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Disruption of lens fiber cell architecture in mice expressing a chimeric AQP0-LTR protein

ALAN SHIELS^*,1, DONNA MACKAY^*, STEVEN BASSNETT^*,, KRISTIN AL-GHOUL^§,¶ and JER KUSZAK^§,¶

^* Departments of Ophthalmology and Visual Sciences,
Genetics and
Cell Biology and Physiology, Washington University School of Medicine, St. Louis 63110, Missouri, USA; and Departments of
^§ Pathology and
^¶ Ophthalmology, Rush-Presbyterian-St. Luke’s Medical Center, Chicago, Illinois 60612, USA

¹Correspondence: Ophthalmology and Visual Science, Box 8096, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110, USA. E-mail: shiels{at}vision.wustl.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Aquaporin-0 (AQP0) is the major intrinsic protein of lens fiber cells and the founder member of the water channel gene family. Here we show that disruption of the AQP0 gene by an early transposon (ETn) element results in expression of a chimeric protein, comprised of 75% AQP0 and 25% ETn long terminal repeat (LTR) sequence, in the cataract Fraser (Cat^Fr) mouse lens. Immunoblot analysis showed that mutant AQP0-LTR was similar in mass to wild-type AQP0. However, immunofluorescence microscopy revealed that AQP0-LTR was localized to intracellular membranes rather than to plasma membranes of lens fiber cells. Heterozygous Cat^Fr lenses were similar in size to wild-type but displayed abnormal regions of translucence and light scattering. Scanning electron microscopy further revealed that mature fiber cells within the core of the heterozygous Cat^Fr lens failed to stratify into uniform, concentric growth shells, suggesting that the AQP0 water channel facilitates the development of the unique cellular architecture of the crystalline lens.—Shiels, A., Mackay, D., Bassnett, S., Al-Ghoul, K., Kuszak, J. Disruption of lens fiber cell architecture in mice expressing a chimeric AQP0-LTR protein.

Key Words: lens • major intrinsic protein • water channel • cataract • mouse

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
THE AQUAPORIN (AQP) gene superfamily encodes over 150 transmembrane proteins that facilitate the transport of water and certain neutral solutes (e.g., glycerol) across the plasma membranes of microorganisms, plants and animals (1) . At least 10 AQP genes are expressed in physiologically diverse mammalian tissues including kidney, airways, and brain and several of these genes have been directly associated with certain clinical phenotypes (2) . In humans, mutations in the gene for AQP1 have been associated with loss of the Colton blood group (3) , whereas mutations in the AQP2 gene underlie autosomal recessive (4) and dominant (5) forms of nephrogenic diabetes insipidus, in which patients are unable to concentrate their urine. Similarly in mice, null mutations in the genes for AQP1 or AQP4 impair ability to concentrate urine (6 , 7) , whereas deficiency of AQP5 results in defective secretion of saliva (8) .

Recently, mutations in the gene for AQP0, or lens major intrinsic protein (MIP), have been associated with autosomal dominant cataracts that map to human chromosome 12q (9) . In addition, two spontaneous mutations in the mouse gene for AQP0 (Aqp0) on chromosome 10 have been associated with autosomal dominant cataracts (10 11 12) , and these mutant mice provide valuable model systems for AQP0-related cataract in humans. The lens opacity (Cat^lop) mouse inherits a missense mutation that is associated with impaired targeting of mutant AQP0 to the cell surface and severe congenital cataracts in homozygotes. The cataract Fraser mutation (Cat^Fr), formerly called Shriveled (Svl), arose in the A/Jax mouse strain and is believed to be allelic with the cataracta congenita subcapsularis (Cat) mutation (13 , 14) . Homozygous Cat^Fr mutants also inherit severe congenital cataracts (15 , 16) with early cytological changes commencing during embryogenesis (17 , 18) . Mutation analysis of AQP0 transcripts from the Cat^Fr lens has indicated that they are truncated (19) and mis-spliced to the long terminal repeat (LTR) sequence of an early transposon (Etn) element (12) . Here we determine the genomic organization of the Cat^Fr mutation, localize the resulting Cat^Fr fusion protein, and describe its effects on lens morphology in the heterozygous state.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Mice
Cat^Fr mice were barrier-housed in 12 h light and dark cycles with free access to food and water, in accordance with the animal studies regulations at Washington University. Heterozygous mutants were bred by crossing homozygotes with descendants of the parental wild-type strain (A/J). Mice were killed by CO₂ inhalation, followed by cervical dislocation or decapitation. Eyes were enucleated and lenses dissected, then photographed under a dissecting microscope (Zeiss, Stemi 2000).

