocrine cells require a cell-specific mechanism(s) to reduce migration rate relative to neighbouring goblet and absorptive cells in the epithelial sheet migrating from crypt base to lumenal surface. Could the production of collagen IV and assembly of a collagen-IV-rich pericellular matrix alter the migratory phenotype of enteroendocrine cells? This possibility is supported by the finding that collagen synthesis has been shown to be involved in intestinal epithelial migration. Enterocytes preferentially bind and spread on type-IV collagen (Moore et al., 1994) and inhibition of collagen synthesis inhibits intestinal epithelial cell migration in vitro (Moore et al., 1992; Goke et al., 1996). Furthermore in vitro studies with other cell types have shown that matrix protein concentration modulates integrin expression and cell motility (Palecek et al., 1997; Condic and Letourneau, 1997), and also the motility response of cells to growth factors (Ware et al., 1998). High concentrations of ligand lead to strong adhesion with reduced migration (Palecek et al., 1997). Therefore, it is plausible to suggest that the assembly of a type-IV-collagen-rich pericellular environment could reduce enteroendocrine cell motility, enabling absorptive and goblet cells to bypass them in this rapidly migrating epithelial sheet. The concept of a lineage-specific pericellular matrix composition defining patterns of migration warrants further study as it has wide ranging implications for understanding cell anchorage in normal epithelia and the aberrent migratory behaviour of neoplastic cells.
Recent studies have highlighted the importance of cell-surface matrix composition in regulating cell behaviour via cell-matrix signalling (Basbaum and Werb, 1996). Our work indicates that ECM synthesis is also a contributory factor in defining the cell-surface matrix composition of individual cells. Lineage-specific pericellular matrix composition would explain how single cells could maintain specific cell-matrix signalling even while migrating in heterogeneous sheets over a shared basement membrane. Cell-matrix interactions have been shown to regulate the differentiation of epithelial cells (Roskelley et al., 1995), including those of the intestine (Simon-Assmann et al., 1995). Therefore the ability of colorectal epithelial cells to maintain differential matrix synthesis has important implications for understanding cell-matrix regulation of the epithelial phenotype in the human colorectal crypt.
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ACKNOWLEDGMENTS |
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adams, J. C. and Watt, F. M. (1993). Regulation of development and differentiation by the extracellular matrix. Development 117, 1183-1198.
Andrew, A., Kramer, B. and Rawdon, B. B. (1982). The embryonic origin of endocrine cells of the gastrointestinal tract. Gen. Comp. End. 47, 249-265.
Basbaum, C. B. and Werb, Z. (1996). Focalized proteolysis: spatial and temporal regulation of extracellular matrix degradation at the cell surface. Curr. Opin. Cell Biol. 8, 731-738.[Medline]
Beaulieu, J. F. (1992). Differential expression of the VLA family of integrins along the crypt-villus axis in the human small intestine. J. Cell Sci. 102, 427-436.[Abstract]
Beaulieu, J. F. and Vachon, P. H. (1994). Reciprocal expression of laminin A-chain isoforms along the crypt-villus axis in the human small intestine. Gastroenterology 106, 829-839.[Medline]
Beaulieu, J. F., Vachon, P. H., Herring-Gillam, F. E., Simoneau, A., Perreault, N., Asselin, C. and Durand, J. (1994). Expression of the 5 (IV) collagen chain in the fetal human small intestine. Gastroenterology 107, 957-967.[Medline]
Brazel, D., Oberbaumer, I., Dieringer, H., Babel, W., Glanville, R. W., Deutzmann, R. and Kuhn, K. (1987). Completion of the amino acid sequence of the 1 chain of human basement membrane collagen (type IV) reveals 21 non-triplet interruptions located within the collagenous domain. Eur. J. Biochem. 168, 529-536.[Abstract]
Butlowski, R. J., Wieslander, J., Kleppel, M., Michael, A. F. and Fish, A. J. (1989). Basement membrane collagen in the kidney: regional localisation of novel chains related to collagen IV. Kidney Int. 35, 1195-1202.[Medline]
Cheng, H. and Leblond, C. P. (1974). Origin, differentiation and renewal of the four main epithelial types in the mouse small intestine. V. Unitarian theory of the origin of the four epithelial cell types. Am. J. Anat. 141, 537-567.[Medline]
Condic, M. L. and Letourneau, P. C. (1997). Ligand-induced changes in integrin expression regulate neuronal adhesion and neurite outgrowth. Nature 389, 852-856.[Medline]
DeArcangelis, A., Neuville, P., Boukamel, R., Lefebvre, O., Kedinger, M. and Simon-Assmann, P. (1996). Inhibition of laminin 1-chain expression leads to alteration of basement membrane assembly and cell differentiation. J. Cell Biol. 133, 417-430.[Abstract]
DeBruine, A. P., Dinjens, W. N. M., Zijlema, J. H. L., Lenders, M. H. and Bosman, F. T. (1992). Renewal of enterochromaffin cells in the rat caecum. Anat. Rec. 233, 75-82.[Medline]
Goke, M., Zuk, A. and Podolsky, D. (1996). Regulation and function of extracellular matrix in intestinal epithelial restitution in vitro. Am. J. Physiol. 271, G729-G740.
