Rap1 GTPase is required for mouse lens epithelial maintenance and morphogenesis

Abstract

Rap1, a Ras-like small GTPase, plays a crucial role in cell-matrix adhesive interactions, cell-cell junction formation, cell polarity and migration. The role of Rap1 in vertebrate organ development and tissue architecture, however, remains elusive. We addressed this question in a mouse lens model system using a conditional gene targeting approach. While individual germline deficiency of either Rap1a or Rap1b did not cause overt defects in mouse lens, conditional double deficiency (Rap1 cKO) prior to lens placode formation led to an ocular phenotype including microphthalmia and lens opacification in embryonic mice. The embryonic Rap1 cKO mouse lens exhibited striking defects including loss of E-cadherin- and ZO-1-based cell-cell junctions, disruption of paxillin and β1-integrin-based cell adhesive interactions along with abnormalities in cell shape and apical-basal polarity of epithelium. These epithelial changes were accompanied by increased levels of α-smooth muscle actin, vimentin and N-cadherin, and expression of transcriptional suppressors of E-cadherin (Snai1, Slug and Zeb2), and a mesenchymal metabolic protein (Dihydropyrimidine dehydrogenase). Additionally, while lens differentiation was not overtly affected, increased apoptosis and dysregulated cell cycle progression were noted in epithelium and fibers in Rap1 cKO mice. Collectively these observations uncover a requirement for Rap1 in maintenance of lens epithelial phenotype and morphogenesis.

Keywords: Cell adhesion; Epithelial plasticity; Lens morphogenesis; Polarity; Rap1 GTPase.

Fig. 1

Expression and distribution profile of Rap1 GTPase and its effectors in mouse lens. A. RT-PCR analysis-based assessment of expression of Rap1a and Rap1b and their different effector and interacting proteins in P2 and P24 mouse lenses, using GAPDH expression for cDNA normalization. B & C show the qRT-PCR amplification traces for Rap1a and Rap1b expression relative to GAPDH, and the relative fold difference in expression in P2 mouse lenses, respectively. D. Distribution of Rap1 (Rap1a/Rap1b) protein in the soluble fraction (SF) and membrane enriched fraction (MEF) of P24 mouse lens based on immunoblotting analysis. E. Distribution of Rap1 in the developing (embryonic day10.5) and P21 mouse lens based on immunofluorescence analysis. The smaller rectangular box in the right panel is a magnification of the area shown as an inset. In the mature lens fibers (insert) Rap1 is localized predominantly at the cell membrane. In panel C, the error bar indicates SEM based on values derived from three independent analyses. In panel E bars represent image magnification.

Fig. 2

Ocular phenotype in the Rap1 cKO mouse. A & B. Intact lenses (membrane-enriched fraction) derived from three representative E17.5 Rap1 cKO mouse embryos showed a dramatic reduction (by ~ 90%; *P<0.05; n=4) of Rap1 (Rap1a/Rap1b-total) protein compared with WT controls based on immunoblot quantification. C. Reduction of Rap1 in lenses from E11.5 and E17.5 Rap1 cKO mouse embryos based on immunofluorescence analysis. The top panel shows the distribution profile of GFP (green fluorescent protein) only in the lens vesicle of Rap1 cKO mouse embryos (E11.5) confirming Cre expression relative to the WT control. The middle and lower panels show reduction of Rap1 in the E11.5 lens vesicle and E17.5 lens, respectively, derived from Rap1 cKO mice and relative to the WT controls based on immunofluorescence analysis. D. Conditional deletion of Rap1 (Rap1a/Rap1b) in the lens results in microphthalmic eyes with lens opacity in the embryonic and neonatal mice. The images shown are with eye lids removed from the E17.5 embryos. Bars in panel C represent image magnification. LV: Lens vesicle; LE: Lens epithelium; LF: Lens fibers.