PCR genotyping
Genomic DNA was prepared from tail snips using a QIAamp tissue kit (Qiagen, Chatsworth, Calif.). Polymerase chain reaction (PCR) amplification was performed using a sense ‘anchor’ primer (5'-tattacacactggtgcggggatg) in exon 3 (codons 177–183) and two antisense primers (5'-ttacagggcctgagtcttcag) in exon 4 (codons 258–263/stop) and (5'-tctcgggtccagtagaaaggt) in the LTR sequence (codons 231–238). PCR conditions were as follows: 1 cycle at 94°C for 30 s; 30 cycles at 94°C for 30 s, 62°C for 30 s, and 68°C for 8 min using the Elongase amplification kit (Gibco BRL, Grand Island, N.Y.) and a GeneAmp 2400 thermal cycler (Perkin Elmer, Norwalk, Conn.).

Immunoblot analysis
Lens membrane proteins were prepared by urea extraction essentially as described previously (20) and protein concentration quantified by nano-orange (Molecular Probes, Eugene, Oreg.) using a fluorometer (Bio-Rad, Hercules, Calif.). Proteins were separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad) and blotted onto Hybond ECL nitrocellulose membrane (Amersham, Arlington Heights, Ill.) using a Mini Protean II system (Bio-Rad). Blots were blocked (1 h) with 5% non-fat milk in phosphate-buffered saline (PBS) containing 0.1% Tween (PBS-T), then probed with AQP0 antibody (1 h) or a commercially prepared (Quality Controlled Biochemicals, Inc., Gaithersburg, Md.) affinity-purified antibody (16 h) raised against a synthetic peptide (acetyl-NLRSHTRAPFYWTRDYSAC-amide) encoded by the LTR domain (see Fig. 2A ). Blots were washed (2x15 min) in PBS-T and incubated (1 h) with horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulin G (IgG) (Amersham), then rewashed in PBS-T, stained with enhanced chemiluminescence reagents and exposed to Hyperfilm MP (Amersham).

View larger version (27K): [in this window] [in a new window]   Figure 2. The CatFr antigen. A) Schematic topology of mouse AQP0 showing six transmembrane domains (boldface) and two extracellular domains (A, C). Domains B and E contain the NPA core motifs, characteristic of the aquaporin gene family, which are believed to function in pore formation (24) . Domain D, along with the amino and carboxyl termini, are cytoplasmic. In the CatFr mutant, the predicted LTR sequence (hyphenated amino acids; 203–261) replaced most of the sixth transmembrane domain and all of the carboxyl-terminal domain of wild-type AQP0 (amino acids 203–263), including an in vivo phosphorylation site at serine 235 (25) . An asterisk indicates an in-frame, translation stop codon within the LTR sequence. The synthetic peptide sequence (18 mer, codons 224–241) used to raise an LTR-specific antibody is underlined. B) Immunoblot analysis showing that the AQP0 antibody detects a protein of 28 kDa from wild-type (+/+) but not mutant (Fr/Fr) lenses, whereas the LTR antibody detected a protein of 28 kDa from mutant but not wild-type lenses.

Immunofluorescence microscopy
Newborn (P1) lenses were fixed (1–2 h) in 4% paraformaldehyde in PBS (pH 7.4). Fixed lenses were embedded in 4% agar/PBS and cut into 100 µm sections using a Vibratome (TPI; series 1000), either along the optical axis (polar plane) or perpendicular to the optical axis (equatorial plane). Vibratome sections were permeabilized (30 min) with 0.1% Triton X-100/PBS, then blocked (1 h) with 10% goat serum/1% bovine serum albumin/PBS and incubated with AQP0 antibody (1 h) or LTR antibody (16 h). Sections were washed (30 min) in PBS, then incubated (1 h) with fluorescein-conjugated goat anti-rabbit IgG (Sigma, St. Louis, Mo.), rewashed, mounted in Slow-Fade (Molecular Probes), and viewed on a laser scanning confocal microscope (Zeiss LSM 410).

