ARTICLE |
Correspondence to: Ann Harris, Paediatric Molecular Genetics, Inst. of Molecular Medicine, Oxford Univ., John Radcliffe Hospital, Oxford, OX3 9DU, UK.
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Summary |
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The MUC 6 mucin cDNA was isolated from a human stomach cDNA library and has been shown to be expressed in a number of other tissues in the gastrointestinal tract, including the gallbladder, pancreas, and parts of the ileum and colon. Here we establish that MUC 6 is expressed transiently in the nephrogenic zone of the kidney in the early mid-trimester of development. MUC 6 transcripts were detected in the epithelium of ureteric buds at 13 weeks and at lower levels from 17 to 23 weeks of gestation. Traces of MUC 6 mRNA were seen in the collecting ducts but not elsewhere in the developing kidney, and MUC 6 glycoprotein was detected in the epithelium of ureteric buds and collecting ducts. MUC 6 transcripts were absent from adult kidney. This pattern of expression of MUC 6 in the developing kidney suggests a role in epithelial organogenesis. MUC 6 transcripts were also present at low levels in mid-trimester epididymal epithelium. (J Histochem Cytochem 47:817821, 1999)
Key Words: MUC 6, mucin, kidney development
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Introduction |
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The family of human genes that encode the mucins (mucous glycoproteins) expressed by most epithelia includes nine genes, MUC 14, 5AC, 5B, and 68, although only MUC 1, 2, and 7 have been fully cloned (reviewed in
The MUC 6 cDNA was isolated by expression cloning from a human stomach library (
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Materials and Methods |
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Tissues from mid-trimester terminations were obtained with local ethical committee approval and age was determined on the basis of foot length. Data presented here are derived from two female fetuses aged 17 and 23 weeks and four male fetuses aged 13, 16.5, 18, and 19.5 weeks. Adult kidney was normal tissue obtained from a 65-year-old man undergoing nephrectomy for a renal tumor. Tissues for in situ hybridization were fixed directly in 4% paraformaldehyde (pH 9.5) overnight at 4C, embedded, and frozen in liquid nitrogen before cutting 10-µm sections. Frozen sections were mounted on Vectabond-treated slides (Vector Laboratories; Burlingame, CA) and stored dessicated at -20C until used.
In Situ Hybridization
In situ hybridization was carried out as described previously (
Tissue sections were digested with 10 µg/ml proteinase K for 2 min at 37C, then treated with 25 mM acetic anhydride in 0.1 M triethanolamine (pH 8.0) for 10 min at room temperature (RT), briefly rinsed in 2 x SSC before rapid dehydration through a graded ethanol series, and air-dried. As a control for nonspecific binding of the antisense probe, some sections were treated with 20 µg/ml RNase A for 15 min at RT before incubation with proteinase K.
Hybridization was done overnight at 5560C. After hybridization, slides were washed four times in 4 x SSC at RT and digested with RNase A for 30 min at 37C. Sections were washed at a final stringency of 0.1 x SSC at 60C or 70C for 30 min and dehydrated. Slides were exposed to Kodak Nuclear Tracking (NTB-2) liquid emulsion for 1014 days at 4C. The slides were developed, fixed, and counterstained with hematoxylin and eosin. MUC 6 mRNA expression is seen as black dots on brightfield sections and as white dots on darkfield sections.
Immunocytochemistry
Sections 6 µm thick were cut from equivalent paraformaldehyde-fixed frozen tissues that were used for in situ hybridization experiments. Sections were rehydrated in PBS and then treated with 0.225% H2O2 in 80% methanol for 20 min. Sections were subsequently exposed to (a) 1:3000 diluted M6P (anti-MUC 6) chicken antipeptide polyclonal serum, preimmune serum or PBS for 1 hr, (b) 1:1000 diluted peroxidase-conjugated rabbit anti-chicken immunoglobulins for 1 hr, and (c) biotinylated swine anti-rabbit immunoglobulins for 1 hr. The M6P antibody recognizes a 23-amino-acid synthetic peptide (KPPFTTHSPPTGSSPFSSTGPMT) located within the 169-amino-acid tandem repeat unit of the MUC 6 mucin. Preabsorption of the M6P antibody with MUC 6 synthetic peptide largely abolished its reactivity. Peroxidase label was then visualized with the StreptABC complex/HRP kit (Dako; Glostrup, Denmark) according to the manufacturer's instructions. Tissue sections were stained with hematoxylin.
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Results |
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MUC 6 expression was analyzed in the kidney at 13, 15, 17, 18, and 23 weeks of gestation, and in the epididymis at 16.5, 18, and 19.5 weeks.
Expression of MUC 6 in the Kidney
In the kidney, MUC 6 mRNA was detected in the ureteric bud epithelium at 13 weeks of gestation (Figure 1A and Figure 1B) and at lower levels in the epithelium of some collecting ducts. By 17 weeks of gestation the pattern of expression of MUC 6 was rather more diffuse with transcripts detectable in the epithelium of ureteric buds and collecting ducts, although with a less clear differential distribution (Figure 1C1E). The pattern of expression of MUC 6 at 23 weeks of gestation was similar to that seen at 17 weeks, again with most mRNA being present in the ureteric buds (not shown). Figure 2 shows MUC 6 glycoprotein in the epithelium of the ureteric buds and collecting ducts in 15-week (Figure 2A and Figure 2C) and 18-week fetal kidney (Figure 2D).