Griffiths, D. F. R., Davies, S. J., Williams, D., Williams, G. T. and Williams, E. D. (1988). Demonstration of somatic mutation and colonic crypt clonality by X-linked enzyme histochemistry. Nature 333, 461-463.[Medline]
Henderson, K. and Kirkland, S. C. (1996). Multilineage differentiation of cloned HRA-19 cells in serum-free medium: a model of human colorectal epithelial differentiation. Differentiation 60, 259-268.[Medline]
Kirkland, S. C. and Bailey, I. G. (1986). Establishment and characterisation of six human colorectal adenocarcinoma cell lines. Br. J. Cancer 53, 779-785.[Medline]
Kirkland, S. C. (1988). Clonal origin of columnar, mucous and endocrine cell lineages in human colorectal epithelium. Cancer 61, 1359-1363.[Medline]
Kivirikko, K. I. and Myllyharju, J. (1998). Prolyl 4-hydroxylases and their protein disulfide isomerase sub-unit. Matrix Biol. 16, 357-368.[Medline]
Koretz, K., Sclag, P., Boumsell, L. and Moller, P. (1991). Expression of VLA-2, VLA-
6 and VLA-ß1 chains in normal mucosa and adenomas of the colon, and in colon carcinomas and their liver metastases. Am. J. Path. 138, 741-750.[Abstract]
Koukoulis, G. K., Virtanen, I., Moll, R., Quaranta, V. and Gould, V. E. (1993). Immunolocalisation of integrins in the normal and neoplastic colonic epithelium. Virchows Arch. B 63, 373-383.[Medline]
Lauffenburger, D. A. and Horwitz, A. F. (1996). Cell migration: a physically integrated molecular process. Cell 84, 359-369.[Medline]
Lloyd, R. V. and Wilson, B. S. (1983). Specific endocrine tissue marker defined by a monoclonal antibody. Science 222, 628-630.[Medline]
Lundberg, J. M., Tatemoto, K., Terenius, L., Hellstrom, P. M., Mutt, V., Hokfelt, T. and Hamberger, B. (1982). Localisation of peptide YY(PYY) in gastrointestinal endocrine cells and effects on intestinal blood flow and motility. Proc. Natl. Acad. Sci. USA 79, 4471-4475.[Abstract]
Moore, R., Madri, J., Carlson, S. and Madara, J. L. (1992). Collagens facilitate epithelial migration in restitution of native guinea pig intestinal epithelium. Gastroenterology 102, 119-130.[Medline]
Moore, R., Madara, J. L. and MacLeod, R. J. (1994). Enterocytes adhere preferentially to collagen IV in a differentially regulated divalent cation-dependent manner. Am. J. Physiol. 266, G1099-G1107.