Fig. 3

Histological and actin cytoskeletal changes in the developing and neonatal Rap1 cKO mouse eye. A. To determine the effects of conditional Rap1 deficiency on lens changes during development, ocular specimens derived from the E11.5, 13.5, 15.5, 17.5 embryos and day 1 neonatal Rap1 cKO mice were fixed, sectioned and stained with H&E. The tissue sections were imaged using a light microscope. Although the lens vesicle in E11.5 Rap1 cKO mouse embryos showed no overt changes in formation and separation, starting from E13.5 to P1, there was a progressive disruption in fiber cell apical attachment with lens epithelial apical ends (indicated with arrows), resulting in an empty space between the epithelium and fibers in Rap1 cKO specimens compared with WT controls. In E17.5 and P1 Rap1 cKO lenses, there was a gross disorganization of fibers with noticeable disruption at the lens fulcrum (arrow heads) compared with WT controls. In E13.5 specimens derived from the Rap1 cKO embryos, fiber cell migration pattern at the fulcrum is noticeably different from the WT controls (arrow heads) and in E15.5 and 17.5 specimens, the nuclei spread to below the transitional zone and are retained at the posterior ends of the fibers (arrow heads). B. To further evaluate lens defects in the Rap1 cKO mice, cryotissue sections derived from the E11.5 to E17.5 stages from Rap1 cKO and WT mice described above were stained for F-actin with rhodamine phalloidin, prior to image capture by confocal microscopy. Similar to the histological changes noted in Fig. 3A, defective cell-cell attachment between the lens epithelial apical ends and fiber cell apical ends was clearly noted in embryonic Rap1 cKO mouse specimens, in E13.5 and 15.5 specimens, and is indicated with arrows. Additionally, arrow heads indicate defects in the lens fulcrum formation in E13.5 and E15.5 Rap1 cKO embryos. F-actin distribution in the lens vesicle at the apical ends of cells shows reduced staining and extending a similar reduction at the apical junctions of epithelial and fiber cells in E13. 5, 15.5 and 17.5 specimens in the Rap1 cKO embryos compared with WT controls. In E17.5 Rap1 cKO lens specimens, there is a gross disruption in fiber cell organization compared with WT controls. LV: Lens vesicle, LE: Lens epithelium, LF: Lens fibers. Bars in A and B represent image magnification.

Fig. 3

Histological and actin cytoskeletal changes in the developing and neonatal Rap1 cKO mouse eye. A. To determine the effects of conditional Rap1 deficiency on lens changes during development, ocular specimens derived from the E11.5, 13.5, 15.5, 17.5 embryos and day 1 neonatal Rap1 cKO mice were fixed, sectioned and stained with H&E. The tissue sections were imaged using a light microscope. Although the lens vesicle in E11.5 Rap1 cKO mouse embryos showed no overt changes in formation and separation, starting from E13.5 to P1, there was a progressive disruption in fiber cell apical attachment with lens epithelial apical ends (indicated with arrows), resulting in an empty space between the epithelium and fibers in Rap1 cKO specimens compared with WT controls. In E17.5 and P1 Rap1 cKO lenses, there was a gross disorganization of fibers with noticeable disruption at the lens fulcrum (arrow heads) compared with WT controls. In E13.5 specimens derived from the Rap1 cKO embryos, fiber cell migration pattern at the fulcrum is noticeably different from the WT controls (arrow heads) and in E15.5 and 17.5 specimens, the nuclei spread to below the transitional zone and are retained at the posterior ends of the fibers (arrow heads). B. To further evaluate lens defects in the Rap1 cKO mice, cryotissue sections derived from the E11.5 to E17.5 stages from Rap1 cKO and WT mice described above were stained for F-actin with rhodamine phalloidin, prior to image capture by confocal microscopy. Similar to the histological changes noted in Fig. 3A, defective cell-cell attachment between the lens epithelial apical ends and fiber cell apical ends was clearly noted in embryonic Rap1 cKO mouse specimens, in E13.5 and 15.5 specimens, and is indicated with arrows. Additionally, arrow heads indicate defects in the lens fulcrum formation in E13.5 and E15.5 Rap1 cKO embryos. F-actin distribution in the lens vesicle at the apical ends of cells shows reduced staining and extending a similar reduction at the apical junctions of epithelial and fiber cells in E13. 5, 15.5 and 17.5 specimens in the Rap1 cKO embryos compared with WT controls. In E17.5 Rap1 cKO lens specimens, there is a gross disruption in fiber cell organization compared with WT controls. LV: Lens vesicle, LE: Lens epithelium, LF: Lens fibers. Bars in A and B represent image magnification.

