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Results: 8

1.
Fig. 6

Fig. 6. From: Rac1 GTPase -deficient mouse lens exhibits defects in shape, suture formation, fiber cell migration and survival.

Conditional Rac1 deficient mouse lenses show abnormalities in distribution and deficits in levels of Rac1 downstream proteins involved in actin polymerization and branching A. Lens cryosections derived from the E15.5 and E17.5 Le-Cre/Rac1 and MLR10/Rac1 cKO lenses, respectively, immunostained for WAVE-2 and Abi-2 show altered distribution patterns both in the epithelium (Epi) and fiber cells (LF), with the staining being decreased relative to littermate WT lenses. B and C. Lens homogenates (800×g supernatants) derived from P1 MLR10/Rac1 cKO mice, and analyzed by immunoblotting, revealed significant decreases in levels of WAVE-2, Abi-2 and Nap1 as compared to WT lenses. In contrast, levels of phosphorylated-cofilin were increased significantly in the Rac1 deficient lenses compared to WT samples. The values represent average ± SEM values for three independent analyses (* P < 0.05). Scale bar: 20µm.

Rupalatha Maddala, et al. Dev Biol. ;360(1):30-43.
2.
Fig. 4

Fig. 4. From: Rac1 GTPase -deficient mouse lens exhibits defects in shape, suture formation, fiber cell migration and survival.

Increased apoptosis in Rac1 deficient mouse lens epithelium and fiber cells. Cryosections derived from the Le-Cre/Rac1 (E14.5) and MLR10/Rac1 (E17.5) ckO mouse lenses immunostained for TUNEL positive cells (red fluorescence in Le-Cre/Rac1 and green fluorescence in MLR10/Rac1 cKO specimens) revealed a significant increase in apoptosis in the epithelium (arrows) and fiber cells (arrow heads) compared to littermate WT lenses. The blue (top panel) and red (lower panel) staining in panel A shows nuclei counterstaining with Hoechst and propidium iodide, respectively. Green fluorescence in Le-Cre/Rac1 specimens indicates GFP distribution which in turn represents Cre expression and distribution pattern. B. Manual quantification of TUNEL positive cells (bar graphs) per specimen of lens epithelium and fiber mass showed a significant increase in both these regions in the Rac1 deficient lenses compared to the WT specimens. The values represent averages ± SEM from 4 to 5 independent analyses (*P < 0.05). Scale bar: Top panel: 40µm, Lower panel: 20 µm.

Rupalatha Maddala, et al. Dev Biol. ;360(1):30-43.
3.
Fig. 3

Fig. 3. From: Rac1 GTPase -deficient mouse lens exhibits defects in shape, suture formation, fiber cell migration and survival.

Conditional Rac1 deficient mice reveal abnormal lens shape, fiber cell migration and suture formation with considerable variations within each group. A. Schematic illustration of representative shape changes in lens and abnormal fiber cell (LF) migration pattern and shortened epithelial sheet (Epi) length in the E15.5 Le-Cre/Rac1 cKO lenses as compared to WT specimens. B and C. Representative histological images (B) and corresponding schematic drawings (C) of lens shape changes, abnormal fiber cell migration and decreased epithelial sheet length in the P1 MLR10/Rac1 cKO mice compared to WT. D. Representative histological images (D) of lenses derived from the P1 MLR10/Rac1 ckO mice exhibiting defects in suture formation (arrows) in addition to fiber cell migration. E. Quantitative differences in lens epithelial sheet length (in µm) in Le-Cre/Rac1 and MLR10/Rac1 cKOs as compared to littermate WT lenses. Line drawings in panels A & C represent only a small number lens fiber cells. The schematics are based on 2D images. The values represent average ± SEM values from six independent specimens (* P<0.05). Scale bars. B and D is 20µm.

Rupalatha Maddala, et al. Dev Biol. ;360(1):30-43.
4.
Fig. 5

Fig. 5. From: Rac1 GTPase -deficient mouse lens exhibits defects in shape, suture formation, fiber cell migration and survival.

