Proc. Nati. Acad. Sci. USA Vol. 82, pp. 8488-8492, December 1985 Cell Biology Can villin be used to identify malignant and undifferentiated normal digestive epithelial cells? (actin/brush border/differentiation marker/development/tumor dassifficadon) S. ROBINE*, C. HUET*, R. MOLLt, C. SAHUQUILLO-MERINO*, E. COUDRIER*, A. ZWEIBAUMt, AND D. LOUVARD* *Unite de Biologie des Membranes, D6partement de Biologie Moldculaire, Institut Pasteur, Paris, France; tPathologisches Institut, Universitatsklinik, Mainz, Federal Republic of Germany; and *Institut National de la Sant6 et de la Recherche Mddicale U-178, H6pital Broussais, Paris, France Communicated by Andri Lwoff, July 22, 1985

ABSTRACT We have investigated the presence of villin (a border. With this in mind, we investigated villin as a marker Ca2+-regulated actin binding protein) in various tissues (nor- for mature, differentiating, and transformed enterocytes mal or malignant) and in established cell lines by using sensitive using specific anti-villin antisera. Villin was detected on immunochemical techniques on cell extracts and immuno- immunoblots of cell extracts that had been electrophoresed fluorescence analysis on frozen sections. Our results show that and transferred to nitrocellulose and by indirect immuno- villin is a marker that can be used to distinguish normal fluorescence cytochemistry on frozen tissue sections. differentiated epithelial cells from the simple epithelia lining the gastrointestinal tract and renal tubules. Villin is found in MATERIALS AND METHODS the absorptive cells ofthe small and large intestines, in the duct cells of pancreas and biliary system, and in the cells of kidney Reagents. Peroxidase-labeled sheep anti-rabbit IgG were proximal tubules. Furthermore, undifferentiated normal and obtained from Biosys (Compiegne, France) and iodinated tumoral cells of intestinal origin in vivo and in cell culture protein A was from New England Nuclear. Rhodamine- express villin. Therefore, expression ofvillin is seen in cells that conjugated sheep anti-rabbit IgG was from Nordic (Tilburg, do not necessarily display the morphological features charac- Netherlands). teristic of their terminally differentiated state, such as the Cell lines, Tumors, and Tissues. The human adenocarci- microvilli-lined brush border. We suggest the possible clinical noma-derived cell lines HT-29 and Caco-2 were obtained implications of using villin as a marker in the diagnosis of from J. Fogh (9) and maintained as described by Pinto et al. metastatic adenocarcinomas. (10, 11). Other cell lines used were the porcine kidney proximal tubule cell line, LLC-PK1 (12, 13); the canine The identification and characterization of cell-type specific kidney distal tubule cell line, MDCK (14); mouse embryo line structural protein markers has been valuable in the study of 3T3; human larynx epidermoid carcinoma-derived HEp-2; cell differentiation in higher eukaryotes. Antibodies against human cervix epithelial carcinoma-derived HeLa; human intermediate filament proteins (e.g., vimentin, keratins, and colonic adenocarcinoma-derived HRT18; human ileocecal neurofilament proteins) are used to identify cell types in vivo adenocarcinoma-derived HCT8R; human colonic adenocar- and in vitro (1, 2). The detection of these cell-type specific cinoma-derived SW480; and Potorous tridactylis kidney protein markers during embryogenesis and terminal cell epithelial-like Pt K2 cell lines were grown using standard differentiation has allowed investigators to define the devel- conditions. Tumors in nude mice were obtained as reported opmental stage or the developmental commitment ofcells (3, (15). The methods used to isolate intestinal epithelial cells 4). In addition to the available markers, such as the keratins, have been described by Raul et al. (16) and Hauri et al. (17). which are especially useful in discriminating between epithe- Immunochemical Experiments. Proteins from cultures or lial and nonepithelial cells, it would be useful to identify other tissues were extracted according to Garrels and Gibson (18). structural proteins exclusive to one cell type or subtype of Electrophoresis was performed according to Laemmli (19) by differentiated epithelial cells (2). using 7.5% NaDodSO4/polyacrylamide slab gels. Electro- cell found in the intestinal transfer ofthe proteins to nitrocellulose and antigen detection The major epithelial type was performed