Developmental Mucin Gene Expression in the Gastroduodenal Tract and Accessory Digestive Glands. II. Duodenum and Liver,Gallbladder,and Pancreas

Abstract

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 (6.5–27 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 spatio–temporal regulation of the mucin genes and suggest a possible regulatory role for mucin gene products in gastroduodenal epithelial cell differentiation.

Several years ago, mucin gene expression was shown to be altered in various epithelial cancers (Kim et al. 1996). Moreover, expression of several mucin genes has been detected by us and others early during embryonic and fetal life, before morphogenesis and cytodifferentiation, in intestine (Chambers et al. 1994; Reid and Harris 1998; Buisine et al. 1998), airways (Chambers et al. 1994; Reid et al. 1997; Buisine et al. 1999), and stomach (Buisine et al. 2000). From these data has arisen the hypothesis that the complex structural and functional program that determines the development of epithelial organs from the primitive in- testine might include the expression of most of the mucin genes. In this program, duodenum and accessory digestive glands (liver, gallbladder, and pancreas), are closely associated with a common origin in the terminal part of the foregut. The caudal part of the foregut just behind the respiratory diverticulum develops to form esophagus, stomach, and duodenum. Liver, gallbladder, and pancreas arise from an evagination of the distal foregut. The midgut and the hindgut develop to form duodenum caudal to the bile duct orifice, jejunum, ileum, and colon (deSa 1991). In the fetus, intestinal mucosae secrete mucins as early as 10 weeks of gestation, some 2 weeks before gastric mucosae (Stauffer et al. 1990).

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 (MUC1–MUC4, MUC5AC, MUC5B, MUC6–7) in 13 human embryos and fetuses (aged 6.5–27 weeks of gestation).

Materials and Methods

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 crown–rump 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 hematoxylin–eosin–safran 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 (Buisine et al. 1998, 1999).

In Situ Hybridization

The hybridization steps were as described previously (Audié et al. 1993; Buisine et al. 2000). Slides were developed 2–3 weeks after exposure and counterstained with methyl green pyronin (Sigma; L'Isle d'Abeau Chesnes, France).

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 ×200); + +, moderate (visible at magnification ×100); + + +, strong (visible at magnification ×40).

Results

Duodenum

Embryos and Fetuses. Mucin gene expression was analyzed in the duodenum of seven fetuses aged 10–27 weeks of gestation, when it could be easily distinguished from jejunum. Hybridization data are summarized in Table 1.

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 (Figure 1A). From Week 12, the signal was located in the majority of goblet cells, both on villi and in crypts (Figure 1B). After this time, weak signal was also detected in developing Brünner's glands.

Table 1

Mucin gene expression in embryonic, fetal, and adult duodenum a

		Probes
Age	Tissues	MUC1	MUC2	MUC3	MUC4	MUC5AC	MUC5B	MUC6
10 wk	Duodenum	S – C –	S + + + C+ + +r	S + + C +	S – C –	S – C –	S – C –	S – C –
10.5 wk	Duodenum	S – C –	S + + C+ +	S + + C –	S – C –	S – C –	S – C –	S – C –
12 wk	Duodenum	S+ C	S + + + C+ + +	S + + C –	S – C –	S – C –	S – C –	S – C +
18 wk	Duodenum	S+ C+	S + + C + +	S + C –	S – C –	S – C +	S – C +	S – C +
26 wk	Duodenum	S – C –	ND	S + C –	S – C –	S – C –	S – C –	S – C + +
	G –	ND	G –	G –	G –	G –	G + +
	Duodenum	S – C –	S + + + C+ +	S + C –	S – C –	S – C –	S – C –	S – C + +
		G –	G +	G –	G –	G –	G –	G + +
27 wk	Duodenum	S+ C+	S + + C + +	S + + C –	S – C –	S – C –	S – C –	S – C –
		G +	G +	G –	G –	G –	G –	G +
Adults	Duodenum	ND	S + + + C+ + +	S + + C +	S – C –	S – C –	S – C –	S – C –
		ND	G +	G –	G –	G –	G –	G + +
	Duodenum	S – C –	S + + + C+ + +	S + + C –	S – C –	S – C –	S – C –	S – C –
		G +	G +	G –	G –	G –	G –	G + +
	Duodenum	S – C –	S + + + C+ + +	S + + C –	S – C –	S – C –	S – C –	S – C –
		G +	G +	G –	G –	G –	G –	G + +

^a Labeling localization: S, surface epithelium; C, crypts; G, glands. Labeling intensity: –, absent; +, weak; ++, moderate; + + +, strong; r, rare epithelial cells; ND, not determined.

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 (Figure 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 (Figure 1D).

