ARTICLE |
Correspondence to: Nicole Porchet, Unité 377 INSERM, Place de Verdun, 59045 Lille Cedex, France. E-mail: jpa@lille.inserm.fr
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Summary |
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Studies were undertaken to provide information regarding cell-specific expression of mucin genes and their relation to developmental and neoplastic patterns of epithelial cytodifferentiation. In situ hybridization was used to study mRNA expression of mucin genes in duodenum and accessory digestive glands (liver, gallbladder, pancreas) of 13 human embryos and fetuses (6.527 weeks' gestation), comparing these with normal and neoplastic adult tissues. These investigations demonstrated that the pattern of mucin gene expression in fetal duodenum reiterated the patterns we observed during gastric and intestinal ontogenesis, with MUC2 and MUC3 expression in the surface epithelium and MUC6 expression associated with the development of Brünner's glands. In embryonic liver, MUC3 was already expressed at 6.5 weeks of gestation in hepatoblasts. As in adults, MUC1, MUC2, MUC3, MUC5AC, MUC5B, and MUC6 were expressed in fetal gallbladder, whereas MUC4 was not. In contrast, MUC4 was strongly expressed in gallbladder adenocarcinomas. MUC5B and MUC6 were expressed in fetal pancreas, from 12 weeks and 26 weeks of gestation, respectively. Surprisingly, MUC3 which is strongly expressed in adult pancreas, was not detected in developmental pancreas. Taken together, these data show complex spatiotemporal regulation of the mucin genes and suggest a possible regulatory role for mucin gene products in gastroduodenal epithelial cell differentiation. (J Histochem Cytochem 48:16671676, 2000)
Key Words: mucin genes, development, differentiation, duodenum, biliary, pancreas, in situ hybridization
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Introduction |
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Several years ago, mucin gene expression was shown to be altered in various epithelial cancers (
The aim of the present study was to characterize mucin gene expression during human fetal development of the duodenum and accessory digestive glands and to correlate this with normal adult tissues and with the alteration of mucin gene expression that has been reported in neoplastic tissues in adults. Using in situ hybridization, we examined the expression of all known mucin genes (MUC1MUC4, MUC5AC, MUC5B, MUC67) in 13 human embryos and fetuses (aged 6.527 weeks of gestation).
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Materials and Methods |
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Tissues
Duodenum, liver, gallbladder, and pancreas were obtained from five human embryos and eight human fetuses after spontaneous or therapeutic abortion with approval of the local ethical committees. The specimens ranged in age from 6.5 to 27 weeks of gestation, dated from the last menstruation (data obtained from clinical records and confirmed by foot and crownrump length). There was no evidence of congenital anomalies in the digestive tract of any of the specimens.
Specimens of normal adult mucosae (duodenum, n = 3; liver, n = 3; gallbladder, n = 3; pancreas, n = 3) were obtained from organ donors and patients without evidence of neoplastic disease and were used as controls. Specimens of gallbladder adenocarcinomas were also obtained from two patients and analyzed.
Each specimen was immediately immersed in fresh 4% paraformaldehyde, and further embedded in paraffin. Three-µm-thick sections were cut, mounted on gelatin-covered slides, and stored at 4C until used. Serial sections were systematically stained with hematoxylineosinsafran and astra blue for histological analysis.
Probes
In situ hybridization was performed using eight 35S-labeled oligonucleotide probes corresponding to each tandem repeat domain of MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC6, and MUC7, as described in earlier studies (
In Situ Hybridization
The hybridization steps were as described previously (
The following controls were performed: (a) fetal tissue sections treated with 50 µg/ml ribonuclease A (Boehringer Mannheim; Meylan, France); (b) fetal tissue sections treated with a large excess of unlabeled oligonucleotide identical to or distinct from the 35S-labeled-probe; and (c) adult and fetal tissue sections were tested in parallel under the same conditions.
The intensity of the hybridization signal was scored semiquantitatively by two independent observers (MPB, LD) as: -, absent; +, weak (visible at magnification x200); ++, moderate (visible at magnification x100); +++, strong (visible at magnification x40).
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Results |
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Duodenum
Embryos and Fetuses.
