ß1-integrin controls cell fate specification in early lens development.

Integrins are heterodimeric cell surface molecules that mediate cell-extracellular matrix (ECM) adhesion, ECM assembly, and regulation of both ECM and growth factor induced signaling. However, the developmental context of these diverse functions is not clear. Loss of ß1-integrin from the lens vesicle (mouse E10.5) results in abnormal exit of anterior lens epithelial cells (LECs) from the cell cycle and their aberrant elongation toward the presumptive cornea by E12.5. These cells lose expression of LEC markers and initiate expression of the Maf (also known as c-Maf) and Prox1 transcription factors as well as other lens fiber cell markers. ß1-integrin null LECs also upregulate the ERK, AKT and Smad1/5/8 phosphorylation indicative of BMP and FGF signaling. By E14.5, ß1-integrin null lenses have undergone a complete conversion of all lens epithelial cells into fiber cells. These data suggest that shortly after lens vesicle closure, ß1-integrin blocks inappropriate differentiation of the lens epithelium into fibers, potentially by inhibiting BMP and/or FGF receptor activation. Thus, ß1-integrin has an important role in fine-tuning the response of the early lens to the gradient of growth factors that regulate lens fiber cell differentiation.

Beta-1 integrin is important for the structural maintenance and homeostasis of differentiating fiber cells.

ß1-Integrin is a heterodimeric transmembrane protein that has roles in both cell-extra-cellular matrix and cell-cell interactions. Conditional deletion of ß1-integrin from all lens cells during embryonic development results in profound lens defects, however, it is less clear whether this reflects functions in the lens epithelium alone or whether this protein plays a role in lens fibers. Thus, a conditional approach was used to delete ß1-integrin solely from the lens fiber cells. This deletion resulted in two distinct phenotypes with some lenses exhibiting cataracts while others were clear, albeit with refractive defects. Analysis of "clear" conditional knockout lenses revealed that they had profound defects in fiber cell morphology associated with the loss of the F-actin network. Physiological measurements found that the lens fiber cells had a twofold increase in gap junctional coupling, perhaps due to differential localization of connexins 46 and 50, as well as increased water permeability. This would presumably facilitate transport of ions and nutrients through the lens, and may partially explain how lenses with profound structural abnormalities can maintain transparency. In summary, ß1-integrin plays a role in maintaining the cellular morphology and homeostasis of the lens fiber cells.

CD44 expression is developmentally regulated in the mouse lens and increases in the lens epithelium after injury.

Hyaluronan is an oligosaccharide found in the pericellular matrix of numerous cell types and hyaluronan-induced signaling is known to facilitate fibrosis and cancer progression in some tissues. Hyaluronan is also commonly instilled into the eye during cataract surgery to protect the corneal endothelium from damage. Despite this, little is known about the distribution of hyaluronan or its receptors in the normal ocular lens. In this study, hyaluronan was found throughout the mouse lens, with apparently higher concentrations in the lens epithelium. CD44, a major cellular receptor for hyaluronan, is expressed predominately in mouse secondary lens fiber cells born from late embryogenesis into adulthood. Surgical removal of lens fiber cells from adult mice resulted in a robust upregulation of CD44 protein, which preceded the upregulation of alpha-smooth muscle actin expression typically used as a marker of epithelial-mesenchyma transition in this model of lens epithelial cell fibrosis. Mice lacking the CD44 gene had morphologically normal lenses with a response to lens fiber cell removal similar to wildtype, although they exhibited an increase in cell-associated hyaluronan. Overall, these data suggest that lens cells have a hyaluronan-containing pericellular matrix whose structure is partially regulated by CD44. Further, these data indicate that CD44 upregulation in the lens epithelium may be an earlier marker of lens injury responses in the mouse lens than the upregulation of alpha-smooth muscle actin.

Conditional deletion of beta1-integrin from the developing lens leads to loss of the lens epithelial phenotype.

