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First published online 28 April 2009
doi: 10.1242/jcs.045203

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The stratified syncytium of the vertebrate lens

Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, 660 S. Euclid Ave, St Louis, MO 63110, USA

View larger version (87K): [in this window] [in a new window]   Fig. 1. Cellular organization of the mouse lens. The lens consists of two cell types: epithelial cells (Epi) located at the anterior surface, and fiber cells (Fib), which comprise the remainder and majority of the tissue. At the lens equator, epithelial cells differentiate into fiber cells. As they differentiate, fibers become highly elongated. The tips of the elongating fibers converge at the anterior and posterior lens sutures (AS and PS, respectively). In cross section, the fiber cells have a flattened hexagonal profile with two broad faces (oriented parallel to the lens surface) and four narrow faces. Initially, all fiber cells are nucleated but, during differentiation, nuclei (N) and other organelles are degraded. As a result, the central region of the lens constitutes an organelle-free zone (OFZ). The innermost cells, termed primary fiber cells (PF), are formed during embryonic development and are less regular in shape and arrangement than the other fiber cells.

View larger version (96K): [in this window] [in a new window]   Fig. 2. The large molecule diffusion pathway (LMDP) is established in elongating fiber cells and persists in mature fiber cells. A-C. Maximum intensity projections of the posterior lens hemisphere from a 33-day-old Cre-ERTM;Z/EG mouse, 10 days after tamoxifen treatment. The lens was incubated in FM4-64 to visualize the membrane architecture. (A) FM4-64 staining reveals the regular arrangement of the fiber cells and the position of the Y-shaped posterior suture (PS). (B) In short, newly differentiated cells near the periphery, GFP is restricted to the cytoplasm of expressing cells (arrows). As fiber elongation proceeds, GFP diffuses from expressing cells into the cytoplasm of neighboring cells (*). Note that intercellular diffusion commences before cells reach the suture (i.e. in actively elongating fiber cells) (see Fig. 1). (C) Co-visualization of GFP (green) and FM4-64 fluorescence (red). (D) Wild-type mouse lens 10 minutes (green) or 2 days (red) after injection of Alexa Fluor 488-dextran (10 kDa) into a fiber cell located in the center of the lens (arrow). Note the extensive intercellular spread of tracer over the 2 day incubation period. Scale bars: 250 µm.

View larger version (30K): [in this window] [in a new window]   Fig. 3. The LMDP preferentially links cells of the same age. (A) X-Z image of the equatorial region of a tamoxifen-treated Cre-ERTM;Z/EG lens. In this orientation, fiber cells are seen in transverse section. Near the surface, individual GFP-expressing cells are evident (arrows). At greater depths (>50 µm), GFP fluorescence is detected in a cluster of cells, suggesting that a GFP-expressing cell (*) has become coupled to its lateral neighbors by the LMDP. (B) Lateral spread of large molecules is also observed following injection of fluorescent dextran. The upper left panel shows a lens in which an individual fiber cell (arrow) was injected with Alexa Fluor 488-Dextran and, at higher magnification (upper right panel), the appearance of the injected fiber cell 10 minutes (red) and 48 hours (green) after injection. The transects indicate the orientation of the optical sections shown in the lower panel. The X-Z section (lower panel) is oriented such that cells are seen in cross section. The distribution of Alexa Fluor 488-Dextran is shown 10 minutes (red) and 2 days (green) after injection. Ten minutes after injection the dextran is confined to the cytoplasm of the injected cell but, after 2 days in organ culture the dextran has spread laterally, into the cytoplasm of a layer of neighboring cells. Cap, capsule. Scale bars: 10 µm (A); 500 µm (B, upper left); 100 µm (B, upper right); 20 µm (B, lower panel).

View larger version (45K): [in this window] [in a new window]   Fig. 4. Long-term GFP labeling reveals the lamellar substructure of the lens. Lenses from Cre-ERTM;Z/EG mice were examined at intervals (6-170 days) after tamoxifen injection. Optical sections were obtained through the lens equator. In this orientation, fiber cells are seen in cross section. Six days after tamoxifen treatment (6 D), scattered GFP-expressing fiber cells are observed near the lens surface. By 16 days (16 D), the fiber cells have become internalized and the innermost GFP-expressing cells are connected by the LMDP. Consequently, GFP is beginning to diffuse from the cytoplasm of expressing cells into the cytoplasm of non-expressing neighbors. The spread of fluorescence is largely between cells located in the same lens stratum, resulting in the formation of a fluorescent annulus which becomes better defined at later ages (arrowed in 21 D, 80 D, 170 D). At later ages (80 D and 170 D), secondary fluorescent rings (complete and incomplete) are present. These result from the periodic incorporation of clusters of highly fluorescent fiber cells (arrowheads in 170 D) into the lens syncytium. Scale bar: 500 µm.