Mucin gene expression was analyzed in the duodenum of seven fetuses aged 1027 weeks of gestation, when it could be easily distinguished from jejunum. Hybridization data are summarized in Table 1.
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MUC2 was expressed at a high level in the fetal duodenum as early as 10 weeks of gestation. The signal was first located in rare epithelial cells (Fig 1A). From Week 12, the signal was located in the majority of goblet cells, both on villi and in crypts (Fig 1B). After this time, weak signal was also detected in developing Brünner's glands.
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MUC3 mRNAs were also detected from 10 weeks of gestation in fetal duodenum. Labeling of moderate intensity was continuous and homogeneous throughout the undifferentiated epithelium (Fig 1C). In older fetuses, the signal was present in both goblet and absorptive cells on villi. Weak signal was also found in crypt cells.
MUC6 mRNAs were detected from 12 weeks of gestation. The labeling was weak and was located within crypts. After this time, the labeling was restricted to the developing Brünner's glands (Fig 1D).
MUC5AC and MUC5B mRNAs were detected at 18 weeks of gestation in fetal duodenum, where the labeling was restricted to the crypts (Fig 1E and Fig 1F). MUC5AC and MUC5B were not expressed at any other gestational age.
MUC1, MUC4 and MUC7 mRNAs were not detected in fetal duodenum at any gestational age.
Adults. In adults, MUC2, MUC3, and MUC6 were the predominant mucin genes expressed in duodenum. In the surface epithelium, MUC2 mRNAs were detected in goblet cells (Fig 1G), whereas MUC3 mRNAs were detected in both goblet and absorptive cells. Moreover, MUC6 and, to a lesser extent, MUC1 and MUC2, were expressed in Brünner's glands (Fig 1H). MUC4, MUC5AC, MUC5B, and MUC7 were not detected in adult duodenum by in situ hybridization.
Liver and Biliary Tract
Embryos and Fetuses.
Mucin gene expression was analyzed in the gallbladder and liver of 11 embryos and fetuses of 6.527 weeks of gestation. Hybridization data are summarized in Table 2.
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MUC3 mRNAs were detected in embryonic liver as early as 6.5 weeks of gestation (Fig 2A and Fig 2B). The labeling was of moderate intensity and was located in the perinuclear region of hepatoblasts. This pattern of MUC3 expression remained constant until 18 weeks (Fig 2C). After this time, the labeling was weak and distributed throughout the cytoplasm. MUC1, MUC2, MUC4, MUC5AC, MUC5B, MUC6, and MUC7 were not detected in hepatoblasts or primordial hepatocytes at any gestational age. Epithelial cells of intrahepatic bile ducts did not express any of the mucin genes at any gestational age.
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In gallbladder, the strongest signal was obtained with the MUC3 probe, from 18 weeks of gestation. The labeling was homogeneously distributed in all biliary cells of the surface epithelium and epithelial folds (Fig 2D). MUC6, MUC5B, and MUC1 gave weak but homogeneous labeling in all epithelial cells (Fig 2E2G). Weak and heterogeneous signal was also observed with the MUC2 and MUC5AC probes in the epithelium. From 26 weeks, with the MUC6 probe, the labeling became largely restricted to the deep epithelial folds. As in intrahepatic bile ducts, MUC4 and MUC7 mRNAs were not detected in fetal gallbladder. This pattern remained constant from 1827 weeks of gestation.
Adults. In adults, strong labeling was observed with the MUC3 probe in large bile ducts. Weak labeling was also found with the MUC5B probe. MUC1, MUC2, MUC5AC, and MUC6 mRNAs were occasionally detected. MUC4 and MUC7 mRNAs were never detected by in situ hybridization. Small bile ducts remained unlabeled whatever the probe. As in the older fetuses, weak and diffuse signal was detected in hepatocytes with the MUC3 probe.