Fig. 4

Disruption of E-cadherin-based AJs and ZO-1associated cell-cell interactions in Rap1 cKO mouse lenses. A. The Rap1 cKO mouse specimens derived from E13.5, 15.5 and 17.5 embryos reveal a progressive disruption in E-cadherin based AJs (green) in lens epithelium associated with altered cell shape and significantly reduced cell width compared to the respective WT controls (A1, A4). While lens epithelial cells (in both central (A2) and equatorial (A3) epithelium) in WT specimens exhibit a long columnar shape (double headed arrow; known to depend on maintenance of apical to basal polarity) and intense E-cadherin-positive AJs, the Rap1 cKO lens specimens exhibit a dramatic reduction in E-cadherin-based AJs both in central (A2) and equatorial (A3) epithelium, and disruption of apical to basal polarity with altered cell shape. A4 shows a significant decrease in lens central epithelial width in the Rap1 cKO specimens compared with WT, based on values (mean ± SEM) derived from 6 independent specimens. *P<0.05. LE: Lens epithelium, LF: Lens fibers, CLE: Central lens epithelium, ELE: Equatorial lens epithelium. B. Similar to AJs shown in Fig. 4A, ZO-1-based cell adhesive interactions were dramatically reduced in the lens of E13.5, E15.5 and E17.5 Rap1 cKO embryos. ZO-1 based cell-cell interactions (in green) were distributed discretely between the apical junctions of fiber cells and epithelium in the WT lenses (arrows). However, in the absence of Rap1, the ZO-1 based cell-cell interactions were dramatically reduced. The loss of cell adhesive interactions was associated with reduction in number and size of nuclei in the lens epithelium of Rap1 cKO specimens relative to WT controls, based on Hoechst staining (blue). Bars represent magnification.

Fig. 4

Disruption of E-cadherin-based AJs and ZO-1associated cell-cell interactions in Rap1 cKO mouse lenses. A. The Rap1 cKO mouse specimens derived from E13.5, 15.5 and 17.5 embryos reveal a progressive disruption in E-cadherin based AJs (green) in lens epithelium associated with altered cell shape and significantly reduced cell width compared to the respective WT controls (A1, A4). While lens epithelial cells (in both central (A2) and equatorial (A3) epithelium) in WT specimens exhibit a long columnar shape (double headed arrow; known to depend on maintenance of apical to basal polarity) and intense E-cadherin-positive AJs, the Rap1 cKO lens specimens exhibit a dramatic reduction in E-cadherin-based AJs both in central (A2) and equatorial (A3) epithelium, and disruption of apical to basal polarity with altered cell shape. A4 shows a significant decrease in lens central epithelial width in the Rap1 cKO specimens compared with WT, based on values (mean ± SEM) derived from 6 independent specimens. *P<0.05. LE: Lens epithelium, LF: Lens fibers, CLE: Central lens epithelium, ELE: Equatorial lens epithelium. B. Similar to AJs shown in Fig. 4A, ZO-1-based cell adhesive interactions were dramatically reduced in the lens of E13.5, E15.5 and E17.5 Rap1 cKO embryos. ZO-1 based cell-cell interactions (in green) were distributed discretely between the apical junctions of fiber cells and epithelium in the WT lenses (arrows). However, in the absence of Rap1, the ZO-1 based cell-cell interactions were dramatically reduced. The loss of cell adhesive interactions was associated with reduction in number and size of nuclei in the lens epithelium of Rap1 cKO specimens relative to WT controls, based on Hoechst staining (blue). Bars represent magnification.

Fig. 5

Rap1 deficiency suppresses β-catenin-based cell-cell junctions and increases N-cadherin levels in mouse lens epithelium. A. β-catenin, a well-characterized component of AJs localizes to the cell-cell junctions of lens epithelium and fiber cells in WT specimens based on immunofluorescence analysis (red) of paraffin embedded sagittal sections. In Rap1 cKO E13.5, E15.5 and E17.5 specimens, there is a progressive and dramatic reduction of β-catenin staining in both lens epithelium and fibers compared to WT specimens. The insets show magnified areas of lens epithelium. B. Unlike β-catenin, N-cadherin distribution (red staining) is relatively intense in fiber cells compared to the epithelium of WT lenses of mouse embryos. However, in the Rap1 cKO mouse lens specimens (E13.5, 15.5 and 17.5), there was a progressive and marked increase in N-cadherin staining in the epithelium with a dramatic and concomitant reduction in the fiber cells. The insets show the magnified area of lens epithelium. Bars represent image magnification. LE: Lens epithelium, LF: Lens fibers.

