C57BL/6 Mice Lacking Muc1 Show No Ocular Surface Phenotype

Abstract

purpose. To test the hypothesis that a membrane-spanning mucin, Muc1, facilitates the spread of tear film and protects against bacterial adherence.

methods. Age-matched, Muc1 null mice and wild-type mice of C57BL/6 genetic background were used for comparison. Eyes were examined by slit lamp biomicroscopy with fluorescein solution to assess epithelial damage and tear film stability. Structure of the ocular surface epithelia was examined by light microscopy, scanning and transmission electron microscopy, and wholemount confocal microscopy. Bacterial adherence assay was performed on in vivo corneas with Pseudomonas aeruginosa containing a plasmid encoding green fluorescent protein, followed by wholemount confocal microscopy. Real-time reverse transcription–polymerase chain reaction was performed using Muc4-specific primers to quantitate Muc4 mRNA expression in ocular surface tissues.

results. No differences were found between Muc1 null and control mice in any parameter tested. Ocular surface epithelia of Muc1 null mice of the C57BL/6 strain had a normal appearance of surface microplicae, a well-developed glycocalyx on the apical cell membrane, and a normal appearance of goblet cell mucin packets. There was no convincing evidence that bacterial adherence on the cornea was increased in Muc1 null mice. Muc4 mRNA expression was not upregulated in Muc1 null mice compared with control. No ocular surface infections were observed in Muc1 null mice of the C57BL/6 strain (n = 204), which were housed in the animal facility over a period of 26 months.

conclusions. Muc1 null mice of C57BL/6 background appeared normal in all respects tested. These data differ from the reported phenotype in the mice of the C57BL/6 x SVJ129 background, which show development of blepharitis and conjunctivitis.

Amucus layer is present along the apical surface of the entire ocular surface epithelium. ¹ The layer is believed to provide a barrier to pathogen penetrance, a mechanism for removal and trapping of particulates, and a means of maintaining hydration at the ocular surface. 

The major constituents of the mucus layer are mucins, which are exceptionally large glycoproteins that have at least half of their mass as O-linked carbohydrate. A second defining character of mucins is the presence in their protein backbone of tandem repeats of amino acids. These repeated segments are rich in serine and threonine, which are sites of attachment of the O-linked sugars. These abundant O-linked carbohydrate side chains provide the very hydrophilic character of mucins. The length and the amino acid sequence of the tandem repeat varies between each mucin gene product, and the number of tandem repeats per mucin molecule can vary in individuals as a result of genetic polymorphism (for review see Gendler and Spicer ² ). 

Thirteen distinct human mucin genes have been reported to date; they can be categorized as either membrane spanning or secreted. Five of the human mucin genes for which sufficient sequence data are available can be categorized as membrane spanning (MUCs 1, 3, 4, 12, and 13). ³ In general, these mucins have short cytoplasmic tails, a hydrophobic membrane-spanning domain, and an extracellular domain, which is primarily the heavily glycosylated tandem repeat region. It has been estimated that MUC1 and MUC4 can extend 200 to 500 μm from the cell surface, depending on the number of tandem repeats present. ^{2 4} These mucins are believed to interact with the large secreted mucins that are derived from goblet cells or submucosal glands, but the nature of the interaction is unknown. Extracellular domains of Muc1 and the rat homologue of Muc4 have been shown to be shed from epithelial surfaces. ^{2 5}  

Previous studies demonstrate that at least three of the human mucin genes (MUC1, MUC4, and MUC5AC) are expressed by the ocular surface epithelium. ^{6 7} MUC1 mucin is a membrane-spanning mucin produced by the entire ocular surface epithelia. MUC4 mucin, which has also been recently characterized as a membrane-spanning mucin, ⁸ is expressed by the human conjunctival epithelium. MUC5AC mucin is a secretory mucin derived on the ocular surface from conjunctival goblet cells. Although the roles of all the mucins are assumed to be protection against desiccation and microbial invasion, the specific role played by each mucin in the tear film remains to be determined. 

We hypothesized that the membrane-spanning mucins, which extend from the apical cell membranes of epithelial cells, facilitate the spread of secreted mucins of the goblet cells and the tear film and protect against adherence of bacteria at the interface between the tear film and the epithelial surface. We used C57BL/6 mice with targeted disruption of the Muc1 gene to test this hypothesis but found no aberrant phenotype. While this report was in preparation, Kardon et al. ⁹ reported that Muc1 null mice of a different genetic background displayed a marked propensity for development of blepharitis and conjunctivitis. These seemingly contradictory results may be due to differences in facility maintenance, strain variation in the role of the mucin, or other environmental or epigenetic differences. 

