Using a tissue-specific conditional gene deletion approach, we demonstrate an essential role for Rac1 GTPase during lens morphogenesis and maturation. We show that Rac1 GTPase regulates crucial aspects of lens morphogenesis and cytoarchitecture including epithelial cell survival, fiber cell elongation, migration and adhesion. This study also reveals the critical role for Rac1 GTPase in establishment of lens shape, suture formation, capsule basement membrane integrity, and fiber cell hexagonal geometry, which are important determinants of lens function.
Rac1 is expressed uniformly throughout the mouse lens including in the epithelium and fiber cells. Lack of Rac1 expression in the developing lens resulted in abnormal lens shape and reduction in size ( & ), with the prominent phenotype being manifested only when Rac1 expression is deleted from both lens epithelium and fibers, as evidenced by the lack of a lens phenotype in MLR39/Rac1 cKO mice in which Rac1 expression is deleted only in the fiber cells (). This observation demonstrates the importance of the epithelium and its interaction with fiber cells in establishment of lens shape and growth. Further, this phenotype appears to be a direct result of shortening of the epithelial sheet, increased apoptosis, and the abnormal fiber cell migration and orientation noted in the Rac1 deficient lenses ( & ). While the molecular basis for the shortened epithelial sheet in Rac1 deleted lenses however, is not entirely clear at present, we speculate that the increase in epithelial cell apoptosis () which is consistent with the known anti-apoptotic activity of Rac (
Murga et al., 2002;
Nishida et al., 1999) could be partly responsible for this phenotype. Additionally, the lens epithelium in Rac1 null mice exhibited abnormal cell cycle progression based on
in vivo BrdU incorporation profile (
Fig. S1) in association with defective E-cadherin-based cell-cell interactions, suggesting compromised cell survival and proliferation in the absence of Rac1 GTPase in lens. However, it is important to point out that the MLR39 Cre transgenic mice start expressing the Cre protein from E12.5, unlike the Le-Cre (at E8.75) and the MLR10 (at E10.5) mice. Further, the promoters driving Cre expression are different in the three Cre transgenic mice used in this study (
Ashery-Padan et al., 2000;
Zhao et al., 2004). Therefore, the possibility that lack of phenotype in the MLR39/Rac1 mutant mice is partly attributable to the differences in amount and onset of Cre expression in fibers cells cannot be completely ruled out. It is possible that manifestation of the abnormal lens shape and fiber cell orientation phenotype result from the absence of Rac1 GTPase expression during early embryonic lens development, perhaps before E12.5.
Rac1 GTPase has been demonstrated to control directional cell migration in various cell types via regulating cell adhesive interactions and the formation of cell membrane protrusions and lamellipodia at the leading edges, by acting downstream of growth factor receptors, integrins, PI3 kinase and its lipid products (
Burridge and Wennerberg, 2004;
Raftopoulou and Hall, 2004;
Ridley, 2001b;
Ridley et al., 2003). However, the role of Rac in cell migration in the context of organogenesis is obscure (
Heasman and Ridley, 2008;
Wang and Zheng, 2007), and this study provides direct evidence for the involvement of Rac1 in fiber cell migration and organization in the intact lens, as shown in . The mouse lens expresses both Rac1 and Rac2 (
Rao et al., 2004) and it is evident from the lens phenotype and other lens changes reported in this study that Rac1 plays a non-redundant role in lens. Rac1 GTPase regulates actin polymerization during lamellipodial extension and membrane ruffle formation by activating the actin–nucleating ARP2/3 complex through WASP-family verprolin-homologous proteins (WAVE) (
Eden et al., 2002;
Ridley, 2011;
Stradal et al., 2004;
Takenawa and Suetsugu, 2007;
Yamazaki et al., 2003). Importantly, WAVE-2 has been demonstrated to regulate ARP2/3 mediated actin assembly and branching in a wide range of systems, acting in concert with Sra1, Nap1, Abi and HSPC300, which exist as a complex with WAVE-2 (
Eden et al., 2002;
Ridley, 2011;
Stradal et al., 2004;
Takenawa and Suetsugu, 2007). Rac1 GTPase interacts with and activates the WAVE-2 complex through the Sra1 subunit (
Kunda et al., 2003;
Pollitt and Insall, 2009;
Stradal et al., 2004;