Fig. 6

Rap1 deficiency disrupts cell-ECM adhesion, β1 integrin activation and PAR complex in mouse lens. A. To determine the influence of Rap1 deficiency on cell-ECM interactions, E15.5 and E17.5 ocular specimens derived from the Rap1 cKO embryos were evaluated for changes in activation status of paxillin based on immunofluorescence analysis of phosphorylation (Tyr118) status. In WT specimens, phospho-paxillin (green) is distributed discretely and intensely to the apical junction of epithelial and fiber cells (arrows) very similar to the distribution pattern of ZO-1. Lens capsule (both anterior and posterior) also appears to exhibit some positive staining. In Rap1 cKO (E15.5 and E17.5) mouse ocular specimens, a dramatic reduction in phospho-paxillin staining is noted at the apical junctions of epithelial and fiber cells compared with WT controls. B. Immunofluorescence analysis shows that weak staining for β1-integrin (detected using a monoclonal antibody which recognizes the activated epitope of β1-integrin) was found to be distributed throughout the WT lens, in both the epithelium and fibers (red). These specimens were also labeled for cell nuclei using Hoechst (blue). In contrast, the lens epithelium of Rap1 cKO mouse embryos showed a robust increase in β1-integrin specific staining (arrows). In lens fibers of Rap1 cKO mouse embryos, there appears to be some decrease in the staining for β1-integrin relative to WT controls. Insets depict magnified areas of the central epithelium. Bars in both A and B represent image magnification. LE: Lens epithelium, LF: Lens fibers. C. To determine the status of PAR complex activity in Rap1 cKO mouse lens specimens, the levels of aPKC (both aPKCλ and aPKCς), a well-characterized component of PAR, were examined by immunoblot analysis in E17.5 lenses and compared with respective WT lenses. The levels of both aPKCλ and aPKCς were decreased significantly in the Rap1 cKO lenses (total lysates) compared to WT controls. Immunoblots of three individual representative specimens from both WT and Rap1 cKO are shown. α-tubulin was immunoblotted as a loading control.

Fig. 6

Rap1 deficiency disrupts cell-ECM adhesion, β1 integrin activation and PAR complex in mouse lens. A. To determine the influence of Rap1 deficiency on cell-ECM interactions, E15.5 and E17.5 ocular specimens derived from the Rap1 cKO embryos were evaluated for changes in activation status of paxillin based on immunofluorescence analysis of phosphorylation (Tyr118) status. In WT specimens, phospho-paxillin (green) is distributed discretely and intensely to the apical junction of epithelial and fiber cells (arrows) very similar to the distribution pattern of ZO-1. Lens capsule (both anterior and posterior) also appears to exhibit some positive staining. In Rap1 cKO (E15.5 and E17.5) mouse ocular specimens, a dramatic reduction in phospho-paxillin staining is noted at the apical junctions of epithelial and fiber cells compared with WT controls. B. Immunofluorescence analysis shows that weak staining for β1-integrin (detected using a monoclonal antibody which recognizes the activated epitope of β1-integrin) was found to be distributed throughout the WT lens, in both the epithelium and fibers (red). These specimens were also labeled for cell nuclei using Hoechst (blue). In contrast, the lens epithelium of Rap1 cKO mouse embryos showed a robust increase in β1-integrin specific staining (arrows). In lens fibers of Rap1 cKO mouse embryos, there appears to be some decrease in the staining for β1-integrin relative to WT controls. Insets depict magnified areas of the central epithelium. Bars in both A and B represent image magnification. LE: Lens epithelium, LF: Lens fibers. C. To determine the status of PAR complex activity in Rap1 cKO mouse lens specimens, the levels of aPKC (both aPKCλ and aPKCς), a well-characterized component of PAR, were examined by immunoblot analysis in E17.5 lenses and compared with respective WT lenses. The levels of both aPKCλ and aPKCς were decreased significantly in the Rap1 cKO lenses (total lysates) compared to WT controls. Immunoblots of three individual representative specimens from both WT and Rap1 cKO are shown. α-tubulin was immunoblotted as a loading control.