View larger version (69K): [in this window] [in a new window]   Fig. 5. Age-dependent changes in clonal expansion of fiber cell progenitors accounts for the presence of secondary rings. (A) Maximum intensity projection of the lens equatorial region from a 29-day-old Cre-ERTM;Z/EG mouse 7 days (7 D) after tamoxifen treatment. The position of the lens equator (Eq.) and individual GFP-expressing epithelial cells (Epi) is indicated. Note that GFP-positive epithelial cells are apparently randomly distributed in the lens epithelium and many, especially those located near the equator, are present as cell pairs. GFP-expressing fiber cells (Fib) are evenly distributed in the cortical lens tissue beneath the equator. (B) Maximum intensity projection of the lateral aspect of a lens from a Cre-ERTM;Z/EG mouse 147 days (147 D) after tamoxifen treatment. Immediately anterior to the lens equator, clusters of GFP-positive epithelial cells are present (one cluster is boxed in B and shown at higher magnification in C). Diffusely labeled bundles of fiber cells are present interspersed with unlabeled fibers, resulting in a characteristic striped fluorescence pattern. (C) Clusters of GFP-positive epithelial cells contain 20-30 cells. (D) Equatorial optical sections of the lens at 147 days reveal the spatial relationship between the fluorescent fiber bundles and the pattern of secondary fluorescent rings. Near the periphery, discretely labeled bundles of GFP-positive cells (typically containing 20-30 fluorescent fiber cells) are present (arrowheads). As the fiber bundles are overlaid with more recently differentiated cells, GFP begins to diffuse into the cytoplasm of neighboring cells located in the same stratum of the lens as the GFP bundles (arrows). Diffusion of GFP throughout an entire stratum results in the formation of secondary fluorescent rings. Scale bars: 250 µm (A); 500 µm (B,D); 50 µm (C).

View larger version (100K): [in this window] [in a new window]   Fig. 6. Expression of Lim2 in the cortical region of 1-month-old mouse lenses. Lenses were sectioned in the mid-sagittal (A) or equatorial (B,C) plane. The position of the lens epithelium (Epi) is indicated by dashed lines. In wild-type mouse lenses (A,B), Lim2 fluorescence is first detected in the membranes of fiber cells located approximately 50 µm beneath the lens surface (arrows) and increases in intensity in the older, central cells. No membrane fluorescence was observed in Lim2-null mouse lenses, although granular background staining was present in the cytoplasm of epithelial cells (Epi) and superficial fibers. Scale bars: 20 µm.

View larger version (90K): [in this window] [in a new window]   Fig. 7. Formation of the LMDP requires Lim2. Lim2Gt/Gt (right semicircles) or wild-type (WT) mice (left semicircles) were crossed with the inducible reporter strain Cre-ERTM;Z/EG (A-C) or TgN(GFPU)5Nagy (D), a strain in which GFP is spontaneously expressed by scattered lens fiber cells. In Cre-ERTM;Z/EG mice, fluorescence was examined 3 months after tamoxifen treatment (A-C). As noted elsewhere (see Figs 4 and 5), on a wild-type background, bundles of GFP-expressing fibers are present in the lens cortex. As these become incorporated into the lens syncytium, GFP diffuses into neighboring cells, resulting initially in the formation of broad diffuse stripes of fluorescence (*) in anterior (A) and posterior projections (B) and, at later time points, the formation of characteristic fluorescence rings in equatorial optical sections of the lens (C). In Lim2-deficient mice, intercellular diffusion in the lens was not observed. Numerous discretely labeled fibers were evident throughout the anterior (A) and posterior (B) hemispheres of lenses in Lim2-null mice. In equatorial sections (C), where fibers are seen in cross section, individual labeled fibers were noted (arrowheads), extending to a depth of approximately 300 µm below the lens surface (corresponding to the tissue stratum in which GFP expression was first induced). In TgN(GFPU)5Nagy mice, GFP is expressed in the lens throughout development, enabling the formation of the LMDP to be visualized in the center of young (5-day-old) lenses (D). In wild-type mice, the LMDP is established in fibers located 100-200 µm below the lens surface (arrow). However, in Lim2-deficient animals, the LMDP does not form and the central region of the lens has a checkerboard appearance as a result of the presence of uncoupled GFP-expressing and non-expressing cells. Epi, epithelial cells. Scale bars: 500 µm (A-C); 250 µm (D).

View larger version (152K): [in this window] [in a new window]   Fig. 8. Conduits for intercellular diffusion of macromolecules might be Lim2-dependent cell fusions. Living, intact lenses were imaged by confocal microscopy following incubation in the lipophilic fluorescent dye, FM4-64. The ordered arrangement of the fiber cells in the vicinity of the anterior suture is evident but, in wild-type lenses, numerous cellular dilations are also present (arrows). Dilations are distributed throughout the tissue volume in this region, as shown by three representative sections from a z-stack collected beneath the anterior pole of the lens. A similar region (note the presence of the Y-shaped suture in each case) from a Lim2Gt/Gt mouse lens is shown in the lower panels. Cellular dilations are not present in the Lim2Gt/Gt lens. Scale bar: 50 µm.

View larger version (84K): [in this window] [in a new window]   Fig. 9. Regions of cellular dilation correspond to zones of limited cell fusion. The anterior polar regions of lenses from 3-week-old wild-type mice were processed for array tomography. Ultrathin (80 nm) sections were incubated with an antibody against Aqp0, an abundant integral protein of lens fiber membranes. In longitudinal sections (left panel) numerous examples of cellular fusions were observed (arrows) adjacent to the anterior suture (visible as a vertical discontinuity in the cell packing arrangement). In transverse sections (center panel), large caliber cells were identified which, on volumetric reconstruction, were found to correspond to zones of fusion between neighboring fibers. The four images in the center panel are selected from a z-series of 150 serial ultrathin sections. The numbers represent the position (in µm) of sections in the image stack. Note that the membrane septum dividing the cells at position 8.00 µm has disappeared by position 2.00 µm and, as a result, the cells are fused in this region. The right panel shows an equivalent region from a Lim2Gt/Gt mouse lens. Note the more regular arrangement of the cells (compare with wild-type lens in the left panel), and the absence of dilated or fused regions. Scale bars: 20 µm.

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