Conditional Rac1deficient mouse lenses exhibit alterations in F-actin distribution. A, B. Both Le-Cre/Rac1 and MLR10/Rac1 cKO lenses (cryosections) stained for F-actin with phalloidin- rhodamine exhibit a progressive decrease in F-actin immunofluorescence staining in epithelium (Epi) and fiber (LF) cells starting from E12.5 to day1 as compared to littermate WT specimens. C. In Le-Cre/Rac1 and MLR10/Rac1 cKO lenses at E14.5 and day1, respectively, F-actin levels were significantly decreased in both the epithelium and in fiber cells, as determined by F-actin pixel intensity measurements using ImageJ (C; Left panel) or Metamorph (C; Right panel). The values represent average ± SEM values from three independent analyses (* P < 0.05). Scale bars: A: 40 µm, B; 20µm. Green fluorescence derived from the GFP in panel A shows the expression and distribution profile of Cre recombinase in the Le-Cre/Rac1 cKO lenses. Blue staining in Le-Cre/Rac1 mutant specimens show cell nuclei stained with Hoechst. lv; lens vesicle, pfc: primary fiber cells, sfc: secondary fiber cells.

Rupalatha Maddala, et al. Dev Biol. ;360(1):30-43.
5.
Fig. 2

Fig. 2. From: Rac1 GTPase -deficient mouse lens exhibits defects in shape, suture formation, fiber cell migration and survival.

Lens phenotype in Rac1 deficient mice. A. Le-Cre/Rac1 cKO lenses exhibit reduction in size and impaired elongation of primary fiber cells (arrows) at E12.5. At E14.5, the lens epithelial sheet length becomes markedly reduced and fiber cells display abnormal organization along with abnormal lens shape in the Rac1 cKOs as compared to lenses from WT littermates. B. The Le-Cre/Rac1 cKO lenses exhibit significantly reduced size and progressive degeneration of fiber cells and lens material leaking into the vitreous body in association with ruptured capsules starting from E15.5 to postnatal day1. In contrast to Le-Cre/Rac1 cKOs, the MLR10/Rac1 cKO lenses exhibit early and noticeable histological changes starting at E15.5. At E15.5, the lens is slightly smaller and the secondary fiber cell organization is noticeably different from the WT controls. Subsequently at E17.5 and P1, the epithelial sheet (Epi) length was found to be much shorter in the MLR10/Rac1 cKO lenses with the fiber cells (LF) exhibiting noticeable abnormalities in migration pattern and organization. These changes were found to be progressive, resulting finally in change of lens shape and size as compared to WT control lenses. Scale bars: A and B 20µm.

Rupalatha Maddala, et al. Dev Biol. ;360(1):30-43.
6.
Fig. 1

Fig. 1. From: Rac1 GTPase -deficient mouse lens exhibits defects in shape, suture formation, fiber cell migration and survival.

Generation of lens specific Rac1 deficient mice using the LoxP-Cre system. A and B, depict PCR based genotyping of Le-Cre/Rac1 and MLR10/Rac1 conditional mutant mice, respectively, using genomic DNA. C and D reveal a significant reduction in Rac1 protein levels in the different cKO lenses (at P1) based on immunoblot analysis (C), and subsequent densitometry based quantification (D). The values represent average ± SEM of three independent analyses (* P < 0.05). β-tubulin was immunoblotted (C) to confirm equal loading of protein. Lanes 1 and 2 in panel C represent two independent specimens from each group analyzed. E, Distribution of Rac1 in WT and Rac1 cKO mouse lenses based on immunofluorescence staining. Both Le-Cre/Rac1 and MLR10/Rac1 cKO lenses exhibit lack of Rac1 expression in the epithelium (Epi) and fibers (LF) relative to other ocular tissues. A line drawing in the Le-Cre/Rac1 specimen depicts the location of lens. The MLR39/Rac1 cKOs in contrast, exhibit a much reduced expression of Rac1 in lens fibers but not in the lens epithelium. Scale bar, 20µm.

Rupalatha Maddala, et al. Dev Biol. ;360(1):30-43.
7.
Fig. 7

Fig. 7. From: Rac1 GTPase -deficient mouse lens exhibits defects in shape, suture formation, fiber cell migration and survival.