according to Burnette (20) as modified by mucosa is the absorptive enterocyte. This epithelia has a Coudrier et al. (21). brush border, a specialized domain of the plasma membrane Antisera. Using purified porcine villin as immunogen (a gift composed of microvilli facing the lumen of the intestine that from V. Gerke and K. Weber; ref. 22), antibodies were raised is assembled during the terminal differentiation of as described (23). The specificity of the antisera was checked enterocytes. The axial-microfilament bundle of a microvillus on immunoreplicas of intestinal mucosa homogenate or by contains a Ca2+-regulated actin-binding protein, villin (Mr indirect immunofluorescence on intestinal frozen sections. 95,000), which is the best-characterized protein of the The antibodies used in the present work did not cross-react cytoskeleton of intestinal microvilli (5, 6). Indirect im- with the 110-kDa protein of the microvilli (23). munofluorescence using anti-villin antisera on frozen sec- Immunocytochemistry. Large and small rat intestines were tions localizes villin at the apical pole ofcolumnar cells in the fixed and prepared for cryosectioning according to Brown intestine and at the apical pole ofproximal tubule cells in the and Farquhar (24). Other tissues were cut without prior kidney, which is the location ofthe brush border. In contrast, aldehyde fixation, and sections were fixed with cold (-200C) villin was not detected in microvilli ofother epithelia that lack acetone. Immunofluorescent labeling was performed as de- a highly organized brush border (7, 8). Thus, the presence of scribed by Reggio et al. (25). villin may be correlated with the existence of a mature brush RESULTS The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Villin Expression in Epithelia. Frozen sections of rat or in accordance with 18 U.S.C. §1734 solely to indicate this fact. human small intestines showed strongly labeled brush bor- 8488 Downloaded by guest on September 27, 2021 CeH Biology.- Robine et al. Proc. NatL. Acad. Sci. USA 82 (1985) 8489

FIG. 1. Villin localization on frozen sections. (A) Rat small intestine. (B) Rat large intestine. The brush borders (bb) are heavily labeled and a significant diffuse staining is seen in the cytoplasm of the adsorptive cells. By this technique, vilhin was not detected in goblet cells (gc). The stroma (str) shows a strong autofluorescence presumably due to histamine (26). Tissue was fixed by the paraformaldehyde/lysine/periodate procedure (24). (C) Human pancreas. Villin could be observed only in cells from the main duct (md) or from interlobular ducts (id). It was absent in connective tissue (ct). (D) Human liver. Large bile ducts (bd) contained villin. Liver connective tissue (ct) were negative. Cells were fixed with cold (-200C) acetone. (Bar = 0.5 Aum.)

ders by indirect immunofluorescence (Fig. LA). We observed and has been shown to express high levels of digestive a similar pattern of decoration in cells of the large intestine enzymes (e.g., alkaline phosphatase, aminopeptidase, and (Fig. 1B). Intestine, when prepared as described above (24), sucrase). In addition, electron microscopy studies have exhibited a diffuse labeling in the cytoplasm, but when the shown a brush border at the upper cell surface (11). As shown technique of Bretscher et al. (7) was used, this diffuse in Fig. 2 (lane 6), Caco-2 cells expressed a high level of villin labeling was not seen. Other cells of the simple epithelia of (about 0.1% of total protein as measured by an ELISA the human digestive tract were also found to contain villin. developed in our laboratory). Labeling was observed on the apical side of cells in the main The relationship between terminal differentiation and villin and interlobular ducts of the pancreas (Fig. 1C), but no expression in cells derived from intestinal epithelium was significant labeling of the acini was observed. Anti-villin further studied by using a subclone of the cell line HT-29 (9), antisera decorated the large bile ducts (Fig. 1D) whereas isolated in our laboratory. In this cell line, the control of small bile ducts and bile canaliculi were usually not labeled. enterocytic differentiation is under nutritional control. When By immunoreplica techniques, large amounts ofvillin were grown in standard culture medium, Dulbeccos-Vogt modi- found in cells of the large and small intestines (Fig. 2, lanes fied Eagle's medium