MUC5AC and MUC5B mRNAs were detected at 18 weeks of gestation in fetal duodenum, where the labeling was restricted to the crypts (Figure 1E and 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 (Figure 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 (Figure 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 6.5–27 weeks of gestation. Hybridization data are summarized in Table 2.

MUC3 mRNAs were detected in embryonic liver as early as 6.5 weeks of gestation (Figure 2A and 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 (Figure 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.

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 (Figure 2D). MUC6, MUC5B, and MUC1 gave weak but homogeneous labeling in all epithelial cells (Figures 2E–2G). 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 18–27 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 (Figure 2H). MUC4 was not expressed in normal liver or biliary tract. In contrast, strong labeling was observed with the MUC4 probe in gallbladder adenocarcinomas (Figure 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 9.5–27 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 pancreato–biliary 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 (Figure 2J and 2K). The signal intensity was stronger in interlobular ducts than in main pancreatic ducts or intralobular ducts (Figure 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 (Figure 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 (Figure 2M). Endocrine cells of islets remained unlabeled. MUC2, MUC4, MUC5AC, and MUC7 were never detected in normal adult pancreas by in situ hybridization.

Figure 1

In situ hybridization for mucin gene mRNAs in embryonic, fetal, and adult duodenum. (A) Embryonic duodenum at 8 weeks of gestation with the MUC2 probe. The labeling is located in rare epithelial cells (arrows). Original magnification ×270. (B) Fetal duodenum at 12 weeks of gestation with the MUC2 probe. The labeling is located in the majority of goblet cells, both on villi and in crypts. Original magnification ×270. (C) Embryonic duodenum at 8 weeks of gestation with the MUC3 probe. The labeling is of moderate intensity and homogeneous throughout the epithelium. Original magnification ×270. (D-F) Fetal duodenum at 18 weeks of gestation. (D) With the MUC6 probe: the labeling is restricted to developing Brünner's glands. Original magnification ×630. (E) With the MUC5AC probe. Original magnification ×360. (F) With the MUC5B probe: the labeling is located in crypts. Original magnification ×630. (G,H) Adult duodenum. (G) With the MUC2 probe: the signal is strong and located in goblet cells, both on villi and in crypts. Original magnification ×130. (H) With the MUC6 probe: the labeling is restricted to the Brünner's glands. Original magnification ×180.

Discussion

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 (Hilkens et al 1995; Ho and Kim 1995). Moreover, a possible role of mucin glycoproteins in cellular adhesion mechanisms during organogenesis has been proposed (Braga et al. 1992).

We have previously found a complex spatio–temporal expression pattern of mucin genes in developing intestine and airways (Buisine et al. 1998, 1999). A possible role of some mucin genes in epithelial cytodifferentiation, elongation, and branching was proposed.

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 (Buisine et al. 1998), with MUC2 being expressed at a high level within goblet cells, both on villi and in crypts, MUC3 within goblet and absorptive cells, and no MUC4 expression. However, in fetal duodenum we did not observe the large predominance of MUC2 expression within the developing crypts that had been observed in embryonic and fetal jejunum and ileum between 9 and 23 weeks of gestation.

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 (Moxey and Trier 1978; Botros et al. 1990). MUC6 expression is therefore initiated during this developmental stage, when it may play a role in regulating the formation of glands. After this stage, MUC6 expression is restricted to the Brünner's glands, which can be distinguished by 16 weeks of gestation (Moxey and Trier 1978; Botros et al. 1990; deSa 1991). These data confirm previous observations of MUC6 apomucin expression in fetal duodenum (Bartman et al. 1998). This expression pattern of MUC6 is reminiscent of MUC5B in the developing respiratory tract, where it is associated with MUC5AC with the development of submucosal mucous glands (Buisine et al. 1999). This pattern of MUC6 expression is also very close to that we described in the developing gastric glands (Buisine et al. 2000).

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 (Moxey and Trier 1978; deSa 1991). Moreover, although MUC5B is not expressed in fetal jejunum and ileum, MUC5B is expressed at 18 weeks of gestation within duodenal crypts in a similar fashion to MUC5AC.

In the preceding report (Buisine et al. 2000), we have shown that MUC5B is expressed in the fetal stomach. MUC5B expression therefore seems to be specific for gastroduodenal ontogenesis. This might indicate that epithelial cells in fetal stomach and duodenum respond to similar stimuli and possess similar mechanisms of turning on and off MUC5B mRNA expression. It is tempting to conclude that, during development, whereas Brünner's glands arise from the base of the duodenal crypts, the cell lineage that gives rise to these glands is closer to the one that gives rise to the cardial and antral glands in stomach than to the cell lineage that gives rise to the goblet and absorptive cells in intestine.