Fig. 6

Rap1 deficiency disrupts cell-ECM adhesion, β1 integrin activation and PAR complex in mouse lens. A. To determine the influence of Rap1 deficiency on cell-ECM interactions, E15.5 and E17.5 ocular specimens derived from the Rap1 cKO embryos were evaluated for changes in activation status of paxillin based on immunofluorescence analysis of phosphorylation (Tyr118) status. In WT specimens, phospho-paxillin (green) is distributed discretely and intensely to the apical junction of epithelial and fiber cells (arrows) very similar to the distribution pattern of ZO-1. Lens capsule (both anterior and posterior) also appears to exhibit some positive staining. In Rap1 cKO (E15.5 and E17.5) mouse ocular specimens, a dramatic reduction in phospho-paxillin staining is noted at the apical junctions of epithelial and fiber cells compared with WT controls. B. Immunofluorescence analysis shows that weak staining for β1-integrin (detected using a monoclonal antibody which recognizes the activated epitope of β1-integrin) was found to be distributed throughout the WT lens, in both the epithelium and fibers (red). These specimens were also labeled for cell nuclei using Hoechst (blue). In contrast, the lens epithelium of Rap1 cKO mouse embryos showed a robust increase in β1-integrin specific staining (arrows). In lens fibers of Rap1 cKO mouse embryos, there appears to be some decrease in the staining for β1-integrin relative to WT controls. Insets depict magnified areas of the central epithelium. Bars in both A and B represent image magnification. LE: Lens epithelium, LF: Lens fibers. C. To determine the status of PAR complex activity in Rap1 cKO mouse lens specimens, the levels of aPKC (both aPKCλ and aPKCς), a well-characterized component of PAR, were examined by immunoblot analysis in E17.5 lenses and compared with respective WT lenses. The levels of both aPKCλ and aPKCς were decreased significantly in the Rap1 cKO lenses (total lysates) compared to WT controls. Immunoblots of three individual representative specimens from both WT and Rap1 cKO are shown. α-tubulin was immunoblotted as a loading control.

Fig. 7

Rap1 deficiency induces EMT in mouse lens. A. To determine the effects of Rap1 deficiency on lens epithelial plasticity, E13.5, 15.5 and 17.5 ocular specimens derived from Rap1 cKO mouse embryos were evaluated for changes in αSMA by immunofluorescence analysis using an anti-αSMA monoclonal antibody conjugated with Cy3™. While the respective WT specimens show absence of αSMA in lens epithelium, the Rap1 cKO mouse lens specimens exhibit progressively increased levels of αSMA in the epithelium of E13.5, E15.5 and E17.5 specimens (indicated with arrows; red staining). Both WT and Rap1 cKO mouse specimens exhibit αSMA staining in the presumptive ciliary body (PCB) and iris. B. In addition to αSMA, changes in vimentin were examined in the Rap1 cKO mouse ocular specimens by immunofluorescence analysis. In E17.5 WT specimens, fibers stain intensely for vimentin (green) with very little positive staining in the lens epithelium (see inserts). Rap1 cKO mouse lens specimens in contrast, exhibit a marked increase in vimentin staining in the lens epithelium (arrows), with a concomitant decrease in the fiber cells. Insets show magnified areas of lens epithelium and fibers. Red staining shows propidium iodide (PI)-based nuclei distribution. LE; Lens epithelium, LF: Lens fibers. Bars in A and B represent image magnification.

Fig. 7

Rap1 deficiency induces EMT in mouse lens. A. To determine the effects of Rap1 deficiency on lens epithelial plasticity, E13.5, 15.5 and 17.5 ocular specimens derived from Rap1 cKO mouse embryos were evaluated for changes in αSMA by immunofluorescence analysis using an anti-αSMA monoclonal antibody conjugated with Cy3™. While the respective WT specimens show absence of αSMA in lens epithelium, the Rap1 cKO mouse lens specimens exhibit progressively increased levels of αSMA in the epithelium of E13.5, E15.5 and E17.5 specimens (indicated with arrows; red staining). Both WT and Rap1 cKO mouse specimens exhibit αSMA staining in the presumptive ciliary body (PCB) and iris. B. In addition to αSMA, changes in vimentin were examined in the Rap1 cKO mouse ocular specimens by immunofluorescence analysis. In E17.5 WT specimens, fibers stain intensely for vimentin (green) with very little positive staining in the lens epithelium (see inserts). Rap1 cKO mouse lens specimens in contrast, exhibit a marked increase in vimentin staining in the lens epithelium (arrows), with a concomitant decrease in the fiber cells. Insets show magnified areas of lens epithelium and fibers. Red staining shows propidium iodide (PI)-based nuclei distribution. LE; Lens epithelium, LF: Lens fibers. Bars in A and B represent image magnification.