Scanning electron microscopy
Lenses were fixed in 2.5% glutaraldehyde in 0.07 M sodium cacodylate buffer (pH 7.2) at room temperature for 5 days with daily changes of fresh fixative (21) . After an overnight buffer wash, lenses were split along their polar axis to reveal fiber organization in radial cell columns and then osmicated overnight in 1% aqueous OsO₄. After an additional overnight buffer wash, the tissue water was removed by dehydration through a graded series of ethanol to absolute ethanol. Ethanol was then replaced through a graded series of Freon 113/absolute ethanol to 100% Freon 113. Lens pieces were critical point dried in Freon 113 in a Balzers CPD 020 (Balzers, Hudson, N.H.). Critical point dried pieces were secured onto aluminum stubs with conductive silver paint. Specimens were mounted on their convex surfaces so that the exposed radial cell columns were oriented at essentially 90° to the direction of the electron beam. All specimens were sputter coated with gold in vacuo, then examined in a JEOL JSM 35c scanning electron microscope (Peabody, Mass.) at 15 kV. Micrographs were taken with a Polaroid (Cambridge, Mass.) camera system at F11.

	RESULTS

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Genomic organization of the Cat^Fr mutation
Our previous cDNA sequence data suggested that the ETn element had inserted downstream (3') from the third exon of the mouse AQP0 gene (Aqp0), resulting in the mis-splicing of the final (fourth) exon (12) . To locate exon 4 and determine the ETn integration site, we designed gene-specific PCR primers to exons 3 and 4 of wild-type Aqp0 and to the 5'-LTR sequence of the ETn element (Fig. 1A ). Genomic PCR analysis showed that the distance between the exon 3 primer (codons 177–183) and the exon 4 primer (codons 258–263*) increased from 3 kb in the wild-type gene to 8 kb in the mutant gene (Fig. 1B ), consistent with the size (5–6 kb) of an ETn element (22) . The distance between the exon 3 primer and the LTR primer (codons 231–238) in the mutant gene was 2 kb. However, no such PCR product could be generated from the wild-type gene. The PCR data indicated that exon 4 of Aqp0 was present in the Cat^Fr genome and that an ETn element had inserted into the third intron of Aqp0, placing an LTR sequence 1.8 kb downstream from the end of exon 3. Sequence analysis revealed that the novel junction between intron 3 and the LTR represented a consensus splice acceptor site (tctc₄tc₄tag) along with a consensus branch sequence (ctgag) located 15 nucleotides upstream (Fig. 1A ). Thus, the combined PCR and sequence data are consistent with aberrant splicing of an LTR sequence to the first three exons of Aqp0 and skipping of exon 4. The resulting AQP0-LTR fusion transcripts appeared truncated on RNA blots (19) , consistent with the presence of a consensus polyadenylation signal (aataaa) in the LTR sequence located 100 nucleotides upstream from the equivalent signal in the wild-type AQP0 transcript (12) .

View larger version (20K): [in this window] [in a new window]   Figure 1. The CatFr mutation. A) Schematic diagram of the wild-type AQP0 gene (+/+) showing the proposed integration site of an early transposon (ETn) element in the CatFr mouse mutant (Fr/Fr). Codons at exon/intron boundaries are numbered. Codon sequences used for design of PCR primers are indicated with arrows. An asterisk indicates the translation stop codon of the wild-type gene. B) PCR analysis of the AQP0 gene from wild-type (+/+) and mutant (Fr/Fr) mice. The intron separating exons 3 and 4 of the wild-type gene was 3 kb. The same intron in the mutant gene was 8 kb. The distance between exon 3 and the nearest ETn LTR in the mutant gene was 2 kb. Sequence analysis detected a splice acceptor site (tctc4tc4tag) at the junction of intron 3 and the LTR with a consensus polyadenylation signal (aataaa) located 161 nucleotides downstream.

Immunodetection of the Cat^Fr antigen
Previous attempts to detect the putative AQP0-LTR antigen in situ using a polyclonal antibody to AQP0 were unsuccessful (20) , presumably because the LTR sequence abolished critical epitopes at the carboxyl terminus recognized by the wild-type antibody. Therefore, to detect the chimeric AQP0-LTR protein we raised an affinity-purified antibody directed against the carboxyl-terminal LTR domain (Fig. 2A ). We selected a potentially antigenic peptide of 18 amino acids between codons 224 and 241 of the AQP0-LTR mutant. Alignment of this peptide in the SwissProt database, using the BLAST algorithm (23) , failed to detect significant homology with other known proteins. Immunoblotting of lens membrane proteins from 3–4 wk old mice using the LTR peptide antibody showed that mutant AQP0-LTR was similar in mass (28 kDa) to that of wild-type AQP0 (Fig. 2B ). This value was consistent with that predicted from the AQP0-LTR cDNA sequence, which encoded a mutant protein only two amino acids shorter than wild-type AQP0 (12 and Fig. 2A ).

