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doi: 10.1242/10.1242/jcs.00325

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Reduced survival of lens epithelial cells in the A-crystallin-knockout mouse

¹ Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St Louis, MO 63110, USA
² Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO 63110, USA

View larger version (102K): [in a new window]   Fig. 1. Expression of A in lens epithelial wholemounts by immunofluorescence. Wholemounts prepared from wild-type and A–/– lenses were stained with a monoclonal antibody to A and an Alexa568-labeled secondary antibody (red). The DNA-binding dye TOTO-1 (green) was used to stain the epithelial cell nuclei. (A) Merged confocal micrographs of A and nuclei visualized in the wholemount of a wild-type mouse lens. (B) Lens epithelial wholemount from an A–/– mouse. No A immunofluorescence was detected from the A–/– lens epithelial wholemount. Bar, 25 µM.

View larger version (102K): [in a new window]   Fig. 2. Confocal micrographs of BrdU and TOTO-1 labeling in lens epithelial wholemounts 3 hours after injection of BrdU. Seven-day-old wild-type or A–/– mice were injected with BrdU and wholemounts of lens epithelium were fixed 3 hours after the BrdU injection. The chromosomes were stained with BrdU. (A,D) S-phase cells labeled with BrdU (red) in wild-type (A) or A-knockout (D) lenses. (B,E) Cells labeled with the DNA stain TOTO-1 in the same wholemounts (green): (B) wild-type; (E), A–/–. (C,F) Merged confocal images of BrdU and TOTO-1: (C) wild-type; (F) A–/–. Note that the majority of the BrdU-labeled cells are single cells. A pair of mitotic cells can be seen in TOTO-1 staining of the wild-type epithelial wholemount (arrowheads). The nuclei of the lens epithelium demonstrate several staining patterns, including uniform light labeling, punctate labeling and uniform intense labeling. Note also that the first labeled mitoses were observed 3 hours after BrdU injection (A-C). (G) Quantitative analysis of the labeling index in the wild-type and A–/– wholemounts 3 hours after the BrdU pulse. Note that 3 hours after the BrdU injection, the labeling index was the same in wild-type and A–/– epithelial cells. Also note that the minor differences seen in the nuclear size of the wild-type and A–/– wholemounts were not consistently observed, and show variation in the spreading out and fixation during preparation of the wholemounts. Bars, 25 µM (A-C); 25 µM (D-F).

View larger version (90K): [in a new window]   Fig. 3. Confocal micrographs of BrdU and TOTO-1 staining in lens epithelial wholemounts 24 hours after injection of BrdU. Seven-day-old wild-type or A–/– mice were injected with BrdU, and wholemounts of the lens epithelium were fixed 24 hours after BrdU injection. In these wholemounts, most of the lens epithelial nuclei are members of cell pairs. (A,D) Cells labeled with BrdU (red): (A) wild-type; (D) A–/– mice. (B,E) Cells labeled with the DNA stain TOTO-1 in the same wholemounts (green): (B) wild-type; (E) A–/–. (C,F) merged confocal images of BrdU and TOTO-1: (C) wild-type; (F) A–/–. (G) The labeling index was determined 24 hours after the BrdU pulse, and it was found to be lower in the A–/– wholemounts than in the wild-type. The minor increase in the internuclear spacing of the A–/– wholemount (E) as compared with the wild-type (B) was not consistently observed, and suggests variability in the sample preparation and spreading out of the epithelial wholemount. Bar, 25 µM (A-C); 25 µM (D-F).

View larger version (58K): [in a new window]   Fig. 4. Merged confocal micrographs of BrdU- and TOTO-1-stained nuclei in the lens epithelial wholemounts. (A) In this micrograph, several BrdU-labeled `tetrads' can be seen (arrows) 24 hours after BrdU injection. Pairs of BrdU-labeled nuclei (red) were identified by their identical staining pattern. The wholemount was stained with TOTO-1 (green) to detect all the nuclei. Note that a majority of the nuclei were not labeled with BrdU. Bar, 25 µM. (B) BrdU and TOTO-1 staining in lens epithelial wholemounts 5 days after injection. Wild-type mice were injected with BrdU and wholemounts were fixed 5 days later. BrdU immunofluorescence (red) in the central region of the wholemount is shown. TOTO-1 (green) was used to label the nuclei of all the cells. Note that the BrdU-labeled cells were all members of cell pairs (arrows). Note also that, in some cases, members of a pair of BrdU-labeled nuclei were separated by two or more nuclei. Bar, 10 µM. (C) BrdU and TOTO-1 labeling in the periphery of a 7-day-old wild-type lens epithelial wholemount 24 hours after BrdU injection. Near the periphery (germinative region) of the lens epithelium, the labeling index was 2-3-fold higher as compared with the central region (Fig. 4A,B). In many cases, pairs of BrdU-labeled nuclei (red) that were very close to each other could be identified. In other cases, it was difficult to ascertain if two adjacent BrdU-labeled nuclei were members of a pair or not. TOTO-1 staining (green) was used to visualize all the nuclei. Bar, 25 µM.

View larger version (21K): [in a new window]   Fig. 5. BrdU labeling index in the lens epithelial wholemounts at successive times after BrdU injection. Seven-day-old wild-type or A–/– mice were injected with BrdU and wholemounts were fixed at successive times after the BrdU injection. The BrdU labeling index was determined at 1, 3, 8, 16 and 24 hours, and 2, 3 and 5 days after the BrdU injection. Four lenses were used at each time point. Note that the labeling index of the A–/– wholemount lagged the labeling index of the wild-type between 16 hours and 5 days.