Table 2

Mucin gene expression in embryonic, fetal, and adult accessory digestive glands a,c

		Probes
Age	Tissues	MUC1	MUC2	MUC3	MUC4	MUC5AC	MUC5B	MUC6
6.5 wk	Liver	H–	H–	H+	H–	H–	H–	H–
		D–	D–	D–	D–	D–	D–	D–
8 wk	Liver	H–	H–	H++	H–	H–	H–	H–
		D–	D–	D–	D–	D–	D–	D–
9.5 wk	Liver	H–	H–	H++	H–	H–	H–	H–
		D–	D–	D–	D–	D–	D–	D–
	Pancreas	A–	ND	A–	A–	A–	A–	A–
10 wk	Liver	H–	H–	H++	H–	H–	H–	H–
		D–	D–	D–	D–	D–	D–	D–
	Pancreas	A–	A–	A–	A–	A–	A–	A–
10 wk	Liver	H–	H–	H++	H–	H–	H–	H–
		D–	D–	D–	D–	D–	D–	D–
	Pancreas	A–	A–	A–	A–	A–	A–	A–
10.5 wk	Liver	H–	H–	H+	H–	H–	H–	H–
		D–	D–	D–	D–	D–	D–	D–
	Pancreas	A–	A–	A–	A–	A–	A–	A–
		D–	D–	D–	D–	D–	D–	D–
10.5 wk	Liver	H–	H–	H++	H–	H–	H–	H–
		D–	D–	D–	D–	D–	D–	D–
12 wk	Pancreas	A–	A–	A–	A–	A–	A–	A–
		D–	D–	D–	D–	D–	D–	D–/+
13 wk	Liver	H–	H–	H++	H–	H–	H–	H–
		D–	D–	D–	D–	D–	D–	D–
18 wk	Liver	H–	H–	H+	H–	H–	H–	H–
		D–	D–	D–	D–	D–	D–	D–
	Gallbladder	S+ F+	S+ F+	S+ F+	S- F–	S+ F+	S+ F+	S- F+
	Pancreas	A–	A–	A–	A–	A–	A–	A+
		D–	D–	D–	D–	D–	D–	D-/+
23 wk	Liver	H–	H–	H+	H–	H–	H–	H–
		D–	D–	D–	D–	D–	D–	D–
26 wk	Pancreas	A–	A–	A–	A–	A–	A–	A+
		D–	D–	D–	D–	D–	D–	D–/+
26 wk	Pancreas	A–	A–	A–	A–	A–	A–	A+
		D–	D–	D–	D–	D–	D–/+	D–/+
27 wk	Liver	H–	H–	H+	H–	H–	H–	H–
		D–	D–	D–	D–	D–	D–	D–
	Gallbladder	S+ F+	S+ F+	S++ F++	S– F–	S+ F+	S+ F+	S– F+
	Pancreas	A–	A–	A–	A–	A–	A–	A+
		D–	D–	D–	D–	D–	D–	D–/+
Adult:	Normal tissues
	Liver	H–	H–	H+	H–	H–	H–	H–
		D–/+	D–/+	D–/+++	D–	D–/+	D–/++	D–/+
	Liver	H–	H–	H+	H–	H–	H–	H–
		D–	D–	D-/++	D–	D–	D–/+	D–/+
	Liver	H–	H–	H+	H–	H–	H–	H–
		D–/+	D–/+	D–/+++	D–	D–/+	D–/++	D–/+
	Gallbladder	S+ F+	S+ F+	S+++ F+++	S– F–	S+ F+	S++ F++	S+ F+++
	Gallbladder	S+ F+	S+ F+	S+++ F+++	S– F–	S+ F+	S++ F++	S+ F++
	Gallbladder	S+ F+	S+ F+	S+++ F+++	S– F–	S+ F+	S++ F++	S+ F++
	Pancreas	A–	A–	A–	A–	A–	A–	A+
		D–/+	D–	D–/+++	D–	D–	D–/++	D–/+
	Pancreas	A–	A–	A–	A–	A–	A–	A+
		D–/+	D–	D–/+++	D–	D–	D–/+	D–/+
	Pancreas	A–	A–	A–	A–	A–	A–	A+
		D–/+	D–	D-/+++	D–	D–	D–/+	D–/+
	Gallbladder adenocarcinomas
	Well-differentiated	+	++	++	+++	+	+	+
	Well-differentiated	+	++	++	++	+	+	+
	Well-differentiated	ND	ND	ND	++	ND	ND	ND
	Well-differentiated	ND	ND	ND	+	ND	ND	ND
	Moderately differentiated	ND	ND	ND	+	ND	ND	ND
	Moderately differentiated	ND	ND	ND	+++	ND	ND	ND

^a Labeling localization: H, hepatocytes; D, duct cells; A, acinar cells; S, surface epithelium; F, epithelial folds. Labeling intensity: –, absent; +, weak; ++, moderate; +++, strong; D–/^*, labeling intensity – to ^* depending on the ducts (see text for additional information); ND, not determined.