Takenawa and Suetsugu, 2007). WAVE-2 and Abi-2 were found to be abundantly expressed in lens fibers relative to the epithelium and other ocular tissues based on distribution analysis (). Moreover, distribution analysis of actin filaments, WAVE-2 and its associated proteins Abi-2 and Nap1 demonstrated reduced actin filament staining and downregulation of WAVE-2, Abi-2 and Nap1, in the lens epithelium and fiber cells of Rac1 cKO mice, implying impairment in actin nucleation possibly via the ARP2/3 complex. These observations, taken together with the earlier report on the lens phenotype of defective fiber cell migration and cell-cell interactions in the Abi-2 null mice (
Grove et al., 2004), confirm the importance of Rac1 GTPase regulated WAVE-2 complex activity in controlling fiber cell migration. Abi-1 and Abi-2 both have been found to exist as part of the WAVE protein complex and to regulate actin dynamics (
Eden et al., 2002;
Soderling et al., 2002).
Stimulation of PAK kinase and LIM kinase by Rac1 GTPase results in the phosphorylation-dependent inactivation of cofilin (
Arber et al., 1998;
Huang et al., 2006;
Yang et al., 1998), and leads to decreased actin filament severing and cell movement under normal circumstances. Intriguingly, deletion of Rac1 expression did not result in the expected decrease in PAK and LIM kinase-mediated cofilin phosphorylation, with Rac1 KO lenses exhibiting an increase in cofilin phosphorylation (). This observation suggests the involvement of a Rac1-independent pathway in the inhibition of cofilin activity in the Rac1 KO lenses. Such a pathway is likely upregulated as an adaptive response to the Rac1 deficiency-induced alterations in actin dynamics and cell mobility in the Rac1 KO lenses. We have not analyzed the status of Slingshot phosphatase activity, which is known to dephosphorylate and control cofilin activity (
Huang et al., 2006), in Rac1 cKO lenses.
It is important to note that, in addition to involvement of Rac GTPase downstream signaling pathways in fiber cell actin dynamics and migration discussed above, the fact that Rac1 deficient lenses and Sfrp2 (secreted WNT antagonist) overexpressing lenses (
Chen et al., 2008) appear to share a striking number of abnormalities including changes in shape, shortened epithelial sheet length, and abnormal fiber cell migration and cytoskeletal organization, further suggests that Rac1 is also an important component of the WNT/PCP signaling pathway in the lens.
Another prominent phenotype in the Rac1 deficient lenses was disorganization of fiber cells, defective suture formation and disruption of the prismoid morphology of fiber cells. Lens suture formation depends on fiber cell migration, spreading and turnover of cell-cell adhesive interactions at the interface of apical fiber tips and epithelium, the basal tips of fiber cells and basement membrane of capsule (
Kuszak, 2004), and between the lateral membranes of fiber cells. As discussed above, in addition to defective fiber cell migration, N-cadherin/β-catenin and Rap1/Nectin mediated cell-cell interactions were disrupted in the Rac1 deficient lenses. E-cadherin-based cell-cell junctions regulate Rac activity (
Fukuyama et al., 2006;
Kawakatsu et al., 2002;
Sander et al., 1998;
Yap and Kovacs, 2003) and activated Rac in turn regulates actin cytoskeletal interactions with the adherens junctions (
Baum and Georgiou, 2011;
Fukata and Kaibuchi, 2001), which are important for the stabilization of the adherens junctions (
Baum and Georgiou, 2011;
Fukata and Kaibuchi, 2001;
Halbleib and Nelson, 2006;
Hordijk et al., 1997;
Takai et al., 2008). Similarly, Rap1/Nectin, which act upstream of Rac GTPase, collectively regulate adherens junctions in various other cell types (
Kooistra et al., 2007;
Takai et al., 2008). The disruption of fiber cell organization and hexagonal shape in Rac1 deficient lenses as assessed by TEM () therefore implies that the deficits in N-cadherin/β-catenin and Rap1/Nectin mediated cell-cell interactions noted in these lenses likely compromise lens suture formation and cytoarchitecture. It is not clear however, why and how the levels of Rap1 and Nectin-1 are decreased in Rac1 KO lenses. It is unlikely that these changes are secondary to the primary insult since we not only used intact lenses for the analyses but also noted that the Rac1 deficient lenses derived from the MLR10-Cre mice at P1 did not exhibit the typical secondary changes such as extensive vacuole accumulation and degeneration of fiber cells, as noted for many types of cataractous lenses (
Chen et al., 2008;
Cooper et al., 2008;
Simirskii et al., 2007).