View larger version (123K): [in a new window]   Fig. 6. Confocal micrographs of TUNEL staining in lens epithelial wholemounts. Seven-day-old mouse lens epithelial wholemounts were fixed and stained with the TUNEL staining kit. Propidium iodide was used to stain the DNA of all the nuclei in the wholemounts: (A) wild-type lens epithelium; (B,C) A–/– lens epithelium. In the wild-type wholemounts, TUNEL staining was rarely detected. Pairs of TUNEL-labeled cells (green) were scattered throughout the A–/– wholemounts. Note that the TUNEL-positive nuclei were nearly always seen as pairs. (D) Quantitative analyses of cell death in lens epithelial wholemounts. TUNEL-positive nuclei were counted in wild-type or A–/– lens epithelial wholemounts. In the wild-type lens epithelium, there was an average of one TUNEL stain in the central region. In the A–/– wholemount, there was an average of six TUNEL-stained nuclei. Data represent average of six wild-type and six A–/– wholemounts. Bars, 25 µM (A-C).

View larger version (118K): [in a new window]   Fig. 7. Confocal images of TUNEL and BrdU staining in lens epithelial wholemounts. Seven-day-old wild-type and A–/– mice were injected with BrdU, and wholemounts were fixed 24 hours later. (A,D,G) BrdU-labeled cells. (B,E,H) TUNEL-labeled cells. (C,F,I) Merged confocal images of BrdU- and TUNEL-labeled cells in (A,D,G) and (B,E,H). In the A–/– lens epithelial wholemounts (A,D), several BrdU-labeled nuclei, smaller in size than normal, are scattered among the pairs of normal-sized BrdU-labeled nuclei. These smaller nuclei were often seen as distinct members of a pair (arrows). The smaller nuclei were intensely stained with TUNEL reagents (B,C,E,F). Note that the BrdU-labeled nuclei having normal dimensions were not stained with the TUNEL label. In the wild-type wholemounts, smaller BrdU-labeled nuclei were not detected often (G), and the TUNEL labeling was negligible (H,I). Bars, 10 µM (A-H).

View larger version (103K): [in a new window]   Fig. 8. Visualization of ß-tubulin in lens epithelial wholemounts by immunofluorescence and confocal microscopy in different cell-cycle phases. Seven-day-old wild-type or A–/– mouse lens epithelial wholemounts were immunostained with an antibody to ß-tubulin (red) and the nuclei were stained with TOTO-1 (green). (A) In the interphase of wild-type cells, ß-tubulin was uniformly distributed around the nuclei. (B,C) In metaphase cells, the bundling of ß-tubulin around the condensed chromosomes could be readily detected. Note that there was no difference in the organization of the metaphase spindles of the wild-type (B) and A–/– (C) lens epithelial wholemounts. (D-F) In anaphase cells of the wild-type epithelium, the spindle was well organized (D). However, there was disorganization of the anaphase spindle of the A–/– lens epithelial wholemounts (E,F). This aberrant spindle phenotype was observed in 45% of the anaphase spindles of the A–/– wholemounts. Twenty metaphase and 20 anaphase spindles in six different lens epithelial wholemounts were analyzed for each genotype. Bars, 5 µM.

View larger version (125K): [in a new window]   Fig. 9. Visualization of A and F-actin in lens epithelial cells during mitosis. Merged confocal images of A immunofluorescence (red) and F-actin staining (green) of wild-type mouse lens epithelial cells. Cell morphology was imaged with differential interference contrast (blue). (A) Cells in anaphase (arrow) or interphase (arrowheads). During interphase, A was distributed throughout the cytoplasm. Note that a pair of recently divided cells remaining close to each other can be seen (arrowheads). (B) Cells in cytokinesis (arrowheads). Note that A immunofluorescence (red) was concentrated in the middle of the dividing cells. Bars, 25 µm (A,B).

View larger version (100K): [in a new window]   Fig. 10. Visualization of A-crystallin and ß-tubulin in lens epithelial cells during mitosis. Wild-type mouse lens epithelial cells were fixed and stained with anti-A and an Alexa488-conjugated secondary antibody (green). Cells were also immunostained with an antibody to ß-tubulin and an Alexa568-conjugated secondary antibody (red) and the nuclei were stained with TOPRO-3 (blue). (A-C) Metaphase, (D-F) anaphase and (GI) cytokinesis. Note that A was concentrated in the region of the centrosome in metaphase and in the intercellular bridge microtubules of the dividing cells during cytokinesis (arrowheads). Bars, 10 µm (A-F); 25 µm (G-I).

View larger version (15K): [in a new window]   Fig. 11. Lens dimensions in wild-type and A–/– lenses. Mid-sagittal sections (4 µm) of wild-type and A-knockout lenses were stained with hematoxylin and eosin. The lens sections were examined by confocal microscopy. The equatorial diameter of the lens was determined at different postnatal ages. The equatorial diameter of the A–/– lenses was 30% smaller at birth and lagged in growth compared with the wild-type throughout the early postnatal period.

View larger version (126K): [in a new window]   Fig. 12. Cross-sectional area of fiber cells in wild-type and A–/– mouse lenses. Lens slices were cut in the equatorial plane and stained with an antibody to MIP (AQP0) to visualize fiber cell membranes. The distance from the center of the lens was 300 µm. The organization of the fiber cells in these equatorial sections of wild-type and A–/– mice is different, and the membranes of the wild-type lens fiber cells appear smoother than those of the A–/–. Variation in the cross-sectional areas of the different fiber cells was noted for both wild-type and A–/– cells. However, the distribution of the cross-sectional areas of the fiber cells was similar in wild-type and A–/– lenses. Bar, 10 µM (A,B).

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