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First published online 2 September 2003
doi: 10.1242/jcs.00738

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Development of a macromolecular diffusion pathway in the lens

Valery I. Shestopalov^1,*, and Steven Bassnett^1,2

¹ Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, Saint Louis, MO 63110, USA
² Department of Cell Biology and Physiology, Washington University School of Medicine, Saint Louis, MO 63110, USA

View larger version (70K): [in a new window]   Fig. 1. Variegated expression of a GFP transgene in the lens of hemizygous TgN(GFPU)5Nagy mice. (A) En face confocal view of the lens epithelium. Individual GFP-expressing cells are evident (arrows). Nonexpressing cells occupy the spaces between the fluorescent cells. The proportion of GFP-expressing epithelial cells varied from 20-50%. (B) Confocal image of a living, intact, P1 lens imaged in the equatorial plane. Note the variegated GFP expression pattern in the outer cortex (arrows) and the reduced, but uniform, fluorescence in the lens core (*). Bars, A, 25 µm; B, 100 µm.

View larger version (91K): [in a new window]   Fig. 2. Three-dimensional reconstruction of GFP cellular fluorescence in the outer cortex of a one-month-old TgN(GFPU)5Nagy hemizygous mouse lens. (A) Viewed end on, the variegated expression of GFP in superficial fiber cells results in a checkerboard fluorescence pattern (arrows) due to the interposition of nonexpressing cells between GFP-expressing cells. In the deeper cell layers the fluorescence is uniform (*). (B) By rotating the volume, the elongated morphology of the fiber cells becomes evident. Note the sharp transition (arrow) between the discrete labeling pattern of individual fibers in the superficial tissue and the homogeneous fluorescence of cells in the deep cortex. (C) GFP-expressing cells are uniformly fluorescent along their lengths, although regions of the fibers that contact the surface of the reconstructed volume appear somewhat brighter. (D) Viewed from beneath; the cytoplasmic fluorescence in the fibers is uninterrupted. At this depth (approximately 125 µm), all fibers contain GFP. Bar, 100 µm

View larger version (119K): [in a new window]   Fig. 3. Covisualization of GFP fluorescence and AQP0 immunofluorescence in a fixed, equatorial lens slice from a P7 TgN(GFPU)5Nagy mouse. (A) GFP expression in fiber cells in the outer cortex. Near the lens surface (S), GFP-expressing fiber cells are interspersed with nonexpressing cells. Approximately 150 µm below the surface there is a transition (arrowhead) to a diffuse pattern of fluorescence in which all cells are weakly fluorescent. (B) Immunofluorescence detection of AQP0, an intrinsic lens membrane protein, highlights the membrane organization in this region of the lens. In equatorial sections lens fiber cells are seen in cross section. (C) Merged image of endogenous GFP fluorescence (green) and AQP0 immunofluorescence (red). Note that the transition (arrowhead) from the mosaic GFP fluorescence pattern that characterizes the superficial cells to the diffuse distribution in the inner cells is not associated with a marked change in the cross-sectional profiles of the fibers. (D) Pixel intensity histogram of GFP fluorescence collected along the line indicated in A. The average fluorescence intensity does not differ markedly in cells located either side of the transition region (arrowhead). In this example, at pixel positions >385, the average fluorescence intensity was 70.9±71.0 At pixel positions <385, the average intensity was 52.5±14.9. Bars in A,B,C, 50 µm.

View larger version (21K): [in a new window]   Fig. 4. GFP proteolysis does not occur during fiber cell differentiation. Lenses from P14 or P30 TgN(GFPU)5Nagy mice or wild-type controls were separated into three regions. The peripheral sample (p) contained the region of variegated GFP expression. The core sample (cr) was from the center of the lens and contained cells of uniform fluorescence. A cortex sample (cx) was obtained from a region intermediate between the peripheral and core samples. Blotted samples were probed with antibodies against the mitochondrial marker SDH(Fp) or GFP (see text for details). SDH(Fp) was abundant in the peripheral samples from transgenic or wild-type lenses, reduced in the cortex and absent from the core. The GFP antibody identified a single band of the expected size (27 kDa) in samples from the TgN(GFPU)5Nagy lenses. This band was absent from the wild-type controls. Full-sized GFP was present in samples from each region of the transgenic lenses, including the oldest cells in the lens, those of the core. No evidence of proteolytic breakdown products was observed. The results are representative of three experiments.