Palecek, S. P., Loftus, J. C., Ginsberg, M. H., Lauffenburger, D. A. and Horwitz, A. F. (1997). Integrin-ligand binding properties govern cell migration speed through cell-substratum adhesiveness. Nature 385, 537-540.[Medline]
Perreault, N., Herring-Gillam, F. E., Desloges, N., Belanger, I., Pageot, L. P. and Beaulieu, J. F. (1998). Epithelial vs Mesenchymal contribution to the extracellular matrix in the human intestine. Biochem. Biophys. Res. Commun. 248, 121-126.[Medline]
Ponder, B. A. J., Schmidt, G. H., Wilkinson, M. M., Wood, M. J., Monk, M. and Reid, A. (1985). Derivation of mouse intestinal crypts from single progenitor cells. Nature 313, 689-691.[Medline]
Potten, C. S., Booth, C. and Pritchard, D. M. (1997). The intestinal epithelial stem cell: the mucosal governor. Int. J. Exp. Pathol. 78, 219-243.[Medline]
Richman, P. I. and Bodmer, W. F. (1987). Monoclonal antibodies to human colorectal epithelium: markers for differentiation and tumour characterisation. Int. J. Cancer 39, 317-328.[Medline]
Richman, P. I., Tilly, R., Jass, J. R. and Bodmer, W. F. (1987). Colonic pericrypt sheath cells characterisation of cell type with new monoclonal antibody. J. Clin. Pathol. 40, 593-600.[Abstract]
Roskelley, C. D., Srebrow, A. and Bissell, M. J. (1995). A hierarchy of ECM-mediated signalling regulates tissue-specific gene expression. Curr. Opin. Cell Biol. 7, 736-747.[Medline]
Simo, P., Simon-Assmann, P., Bouziges, F., Leberquier, C., Kedinger, M., Ekblom, P. and Sorokin, L. (1991). Changes in the expression of laminin during intestinal development. Development 112, 477-487.[Abstract]
Simon-Assmann, P., Bouziges, F., Arnold, C., Haffen, K. and Kedinger, M. (1988). Epithelial-mesenchymal interactions in the production of basement membrane components in the gut. Development 102, 339-347.[Abstract]
Simon-Assmann, P., Duclos, B., Orian-Rousseau, V., Arnold, C., Mathelin, C., Engvall, E. and Kedinger, M. (1994).Differential expression of laminin isoforms and 6-ß4 integrin subunits in the developing human and mouse intestine. Dev. Dyn. 201, 71-85.[Medline]
Simon Assmann, P., Kedinger, M., De Arcangelis, A., Rousseau, V. and Simo, P. (1995). Extracellular matrix components in intestinal development. Experentia 51, 883-899.[Medline]
Thompson, E. M., Price, Y. E. and Wright, N. A. (1990). Kinetics of enteroendocrine cells with implications for their origin: a study of the cholecystokinin and gastrin subpopulations combining tritiated thymidine labelling with immunocytochemistry in the mouse. Gut 31, 406-411.[Abstract]
Trier, J. S., Allan, C. H., Abrahamson, D. R. and Hagen, S. J. (1990). Epithelial basement membrane of mouse jejunum. Evidence for laminin turnover along the entire crypt-villus axis. J. Clin. Invest. 86, 87-95.[Medline]
Tsubouchi, S. and Leblond, C. P. (1979). Migration and turnover of enteroendocrine and caveolated cells in the epithelium of the descending colon, as shown by radioautography after continuous infusion of 3H-thymidine into mice. Am. J. Anat. 156, 431-452.[Medline]
Ware, M. F., Wells, A. and Lauffenburger, D. A. (1998). Epidermal growth factor alters fibroblast migration speed and directional persistence reciprocally and in a matrix dependent manner. J. Cell Sci. 111, 2423-2432.
Weiser, M. M., Sykes, D. E. and Killen, P. D. (1990). Rat intestinal basement membrane synthesis. Epithelial versus nonepithelial contributions. Lab. Invest. 62, 325-330.[Medline]
Whitehead, R. H., Brown, A. and Bhathal, P. S. (1987). A method for the isolation and culture of human colonic crypts in collagen gels. In vitro Cell. Dev. Biol. 23, 436-442.[Medline]
Wilson, T. J., Ponder, B. A. and Wright, N. A. (1985). Use of a mouse chimaeric model to study cell migration patterns in the small intestinal epithelium. Cell Tissue Kinet. 18, 333-344.[Medline]
Wilson, A. J. and Gibson, P. R. (1997). Epithelial migration in the colon: filling in the gaps. Clin. Sci. 93, 97-108.[Medline]
Winton, D. J., Blount, M. A. and Ponder, B. A. J. (1988). A clonal marker induced by mutation in mouse intestinal epithelium. Nature 333, 463-466.[Medline]
Zhu, A. J., Haase, I. and Watt, F. M. (1999). Signaling via ß1 integrins and mitogen-activated protein kinase determines human epidermal stem cell fate in vitro. Proc. Natl. Acad. Sci USA 96, 6728-6733.