Materials and Methods

Animals

Muc1 null mice of C57BL/6 genetic background were generated by homologous recombination ¹⁰ and generously provided by Sandra J. Gendler (Mayo Clinic, Scottsdale, AZ). Wild-type C57BL/6 mice were obtained from Taconic Farm (Germantown, NY) and used for control. Mice were housed and fed ad libitum in our conventional animal facility at the Schepens Eye Research Institute, which is licensed by the US Department of Agriculture and uses the Animal Care Assurance Statement of the Public Health Service. The original breeder mice were confirmed to be Muc1 null by polymerase chain reaction (PCR) using tail genomic DNA samples (data not shown). To maintain the Muc1 null strain, the Muc1 null mice were intercrossed. During this study, August 20, 1997, to date, 204 Muc1 null mice have been weaned. All procedures conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Assessment of Ocular Surface Integrity

Eyes of 9-week-old mice were examined without anesthesia with a slit lamp biomicroscope. To assess epithelial damage and tear film stability, fluorescein (Sigma, St. Louis, MO) diluted 2% in phosphate-buffered saline (PBS; pH 7.4), was instilled onto the cornea, followed by observation using a blue filter. 

Morphologic Assessment

After death, eyes, including lids, of mice aged 9 weeks were fixed in situ with half-strength Karnovsky’s fixative, dissected, and placed in the fixative. Tissues for scanning electron microscopy were processed as described previously. ¹¹ For transmission electron microscopy, they were processed and embedded as described previously. ¹² Thick sections were stained with toluidine blue and viewed by light microscopy. 

Bacterial Adherence Assay

Bacterial adherence assays were performed in situ on corneas of anesthetized mice aged 6 to 7 months, with Pseudomonas aeruginosa strain 19660 (American Type Culture Collection[ ATCC], Rockville, MD) containing a plasmid, pANT4, ¹³ that encodes green fluorescent protein (GFP). pANT4 was introduced into P. aeruginosa ATCC 19660 by electroporation as described by Diver et al. ¹⁴ Preparation of bacterial inoculum was as described previously, ¹⁵ except that bacterial cultures were grown in peptone-tryptic soy broth medium containing 100 μg/ml carbenicillin and 50 μg/ml kanamycin. Five or 10 μl of bacteria solution corresponding to 1 × 10⁷ or 2 × 10⁷ colony-forming units (CFU), respectively, was placed in situ on corneas of anesthetized mice for 1 hour, with the help of surface tension. After death, eyes were enucleated carefully to avoid touching the corneas. Eyes were rinsed three times in either saline or PBS, fixed in 4% paraformaldehyde, labeled with rhodamine phalloidin, and processed for wholemount confocal microscopy, as previously described. ¹⁶ Dual detection was performed using fluorescein isothiocyanate (FITC) and tetrarhodamine isothiocyanate (TRITC) detector channels. 

PCR Verification of Muc1 Null Mutation

Tail genomic DNA was isolated using a kit (QIAamp; Qiagen, Valencia, CA), according to the manufacturer’s instructions. It was then used for PCR with Muc1-specific primers ¹⁰ : sense 5′-ACCTCACACACGGAGCGCCAG-3′ (GenBank accession M64928, nucleotides [nt] 463-483) and antisense 5′-TCCCCCCTGGCACATACTGGG-3′ (nt 724-704) to confirm the homozygous null mutation for the Muc1 gene. Forty cycles of PCR amplification were performed. Amplified products were electrophoresed on a 2% agarose gel and visualized with ethidium bromide. 

Real-Time RT-PCR to Determine Relative Muc4 Expression

Techniques for real-time reverse transcription–polymerase chain reaction (RT-PCR) were essentially the same as previously reported. ¹⁷ Total RNA of the ocular surface, including cornea and conjunctiva, was isolated from 1-year-old Muc1 null mice and wild-type control mice using a commercial reagent (TRIzol; Life Technologies, Grand Island, NY). The first strand of cDNA was synthesized from 1 μg of the RNA (DNase treated) with random primers using a commercial reverse transcriptase (Superscript; Life Technologies). Real-time PCR was performed using a commercial system (TaqMan PCR GeneAmp 5700 Sequence Detection System; PE Biosystems, Foster City, CA). The Muc4-specific primers were designed from the sequence in GenBank (accession AF218265). The sense primer was 5′-CTCCAAGAAATGTAGTGGCTTTCAG-3′ (nt 2925-2949), the antisense 5′-CACGGTCTTGGGCTGGAGTA-3′ (nt 3066-3047), and the TaqMan probe sequence was 5′-AACATCCCCAGAAGCGTGTACCCTGG-3′ (nt 2982-3007). The internal standard control gene was amplified (TaqMan Rodent GAPDH Control Reagents; PE Biosystems). To verify the validity of using GAPDH as the internal calibration standard, the efficiencies of the Muc4 and GAPDH amplifications were compared and found to be equivalent. To verify the identity of the Muc4 PCR product, it was sequenced at the DNA Sequencing Core Facility of Massachusetts General Hospital, Boston. 