In normal adult gallbladder, the hybridization pattern was similar to that we observed in fetal gallbladder, with a strong and homogeneous signal with the MUC3 probe, a moderate signal with the MUC5B and MUC6 probes, and a weak signal with the MUC1, MUC2, and MUC5AC probes. A marked gradient in labeling intensity was observed with the MUC6 probe, with a progressively stronger signal from the surface epithelium to the deep epithelial folds (Fig 2H). MUC4 was not expressed in normal liver or biliary tract. In contrast, strong labeling was observed with the MUC4 probe in gallbladder adenocarcinomas (Fig 2I). MUC2, MUC3 and, to a lesser extent, MUC1, MUC5AC, and MUC5B mRNAs were also detected in gallbladder adenocarcinomas.
Pancreas
Embryos and Fetuses.
Pancreas was available from nine embryos and fetuses of 9.527 weeks of gestation.
MUC6 mRNAs were first detected at 12 weeks of gestation. The labeling was weak and was located in epithelial cells of a large pancreatobiliary duct and other pancreatic ducts. After this age, a signal was constantly found in pancreatic ducts and acini, with a large predominance in centroacinar cells (Fig 2J and Fig 2K). The signal intensity was stronger in interlobular ducts than in main pancreatic ducts or intralobular ducts (Fig 2J). MUC5B mRNAs were detected from 26 weeks of gestation in fetal pancreas, in which the labeling was located in epithelial cells of interlobular ducts. Endocrine cells grouped into islets of Langherans did not express MUC6 or MUC5B. MUC3 and other mucin genes were not detected in fetal pancreas at any gestational age.
Adults. In adults, the MUC3 probe showed a strong but heterogeneous signal in epithelial cells of interlobular pancreatic ducts (Fig 2L). Weak but homogeneous hybridization signal was observed with the MUC1, MUC5B, and MUC6 probes in interlobular ducts. Weak signal was also occasionally detected in smaller pancreatic ducts with MUC1 and MUC6 probes. MUC6 mRNAs were also detected in acini, in which the signal was largely restricted to centroacinar cells (Fig 2M). Endocrine cells of islets remained unlabeled. MUC2, MUC4, MUC5AC, and MUC7 were never detected in normal adult pancreas by in situ hybridization.
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Discussion |
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Major changes in the normal pattern of mucin expression, quantitatively and/or qualitatively, have been described in various preneoplastic and neoplastic tissues. There is evidence that mucins expressed by neoplastic cells play diverse roles in addition to protective functions. More particularly, mucins may be implicated in progression of human carcinomas and in promotion of tumor cell metastasis because of alterations in cell growth regulation, cellular adhesion and immune recognition (
We have previously found a complex spatiotemporal expression pattern of mucin genes in developing intestine and airways (
Duodenum
In this study, we have examined the expression of all mucin genes in adult duodenum and in fetal duodenum, when it could be easily distinguished from jejunum. Analysis of developmental mucin gene expression in the duodenum warrants separate consideration, not only because the organ is derived from both the caudal foregut and the cephalic midgut but also because it is the site from which the hepatic and pancreatic primordia arise. To our knowledge, this is the first extensive study reporting the pattern of mucin gene expression in both fetal and adult duodenum.
In adults, MUC2, MUC3, and MUC6 are the major mucin genes expressed in duodenum. MUC2 mRNAs are detected in epithelial goblet cells, whereas MUC3 mRNAs are detected in both goblet and absorptive cells. Moreover, MUC6 and, to a lesser extent, MUC1 and MUC2 are expressed in Brünner's glands. MUC4, MUC5AC, MUC5B, and MUC7 are not expressed in duodenum.
In fetuses, data concerning MUC2, MUC3, and MUC4 expression in duodenum were consistent with those we have reported in jejunum and ileum (
Fetal MUC6 expression is detected at 12 weeks of gestation, when it is associated with the development of Brünner's glands. Brünner's glands originate from pluripotent stem cells situated at the crypt base. When the majority of the daughter cells migrate up towards the villous tip, some of them multiply to form a cellular bud which grows into the juxtaposed mesenchyme as a solid cylinder, maintaining continuity with the surface epithelium (
MUC5AC mRNAs are significantly detected in fetal duodenum only at 18 weeks of gestation. The signal is present within crypts. We have reported MUC5AC mRNA expression in the developing intestine (jejunum and ileum) between 8 and 12 weeks, with no MUC5AC expression in older fetuses. The present result confirms the notion that MUC5AC is developmentally expressed during intestinal ontogenesis. However, the expression of MUC5AC is late in the duodenum and in the opposite direction, with the progression of cytodifferentiation from duodenum to colon (
In the preceding report (
MUC1 has been reported to be expressed in fetal intestine between 12 and 19 weeks (
Accessory Digestive Glands
Liver and Biliary Tract.