Immunofluorescence confocal microscopy of the homozygous Cat^Fr lens at birth using the LTR-specific antibody detected strong staining of cortical fiber cells that was not detected in the wild-type lens (Fig. 3A , B ). The lack of LTR staining within the central core of the homozygous Cat^Fr lens (Fig. 3B ) likely reflects the considerable proteolytic damage in this region of the lens. Under higher magnification, the LTR staining of homozygous and heterozygous Cat^Fr cortical fiber cells appeared diffuse or localized to intracellular membranes surrounding cell nuclei (Fig. 3D , F ). In contrast, AQP0 staining was localized to plasma membranes of fiber cells in wild-type and heterozygous Cat^Fr lenses (Fig. 3C , E ).

View larger version (160K): [in this window] [in a new window]   Figure 3. Confocal microscopy of AQP0 and LTR immunofluorescence (red) in the newborn (P1) CatFr lens. A) Optical section (polar plane) of wild-type lens (anterior pole up) showing that the LTR peptide antibody fails to cross-react with fiber cells. Cell nuclei have been stained blue with propidium iodide. Scale bar, 100 µm. B) Similar optical section of the CatFr/Fr lens showing diffuse LTR staining of fiber cells in the equatorial cortex. Inset locates region of higher magnification. C) Equatorial cortex region (polar plane) of wild-type lens showing intense AQP0 staining of fiber cell plasma membranes. Scale bar, 10 µm. D) Similar equatorial cortex region of the CatFr/Fr lens showing diffuse intracellular and perinuclear (arrows) staining with LTR antibody. E) Cross section (equatorial plane) of the CatFr/+ lens showing intense staining of fiber cell plasma membranes with AQP0 antibody. F) Similar cross section of the CatFr/+ lens showing perinuclear staining (arrows) of fiber cells with LTR antibody.

Pathology of the Cat^Fr lens
To evaluate the pathological properties of AQP0-LTR, we compared lenses from mice that were either homozygous (Cat^Fr/Fr) or heterozygous (Cat^Fr/+) for the LTR mutation with those of the parental wild-type strain. Cat^Fr/Fr mice presented with bilateral cataracts when the eyes opened around postnatal day 14 (P14), consistent with a congenital onset (data not shown). On P21, Cat^Fr/Fr lenses were 30% smaller than wild-type (n=12) and displayed a dense cataract located within the anterior subpolar region (Fig. 4C ). In contrast, Cat^Fr/+ mice did not develop frank opacities until 6 wk of age and lens size was similar to that of age-matched wild types. Close inspection of the P21 Cat^Fr/+ lens under oblique illumination, however, revealed a striking zone of translucence that was not present in the wild-type lens (Fig. 4A , B ). An equatorial view of this zone showed that the anterior subpolar region and posterior polar axis of the Cat^Fr/+ lens were grossly disturbed. When viewed from the anterior or posterior poles under intense illumination, the P21 Cat^Fr/+ lens displayed an abnormal pattern of light scattering (Fig. 5D ). In contrast, similar illumination of the P21 wild-type lens revealed the subtle Y-shaped lines of discontinuity, or sutures, that define the normal overlap of fiber cell ends within the anterior and posterior subpolar regions of the lens (Fig. 5A ).

View larger version (48K): [in this window] [in a new window]   Figure 4. Gross pathology of the CatFr lens at 3 wk of age. A) Dissecting microscope image (equatorial view) of wild-type lens (anterior pole up) with oblique illumination. B) CatFr/+ lens showing the translucent anterior subpolar region. C) CatFr/Fr lens showing the anterior subpolar opacity.

View larger version (93K): [in this window] [in a new window]   Figure 5. Fiber cell disorganization in the heterozygous CatFr lens at 3 wk of age. A) Dissecting microscope image of a wild-type lens showing the posterior Y-suture and D) posterior view of a CatFr/+ lens showing aberrant light scattering under intense illumination. B) Scanning electron micrograph of wild-type lens and (E) CatFr/+ lens split open along the optical (polar) axis. C) Computer-assisted tracing (26) of panel B outlining arc-like fiber cells arranged in concentric growth shells. F) Computer tracing of panel E outlining aberrant curvature of core fiber cells and disorganization of the optical axis.

