The sld mutation is specific for sublingual salivary mucous cells and disrupts apomucin gene expression

M. A. Fallon1, L. R. Latchney1, A. R. Hand2, A. Johar3, P. A. Denny4, P. T. Georgel1, P. C. Denny4 and D. J. Culp1

1 University of Rochester Medical Center, Center for Oral Biology and the Department of Pharmacology and Physiology, Rochester, New York 14642
2 Departments of Pediatric Dentistry
3 BioStructure and Function, University of Connecticut Health Center, School of Dental Medicine, Farmington, Connecticut 06030
4 University of Southern California, Division of Diagnostic Sciences, School of Dentistry, Los Angeles, California 90089


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 References
 
NFS/N-sld mice harbor a spontaneous autosomal recessive mutation, sld (sublingual gland differentiation arrest) and histologically display attenuated mucous cell expression in sublingual glands (Hayashi et al. Am J Pathol 132: 187–191, 1988). Because altered serous demilune cell expression is unknown, we determined the phenotypic expression of this cell type in mutants. Moreover, we evaluated whether absence of glycoconjugate staining in 3-day-old mutant glands is related to disruption in apomucin gene expression and/or to posttranslational glycosylation events. Serous cell differentiation is unaffected, determined morphologically and by serous cell marker expression (PSP, parotid secretory protein; and Dcpp, demilune cell and parotid protein). Conversely, apical granules in "atypical" exocrine cells of mutant glands are PSP and mucin negative, but contain abundant SMGD (mucous granule marker). Age-related appearance of mucous cells is associated with expression of apomucin gene products, whereas SMGD expression is unaltered. "Atypical" cells thus appear specified to a mucous cell fate but do not synthesize mucin glycoproteins unless selectively induced postnatally, indicating the sld mutation disrupts apomucin transcriptional regulation and/or decreases apomucin mRNA stability.

salivary glands; exocrine cells; secretion; mucins; postnatal development


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 References
 
SALIVA PRODUCTION involves the coordinated action of a diverse array of glandular structures, each contributing differentially to the total pool of salivary organic and fluid constituents. In humans, 70% of salivary mucins are contributed by the combined secretions of mucous glands, consisting of the major sublingual glands and the numerous minor mucous glands such as palatine, buccal, and labial glands (14). The remaining 30% of mucins are secreted by submandibular glands, which are mixed glands composed of both serous and mucous acinar structures. Mucins are proposed to play various roles to preserve the integrity of the oral cavity including lubrication, hydration, and the selective clearance or adherence of microorganisms (19).

Elucidation of gene expression by salivary exocrine cells is a primary step in development of cellular therapeutic strategies to treat glands damaged due to radiation treatment for cancer or to autoimmune disease. Accordingly, we initiated studies of NFS/N-sld mice harboring an autosomal recessive mutation (sld, sublingual gland differentiation arrest) that alters expression of the mucous cell phenotype in sublingual glands (9, 12). In mutant neonates, mucous cells are absent, whereas ductal cells and myoepithelial cells appear normal (9). Histologically, other tissues expressing mucous cells (i.e., trachea, stomach, lung, and intestine) appear unaffected, as do parotid and submandibular salivary glands (9). During postnatal development, a subpopulation of mucous cells appear gradually, as assessed by histological staining for glycoconjugates, although mucous cells account for only 30% of the total acinar cell population at 36 wk, the longest time point measured (9).

Tubuloacini of sublingual glands are capped at their distal ends by serous demilune cells that contain a small number of apical secretory granules (11, 18). It is unknown whether the sld mutation has global acinar effects, altering the expression of both serous demilune cells and mucous cells. We therefore conducted morphological and biochemical studies to determine whether the sld mutation disrupts phenotypic expression of serous demilune cells. In addition, the absence of staining for glycoconjugates in histological sections of 3-day-old mutant glands indicates a disruption in mucin glycoprotein expression, either at the level of apomucin gene expression or in posttranslational glycosylation events. We therefore performed biochemical studies to better delineate effects of the sld mutation on mucin glycoprotein expression. Results demonstrate mucous cells are affected specifically and that a major consequence of the sld mutation is attenuation of sublingual apomucin gene expression.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 References
 

Materials.
All materials were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. Antisera against purified rat sublingual mucins from two rabbits were pooled to derive anti-mucin antisera, as described previously (13). Rabbit IgG antibodies to rat submandibular gland protein (SMGD) (1) and rat parotid secretory protein (PSP) (2) were obtained from Dr. William D. Ball, Department of Anatomy, Howard University, College of Medicine, Washington, DC.

Animals and excision of glands.
Three breeding pairs of NFS/N-sld mice were derived from frozen embryos and kindly provided by Drs. Tatsuji Nomura and Kyoji Hioki of the Central Institute for Experimental Animals, Kawasaki, Japan. Mutant mice are normal in appearance, activity, and breeding capacity and thus require no special husbandry. As controls, pregnant female and young adult NFS/NCr mice were purchased from the National Cancer Institute animal program maintained by Charles River Laboratories in Frederick, MD. Mice that eventually mutated to the NFS/N-sld strain were originally obtained from this source. Animals were anesthetized by CO2 inhalation and killed by exsanguination via section of the aorta. Sublingual glands were rapidly excised free of associated connective tissue as well as vascular and neural elements at the hilum. Glands were either quickly blotted with filter paper and weighed (Mettler MT5 microbalance; Mettler Toledo, Columbus, OH), immediately frozen in liquid nitrogen, or homogenized. Mice of 3 days and 8 wk of age were used. At 8 wk, ~10% of acinar cells of mutant mice are reportedly mucous-like (9). We reasoned this age represents the earliest time point at which sufficient numbers of mucous-like cells would be present in mutant glands for our experiments.

