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Integrin ?5/fibronectin1 and focal adhesion kinase are required for lens fiber morphogenesis in zebrafish.

ABSTRACT: Lens fiber formation and morphogenesis requires a precise orchestration of cell- extracellular matrix (ECM) and cell-cell adhesive changes in order for a lens epithelial cell to adopt a lens fiber fate, morphology, and migratory ability. The cell-ECM interactions that mediate these processes are largely unknown, and here we demonstrate that fibronectin1 (Fn1), an ECM component, and integrin ?5, its cellular binding partner, are required in the zebrafish lens for fiber morphogenesis. Mutations compromising either of these proteins lead to cataracts, characterized by defects in fiber adhesion, elongation, and packing. Loss of integrin ?5/Fn1 does not affect the fate or viability of lens epithelial cells, nor does it affect the expression of differentiation markers expressed in lens fibers, although nucleus degradation is compromised. Analysis of the intracellular mediators of integrin ?5/Fn1 activity focal adhesion kinase (FAK) and integrin-linked kinase (ILK) reveals that FAK, but not ILK, is also required for lens fiber morphogenesis. These results support a model in which lens fiber cells use integrin ?5 to migrate along a Fn-containing substrate on the apical side of the lens epithelium and on the posterior lens capsule, likely activating an intracellular signaling cascade mediated by FAK in order to orchestrate the cytoskeletal changes in lens fibers that facilitate elongation, migration, and compaction.

SUBMITTER: Hayes JM

PROVIDER: S-EPMC3521681 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Integrin α5/fibronectin1 and focal adhesion kinase are required for lens fiber morphogenesis in zebrafish.

Hayes Julie M JM Hartsock Andrea A Clark Brian S BS Napier Hugh R L HR Link Brian A BA Gross Jeffrey M JM

Molecular biology of the cell 20121024 24

Lens fiber formation and morphogenesis requires a precise orchestration of cell- extracellular matrix (ECM) and cell-cell adhesive changes in order for a lens epithelial cell to adopt a lens fiber fate, morphology, and migratory ability. The cell-ECM interactions that mediate these processes are largely unknown, and here we demonstrate that fibronectin1 (Fn1), an ECM component, and integrin α5, its cellular binding partner, are required in the zebrafish lens for fiber morphogenesis. Mutations co ...[more]

PMID: 23097490

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Project description:PURPOSE: Regulation of lens development involves an intricate interplay between growth factor (e.g. FGF and TGFbeta) and extracellular matrix (ECM) signaling pathways. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that plays key roles in transmitting ECM signals by integrins. In this study, we delineated patterns of FAK expression and tyrosine phosphorylation (Y397) in the developing lens and investigated its regulation by FGF2. We also examined FAK expression and activation during disrupted fiber differentiation in mice expressing a dominant-negative TGFbeta receptor. METHODS: FAK expression and activation (phosphorylation on Y397) was studied in embryonic and postnatal rodent lenses by in situ hybridization, immunofluorescence, and western blotting. Rat lens explants were used to investigate the effects of FGF2 on FAK expression and activation. Immunofluorescence and western blotting were used to examine FAK expression and phosphorylation in transgenic mice that express a dominant-negative TGFbeta receptor. RESULTS: FAK is widely expressed and phosphorylated during embryonic stages of lens morphogenesis and differentiation. However, in postnatal lenses its expression and activation becomes restricted to the posterior germinative zone and the transitional zone at the lens equator. While both NH2- and COOH-terminal antibodies revealed cytoplasmic and membrane-associated staining in lens cells, the NH2-terminal antibody also showed FAK was present in fiber cell nuclei. In vitro, FAK expression and phosphorylation on Y397 were increased by concentrations of FGF2 that initiate lens epithelial cell migration (10 ng/ml) and differentiation (50 ng/ml) but not proliferation (5 ng/ml). Moreover, reactivity for Y397 phosphorylated FAK is prominent in the nuclei of differentiating fibers both in vivo and in vitro. Disruption of TGFbeta-like signals by ectopic expression of a dominant-negative TGFbeta receptor (TbetaRII(D/N)) results in abnormal lens fiber differentiation in transgenic mice. While FAK expression is initiated normally in the posterior germinative zone of TbetaRII(D/N) transgenic lenses, as fiber differentiation proceeds, FAK becomes localized to a perinuclear compartment, decreases its association with the cytoskeleton and is poorly phosphorylated on Y(397). CONCLUSIONS: FAK is widely expressed and activated during early lens morphogenesis. During secondary lens fiber differentiation, FAK is expressed and phosphorylated on Y397 as epithelial cells exit the cell cycle, initiate migration at the equator, and undergo differentiation in the transitional zone. During terminal fiber differentiation an NH2-terminal fragment of FAK, including Y397, is translocated to the nucleus. The expression, activation, and nuclear localization of FAK are regulated, at least partly, by FGF2. FAK activity and subcellular localization are also modulated by TGFbeta-like signals. In fiber cells of TbetaRII(D/N) transgenic lenses, FAK is abnormally retained in a perinuclear compartment, loses its association with the cytoskeleton, and is poorly phosphorylated. These data suggest that integrin signaling via FAK plays important roles during lens differentiation, mediated by FGFs and TGFbeta-superfamily signals.

Dataset Information

Integrin ?5/fibronectin1 and focal adhesion kinase are required for lens fiber morphogenesis in zebrafish.

Publications

Integrin α5/fibronectin1 and focal adhesion kinase are required for lens fiber morphogenesis in zebrafish.

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