In fetal liver, bile ducts did not express any of the mucin genes at any gestational age. In adult liver, only large bile ducts expressed MUC3, MUC5B, MUC1, MUC2, MUC5AC, and MUC6 mRNAs, in decreasing order of labeling intensity. Unfortunately, none of the fetal liver samples we studied, except for one, contained large bile ducts, and therefore we were unable to show if the MUC3 gene and, to a lesser extent, MUC5B, MUC1, MUC2, MUC5AC, and MUC6 are expressed in fetus as they are in adults. Likewise, using immunohistochemistry,
We have reported that MUC3 is expressed at a low level in hepatocytes in adult liver (
In gallbladder, the same pattern of mucin gene expression was observed in fetuses and normal adults, with MUC3, MUC5B, MUC1, MUC2, and MUC5AC expression in all epithelial cells and MUC6 expression in epithelial folds. MUC4 and MUC7 are not expressed in normal gallbladder. In contrast, MUC4 is strongly expressed in gallbladder adenocarcinomas and may therefore be a marker for gallbladder adenocarcinomas.
Pancreas.
In adults and fetuses from 12 weeks of gestation, MUC6 and MUC5B are expressed in epithelial cells of pancreatic ducts. MUC6 is also expressed in some acinar cells, essentially in developing centroacinar cells. These data are in accordance with our previous report of MUC6 mRNA and peptide expression in adult and fetal pancreas (
We have shown that the MUC4 gene is abnormally expressed in gallbladder adenocarcinomas. Likewise, MUC4 is expressed in pancreas cancers but not in normal pancreas (
The occurrence of heterotypic islands of small intestine-type mucosa, Brünner's glands, or pancreatic tissues in embryonic and fetal digestive tract, and also the gastric and intestinal metaplasia that can be displayed by a regenerating or neoplastic mechanism, both suggest that only a few gene products distinguish the epithelial cells of accessory digestive glands (liver, gallbladder, pancreas) from those of the surrounding tissues of stomach and duodenum. This hypothesis is reinforced by the pattern of MUC5B and MUC6 expression in the gastroduodenal tract. We have shown that MUC5B is developmentally expressed during gastric and duodenal ontogenesis. Moreover, MUC6 is expressed in gastroduodenal mucous glands and with MUC5B in gallbladder and pancreas. This pattern is very close to that of MUC5B in the respiratory tract, where it is associated with MUC5AC in the development of glandular ducts and submucosal glands. Therefore, MUC5B and MUC6 may play a role in regulating the formation of gastroduodenal mucous glands and accessory digestive glands.
Taken together, our data show that, during embryonic and fetal life, epithelial cells of the developing organs derived from the primitive gut are able, even poorly differentiated, to express important amounts of mucin gene messengers according to complex spatiotemporal patterns. Some common features are noteable, such as the expression of MUC6/MUC5B concomitant with the formation of mucous glands. Conversely, some differences must be noted, suggesting various roles for a given apomucin depending on the organ: early MUC2 expression before cytodifferentiation of surface epithelial goblet cells in intestine and airways, and, in contrast, late MUC2 expression during terminal differentiation of mucous glands in airways and stomach. Moreover, all these observations show that, in epithelial organs coming from the primitive gut, each specific cell lineage exhibits co-expression of a typical set of mucin genes, therefore strongly suggesting cooperative but specific roles for their products.
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Acknowledgments |
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Supported by grants from the Association pour la Recherche sur le Cancer (contract no. 9643), the EC Concerted Action: Mucins in inflammatory disease, and the CHRU de Lille (contract no. 96/29/9595).
We thank Elisabeth Deschodt for excellent technical assistance and ICARE Multimédi@, particularly Gérard Espouy, for color prints.
Received for publication February 21, 2000; accepted June 14, 2000.
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