SDS-PAGE.
Sublingual glands (one 8 wk or ten 3 day) in liquid nitrogen were immediately placed into siliconized glass tubes (13 x 75 mm) and sonicated (Sonifier model 450; Branson Ultrasonic, Danbury, CT) in 300 µl of 1% dithiothreitol (DTT, US Biochemical), 2% SDS, 0.002% bromophenol blue (Bio-Rad Laboratories, Hercules, CA), and 10% (wt/vol) glycerol in 63 mM Tris·HCl (pH 6.8). DTT was then added (3% final concentration), and the samples were boiled 10 min and centrifuged (12,000 g, for 20 min). Supernatants were applied directly to SDS-PAGE gels or stored at -20°C. This method of sample preparation was found necessary to prevent complexation and rapid degradation of glandular proteins. Subsequent protein estimation of homogenates is prevented due to interference with protein assays by constituents in the sonicate. Thus we initially applied 15 µl of each sample to a 2–15% gradient gel (Owl Separation Systems, Portsmouth, NH), then stained for protein, and a digital image was obtained. The pixel density of each lane was estimated (NIH Image v1.62, http://rsb.info.nih.gov/nih-image/), and sample volumes were adjusted to provide equivalent pixel densities for each lane. A gland homogenate prepared similarly, but in phosphate buffer plus protease inhibitor cocktail (Sigma-Aldrich), contained about 50 µg protein per 15 µl of homogenate (BCA protein assay; Pierce Chemical, Rockford, IL).

Electrophoresis was performed using a Hoefer Mighty Small minigel system (Amersham Biosciences, Piscataway, NJ). Precast gels were obtained from either Invitrogen (Carlsbad, CA) or Owl Separation Systems. Gradient gels (10 x 10 cm) included NuPAGE Tris-acetate 3–8% gels (Invitrogen) and PAGE-ONE 10–20% and 2–15% (Owl Separation Systems). Gels were run at 30–40 mA per gel in either Tris-acetate-SDS buffer (NuPAGE 3–8% gels) or Tris-glycine-SDS buffer (PAGE-ONE gels) under reducing conditions (NuPAGE antioxidant) according to each manufacturer’s instructions. Gels were stained either for proteins with Coomassie brilliant blue (Bio-Rad) or for glycoproteins with Alcian blue (10). In both cases, dye staining was enhanced with subsequent silver staining as described previously (10), except the method of Morrissey (17) was used. Molecular weight standards (MultiMark Multi-Colored standard, Invitrogen) included myosin (250 kDa), phosphorylase B (148 kDa), glutamic dehydrogenase (60 kDa), carbonic anhydrase (42 kDa), and myoglobin (30 and 22 kDa). Digital images of stained gels were obtained using a ChemiImager ready System (Alpha Innotech, San Leandro, CA).

Western blotting.
Proteins in 3–8% gels were transferred overnight onto Immobilon-PVDF membranes (Millipore, Milford, MA) at 200 mA in NuPAGE transfer buffer (Invitrogen) containing 0.1% NuPAGE sample antioxidant. Membrane strips were rinsed twice and incubated in blotting solution (0.2% Triton X-100, 5% non-fat dry milk in PBS) at room temperature for 1 h prior to overnight incubation with individual antibody diluted in blotting solution. Rabbit anti-PSP and anti-SMGD IgG were each used at 5 µg/ml, and rabbit anti-mucin was diluted 500-fold. Strips were rinsed and incubated 2 to 3 h with 500,000 cpm/ml 125I-labeled rProtein A (>80 µCi/µg; PerkinElmer Life Sciences, Boston, MA), rinsed, and subjected to autoradiography using Kodak Biomax MS or MR film (Eastman Kodak, Rochester, NY) at -80°C. Films were developed with a Kodak X-OMAT 3000RA processor and scanned with a high-resolution flatbed scanner (ScanMaker 9600XL; Microtek, Redondo Beach, CA).

Immunolocalization of proteins.
For immunoperoxidase labeling, tissue was fixed overnight in 10% buffered formalin (pH 7.5) and embedded in paraffin. To expose antigens, sections (5 µm) were incubated 10 min in 5% urea (95–99°C), allowed to cool 10 min, then incubated 20 min in 0.4% Tween 20 in PBS. Endogenous peroxidase was inhibited (0.3% H2O2 in methanol, 10 min), and sections incubated 30 min in 10% normal horse serum (NHS) in PBS followed by 1 h in primary antibody diluted in 10% NHS in PBS. Rabbit anti-SMGD IgG and nonimmune rabbit IgG were used at 8 µg/ml. Rabbit anti-mucin and preimmune serum were diluted 500-fold. Sections were washed (PBS + 0.05% Tween 20) and incubated 1 h with 5 µg/ml biotinylated horse anti-mouse/rabbit (Vector Laboratories, Burlingame, CA) in PBS. Immunodetection was performed using the avidin-biotin-peroxidase complex method (Vectastain Elite kit, Vector Laboratories) with 3,3’-diaminobenzidine tetrahydrochloride (DAB) as peroxidase substrate, either with or without Ni enhancement. Sections were either mounted in Refrax (Anatech, Battle Creek, MI) or counterstained in hematoxylin and mounted in Permount (Fisher Scientific, Fair Lawn, NJ). Sections were examined with an Eclipse E800 microscope (Nikon, Melville, NY) under either bright-field or differential-interference contrast (DIC) optics. Digital images were captured with a Spot 2 digital camera and software (Diagnostic Instruments, Sterling Heights, MI).