Each PCR reaction contained equivalent amounts of cDNA. Assays were performed in quadruplicate using a kit (TaqMan PCR; PE Biosystems) according to the manufacturer’s recommendations. Quantitation and comparison of amounts of Muc4 mRNA in Muc1 null mice and wild-type control animals (n = 5 per group) were as previously described. ¹⁷  

Results

Gross Appearance of Muc1 Null Mice

The gross views of eyes by slit lamp biomicroscopy appeared normal in Muc1 null mice of C57BL/6 genetic background. In 10 Muc1 null as well as 4 wild-type mice, punctate fluorescein staining was detected on all the corneas, indicating that the punctate stains were not characteristic of Muc1 null mutation (Fig. 1) . No breakup of the tear film was observed for at least 30 seconds. During the period of this study, August 20, 1997, to date, 204 Muc1 null mice were weaned, and the age of the mice ranged to at least a year and a half. No ocular abnormalities—including conjunctivitis or blepharitis—were found under routine animal care. 

Morphologic Examinations

As demonstrated by scanning electron microscopy, Muc1 null mice possessed normal-appearing surface microplicae, comparable to those of wild-type control mice, on corneas (Fig. 2A ) as well as on conjunctiva (not shown). Transmission electron microscopy demonstrated that glycocalyces on the apical epithelial cells (Fig. 2B) and mucin packets in the goblet cells (Fig. 2C) in Muc1 null mice appeared well developed and were comparable to those of wild-type control mice. Light microscopy demonstrated that epithelia of cornea (not shown) and conjunctiva (Fig. 2D) from Muc1 null mice were comparable and similar to those of wild-type control mice, with no inflammation and goblet cell hypertrophy. 

Bacterial Adherence Assay

In 16 of 20 Muc1 ^−/− eyes (Fig. 3A ) and 8 of 10 wild-type control eyes (data not shown), there was no adherence of the GFP-expressing P. aeruginosa on in vivo corneas. On several eyes, a few clusters of adherent bacteria were observed (Fig. 3B) , apparently from nonspecific attachment, but no distinct difference was seen between these clusters on Muc1 null and control mice. Several different rinsing procedures were used, including vigorous rinse, gentle rinse, and fixation before rinse, yielding similar results. To make sure that the P. aeruginosa used in this study was capable of adhering to tissues, the bacteria were confirmed to adhere to the wound bed of wounded corneas (data not shown), which was consistent with results described previously. ¹¹ The P. aeruginosa strain (ATCC 19660) has been shown to infect mice of the C57BL/6 strain. ¹⁵ We could not find convincing evidence that bacterial adherence was increased on corneas of Muc1 null mice. 

Muc4 mRNA in Muc1 Null Mice

To determine whether another membrane-spanning mucin, Muc4, is upregulated to compensate for loss of Muc1, we compared their mRNA levels using real-time PCR and PCR primers designed from the carboxyl terminal region of the mouse MUC4 homologue (GenBank accession AF218265). These primers amplified a 142-bp product by RT-PCR of mouse ocular surface RNA. Sequencing of this PCR product showed it to be identical with the GenBank sequence for Muc4. The relative amounts of Muc4 expression in Muc1 null mice compared with that in control animals did not differ (−/− = 0.93, range 0.49–1.75, n = 5; +/+ = 1.0, 0.54–1.86, n = 5). Rodent GADPH was used as the internal standard. These data indicate that mRNA expression of another membrane-spanning mucin, Muc4, is not upregulated in Muc1 null mice. 