containing 25 mM glucose, cells remain 2b and 3). By quantitative analysis (27), an equivalent amount undifferentiated. However, when glucose is removed or of villin was detected in both tissues. In extracts of stomach replaced by 5 mM galactose, these cells become polarized mucosa (Fig. 2, lane 4) villin could not be detected. Negative and express an enterocyte-like phenotype (10, 29). Fig. 2 results (i.e., less than 5 ng of villin in 200 ,ug of cell protein) (lanes 7 and 8) shows that villin was detected in differentiated were also obtained in other tissues containing epithelial cells and undifferentiated HT-29 cells, although the undifferenti- such as lung, skin, ovary, bladder, and endometrium. Our ated HT-29 cells contain only about 10% of the villin found results and those of others (Table 1) demonstrate that villin in differentiated cells. Morphological studies of the cells can be detected either by immunofluorescence or by im- grown in galactose (5 mM) or in the absence of hexose have munoreplica procedures in simple epithelia of the gastroin- shown a well-defined brush border; each microvillus contains testinal and urogenital tracts. a core bundle of microfilaments that extends into the cyto- Villin Expression in Cell Cultures. We investigated the plasm (10). Ultrastructural examination of the cells grown in presence ofvillin in an established intestinal cell line, Caco-2, glucose shows that the cells remain undifferentiated; micro- which was derived from a human colonic adenocarcinoma villi occur irregularly and are not organized into a brush Downloaded by guest on September 27, 2021 8490 Cell Biology: Robine et al. Proc. Natl. Acad. Sci. USA 82 (1985) CELL Table 1. Villin expression in epithelial tissues ORGANS CULTURES TUMORS I 0 a Immunochemical Immuno- MW L2a2b 3 4 5 6 7 8 9 10 11 12 Tissue detection localization Gastrointestinal tract m- Small intestine + (7) + (7, 8) Large intestine + Gall bladder ND Pancreas _...... - Duct ND Acini ND Tissue homogenate + (28) Liver Large bile duct ND Hepatocytes ND Tissue homogenate* Stomach Oesophagus ND FIG. 2. Immunodetection of villin. Lanes: 1, purified pig villin Urogenital tract (Coomassie blue); 2a, protein pattern of human colon mucosal Kidney extract (Coomassie blue). Lanes 2b-12, villin revealed after im- munoreplica with anti-villin antiserum and peroxidase-conjugated Proximal tubule ND + (7) Fab fragment of sheep anti-rabbit IgG (both at 20 ug/ml)-2b, Distal tubule ND extracts from adult human colon; 3, human small intestine; 4, rat Cortex homogenate + stomach; 5, intestinal tube of an 8-week-old human fetus; 6, Caco-2; Epididymist 7, differentiated HT-29 cells grown in 5 mM galactose medium; 8, Ductus epididymidis ND undifferentiated HT-29 grown in 25 mM glucose medium; 9, Hela Ductuli efferentes ND cells; 10, human colonic primary tumor; 11, a tumor induced in nude Tissue homogenate mice with an SW480 cell line; 12, a human epidermoid oesophageal Testis carcinma.. The total amount of protein used in each experiment was Oviduct 100-200 ,ug. In these experimental conditions, the intensity of the reaction can be correlated to the extent of expression of villin in the Uterus endometrium extract. Molecular size markers (MW): myosin (180 kDa), ,- Bladder urothelium ND galactosidase (116 kDa), phosphorylase b (92 kDa), bovine serum Prostate glands albumin (68 kDa), and actin (43 kDa). Other tissues Lung border (ref. 10 and unpublished results). Furthermore, Bronchial epithelium ND sucrase is not detected (10). Therefore, our results show that Alveolar epithelium ND morphologically undifferentiated HT-29 cells produce a sig- Tissue homogenate nificant amfount of villin, even before they construct a typical Skin brush border. Epidermis ND ND The presence of villin in extracts of established cell lines Dermis ND ND derived from kidney epithelia was also tested. Cells that Sweat glands ND exhibited characteristic enzymatic markers or tralsport ac- Tissue homogenate tivities corresponding to the proximal tubule (LLC-PK1) (12, Chloroid plexus ND 13) or the distal tubule (MDCK) (14) were chosen. The These results were obtained by two different methodologies: (i) presence of a low but measurable amount of villin in LLC- Immunolocalization of villin by immunofluorescence techniques PK1 cells and its absence in MDCK cells are consistent with [results from the literature (7, 8) and our observations] and (ii) the origin