For immunogold labeling of 1-µm sections, freshly excised sublingual glands were diced ({approx}1 mm3) on ice, fixed overnight in 10% buffered formalin (pH 7.5), embedded in LR Gold resin (London Resin, Basingstoke, UK), and polymerized in UV light (365 nm) at -20°C. Sections (1 µm) were rinsed, treated with 1% bovine serum albumin (BSA) plus 5% normal goat serum (NGS) in PBS and incubated 90 min with either primary rabbit IgG antibody (anti-PSP, 0.1 µg/ml; or anti-SMGD, 5 µg/ml) or rabbit nonimmune IgG diluted with 1% BSA, 5% NGS in PBS. Sections were rinsed, incubated 60 min with gold-labeled goat-anti-rabbit IgG (5 nm; Amersham Biosciences) diluted in 1% BSA in PBS, then rinsed, and the bound gold was visualized by silver enhancement (British BioCell International, Cardiff, UK). Sections were lightly stained with 1% methylene blue, 1% azure II, mounted with DPX (BDH Laboratory Supplies, Poole, Dorset, UK), and photographed in a Leitz Orthoplan microscope.

For electron microscopic immunogold labeling, tissue (~1 mm3) was fixed 1 h in ice-cold 1% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, and embedded in LR Gold resin. Thin sections were collected on Formvar-coated nickel grids, then incubated overnight with primary antibody or nonimmune serum. Bound primary antibody was detected with 10-nm diameter gold-labeled goat anti-rabbit IgG (Amersham). Sections were then stained with uranyl acetate and lead citrate and examined in a JEOL 100CX transmission electron microscope.

For all histological and immunolocalization studies, we examined a minimum of four sections from each tissue block prepared from at least three different animals for each strain and age group of mice.

Electron microscopy.
Gland fragments (~1 mm3) were fixed for 3 h at room temperature with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, rinsed in cacodylate buffer, postfixed in 1% OsO4, 0.8% potassium ferricyanide in 0.1 M cacodylate buffer, stained in block with 0.5% aqueous uranyl acetate, and embedded in Polybed epoxy resin (Polysciences, Warrington, PA). Thin sections were cut with a diamond knife, collected on copper grids, and examined with a JEOL 100CX transmission electron microscope.

Northern analysis.
Total RNA was isolated from glands frozen in liquid nitrogen immediately after excision. Frozen glands were ground to a fine powder under liquid nitrogen using a mortar and pestle and resuspended in TRIzol (Invitrogen). Subsequent steps were according to the manufacturer specifications but without the addition of glycogen. In some cases, modifications were made to reduce mechanical fragmentation of extremely large apomucin RNA molecules. These modifications include the following: 1) gently resuspending powdered glands in TRIzol using a truncated 200-µl pipette tip (Laboratory Product Sales, Rochester, NY) until homogeneous (about 30 s); 2) after centrifugation of the TRIzol-gland mixture, incubating the supernatant at 30°C for 5 min; 3) mixing with chloroform by shaking the tube gently by hand until the sample is uniformly opaque, followed by incubation at 30°C for 5 min; and 4) the use of truncated pipette tips for all steps to resuspend and transfer samples.

The concentration and purity of RNA was determined from the A260/280 absorption values, with a ratio of 1.9–2.0 considered acceptable. RNA samples (5 or 10 µg/lane) were separated on agarose gels under formaldehyde denaturing conditions and transferred to nylon membranes. Probes to mouse sublingual gland apomucin transcripts were generated using [{alpha}-32P]dCTP (>3,000 Ci/mmol, PerkinElmer Life Sciences) and either nick translation (Nick Translation System, Invitrogen) or random-primed synthesis (Random Primers DNA Labeling System, Invitrogen) of the 1,457-nucleotide putative apomucin cDNA inserted in pBluescript SK(+/-) to yield pSL1-12. An EcoRV truncated version (803 bp) of pSL1-12 was also used and excludes 654 nucleotides of the cDNA 3’ end. The probe for demilune cell and parotid protein (Dcpp) (32P-labeled cRNA) was transcribed from pSLGp203, as described previously (3). Component solutions of the NorthernMAX kit (Ambion) were used for prehybridization, hybridization (42°C overnight), and washing of blots (2 washes of 30 min at 23°C in low-stringency solution followed by 4 washes of 30 min at 65°C in high-stringency solution). After autoradiography using Kodak MR film (Eastman Kodak), blots were stripped and reprobed with 32P-labeled internal standards consisting of either antisense RNA generated by transcription of the pTRI RNA 28S antisense template (Ambion, Austin, TX) or DNA produced by random-primed synthesis from the DECA template for mouse 18S RNA (Ambion).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 References
 