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In adult kidney, no MUC 6 expression was seen, although the integrity of RNA within the tissue was confirmed with a cDNA probe for vascular endothelial growth factor (VEGF)/vascular permeability factor. VEGF mRNA expression in the adult kidney sections was localized to glomeruli (not shown), as shown in a previous report (
Expression of MUC 6 in Male Genital Ducts
Low levels of MUC 6 mRNA were detected in the epithelium of the epididymis at 16.5, 18 (Figure 3), and 19.5 weeks. No MUC 6 expression was detected elsewhere in the testis at any gestational age examined.
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Discussion |
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Mucins are known to have a primary role in lubricating and protecting epithelial surfaces. It has also been suggested that they may be involved in epithelial organogenesis, primarily because of the predicted functions of the MUC 1 mucin (
The detection of MUC 6 mucin gene expression in the epididymal epithelium is also of interest. The maturation of sperm and their passage from the testis through the male genital duct system is dependent on the secretion of fluids, salts, and maturation factors from the epididymal epithelium. Among diseases associated with male genital duct obstruction or absence is cystic fibrosis (CF). In CF males, although often anatomically normal during the mid-trimester of human gestation, the male genital ducts become either obstructed or destroyed later in gestation, resulting in male infertility. The pathology of CF in many organ systems involves a failure to clear mucous secretions. We have previously examined the developmental expression all nine mucin genes in the human male genital duct system and have established that MUC 6 is the only mucin gene that is expressed in this tissue during prenatal development. This observation is of importance because it identifies the appropriate gene product to analyze in order to elucidate the biochemical cause of mucin deposition in the CF male genital duct system.
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Acknowledgments |
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Supported by the Cystic Fibrosis Research Trust and grant DK46589 from the National Institutes of Health.
We thank Drs David Cranston, Stephen Gould, Christopher Pugh, and Patrick Maxwell for their assistance, and Dr Sam Ho for the M6P antibody.
Received for publication April 22, 1998; revised December 21, 1998; accepted December 29, 1998.
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Literature Cited |
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Brown LF, Berse B, Tognazzi K, Manseau EJ, Van de Water L, Senger DR, Dvorak HF, Rosen S (1992) Vascular permeability factor mRNA and protein expression in human kidney. Kidney Int 42:1457-1461[Medline]
Chambers JA, Hollingsworth MA, Trezise A, Harris A (1994) Developmental expression of mucin genes MUC1 and MUC2. J Cell Sci 107:413-424
De Bolos C, Garrido M, Real FX (1995) MUC 6 apomucin shows a distinct normal tissue distribution that correlates with Lewis antigen expression in the stomach. Gastroenterology 109:723-734[Medline]
Gleadle JM, Ebert BL, Firth JD, Ratcliffe PJ (1995) Regulation of angiogenic growth factor expression by hypoxia, transition metals, and chelating agents. Am J Physiol 268:C1362-1368
Ho S, Robertson AM, Shekels LL, Lyftogt CT, Niehans GA, Toribara NW (1995) Expression cloning of gastric mucin complementary cDNA and localization of mucin gene expression. Gastroenterology 109:735-747[Medline]
Kispert A, Vainio S, Shen L, Rowitch DH, McMahon AP (1996) Proteoglycans are required for maintenance of Wnt-11 expression in the ureter tips. Development 122:3627-3637
Lan MS, Batra SK, Qi W-N, Metzgar RS, Hollingsworth MA (1990) Cloning and sequencing of a human pancreatic tumor cDNA. J Biol Chem 265:15294-15299
Reid CJ, Gould S, Harris A (1997a) Developmental expression of mucin genes in the human respiratory tract. Am J Respir Cell Mol Biol 17:592-598
Reid CJ, Harris A (1998) Developmental expression of mucin genes in the human digestive system. Gut 42:220-226
Reid CJ, Hyde K, Ho SB, Harris A (1997b) Cystic fibrosis of the pancreas: involvement of MUC6 mucin in obstruction of pancreatic ducts. Mol Med 3:403-411[Medline]
Spicer AP, Rowse GJ, Lidner TK, Gendler SJ (1995) Delayed mammary tumour progression in Muc-1 null mice. J Biol Chem 270:30093-30101
Toribara NW, Robertson AM, Ho S, Kuo WL, Gum J, Hicks E, Byrd JC, Sidikki B, Kim YS (1993) Human gastric mucin. J Biol Chem 268:5879-5885
Woolf AS, KolatsiJoannou M, Hardman P, Andermarcher E, Moorby C, Fine LG, Jat PD, Noble MD, Gherardi E (1995) Roles of hepatocyte growth factor/scatter and the Met receptor in the early development of the metanephros. J Cell Biol 128:171-184[Abstract]