MUC1 has been reported to be expressed in fetal intestine between 12 and 19 weeks (Chambers et al. 1994). In contrast, we did not detect MUC1 mRNAs in fetal duodenum (the present study) nor in jejunum, ileum, or colon (data not shown). However, our results are in accordance with a study of the Muc1 expression in the developing mouse (Braga et al. 1992).

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, Sasaki et al. (1995) have shown that in the fetal liver new bile ducts do not express MUC3. By contrast, in the postnatal liver, the biliary epithelial cells of intrahepatic large bile ducts consistently express MUC3 apomucin, whereas those of small bile ducts do not. Therefore, absence of detection of MUC3 apomucin does not result from masking of immunoreactive epitopes due to altered or incomplete glycosylation in fetal liver. Synthesis of MUC3 apomucin in postnatal liver results from the upregulation of the MUC3 gene.

We have reported that MUC3 is expressed at a low level in hepatocytes in adult liver (Vandenhaute et al. 1997). This datum was confirmed in the present study. MUC3 mRNAs were also detected in embryonic liver as early as 6.5 weeks of gestation. It is noteworthy that, early in development (6.5–18 weeks), MUC3 is expressed in hepatocytes at a higher level compared to older fetuses and adults. The level of MUC3 mRNA expression is close to that we observed in a post-hepatitis cirrhotic liver (Vandenhaute et al. 1997). The presence of MUC3 mRNAs in hepatocytes is not surprising because it is known that hepatocytes and epithelial cells of bile ducts have a common origin, differentiating from periportal primordial hepatocytes (Van Eyken et al. 1988; Shah and Gerber 1989; Shiojiri et al. 1991; Gerber and Thung 1993). However, it appears that epithelial cells of inter- and intralobular bile ducts (small bile ducts) lose their capacity to express the mucin genes during cytodifferentiation, whereas epithelial cells of main bile ducts retain it.

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 (Bartman et al. 1998). Surprisingly, although MUC3 expression is expressed at a high level in epithelial cells of pancreatic ducts in adults, we did not detect MUC3 mRNAs in developing 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 (Balagué et al. 1995). MUC4 is also expressed in freshly isolated and routinely cultured human gallbladder epithelial cells (Campion et al. 1995). Moreover, MUC4 is expressed in the primitive intestine as early as 6.5 weeks of gestation, long before the appearance of the first differentiation products (Buisine et al. 1998). Therefore, abnormal expression of MUC4 in both gallbladder and pancreas neoplastic situations is reminiscent of their common origin and, although MUC4 was not detected in fetal biliary tract and pancreas, it is likely to result from the reactivation of a very early developmental gene. However, further analyses of mucin gene expression in human embryos will be necessary to draw any firm conclusions.

Figure 2

in situ hybridization for mucin gene mRNAs in embryonic, fetal, and adult liver, gallbladder, and pancreas. (A,B) Embryonic liver at 6.5 weeks of gestation with the MUC3 probe. (A) With the ³⁵S-labeled MUC3 probe: the labeling is weak and located in hepatoblasts. (B) With the ³⁵S-labeled MUC3 probe and a large excess of unlabeled MUC3 probe: hybridization signal is absent. (C) Fetal liver at 18 weeks of gestation with the MUC3 probe. The labeling is weak and located in primordial hepatocytes. (D–G) Fetal gallbladder at 18 weeks of gestation. (D) With the MUC3 probe. (E) With the MUC6 probe. (F) With the MUC5B probe. (G) With the MUC1 probe: the labeling is located in all biliary epithelial cells. Original magnification ×360. (H) Normal adult gallbladder with the MUC6 probe. A marked gradient in the labeling intensity is observed, with a progressively stronger signal from the surface epithelium to the deep epithelial folds. Original magnification ×250. (I) Gallbladder adenocarcinoma with the MUC4 probe. The signal is strong in tumor cells (arrows), whereas normal epithelial cells are unlabeled. Original magnification ×180. (J,K) Fetal pancreas at 26 weeks of gestation with the MUC6 probe. (J) The labeling is located in epithelial cells of interlobular pancreatic ducts (arrows). Original magnification ×360. (K) A signal is also present in developing acini with a large predominance in centroacinar cells. Original magnification ×730. (L,M) Normal adult pancreas. (L) With the MUC3 probe: the signal is strong and heterogeneous in pancreatic ducts; islets (i) are unlabeled. Original magnification ×180. (M) With the MUC6 probe: the signal is located in pancreatic ducts and acini, with a large predominance in centroacinar cells. Original magnification ×310.

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 spatio–temporal 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.