Sublingual gland mass.
Because the sld mutation reportedly diminishes expression of the mucous cell phenotype, we initially asked whether this may reflect, in part, a more global defect in the developmental expansion of acinar cells, as manifested by a decrease in gland mass. As shown in Table 1, no significant differences were found in absolute and normalized gland mass between 3-day-old controls and mutants, although glands were markedly larger in males than females. By 8 wk, sexual differences in absolute gland mass are much less distinct in controls and absent in mutants. When normalized to body weights, gland mass is more than 25% larger in mutants compared with controls at 8 wk. This difference is not due to an obvious selective increase in ductal or acinar cells, at least as determined by eye in the subsequent examination of many histological sections (see below). Nevertheless, the developmental expansion of acinar cells does not appear to be compromised in mutant glands.


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Table 1. Comparisons between sexes and strains of mice with respect to sublingual gland wet weights, body weights, and to gland weights normalized to body weights

 
Histological comparison of mucous and serous cells in mutant and control glands.
Rat sublingual glands are composed primarily of mucous acini with a minor component of serous demilunar cells and a simple ductal system. Serous demilune cells are readily observed in both mutant and control glands of both age groups (Fig. 1, arrows). We used Alcian blue to stain acidic mucous glycoproteins localized to mucous cell secretion granules. Glands of control mice at 3 days and 8 wk of age are composed mostly of dark Alcian blue staining mucous cells (Fig. 1, A and C). In contrast, no mucous cells can be identified by Alcian blue staining in 3-day-old mutants (Fig. 1B), whereas at 8 wk, glands display islands of Alcian blue-stained cells (Fig. 1D). With age, mutant glands remain grossly deficient of Alcian blue-stained cells, even after 1 yr (not shown). There are no obvious histological differences between male and female sublingual glands within each strain and age group (not shown), consistent with previous reports (9, 18).



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Fig. 1. Paraffin sections (5 µm) of sublingual glands from control (A and C) and mutant (B and D) mice at 3 days (A and B) and 8 wk (C and D) of age. Sections were stained with Alcian blue for acidic glycoconjugates and counterstained with nuclear Fast red using standard histological methods. Arrows indicate serous demilune cells; d, ductal structures. Scale bar in A = 35 µm.

 
Ultrastructural comparisons of acinar cells in mutant and control glands.
Acinar cells of control mice at 3 days and 8 wk of age are similar in appearance in transmission electron micrographs, displaying features typical of mucous and serous demilune cells. Mucous cells are pyramidal in shape and have an apical cytoplasm packed with large electron-translucent granules, prominent Golgi, and a nucleus compressed against the basal cell border (Fig. 2A). Serous demilune cells are denoted by their peripheral orientation within an acinus, a central large nucleus with dense chromatin, and by abundant rough endoplasmic reticulum. Demilune cells contain a sparse population of small electron-dense secretion granules localized to the apical cytoplasm. These cells can also be observed extending between mucous cells to reach the acinar lumen (Fig. 2B). In acini of 3-day-old mutants, the great majority of cells are atypical in appearance, although they have characteristics of exocrine cells (Fig. 2C). These "atypical" cells contain 1) few and small electron-dense granules in their apical cytoplasm adjacent to the acinar lumen; 2) a large basally oriented nucleus containing scant dense chromatin and surrounded by a modest amount of rough endoplasmic reticulum; 3) tight junctions; and 4) accumulations of glycogen, either well preserved as small dense particles or extracted and/or poorly contrasted to look more like vacuoles. In contrast, serous demilune cells of mutant glands appear normal. These cells are arranged at the end of acini and contain a more centrally located nucleus that is surrounded by abundant rough endoplasmic reticulum. Secretion granules of serous demilune cells are 50% to 100% larger and slightly less electron dense than those of "atypical" exocrine cells (Fig. 2D).



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Fig. 2. Transmission electron micrographs of sublingual acinar cells in control and mutant glands. A: normal mucous cell in an 8-wk-old control gland. The cell is filled with electron-lucent granules, contains a basal and compressed nucleus (N) and moderate rough endoplasmic reticulum (arrow). L, lumen. B: typical serous demilune cell (8-wk-old control gland) with an apical extension between two adjacent mucous cells (M), a large central nucleus (N) containing dense chromatin, and few electron-dense secretion granules. C: "atypical" exocrine cells of a 3-day-old mutant gland display a triangular shape, a large round basally oriented nucleus (N), tight junctions (arrowhead), and dark granular deposits of glycogen throughout the cytoplasm (arrows). Small, moderately dense, secretion granules are present in the apical cytoplasm adjacent to the lumen (L). D: serous demilune cell (S) displaying normal features in a 3-day-old mutant gland. Note the abundant rough endoplasmic reticulum and the larger size of serous cell granules compared with those of the adjacent "atypical" cell (A). E: mature mucous cell (M) with distorted basal nucleus (N) adjacent to the apical cytoplasm of a "transitional" mucous cell (T) in an 8-wk-old mutant gland. F: "transitional" cell at higher magnification displaying granules with regions of varying electron density (arrows). Bars = 2 µm.