The lens capsule is a basement membrane that surrounds and serves to control the shape, elastic and mechanical properties of the ocular lens. Additionally, the ECM components of the capsule play a crucial role in lens epithelial cell proliferation, differentiation, migration and cell adhesion via engagement of integrin receptors (
Danysh and Duncan, 2009;
Menko S., 2004). Interestingly, the posterior capsule was frequently ruptured very early on (embryonic and neonatal) in the Le-Cre/Rac1 and MLR10/Rac1 cKOs, a phenotype which was found to be associated with decreased content of specific ECM proteins (). ECM components are produced and secreted by the lens epithelium and fiber cells (
Danysh and Duncan, 2009). It is possible that the absence of Rac1 GTPase activity results in impaired transcriptional activity and MAP kinase activation, (
Coso et al., 1995;
Etienne-Manneville and Hall, 2002;
Hill et al., 1995;
Minden et al., 1995) which in turn may be associated with, or partly responsible for, the altered basement membrane composition in the Rac1 deficient lenses. Indeed Rac GTPase has been shown to regulate the activity of several different matrix metalloproteases (
Engers et al., 2001;
Kheradmand et al., 1998). Therefore, the absence of Rac1 in lens appears to have a direct influence on ECM turnover. Importantly, expression of various integrins is downregulated at the level of protein in these Rac1 deficient lenses. Integrins regulate lens epithelial proliferation, survival and differentiation (
Walker and Menko, 2009) and mediate cell adhesive interactions through fiber cell basal membrane complex and through interactions with epithelial cells (
Bassnett et al., 1999;
Simirskii et al., 2007;
Walker and Menko, 2009). ECM and integrins regulate Rac GTPase activity and cell adhesive interactions (
del Pozo et al., 2000;
Hotchin and Hall, 1995;
Price et al., 1998) and conversely, Rac1 and Rap1 control integrin activation and cell adhesion in certain cell types (
D'Souza-Schorey et al., 1998;
Vielkind et al., 2005). Therefore, the changes in the basement membrane protein organization and composition in the Rac1 deficient mice very likely play a key role in the compromised cell survival and defective fiber cell migration and suture formation noted in the Rac1 cKO lenses. Interestingly, although the absolute requirement of Rac1 in cell migration has remained somewhat questionable (
Wells et al., 2004), Rac1 deficient fiber cells in intact lenses exhibit defective cell membrane/cytoplasmic extensions required for cell adhesion and migration, similar to the effects noted in Schwann cells in the peripheral nerves (
Benninger et al., 2007).
In conclusion, this study uncovers a crucial role for Rac1 GTPase activity in lens morphogenesis and architecture via regulation of epithelial cell survival, cell cycle progression, fiber cell migration, cell adhesion, and ECM synthesis. Importantly, Rac1 deficiency significantly impacts lens growth, shape, capsule thickness, cell survival and cytoarchitecture.
Highlights- Conditional Rac1 GTPase deficiency affects lens shape and cytoarchitecture.
- Rac1 GTPase deficiency impairs lens sure formation, fiber cell migration and cell survival.
- Absence of Rac1 GTPase impairs lens capsule basement membrane integrity.
- Rac1 GTPase activity is essential for lens morphogenesis and structural integrity.
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