View larger version (71K): [in a new window]   Fig. 5. Intercellular diffusion of macromolecules is observed between fibers located deep within the lens but not between superficial cells. (A) Injections of 10 kDa TMRD (red) were made into individual fiber cells situated at different depths below the surface of a living P2 lens. The 0 hour time point is a nominal value. It took approximately 10 minutes to complete the injections and photograph the lens. During the interim TMRD had already begun to diffuse from injected cells in the lens core (arrowheads). (B) TMRD injected into surface cells (blue arrows) was well retained, even 12 hours after the injection. By contrast, TRMD was not retained by injected cells in the center of the lens (arrowheads) and, after 12 hours, formed a diffuse cloud of fluorescence in the lens core (*). The results are representative of 12 experiments. Bar, 250 µm.

View larger version (88K): [in a new window]   Fig. 6. Intercellular diffusion of 10 kDa TMRD occurs only in the uniformly labeled core region of TgN(GFPU)5Nagy hemizygous mouse lenses. Four fiber cells located at various depths below the surface of a P2 transgenic lens were injected with TMRD (red). Intrinsic GFP fluorescence (green) and TMRD fluorescence were covisualized during a 10 hour period in organ culture. Cell #1 and cell #2 were located in the variegated layer (arrows) at the lens surface. In these cells, TMRD fluorescence was retained over the course of the experiment. Cell #3 and cell #4 were located deeper below the lens surface. Cell #3 lay just within the region of uniform GFP fluorescence and cell #4 was located approximately 80 µm within the uniform region. Over the 10 hour culture period, TRMD fluorescence was not retained by cell #3 or cell #4. At the end of the experiment, TMRD fluorescence was visible as a diffuse cloud that had spread well beyond the boundaries of the injected cells. Bar, 25 µm.

View larger version (143K): [in a new window]   Fig. 7. Establishment of the macromolecular permeable pathway in the core of the lens during embryonic development. (A) At E14, GFP is expressed in a mosaic pattern. In equatorial sections, discretely labeled individual fiber cells are evident, scattered throughout the lens. This section was colabeled with fluorescent phalloidin (red) to visualize the organization of non-GFP-expressing cells (red). (B) In midsagittal sections, the fiber mass has a striped appearance, due to the juxtaposition of GFP-expressing and nonexpressing cells. (C) By E16, the fluorescence labeling pattern had transformed, such that the central fiber cells (situated between the arrows) are now uniformly labeled. (D) The uniformly fluorescent core region is also evident in mid-sagittal sections. Note that the region of uniform fluorescence contains fiber cells (arrows) that have recently detached from the posterior capsule. c, cornea; ep, lens epithelium. Bar, 50 µm.

View larger version (49K): [in a new window]   Fig. 8. The syncytial region expands during postnatal development. Fluorescence within living lenses from one-day-old (A), one-month-old (B) and six-month-old (C) hemizygous TgN(GFPU)5Nagy mice was imaged in the equatorial plane by confocal microscopy. Although the uniformly labeled lens core expands with age, the thickness of the variegated layer at the periphery (arrows) thins slightly over this period. The fluorescence in the center of the oldest lenses appears to be diminished with respect to the cortical cells. Bar, 250 µm.

View larger version (81K): [in a new window]   Fig. 9. A significant proportion of nucleated fiber cells are contained within the syncytial core of the lens. Vibratome slices of fixed hemizygous TgN(GFPU)5Nagy mouse lenses were incubated with propidium iodide to allow the distribution of fiber cell nuclei (red) and intrinsic GFP fluorescence (green) to be covisualized. At all ages examined, the cytoplasmic fluorescence in the outer cell layers had a striped appearance (arrows) due to the variegated expression of the GFP transgene. The nuclear bow region was clearly visible and propidium-stained nuclei extended well into the region of uniform GFP fluorescence. The position of the last nucleated fiber cell is indicated (arrowhead). A, P1 lens; B, P7 lens; C, adult lens. Bar, 50 µm.

View larger version (17K): [in a new window]   Fig. 10. Developmental dynamics of the syncytial region in the mouse lens. (A) The equatorial diameter of the lens (open symbols) and the uniformly labeled syncytial region (closed symbols) were measured in embryonic and adult lenses. The region expanded during development, closely matching the overall growth rate of the lens. (B) The thickness of the variegated region (filled symbols) decreased during early postnatal development before stabilizing at approximately 90 µm in adult lenses. The thickness of the nucleated cell layer (open symbols) also stabilized in older animals at approximately 180 µm. The layer of nucleated cells within the syncytial region (indicated by the shaded area) was approximately 90 µm thick. Data points represent the mean±s.d. (n>8 lenses for each time point).