 
At 8 wk of age, acini of mutant mice still contain mostly the atypical exocrine cells observed at 3 days of age, and serous demilune cells display typical ultrastructural features (not shown). In addition, acini contain small groups of mature mucous cells as well as cells that appear to be in transition from an atypical cell to a mucous cell (Fig. 2E). This proposed "transitional" cell type is characterized by moderately electron-dense secretion granules that are less compressed within the cytoplasm and display regions of varying electron densities (Fig. 2F). Ductal and myoepithelial cells appear normal in sublingual glands of mutant mice (not shown).

Protein expression.
Figure 3 is a comparison by SDS-PAGE of the patterns of proteins expressed in glands with respect to age, strain, and sex. No differences are noted in the protein patterns between glands of each sex or between mutant and control samples. Near the top of the gel are high-molecular-weight mucins (>250 kDa) that stain faintly by Coomassie brilliant blue after subsequent silver enhancement. Also apparent are the differential expression of at least five proteins (~34, 36, 38, 110, and 160 kDa) that are dependent on age and not the strain of mice.



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Fig. 3. Protein expression in glands of mutant mice and age-matched controls. SDS-PAGE gradient gel (10–20%) was stained with Coomassie blue and subsequent silver enhancement of dye staining. Results are representative of 3 separate experiments.

 
Proteins specific to serous demilune cells.
The histological and ultrastructural evidence presented above suggest serous demilune cells are unaffected by the sld mutation. To compare further the serous demilune cell phenotype between mutant and control glands, we studied proteins restricted to secretion granules of serous demilune cells (1, 7, 15, 16). These include the following PSP (parotid secretory protein) and transcripts for Dcpp (demilune cell and parotid protein, previously termed p20) (3). Cell-free translation of Dcpp transcripts (~700 nucleotides) encode for a 200-kDa protein, in good agreement with the molecular mass (18.4 kDa) calculated for the conceptually translated 170-amino acid protein (3). As shown in Fig. 4A, equivalent levels of Dcpp transcripts are observed in glands from mutant mice compared with age-matched controls, although expression appears to decrease with age in both strains of mice. PSP is a 24-kDa secretory protein of serous demilune cells. In Western blots we find no differences between mutant mice compared with age-matched controls (Fig. 4B). On the other hand, PSP expression levels decrease with age, similar to that reported for rat sublingual glands (16). In immunolocalization experiments, we verified PSP is indeed expressed specifically in secretion granules of serous demilune cells of both control and mutant glands of both age groups (not shown). No differences in the expression levels of Dcpp or PSP are observed between males and females with respect to age or strain (not shown).



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Fig. 4. Comparisons of the expression of demilune cell and parotid protein (Dcpp), parotid secretory protein (PSP), and submandibular gland protein (SMGD) in sublingual glands from male mutant and control mice at 3 days and 8 wk of age. A: Northern analysis of the expression of Dcpp transcripts. Bottom: blot stripped and probed for 28S ribosomal RNA. Mobilities of ribosomal 18S and 28S RNAs are indicated (1.2% agarose gel, 10 µg total RNA/lane). B and C: Western blot of sublingual gland homogenates run on 2–15% gradient gels and probed with antibodies against PSP (B) or SMGD (C). In each of the three comparisons (A, B, and C), similar results were observed for samples from female mice (not shown). See MATERIALS AND METHODS for details. All results are representative of 2 separate experiments.

 
Expression of SMGD in murine sublingual glands and comparisons between mutant and control glands.
SMGD is a 175-kDa protein localized to secretory granules of both serous demilune cells and mucous cells of rat sublingual glands (1). The expression of SMGD in murine sublingual glands has yet to be determined. We therefore conducted Western analysis to first determine whether SMGD is expressed in murine sublingual glands and to then compare expression between control and mutant glands. A single immunoreactive protein band of the appropriate molecular mass is apparent in Western blots of glandular homogenates (Fig. 4C). Protein expression levels are comparable between mutant mice and age-matched controls (Fig. 4C). Immunolocalization studies were conducted to determine whether SMGD may serve as an additional marker to evaluate the phenotypic expression of serous demilune cells and/or mucous cells. In 1-µm sections from glands of 3-day-old controls, little to moderate immunogold labeling for SMGD is localized to serous demilune cells, whereas mucous cells display extremely weak or no labeling (Fig. 5A). In 8-wk-old control glands, both mucous and serous cells are unreactive with anti-SMGD in 1-µm sections (not shown). In contrast, immunogold labeling for SMGD is abundant and localized to the apical cytoplasm near the luminal membrane of "atypical" exocrine cells in 3-day-old mutant glands (Figs. 5B). Furthermore, immunoreactivity of serous cells in these glands is sparse, similar to age-matched controls (compare Fig. 5, A and B). By 8 wk of age, SMGD immunoreactivity in serous demilune cells of mutant glands is absent but remains abundant in atypical cells (Fig. 5C). We confirmed by electron microscopic immunogold labeling of sections from 8-wk-old mutants that SMGD immunoreactivity is localized to the small moderately electron-dense cytoplasmic granules of atypical cells (not shown).