of these cells. We have summarized results immunodetection of villin by using immunoreplica procedures [data established cell lines of various from the literature (refs. 7, 8, and 28) and from our study]. ND: not obtained by using origins determined. (Table 2). Negative results were consistently obtained when *The small number of positive cells in the liver do not allow the cells tested were not ofintestinal or renal epithelial origin. immunochemical detection of villin in an extract of this organ. Villin Expression in Undifferentiated Intestinal Cells. To tDuctus epididymidis does not have a brush border; ductuli ef- determine if villin is generally found in undifferentiated cells ferentes does. capable of differentiation into cells with a well-organized brush border, we studied villin expression along the crypt/vil- lus axis in adult intestine. Frozen sections of rat small characterized by a high level of [3H]thymidine incorporation, intestine cut along the crypt/villus axis showed labeling of a marker for proliferative cells. The amount ofvillin in mature epithelial cells but not the underlying mucosa. Mature enterocytes was at least 10-fold higher than the amount in enterocytes displayed strong apical decoration (the brush immature crypt cells (Fig. 3A). border) as well as diffuse cytoplasmic labeling (Fig. 3B). In We also studied the expression of villin during embryonic the undifferentiated cells of the crypts, villin primarily was development. Villin was detected in a cell extract prepared located near the apical plasma membrane; additionally, from the intestinal tube of an 8-week-old human fetus (Fig. 2, weak, diffuse labeling was seen in the cytoplasm. The lane 5). At this stage of development, the organization and existence of a gradient of villin expression along the differentiation of intestinal tissue has just been initiated (30). crypt/villus axis was shown by using the quantitative im- Between 9 and 11 weeks of gestation, many epithelial cells munoreplica procedure (27). Populations of cells enriched in begin to exhibit microvilli at their apical membranes and at 16 differentiated cells (tip of the villi) or in undifferentiated cells weeks of gestation they display a differentiated brush border (crypts) were separated. Differentiated cells at the tips of the (31). In a 16-week-old fetus, villin was found in the small and villi (25% of the isolated cells) were characterized by a high the large intestine (data not shown). These results demon- level of alkaline phosphatase, a common marker for strate the specificity of villin expression in vivo in a few enterocytes. Crypt cells (25% of the isolated cells) were structurally and/or embryologically related epithelia. In Downloaded by guest on September 27, 2021 Cell Biology: Robine et al. Proc. Natl. Acad. Sci. USA 82 (1985) 8491 Table 2. Villin expression in established cell lines found to express villin (see Fig. 2, lane 10), whereas villin was Immuno- not detected in tumors ofother origins, such as an epidermoid chemical Immuno- esophageal carcinoma (Fig. 2, lane 12), a mesenteric node Cell line detection localization lymphoma, a kidney carcinoma, and an adenocarcinoma of unknown origin metastasized in spleen (data not shown). This From the large intestine suggests that the tissue-specific expression of villin is main- Caco-2 (human colonic tained in primary human colonic tumors. In addition, we adenocarcinoma) + + screened tumors in nude mice for villin expression after the HT-29 (human colonic mouse was injected with one of five different cell lines of adenocarcinoma) + + colonic origin. These tumors were selected for the presence From kidney (HT-29 and Caco-2) or absence (HRT18, HCT8R, and LLC-PK1 (derived from pig kidney SW480) of an enterocyte-like pattern of enzymatic differen- proximal tubule) + ND tiation (e.g., sucrase expression) (15). Villin was detected in MDCK (derived from dog kidney all five tumors. The amount of villin was apparently similar distal tubule) - - from one tumor to another and was independent not only of From other origins the pattern of enterocytic differentiation (e.g., digestive HEp-2 (human esophagus) - ND enzymes) but also of the degree of ultrastructural expression Hela (human cervix) - ofbrush border microvilli, which are numerous in Caco-2 and Chicken embryo fibroblasts - HT-29 tumors (15) but very scarce in the other three tumors 3T3 (murine fibroblasts) ND (unpublished results). These results show that the mainte- Pt K2 (rat kangoroo epithelial cell) nance of villin expression during tumorigenesis is indepen- See legend to Table 1. ND: not determined. dent of morphological differentiation or enzymatic expres- sion. addition, our results emphasize the early expression of villin during development. DISCUSSION Villin Expression in Malignant Tissues of Human Intestinal In this paper, we have substantiated data showing the Origin. To determine whether the specific tissue expression presence ofvillin in brush borders. It is also ofnote that villin of villin also extends to malignant tissues of gastrointestinal is used to construct the microvillar microfilaments both in origin, we investigated its occurrence in various tumors. enterocytes and the epithelia of the kidney proximal tubule, Eleven ofthe 12 human colonic adenocarcinomas tested were cells which have a different embryological origin. Further-

C ryp ts Villi

4_mw ....m m m - A ~~M _~ __mm 4~

FIG. 3. Distribution of villin along the crypt/villus axis in rat small intestinal mucosa. (A) Autoradiograms of immunoreplicas using protein extracts from cell populations isolated stepwise from the tip of the villi to the crypts. Immunodetection of villin was with rabbit anti-villin antiserum and 1125-labeled protein A (6 X 10- cpm/ml). Radioactivity was quantitated according to Howe and Hershey (27). The third fraction from the right (the villi), contains 16,340 cpm whereas the ninth fraction from the right (crypt cells) contains 1950 cpm. (B and B') Two adjacent fields of an axial section of the mucosa from the tip of the villus to the crypt zone (c) and muscular layers (m). The brush borders (bb) of the absorptive cells are intensely labeled and their cytoplasm shows a lower, diffuse staining. The goblet cells (gc) are negative, whereas the stroma (str) of the villus shows some autofluorescence (see D). In the crypt area (c), the apical pole of the immature cells is also labeled. (Bars = 50 Am.) (C) High magnification of a longitudinal section ofa crypt. The apex, facing the lumen (lu) ofthe immature cells, is labeled. The cytoplasm shows a very faint diffuse staining. (D) Control. Tissue sections were incubated with preabsorbed villin antiserum. Antiserum (400 ,ul, diluted 1:800) was incubated with 10 ,ul of villin (10 mg/ml) for 3 hr at room temperature. Samples were processed as in B. Only autofluorescent spots are seen in the stroma (str). The same pattern is seen when the specimen is observed using a fluorescein filter set, indicating an endogenous autofluorescence of cells within the stroma (see Fig. 1). Downloaded by guest on September 27, 2021 8492 Cell Biology: Robine et al. Proc. Natl. Acad. Sci. USA 82 (1985) more, our study suggests that in agiven cell type the presence (1982) Differentiation 22, 90-99. of villin may not necessarily be correlated with the existence 4. Paulin, D., Babinet, C., Weber, K. & Osborn, M. (1980) Exp. of an organized brush border. Cell Res. 130, 297-304. the undifferen- 5. Bretscher, A. & Weber, K. (1979) Proc. Natl. Acad. Sci. USA We report here the expression of villin in 76, 2321-2325. tiated dividing cells of the adult intestinal mucosa and of 6. Bretscher, A. & Weber, K. (1980) Cell 20, 839-847. embryonic intestine. Also, well-differentiated epithelial cells 7. Bretscher, A., Osborn, M., Wehland, J. & Weber, K. (1981) lining the pancreatic and liver ducts, which lack brush Exp. Cell Res. 135, 213-219. borders, have significant amounts of villin localized at their 8. Reggio, H., Coudrier, E. & Louvard, D. (1982) Membranes in apical borders. These simple epithelia and intestinal epithelia Growth and Development (Liss, New York), pp. 89-105. with a brush border are derived from a common epithelial 9. Fogh, J. & Trempe, G. (1975) Human Tumor Cells in vitro sheet, the gut, during embryogenesis (32). In the simple (Fogh, New York), pp. 115-141. epithelia, the presence ofvillin is indicative ofthe cell's origin 10. Pinto, M., Appay, M. D., Simon-Assmann, P., Chevalier, G., Dracopoli, N., Fogh, J. & Zweibaum, A. (1982) Biol. Cell. 44, rather than of the existence of a cellular structure. 