To better interpret the translucence and light scattering of the P21 Cat^Fr/+ lens, we used scanning electron microscopy to examine fiber cell organization. Nascent fiber cells within the equatorial cortex of the Cat^Fr/+ lens had elongated toward the poles but failed to effect the precise arc-like growth curvature characteristic of mature fibers within the core or nucleus of the wild-type lens (Fig. 5E ). Cortical Cat^Fr/+ fibers exhibited an irregular, swollen shape especially in the anterior and equatorial regions (Fig. 6B , D ). Similarly, Cat^Fr/+ fibers in the posterior subpolar region had abnormal end curvature and failed to form sutures (Fig. 6F ). Furthermore, mature fibers within the core of the Cat^Fr/+ lens had failed to stratify into concentric growth shells, resulting in anterior displacement of the nucleus and disorganization of the entire polar, or optical, axis (Fig. 5E , F ).

View larger version (208K): [in this window] [in a new window]   Figure 6. Scanning electron microscopy of fiber cell pathology in the heterozygous CatFr lens at three weeks of age. A) Anterior polar region of wild-type lens and B) CatFr/+ lens showing loss of uniform fiber cell stratification in the mutant. C) Equatorial cortex of wild-type lens and D) CatFr/+ lens showing irregular swollen fibers in the mutant. E) Posterior polar region of wild-type lens and F) CatFr/+ lens showing aberrant end-curvature of fibers in the mutant.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In this study, we have demonstrated that insertion of an ETn element into the gene for AQP0 results in the translation of an LTR fusion protein in the Cat^Fr mouse lens. ETn elements have been shown to disrupt several other mouse genes including those for muscle chloride channel in myotonic (adr) mice (27) , Fas antigen in lymphoproliferation (lpr) mice (28) , and leptin in obese (ob2J) mice (29) . However, the Cat^Fr mutant appears to be the first case where an LTR sequence has been translated into a protein domain. The abnormal distribution of AQP0-LTR within homozygous Cat^Fr lens fiber cells resembled the endoplasmic reticulum-like localization of the AQP0 missense mutant in the Cat^lop lens (12) and is consistent with impaired targeting of the chimeric protein to the cell surface. Similarly, mistargeting to the ER and Golgi compartments has been described for certain AQP2 mutants expressed in Xenopus oocytes and cultured cells (5 , 30 , 31) . However, the strong AQP0 immunostaining of fiber cell plasma membranes throughout the Cat^Fr/+ lens suggests that mutant AQP0-LTR does not severely inhibit targeting of normal AQP0 in the heterozygous state.

The heterozygous Cat^Fr phenotype was considerably less severe than that of the homozygous Cat^Fr mutant. Notably, the Cat^Fr/+ lens was similar in size to wild-type and opacities did not develop until 6 wk after birth. In contrast with wild-type, however, fiber cells within the core or nucleus of the juvenile Cat^Fr/+ lens were severely disordered, suggesting that AQP0 may play a role in the symmetrical organization of fiber cells along the optical axis of the lens. The Cat^Fr/+ fiber cell pathology supports the view that the AQP0-LTR mutant does not exert a strong dominant negative effect on lens growth and transparency. Although we cannot exclude other deleterious gain-of-function effects, such as cytotoxicity of the LTR domain, it is equally possible that AQP0-LTR represents a loss-of-function mutation. The only function that has been empirically determined for AQP0 in the lens is a water channel (32) . Significantly, water permeability measurements of Cat^Fr lens fiber cell membranes revealed a 30% reduction in heterozygotes and a 60% reduction in homozygotes compared with wild-type (32) . This loss of function is consistent with intracellular trapping of the AQP0-LTR mutant; however, further physiological studies will be required in order to determine the precise role of AQP0-mediated water transport in lens transparency and cataract formation.

	ACKNOWLEDGMENTS

 
We thank Sam Zigler for AQP0 antibody. This research was supported by grants EY11411 (A.S.), EY09852 (S.B.), EY06642 (J.K.), and EY0287 from the National Institutes of Health and an unrestricted grant from Research to Prevent Blindness. S.B. is the recipient of an RPB career development award.

Received for publication December 21, 1999. Revision received May 1, 2000.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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