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Fig. 5. Immunolocalization of SMGD in control and mutant sublingual glands. AC: immunogold labeling of 1-µm sections with rabbit anti-SMGD antibodies as described in MATERIALS AND METHODS. A: in 3-day-old controls, serous cells are also low to moderately labeled (arrows), but mucous cells exhibit background labeling. B: at 3 days of age serous cells in mutant glands are low to moderately labeled (arrows), whereas "atypical" exocrine cells display abundant labeling in the apical cytoplasm near the lumen (arrowheads). C: at 8 wk, no labeling of serous demilune cells (arrows) or mucous cells is observed in either mutant (shown) or control (not shown) glands. In mutant glands, strong labeling of "atypical" exocrine cells persists at 8 wk of age. Bars in AC = 10 µm. DG: differential-interference contrast images after immunoperoxidase labeling of paraffin sections from glands of 8-wk-old control (D and F) and mutant mice (E and G). Sections underwent antigen retrieval and antibody detection with Vectastain ABC plus Ni-black enhancement of DAB deposition (no counterstain; see MATERIALS AND METHODS for details). Note the moderate to strong immunoreactivity of granules within the cytoplasm of mucous cells (M) in both control (D) and mutant (E) samples. In E, "atypical" (A) exocrine cells of mutant glands display intense immunoreactivity concentrated in their apical cytoplasm near the acinar lumen (arrowheads). Arrows indicate undetectable immunoreactivity in serous demilune cells of control and mutant glands (C and D). M, mucous cells. Nonimmune rabbit IgG negative controls (F and G). Bar in G = 40 µm.

 
The marked attenuation of immunogold labeling for SMGD in controls compared with mutants is inconsistent with results of Western analysis. To test whether this difference may be due to antigen masking by mucin glycoproteins co-packaged with SMGD, we probed paraffin sections after antigen retrieval (10 min in 5% urea at 95°C). SMGD immunostaining of mucous cells in 3-day-old controls is intense after antigen retrieval (not shown). At 8 wk, moderate to strong immunoreactivity is observed in granules of mucous cells in both control and mutant samples (Fig. 5, D and E, respectively). Furthermore, in atypical exocrine cells of mutant glands, immunoreactivity is concentrated in the apical cytoplasm (Fig. 5E), consistent with immunogold labeling in 1-µm sections (Fig. 5B). SMGD remains undetectable in serous demilune cells of both strains of mice at 8 wk (arrows, Fig. 5, D and E, respectively). The absence of SMGD expression in demilune cells of older mice is unexpected, because in rats SMGD continues to be expressed in this cell type throughout adulthood (22). As a result, SMGD is a specific marker for the mucous cell phenotype in postnatal mice, although in neonates it is more selective for mucous cells compared with demilune cells. Furthermore, the abundance of SMGD expression in granules of atypical exocrine cells coupled with the absence of PSP suggests these cells are of the mucous cell lineage.

Comparisons of mucin expression in mutant and control glands.
Only glycoconjugates of mass much greater than 250 kDa are detected by SDS-PAGE (Fig. 6A). At 3 days of age, no staining is observed in glands from mutant mice, whereas control samples exhibit a thin band of glycoconjugates that just enter 3–8% gradient gels (Fig. 6A, top). To further enhance Alcian blue staining, we subsequently restained gels with silver and developed these until proteins were just detectable (Fig. 6A, bottom). Even under these conditions, no distinct band is apparent in samples from mutant mice. At 8 wk, both mutant and control samples exhibit a single band of glycoconjugates, although much less staining is observed in mutants (Fig. 6A, top and bottom). Interestingly, 3-day-old controls express high-molecular-weight glycoconjugates that do not migrate as far compared with samples from both strains at 8 wk (Fig. 6A, top and bottom). Moreover, total staining is noticeably less in control samples from 3-day vs. 8-wk-old animals (Fig. 6A, top). Staining patterns are similar between males and females within each age group.



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Fig. 6. Expression of glycoconjugates and high-molecular-weight mucin glycoproteins in glands of mutant mice and age-matched controls. A: SDS-PAGE gradient gel (6–8%) stained for glycoconjugates with Alcian blue and subsequent silver enhancement of dye staining (top), followed by further development with silver until proteins begin to stain (bottom). B: Western blot of sublingual gland homogenates from male mice run on a 3–8% gradient gel and probed with antiserum against rat sublingual mucin glycoproteins. As controls, rat sublingual gland homogenate (SLG) and purified rat sublingual mucin glycoproteins (mucins, 15 µg dry wt) were also run. Mucin glycoproteins were isolated as described previously (21). Top: 6-h exposure of blot to Kodak Biomax MS film. Bottom: 20-h exposure of same blot. All results are representative of 3 separate experiments.

 
To more specifically detect mucins, we conducted Western analysis with an antiserum against purified rat sublingual mucins that recognizes primarily carbohydrate mucin constituents (13). As shown in Fig. 6B, rat and mouse glandular homogenates have similar staining patterns, suggesting substantial conservation between rodent sublingual mucins. Results of Western blots are similar to those from Alcian blue-stained gels. At 3 days, staining is absent in mutant samples, even after overexposure of blots (Fig. 6B, top and bottom). At 8 wk, both mutant and control samples exhibit mucin glycoproteins, although mutants contain much less than controls (Fig. 6B). Furthermore, mucins from 3-day-old controls are less abundant and lower in mobility than from 8-wk-old mice.

The immunolocalization of mucin glycoproteins in glands from 3-day and 8-wk-old mice was determined using the same antiserum as for Western blots. As shown in Fig. 7, control glands at both ages display abundant acinar cells expressing mucin glycoproteins (Fig. 7, A and C). In contrast, no immunostaining is present in mutant glands at 3 days of age (Fig. 7B). At 8 wk, there is only a sparse distribution of immunoreactive cells (Fig. 7D).