Beta1-integrins are cell surface receptors that participate in sensing the cell's external environment. We used the Cre-lox system to delete beta1-integrin in all lens cells as the lens vesicle transitions into the lens. Adult mice lacking beta1-integrin in the lens are microphthalmic due to apoptosis of the lens epithelium and neonatal disintegration of the lens fibers. The first morphological alterations in beta1-integrin null lenses are seen at 16.5 dpc when the epithelium becomes disorganized and begins to upregulate the fiber cell markers beta- and gamma-crystallins, the transcription factors cMaf and Prox1 and downregulate Pax6 levels demonstrating that beta1-integrin is essential to maintain the lens epithelial phenotype. Furthermore, beta1-integrin null lens epithelial cells upregulate the expression of alpha-smooth muscle actin and nuclear Smad4 and downregulate Smad6 suggesting that beta1-integrin may brake TGFbeta family signaling leading to epithelial-mesenchymal transitions in the lens. In contrast, beta1-integrin null lens epithelial cells show increased E-cadherin immunoreactivity which supports the proposed role of beta1-integrins in mediating complete EMT in response to TGFbeta family members. Thus, beta1-integrin is required to maintain the lens epithelial phenotype and block inappropriate activation of some aspects of the lens fiber cell differentiation program.

Inbred FVB/N mice are mutant at the cp49/Bfsp2 locus and lack beaded filament proteins in the lens.

PURPOSE: FVB/N is considered an ideal inbred mouse strain for transgenic mouse production because of the ease of pronuclear microinjection and its overall fecundity. It is well established that vertebrate lens fiber cells normally express a modified intermediate filament network consisting of the proteins filensin and CP49, and it was recently reported that the mouse strain 129 harbors mutations in CP49 that have the potential to confound the interpretation of gene knockout studies of the lens. The purpose of this study was to evaluate the status of the CP49/Bfsp2 gene in the FVB/N strain. METHODS: PCR analysis of genomic DNA was used to evaluate the status of the CP49 gene in FVB/N mice procured from the four major US distributors of these animals--Harlan Laboratories, Taconic Farms, Jackson Laboratory, and the NIH/NCI/DCT production facility run by Charles River Laboratories. The structure of the CP49 transcript was evaluated by RT-PCR, and the presence of CP49 protein in the lens was evaluated by immunofluorescence. RESULTS: FVB/N mice obtained from all four US distributors were shown to harbor a 6-kb deletion of the CP49 gene identical with that previously reported in mouse strain 129; C57BL/6 mice did not have this modification. Immunofluorescence demonstrated that FVB/N mice do not have detectable CP49 or filensin protein in the lens, whereas C57BL/6 mice have the expected protein distribution. CONCLUSIONS: In humans, mutations in the CP49/BFSP2 gene have been linked to familial, congenital cataract, demonstrating an important role of this gene in lens transparency. The demonstration that FVB/N mice lack CP49 protein in the lens suggests that it may be necessary to reevaluate the mechanisms underlying lens phenotypes obtained as a result of transgenic manipulation of this strain.

Expression of tissue plasminogen activator during eye development.

Tissue plasminogen activator (tPA) is a serine protease responsible for the activation of plasminogen to plasmin as well as extracellular matrix remodeling. While tPA is used clinically to treat some retinal disorders and it is expressed at low levels in the adult eye, its expression pattern during eye development had never been determined. tPA protein is broadly dispersed in the lens placode and optic vesicle of the mouse eye and it becomes highly localized to the apical surfaces of both the lens pit and the optic cup as they invaginate. In the lens, tPA remains at the apical tips of both lens epithelial and fiber cells from the lens vesicle stage until birth in the mouse, when it begins to downregulate to barely detectable levels in adults. In humans, tPA is found in a similar pattern in the lens vesicle and early lens, however, appreciable protein is also detected in the cytoplasm of lens epithelial cells until adulthood. In the retina, tPA is found at the apical interface between the developing retinal pigmented epithelium and neural retina, then begins to downregulate once the photoreceptors have differentiated. In conclusion, tPA protein is found in a different pattern in embryonic versus adult eyes and may be involved in remodeling of the extracellular environment during eye development.