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First published online 26 January 2006
doi: 10.1242/dev.02262

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Caspase-dependent secondary lens fiber cell disintegration in A-/B-crystallin double-knockout mice

Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Building 7, 7 Memorial Drive, MSC 0704 Bethesda, MD 20892, USA.

View larger version (85K): [in a new window]   Fig. 1. Lens fiber cells disintegrate in A-/B-crystallin double knockout mice. Fixed sections of the lens from a wild-type mouse at the age of 3 months (A), and from DKO mice at the ages of 6 weeks (B), 8 weeks (C), 3 months (D), 10 months (E) and 13 months (F) were stained with Hematoxylin and Eosin. eq, equatorial/bow region of lens; ln, lens nucleus; df, disintegrated fiber cells. Scale bar: 250 µm.

View larger version (70K): [in a new window]   Fig. 2. Immunostaining of DKO mouse lens with anti-active caspase 3 antibodies. Both primary anti-active caspase 3 antibody and secondary anti-rabbit IgG antibodies, conjugated with Alexa Fluor 488, were used for detection (A). The bright-field image was captured (B). As a negative control, the primary antibody was omitted, and only the secondary antibody was used (C). The bright field image (B) and active caspase 3 fluorescent image (A) were merged for morphological comparison (D). Scale bar: 250 µm; A-C are at the same magnification.

View larger version (16K): [in a new window]   Fig. 3. Caspase 3 and caspase 6 activities. Caspase 3 (A,C) and caspase 6 (B,D) activities were measured in the presence (+) or absence (-) of a specific inhibitor (A,B) in lens extracts from wild-type and DKO mice from three age groups. The activities plotted in C and D as a function of animal age represents the difference in caspase activity measurements in the presence or absence of a specific inhibitor. Specific activities are in nmol of pNA/hr/ng of total extract protein for caspase 3 or in nmol of AMC/hr/mg of total extract protein for caspase 6. The error bars represent data error analyzed using linear models in R (Ver. 2.1.1). *P<0.001; **P=0.007; ***P=0.003.

View larger version (30K): [in a new window]   Fig. 4 . Caspase 3 and caspase 6 immunostaining. Immunostaining of the anterior region of secondary lens fiber cells (A) in wild-type (B,D,F,H) and DKO (C,E,G,I) lenses with anti-active caspase 3 (B,C) and anti-caspase 6 (F,G) antibodies. In negative controls (D,E,H,I), primary antibodies were omitted and only the secondary anti-rabbit IgG antibody, conjugated with Alexa Fluor 488, was used. (J) Image-Pro Plus v.5.1 software was used to quantitate fluorescence intensity in sections of lens from wild-type and DKO mice. Scale bar: 25 µm.

View larger version (61K): [in a new window]   Fig. 5. TUNEL labeling of lens secondary fiber cells. Bright-field image (A-D), TUNEL reaction fluorescence (E,F) and merge of both (G,H) sections from 7-week-old wild-type (A,C,E,G,I) and 7.5-week-old DKO (B,D,F,H,J) mouse lenses. In negative controls (I,J), calf thymus terminal deoxynucleotidyl transferase was omitted from the TUNEL reaction. C,D are enlargements of boxed areas in A,B. Arrows in E,F,H indicate TUNEL-positive nuclei. Scale bar: 125 µm in A,B; 25 µm in C-J.

View larger version (41K): [in a new window]   Fig. 6. Secondary lens fiber cell apoptosis. DAPI labeling fluorescence (A,B), TUNEL reaction fluorescence (C,D), merge of both (E,F) and merge of DAPI and TUNEL with bright field (G,H) of lens sections from 7-week-old wild-type (A,C,E,G,I) and 7.5-week-old DKO (B,D,F,H,J) mice. Lenses on all images are oriented such that the upper right corner points towards the bow region and the lower left corner points towards the lens nucleus. In negative controls (I,J), terminal transferase was omitted from the TUNEL reaction. Arrows in E,F indicate nuclei double labeled with DAPI and TUNEL. Arrowheads in E indicate the area of secondary fiber cell maturation. Scale bar: 25 µm.

View larger version (21K): [in a new window]   Fig. 7. Western blots of cobalt chelate gel column elution fractions. Recombinant caspase 6 (0.6 µg, left lane), 30 µl of the elution fraction from the control column, where caspase 6 was omitted (middle lane), and 30 µl of the elution fraction from the caspase 6-Co2+ column (right lane) were separated on a 10% NuPage gel and detected by western blot with anti-caspase 6, anti-B-crystallin or anti-A-crystallin antibodies.