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Fig. 7. Immunolocalization of high-molecular-weight mucin glycoproteins in paraffin sections (5 µm) of glands from control (A and C) and mutant (B and D) mice, at 3 days (A and B) and 8 wk (C and D) of age. Sections were probed with rabbit anti-mucin serum (500-fold dilution), and immunodetection was performed using the avidin-biotin-peroxidase complex method (Vectastain Elite kit) with DAB as peroxidase substrate. Sections were counterstained with hematoxylin. Bar in A = 35 µm.

 
In addition to glycoproteins, we also compared transcript levels for an apomucin expressed in sublingual glands. We have compiled a contiguous 2,642-bp transcript derived from overlapping sequence of 16 cDNA clones from a sublingual lambda-Zap II cDNA library (unpublished observations, GenBank accession no. AY172172). The conceptual translated sequence encodes Ser/Thr-rich tandem repeats (15 residues of one repeat plus three full 163-residue repeats of >98% identity), a 79-residue unique Ser/Thr-rich nonrepeat region, and a 231-residue cysteine-rich 3’ end that contains a von Willebrand factor type C domain and a COOH-terminal cystine knot-like domain (NCBI conserved domain search, http://www.ncbi.nlm.nih.gov:80/Structure/cdd/cdd.shtml). The nucleotide sequence aligns to the mouse genome in region E3 of chromosome 15 (http://www.ncbi.nlm.nih.gov/genome/seq/MmBlast.html) and includes a gene derived by computer modeling termed, "similar to Apomucin" (LOC239611, http://www.ncbi.nlm.nih.gov/LocusLink/list.cgi?Q=loc239611&ORG=Mm&V=0). The cDNA does not align to any other regions of the mouse genome or to transcripts in GenBank for all known mouse or human apomucin genes (MUC1–MUC17; BLASTN, expect 10, word size 7; http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). The closest mucin relative is pig submaxillary mucin (PSM) (6). The 3’ end of PSM has 72% identity to the 231-residue cysteine-rich 3’ end of our conceptually translated sequence (not shown). We first probed blots using cDNA (1,457 bp) from clone pSL1-12 (5). This cDNA contains the most distal tandem repeat (489 bp), the 237-bp unique Ser/Thr-rich nonrepeat region, the 693-bp cysteine-rich 3’ end, and 38 bp of nontranslated sequence. As shown in Fig. 8A, transcripts are barely detectable in 3-day-old mutants compared with controls. Furthermore, transcripts are undetectable in other tissues, suggesting apomucin expression is selective for sublingual glands and further demonstrating the cDNA probe does not hybridize to other mucins expressed in submandibular glands (MUC10) or large intestine (MUC2).



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Fig. 8. Northern analysis of the expression of sublingual apomucin transcripts. A: expression in different tissues from 3-day-old mutant and control mice. The 1,457-nucleotide cDNA probe from pSL1-12 was 32P-radiolabeled by nick translation. This cDNA encodes the most distal tandem repeat (163 amino acids), a 79-residue unique Ser/Thr-rich nonrepeat region, and 231 residues of a cysteine-rich 3’ end of a putative sublingual apomucin gene. Bottom: blot stripped and probed for 18S ribosomal RNA. Mobilities of ribosomal 18S and 28S RNAs are indicated (1% agarose gel, 5 µg total RNA/lane). SLG, sublingual glands; SMG, submandibular glands; Lg Int, large intestines. Results are representative of 2 separate experiments. B: expression in sublingual glands from mutant and control mice at different ages. A truncated version of the pSL1-12 insert (exclusion of all but 77 nucleotides of the cysteine-rich 3’ end) was used as probe and 32P-radiolabeled by random-primed synthesis. Total RNA was isolated by a modified TRIzol method to reduce mechanical fragmentation of extremely large apomucin RNA molecules observed with conventional methodologies (see MATERIALS AND METHODS). Bracket indicates band of nonfragmented mRNA. A 10-fold longer exposure of samples from 3-day-old mice is shown in the far right lanes. Bottom: blot stripped and probed for 18S ribosomal RNA. Mobilities of RNA size markers (Ambion) are displayed on the left (0.8% agarose gel, 5 µg total RNA/lane). Results are representative of 3 separate experiments.

 
Transcripts of extremely large mucins (14–24 kb) are easily fragmented by the physical forces associated with standard RNA isolation procedures, resulting in the typical smear observed in Northern blots and also hindering estimation of full-length transcript size (4). Therefore, to better compare the size and expression levels of transcripts between control and mutant mice, we modified the TRIzol RNA isolation protocol to reduce mechanical fragmentation (see MATERIALS AND METHODS). We initially experimented with the density gradient procedure described by Debailleul et al. (4) but found the final yield prohibitive because of a requirement for large amounts of material (0.5 g) coupled with the small size of neonatal sublingual glands (<1 mg). To further rule out the possibility of our probe cross-hybridizing to cysteine-rich regions of another unknown sublingual apomucin not included in the genomic database, we used an 803-bp EcoRV truncated version of pSL1-12 to exclude all but 77 nucleotides of the cysteine-rich 3’ end. As shown in Fig. 8B, the steady-state transcript level in 3-day mutants is barely detectable. With increased exposure (right two lanes in Fig. 8B), the signal is not only more discernible, but displays an uppermost band with mobility similar to all other samples from mutant and control mice. The estimated size of the uppermost band is ~24 kb. At 8 wk, transcript levels are still markedly less in mutants compared with controls.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 References
 