193-196. Our observations emphasize that villin is found in some 11. Pinto, M., Robine-Leon, S., Appay, M. D., Kedinger, M., simple epithelia and that its distribution is restricted to organs Triadou, N., Dussaulx, E., Lacroix, B., Simon-Assmann, P., of the gastrointestinal and urogenital tracts. However, the Haffen, K., Fogh, J. & Zweibaum, A. (1983) Biol. Cell. 47, absence of villin in stomach mucosa and especially in its 323-330. columnar cells is surprising. It is also striking to notice that 12. Rabito, C. A., Kreiberger, J. I. & Wight, D. (1984) J. Biol. exocrine glandular cells found in these organs do not express Chem. 259, 574-582. significant amounts ofvillin. In this respect our results are not 13. Rabito, C. A. & Ausiello, D. A. (1980) J. Membr. Biol. 54, in agreement with those reported by Drenckhahn and Mann- 31-38. 14. Herzlinger, D. A. & Ojakian, G. K. (1982) J. Cell Biol. 93, herz (28) who have observed, by using similar approaches, 269-277. villin in pancreatic and prostatic acini. 15. Zweibaum, A., Triadou, N., Kedinger, M., Augeron, C., In conclusion, our observations demonstrate the utility of Robine-Leon, S., Pinto, M., Rousset, M. & Haffen, K. (1983) villin as a marker for undifferentiated enterocytes in tissue Int. J. Cancer 32, 407-412. culture and in vivo. In contrast to differentiated enterocytes, 16. Raul, F., Simon, P., Kedinger, M. & Haffen, K. (1977) Cell. no well-characterized protein(s) marker has been identified Tissue Res. 176, 167-178. for crypt cells of the intestinal mucosa. Villin would appear 17. Hauri, H. P., Kedinger, M., Haffen, K., Freibughaus, A., also to be a useful marker in establishing, without reference Grenier, J. F. & Hadorn, B. (1977) Biochim. Biophys. Acta to morphological criteria, the origin ofmalignant tumors such 467, 327-339. 18. Garrels, J. I. & Gibson, W. (1976) Cell 9, 793-805. as colonic tumors [one of the most frequent cancers in 19. Laemmli, U. K. (1970) Nature (London) 227, 680-685. Western countries (33)] or tumors arising from other villin- 20. Burnette, W. N. (1981) Anal. Biochem. 112, 195-203. containing epithelia. Villin antibodies may be used clinically 21. Coudrier, E., Reggio, H. & Louvard, D. (1983) EMBO J. 2, to classify colonic primary tumors and their metastases, and 469-475. villin may be measured by using a sensitive immunoassay in 22. Gerke, V. & Weber, K. (1983) Eur. J. Cell. Biol. 31, 249-255. the serum of patients with gastrointestinal disorders. 23. Coudrier, E., Reggio, H. & Louvard, D. (1981) J. Mol. Biol. 152, 49-66. 24. Brown, W. J. & Farquhar, M. G. (1984) Cell 36, 295-307. We thank Drs. M. Kedinger and B. Lacroix (Institut National de 25. Reggio, H., Webster, P. & Louvard, D. (1983) Methods la Sante et de la Recherche M6dicale U61, Strasbourg, France) for Enzymol. 33, 379-395. providing us with human fetal intestine. Human primary tumors were 26. Ishimara, K., Fujita, H., Ban, T., Matsudaira, H., Sobuc, K. kindly provided by Drs. J. C. Delchier and S. Zafrani (H6pital Henri & Kakiuchi, S. (1984) Cell. Tissue Res. 235, 207-209. Mondor, Creteil, France). We are deeply indebted to Dr. F. Raul 27. Howe, J. G. & Hershey, J. W. H. (1981) J. Biol. Chem. 256, (Institut National de la Sante et de la Recherche Medicale U61) for 12836-12839. teaching S.R. the intestinal cell isolation procedure. Purified porcine 28. Drenckhahn, D. & Mannherz, H. G. (1983) Eur. J. Cell. Biol. villin was a generous gift from Drs. V. Gerke and K. Weber. We 30, 167-176. thank Drs. L. Blair and J. Rubenstein for their fruitful criticisms and 29. Zweibaum, A., Pinto, M., Chevalier, G., Dussaulx, E., Mrs. S. Guesdon for typing. This work was supported by grants from Triadou, N., Lacroix, B., Haffen, K., Brun, J.-L. & Rousset, the Institut National de la Sante et de la Recherche M6dicale M. (1985) J. Cell. Physiol. 122, 21-30. (n°837012), the Ministere de l'Industrie et de la Recherche (n°82-L- 30. Koldovsky, 0. (1969) Development of the Functions of the 1116 and n°83-V-0086), the Fondation pour la Recherche Medicale, Small Intestine of Mammals and Man (Karger, Basel, Switz- and the Association pour la Recherche sur le Cancer (n° 6379). erland). 31. Lacroix, B., Kedinger, M., Simon-Assmann, P. & Haffen, K. (1984) Gut 25, 925-930. 1. Lazarides, E. (1980) Nature (London) 283, 249-256. 32. Balinsky, B. I. (1981) An Introduction to Embryology (Holt- 2. Moll, R., Franke, W. W., Schiller, D. L., Geiger, B. & Saunders, Philadelphia), pp. 524-546. Krepler, R. (1982) Cell 31, 11-24. 33. Weisburger, J. H., Reddy, B. S. & Wynder, E. L. (1977) 3. Paulin, D., Jakob, H., Jacob, J., Weber, K. & Osborn, M. Cancer 40, 2414-2420. Downloaded by guest on September 27, 2021