Conditional Rac1 deficient lenses exhibit defects in cell-cell interactions. A and B. Lens cryosections (sagittal plane) derived from the Le-Cre/Rac1 (E15.5) and MLR10/Rac1 (E17.5 and P1) cKO mice immunostained for E-cadherin reveal markedly reduced localization of E-cadherin to the cell-cell junctions as compared to the littermate WT, indicating disruption of adherens junctions. The insets show areas at a higher magnification (2.5×). C. Immunostaining analysis of paraffin sections of Rac1 deficient lenses (equatorial plane) from P1 MLR10/Rac1 cKO mice reveals disruption in localization of β-catenin, N-cadherin, Nectin-1 and Rap1A/B to the cell-cell junctions. These proteins are localized to the cell membrane of hexagonal fiber cells in WT but not in the Rac1 deficient lenses, indicating defective fiber cell-cell interactions in Rac1 deficient lenses. D and E. Immunoblot and subsequent densitometric analysis, respectively, of Rac1 deficient lenses derived from P1 MLR10/Rac1 cKO mice showed a significant decrease in the levels of β-catenin, N-cadherin and Nectin-1 as compared to WT lenses. The values represent average ± SEM values of three independent analyses (* P < 0.05). Scale bar. A, B and C: 20µm. F. Lens equatorial sections derived from both WT and Le-Cre/Rac1 (E15.5) and MLR10/Rac1 (E17.5) cKOs were examined by transmission electron microscopy. While the WT specimens revealed closely packed fibers with distinct hexagonal shape (with two long arms and 4 short arms indicated with arrows and arrow heads, respectively), Rac1 deficient lenses exhibit disrupted fiber cell shape and asymmetric organization. Representative images of 4 independent specimens were shown. Scale bar: 0.5µm

Rupalatha Maddala, et al. Dev Biol. ;360(1):30-43.
8.
Fig. 8

Fig. 8. From: Rac1 GTPase -deficient mouse lens exhibits defects in shape, suture formation, fiber cell migration and survival.

Conditional Rac1 deficient mouse lenses reveal thinning of the lens capsule in association with decreased ECM proteins and disruptions of fiber cell basal terminal attachment to the lens capsule. A. Lens cryosections (sagittal plane) derived from the Le-Cre/Rac1 (E15.5) and MLR10/Rac1 (P1) cKO mice were immunostained for various ECM proteins. Both the WT and the Rac1 deficient lenses stained positively for laminin, fibronectin and collagen IV, which localized distinctly to the lens capsule, distributing to both anterior and posterior regions. In contrast to the WT lenses however, the Rac1 deficient lenses presented with much thinner capsules, especially in the posterior region. Further, the intensity of staining of individual ECM proteins was found to be markedly reduced in the Rac1 deficient lenses relative to WT lenses. Representative images of 3 independent specimens are shown. Scale bar: 20µm. B. Quantification of posterior lens capsule (central region) thickness showed a significant decrease in both Le-Cre/Rac1 and MLR10/Rac1 cKO specimens compared to the WT lenses. C and D. Immunoblot and subsequent densitimetric analyses, respectively of ECM proteins in lens homogenates of Rac1 deficient and WT specimens confirmed a significant decrease in laminin, fibronectin and collagen IV in the P1 MLR10/Rac1 cKO lens compared to the littermate WT lens. E and F. Analysis of Rac1 deficient lenses derived from P1 MLR10/Rac1 cKO mice for changes in levels of integrins (αVβ1, αVβ3, αVβ5 and β1) by immunoblotting (E) followed by densitometric quantification (F) showed a significant decrease in the levels of these proteins as compared to littermate WT lenses. The values represent average ± SEM values from three independent analyses (* P < 0.05). G. Sagittal lens sections derived from the Le-Cre/Rac1 (E15.5) and MLR10/Rac1 (E17.5) cKO and corresponding WT mice assessed by transmission electron microscopy. These analyses revealed that while the fiber cell posterior membrane and cytoplasmic protrusions were attached firmly and uniformly to the lens capsule in the WT lens images (indicated by arrows), in the Rac1 deficient lenses, the fiber cell posterior protrusions (arrow heads) were found to be shortened and broken with disrupted contacts with the lens capsule, indicating disruptions of cell-ECM interactions and impairment in fiber cell membrane protrusion. Representative images of 4 independent analyses are shown. Scale bar: 0.5µm

Rupalatha Maddala, et al. Dev Biol. ;360(1):30-43.

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