Based upon multiple criteria, the sld mutation does not alter the expression of sublingual serous demilune acinar cells. The morphology of serous demilune cells was similar between mutant and control mice, both in histological and ultrastructural studies. In addition, glands of mutant and control mice were equivalent in their expression with age in the serous cell markers, PSP proteins and Dcpp transcripts, as well as in the immunolocalization of SMGD to serous demilune cells (i.e., labeling is very modest at 3 days and absent at 8 wk).

Our results are consistent with the sld defect specifically disrupting mucin expression in mutant glands. Mucin glycoprotein expression is markedly attenuated at 3 days, but with age there are limited increases in total glandular mucin glycoproteins, in the steady-state levels of apomucin transcripts, and in cells producing mucins. Conversely, expression at 3 days of age of total proteins and of the selective mucous cell granule marker, SMGD, are unaltered in mutant mice. The "atypical" exocrine cells of mutant glands do not express the serous cell marker, PSP, but instead contain secretion granules strongly immunoreactive for SMGD. These atypical exocrine cells thus appear previously specified to the mucous cell fate but are primarily unable to synthesize mucin glycoproteins, unless induced selectively during postnatal development. Moreover, adjacent to mucous cells in mutant glands are cells with large granules of mixed electron densities. Such granules are reminiscent of those found during the initial differentiation of mucous cells in fetal rat sublingual glands, in which the electron-dense and more translucent regions are immunoreactive with anti-SMGD and anti-mucin antibodies, respectively (22). The "transitional" cell type likely represents the recent overriding of the sld defect in atypical cells and the subsequent initiation of mucin expression and the resultant changes in mucous cell granule morphology. Furthermore, because the mobilities of apomucin transcripts and of mucin glycoproteins are similar between mutant and control samples, mucin glycoproteins of mutant glands may be normal biochemically. However, given the extremely large size and complexity of these molecules, we cannot exclude small differences in transcript size or posttranslational modification of apomucin proteins.

Mutant glands consistently have lower steady-state levels of apomucin transcripts than age-matched controls, indicating the sld mutation results in disruption of the transcriptional regulation of the apomucin gene and/or a decrease in the stability of apomucin mRNA. The mutation may thus reside within the regulatory region of the apomucin gene or possibly alter the expression/function of a molecule chiefly involved in expression of the apomucin gene. Genetic mapping of the sld mutation to a 1-Mb segment of chromosome 15 containing the sublingual apomucin gene suggests the sld defect may be within this gene (D. J. Culp, unpublished observations). It is also possible the sld defect additionally alters (either directly or indirectly) translational control of apomucin protein expression and/or subsequent glycosylation.

Mucous cells may arise during postnatal development from either intercalated duct cells situated at the proximal ends of tubuloacini and adjacent to striated ducts, or from acinar cell division (20). In mutant mice, the number and size of mucous cell clusters increases with age. Moreover, in transmission electron micrographs, we observe small clusters of mucous cells situated between serous demilune cells and atypical exocrine cells (not shown). We therefore speculate that once an atypical cell in a mutant gland is induced to express mucin glycoproteins, it becomes a focal point for subsequent cell division and differentiation, or it may induce adjacent cells to express mucins.

Our studies additionally demonstrate early postnatal changes in control murine sublingual mucous cells. Mucin glycoproteins display an age-related increase in mobility during SDS-PAGE, possibly due to different posttranslational processing and/or splice variants of apomucin transcripts (i.e., fewer glycosylation sites). Also, mucin glycoproteins and apomucin transcripts are in greater relative abundance at 8 wk, perhaps reflecting a postnatal increase in mucous cell volume density as reported for sublingual glands of rats (8) and/or an enhanced rate of apomucin gene transcription. Mucous cells that divide and differentiate during normal postnatal development may therefore receive various regulatory cues different from prenatal cells, and these cues may function to partially override the sld defect and promote apomucin gene expression. Elucidation of the sld mutation may hence provide important insights into factors regulating apomucin gene expression.


    DISCLOSURES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 References
 
This study was supported by National Institutes of Health Grants DE-13602 and DE-10480 and by Fellowship HL-07126 (to M. A. Fallon), as well as by funds from the Center for Oral Biology, University of Rochester, and the University of Connecticut Health Center.


    ACKNOWLEDGMENTS
 
We thank Ashley John Grillo, Brian Boesch, Shirley Markant, Sally Chuang, and David R. Serwanski for excellent technical assistance. We also thank Dr. Lawrence A. Tabak for initial encouragement and support for these studies.

Present address for P. T. Georgel: Department of Biological Sciences, Marshall University, 1 John Marshall Drive, Huntington, WV 25755.


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: D. J. Culp, Center for Oral Biology, 601 Elmwood Ave., Box 611, Rochester, NY 14642-8611 (E-mail: david_culp{at}urmc.rochester.edu).

10.1152/physiolgenomics.00151.2002.


    References
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 References
 

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