Discussion

This study showed that the ocular surface of Muc1 null mice of C57BL/6 background appeared normal in all respects tested. These data are at variance with the report by Kardon et al., ⁹ in which a marked propensity for development of blepharitis and conjunctivitis by Muc1 null mice in C57BL/6 x SVJ129 background, was reported. Several possibilities for the differences between our study and theirs include the housing conditions of the animals, mouse strain variation in response to deletion of Muc1, strain variation of pathogens, or other environmental or epigenetic differences. Our conventional animal facility, licensed by the US Department of Agriculture, houses mice that are routinely tested to assure the absence of pathogenic viruses, bacteria, and parasites, and is considered murine pathogen free. The facility housing the animals in the study by Kardon et al. ⁹ is described as“ a conventional animal facility,” but it is not clear whether it is murine pathogen free. Because none of the Muc1 null mice housed in our facility (>200) displayed development of infection of the ocular surface, and because the bacterial adherence assay using P. aeruginosa, a common pathogen seen in eye infections, gave no definitive evidence that Muc1 null mice were more susceptible to bacterial adherence, the difference between the two populations may be due to other factors, including strain variation. Although there appear to be no reports on susceptibility of the inbred SVJ129 strain to infection, it is possible that the combination of C57BL/6 and SVJ129 backgrounds renders Muc1 null mice susceptible to microbial infections, implying that Muc1 mucin plays a more important role in the latter background in protecting against infection. Similar phenotypic differences of null mutation between C57BL/6 and SVJ129 backgrounds are reported, with p53 null mice displaying vitreal opacities, fibrous retrolental tissue, and retinal folds in the former strain but not in the latter. ¹⁸  

Because the ocular surface epithelia of rodents express another membrane-spanning mucin, Muc4, it is possible that this mucin compensates for loss of Muc1 in some strains of mice. However, the mRNA level in the ocular surface tissue of the Muc1 null mice was not upregulated when compared by real-time PCR with that of control mice. These data are consistent with that in a previous paper reporting no difference in expression levels of Muc4 using slot-blot analysis of mRNA of mammary gland, salivary gland, lung, stomach, and colon of Muc1 null mice. ¹⁰ Perhaps the Muc4 protein is sufficiently protective without enhanced expression levels. 

Direct comparison of the two different strains of mice under the same experimental, laboratory, and care and handling conditions would be useful to sort out the variance in response to the null mutation. The comparison was impractical, because it is impossible to “duplicate the mice because they were mixed outbred mice” (Sandra J. Gendler, personal communication, May 1999). The outbred mice were retrieved and maintained for approximately a year in the animal facility of the laboratory that originally developed both Muc1 null strains, they reportedly did not observe the same eye problems, but comment that “our facility is very clean” (Gendler, personal communication, May 1999). Although the data obtained regarding absence of an ocular surface phenotype in the C57BL/6 strain of mice represent a report of negative findings, they indicate that in some circumstances, MUC1 is not necessary for protection of the ocular surface. Whether these data are relevant to the human ocular surface is not known. In conjunctiva and cornea of rats and mice, there is a high level of expression of the membrane-spanning mucin Muc4 (Bartman et al., unpublished data) ^{19 20} In humans, the level of expression of MUC4 in central cornea is much less. ^{5 7} Perhaps the concentration of Muc4 on rodent eyes is so high that loss of Muc1 has a negligible effect. It is possible that, in human ocular surface, MUC1 may have a protective role. 

 Supported by Grants R37-EY03306 (IKG) and R01-EY02986 (LDH) from the National Eye Institute.

 Submitted for publication December 13, 1999; revised May 30 and August 16, 2000; accepted August 22, 2000.

 Commercial relationships policy: N.

 

Figure 1.

 

Slit lamp biomicroscopy of the ocular surface of Muc1 null and wild-type mice after fluorescein instillation. Punctate staining was detected on corneas of both Muc1 null (A) and wild-type control (B) mice.

Figure 2.

 

Morphologic examinations of the ocular surface epithelia derived from Muc1 null (left) and wild-type control (right) mice. (A) Scanning electron microscopy of the apical corneal epithelial cells, demonstrating comparable surface microplicae. Bar, 5 μm. Transmission electron microscopy of the apical surface of corneas (B) and the secretory granules of goblet cells (C), demonstrating well-developed, comparable glycocalyces and mucin packets, respectively. Bar, 0.5 μm. (D) Light microscopy of the conjunctiva demonstrating comparable appearance. Toluidine blue stain. Bar, 100 μm.

Figure 3.

 

Bacterial adherence assay on in vivo corneas of Muc1 null mice with P. aeruginosa containing a plasmid encoding green fluorescent protein. Bacterial adherence was visualized by wholemount confocal microscopy after labeling with rhodamine phalloidin. Adherence of the bacteria was not routinely seen (A). Occasionally, adherent bacteria were observed but no distinct difference was seen between Muc1 null and control mice (B). Stacked images of eight optical sections are shown. Bar, 20 μm.

The authors thank Norm Michaud for technical assistance in scanning electron microscopy, Rob Moccia and Sandra Spur–Michaud for work on the Muc4 cDNA sequence, and Jeffery Hobden for providing the GFP-labeled bacteria.