Fig. 8

Upregulation of E-cadherin suppressing transcription factors and the mesenchymal metabolic marker dihydropyrimidine dehydrogenase (DPYD) in Rap1 cKO mouse lens. A. Lenses derived from the E17.5 Rap1 cKO mice were evaluated for changes in the expression levels of E-cadherin suppressing transcription factors including Snai1, Slug, Zeb1 and Zeb2, and DPYD, based on qRT-PCR analysis in comparison with WT controls. Rap1 cKO embryonic mouse lenses showed a significant increase in the expression of Snai1, Slug, Zeb2 and DPYD compared with WT controls. Values represent mean ± SEM. n=3 (pooled specimens). *P< 0.05. B. To further confirm the changes observed in the expression of E-cadherin suppressing transcription factors, changes in Slug protein distribution was evaluated by immunofluorescence analysis in the E15.5 and E17.5 ocular specimens derived from the Rap1 cKO mouse embryos in comparison with WT controls. Slug protein levels markedly and progressively increased in the epithelium (arrows, green staining) of E15.5 and E17.5 Rap1 cKO specimens. There was also some increase of Slug protein in the fiber cells of Rap1 cKO mouse lens compared with WT controls. Insets show a magnified area of lens epithelium. LE: Lens epithelium; LF: Lens fibers. Bar represents image magnification.

Fig. 9

Rap1 deficiency impairs lens epithelial proliferation and survival. A. To determine the effects of Rap1 deficiency on lens epithelial proliferation and cell cycle progression, in vivo BrdU labeling was performed in conjunction with immunofluorescence analysis using anti-BrdU antibody as described in Methods section. Counting of BrdU positive cells showed a significant decrease (>50%) in the lens central epithelium of Rap1 cKO embryos relative to their WT controls. In contrast to the lens central epithelium, no BrdU-positive cells are noted in the transitional zone of the WT epithelium, where cells exit from the cell cycle and start differentiating into secondary fiber cells (a region just below the line drawn and indicated with arrows). Rap1 cKO mouse lens specimens on the other hand, exhibited a significant increase in the number of BrdU positive cells in the transitional zone epithelium (arrows) indicating defective cell cycle exit in the deficiency of Rap1. B. To test the effects of Rap1 deficiency on lens epithelial and fiber cell survival, we examined for changes in apoptotic cells by TUNEL positive staining of ocular specimens in E15.5 and E17.5 mouse embryos. The Rap1 cKO mouse specimens showed a progressively and significantly increase of apoptotic cells (TUNEL positive cells- in green/yellow) in the lens epithelium and fiber mass compared with WT controls. Values (mean ± SEM) were based on n=6. *P<0.05. Bars show image magnification. LE: Lens epithelium, LF: Lens fibers.

Fig. 9

Rap1 deficiency impairs lens epithelial proliferation and survival. A. To determine the effects of Rap1 deficiency on lens epithelial proliferation and cell cycle progression, in vivo BrdU labeling was performed in conjunction with immunofluorescence analysis using anti-BrdU antibody as described in Methods section. Counting of BrdU positive cells showed a significant decrease (>50%) in the lens central epithelium of Rap1 cKO embryos relative to their WT controls. In contrast to the lens central epithelium, no BrdU-positive cells are noted in the transitional zone of the WT epithelium, where cells exit from the cell cycle and start differentiating into secondary fiber cells (a region just below the line drawn and indicated with arrows). Rap1 cKO mouse lens specimens on the other hand, exhibited a significant increase in the number of BrdU positive cells in the transitional zone epithelium (arrows) indicating defective cell cycle exit in the deficiency of Rap1. B. To test the effects of Rap1 deficiency on lens epithelial and fiber cell survival, we examined for changes in apoptotic cells by TUNEL positive staining of ocular specimens in E15.5 and E17.5 mouse embryos. The Rap1 cKO mouse specimens showed a progressively and significantly increase of apoptotic cells (TUNEL positive cells- in green/yellow) in the lens epithelium and fiber mass compared with WT controls. Values (mean ± SEM) were based on n=6. *P<0.05. Bars show image magnification. LE: Lens epithelium, LF: Lens fibers.

Fig. 10

Lens differentiation is normal under Rap1 deficiency. To test whether Rap1 deficiency influences lens differentiation, E17.5 ocular specimens derived from Rap1 cKO mouse embryos along with their respective WT specimens were examined by immunofluorescence analysis for expression profile of lens fiber specific markers including aquaporin-0 (green) and γ-crystallin (red). As shown in the figure, the distribution profile of various fiber cell differentiation markers was found to be comparable between the Rap1 cKO and WT specimens, indicating that Rap1 deficiency does not impact normal lens differentiation. In some Rap1 cKO specimens, there was aquaporin-0 positive staining at the apical surface of lens epithelium as indicated by the arrows. Bar represents image magnification. LE: Lens epithelium, LF: Lens fibers.