alpha6 Integrin is regulated with lens cell differentiation by linkage to the cytoskeleton and isoform switching.

The developing chicken embryo lens provides a unique model for examining the relationship between alpha6 integrin expression and cell differentiation, since multiple stages of differentiation are expressed concurrently at one stage of development. We demonstrate that alpha6 integrin is likely to mediate the inductive effects of laminin on lens differentiation as well as to function in a matrix-independent manner along the cell-cell interfaces of the differentiating cortical lens fiber cells. Both alpha6 isoform expression and its linkage to the cytoskeleton were regulated in a differentiation-specific manner. The association of alpha6 integrin with the Triton-insoluble cytoskeleton increased as the lens cells differentiated, reaching its highest levels in the cortical fiber region where the lens fiber cells are formed. In this region of the lens alpha6 integrin was uniquely localized along the cell-cell borders of the differentiating fiber cells, similar to beta1. alpha6beta4, the primary transmembrane protein of hemidesmosomes, is also expressed in the lens, but in the absence of hemidesmosomes. Differential expression of alpha6A and alpha6B isoforms with lens cell differentiation was seen at both the mRNA and the protein levels. RT-PCR studies demonstrated that alpha6B was the predominant isoform expressed both early in development, embryonic day 4, and in the epithelial regions of the day 10 embryonic lens. Isoform switching, with alpha6A now the predominant isoform, occurred in the fiber cell zones. Immunoprecipitation studies showed that alpha6B, which is characteristic of undifferentiated cells, was expressed by the lens epithelial cells but was dramatically reduced in the lens fiber zones. Expression of alpha6B began to drop as the cells initiated their differentiation and then dropped precipitously in the cortical fiber zone. In contrast, expression of the alpha6A isoform remained high until the cells became terminally differentiated. alpha6A was the predominant isoform expressed in the cortical fiber region. The down-regulation of alpha6B relative to alpha6A provides a developmental switch in the process of lens fiber cell differentiation.     

Expression of Frizzleds and secreted frizzled-related proteins (Sfrps) during mammalian lens development

Recent studies indicate a role for Wnt signaling in regulating lens cell differentiation (Stump et al., 2003). Here we investigated expression patterns of Wnt receptors, the Frizzleds (Fzs) and the Wnt signaling regulators, the secreted frizzled-related proteins (Sfrps), during rodent lens development. RT-PCR showed that Fz receptors, Fz1-Fz8 are expressed in lens. In situ hybridization showed that all the Fz genes examined have similar expression patterns. Fzs are expressed throughout the early lens primordium. At embryonic day 14.5 (E14.5), Fz gene expression is predominantly localized to the epithelium and elongating cells at the lens equator. Fz expression is absent from lens fibers. This pattern of Fz gene expression continues throughout early postnatal development. Immunolocalization studies showed that Fz protein distribution closely follows that of the mRNAs. In addition, epithelial cells in FGF-treated explants show strongest Fz reactivity in cellular protrusions as they migrate and elongate. Sfrp1- Sfrp5 are expressed and all, except Sfrp2, have similar patterns of expression to each other and to the Fzs during lens development. Sfrp2 is strongly expressed in all lens pit cells but becomes restricted to the presumptive epithelial cells of the lens vesicle. By E14.5, Sfrp2 is only present in a few cells above the lens equator. Sfrp2 is not detected in the lens at E18.5 or at later stages. This study shows that multiple Fz and Sfrp genes are expressed during lens morphogenesis and differentiation. This is consistent with a role for Wnt-Fz signaling during both embryonic and postnatal lens development.     

Pax-6 and Prox 1 expression during lens regeneration from Cynops iris and Xenopus cornea: evidence for a genetic program common to embryonic lens development

Lens regeneration from non-lens ocular tissues has been well documented in amphibians, from the dorsal iris in the newt and from the outer cornea in Xenopus. To understand the early molecular events which govern lens regeneration, we examined the expression of two early marker genes of normal lens development, Pax-6 and Prox 1. In both Cynops (newt) iris and Xenopus cornea, Pax-6 is expressed soon after lentectomy in a region broader than that giving rise to the regenerating lens, indicative of an important role for Pax-6 in determination of the regeneration potential. Then Prox 1 expression begins within the Pax-6-expressing tissue, and these Prox 1-expressing cells give rise to the regenerating lens. This sequence of events also takes place in the lens placode of the embryo, indicating that the presence of the same genetic program operates in both embryonic lens development and lens regeneration, at least partly. In the Cynops iris, Pax-6 expression occurs initially in the entire marginal region of the iris after lentectomy but then becomes restricted to the dorsal region. Further studies are expected to elucidate the mechanism of this long-standing problem of the dorsal-restriction of lens regeneration from the newt iris.     

The pattern of DNA methylation in the delta-crystallin genes in transdifferentiating neural retina cultures

Terminally differentiated lens fibre cells are formed in the vertebrate lens throughout life. Lens fibre cells may also be obtained by an in vitro process termed transdifferentiation, from certain tissues of different developmental origin from lens, such as embryo neural retina. delta-Crystallin is the major protein in the chick embryo lens fibre cells, and also in transdifferentiated lens cells obtained from cultured embryonic neural retina. Lens crystallin proteins and mRNA are present at low levels in the intact embryonic neural retina but are no longer detectable in the early stages of neural retina cell culture. However, levels rise steeply in the later stages and crystallins become the major products in terminally transdifferentiating neural retina cultures. We have used this system to test the hypothesis that the patterns of DNA methylation in particular genes are correlated with gene expression. A number of developmentally regulated genes have been found to be undermethylated in tissues where they are expressed, and methylated in tissues where they are not. However this correspondence does not always hold true. Eight-day-old embryonic neural retina was cultured for the period of time during which crystallin gene expression increases 100-fold. DNA methylation in the delta-crystallin gene region was analysed at several stages of cell culture by using the restriction endonucleases HpaII and MspI which cleave at the sequence CCGG. The former enzyme cannot cleave internally methylated cytosine (CmCGG) while the latter cannot cleave externally methylated cytosine (mCCGG). We detect no change in the methylation of CCGG sites within the delta-crystallin gene regions during transdifferentiation. Since dramatic changes in delta-crystallin gene expression occur during this process we conclude that large scale alterations in the pattern of DNA methylation are not a necessary accompaniment to changes in gene activity.     

Xenopus gamma-crystallin gene expression: evidence that the gamma-crystallin gene family is transcribed in lens and nonlens tissues.

Crystallins, the major gene products of the lens, accumulate to high levels during the differentiation of the vertebrate lens. Although crystallins were traditionally thought to be lens specific, it has recently been shown that some are also expressed at very low levels in nonlens tissues. We have examined the embryonic expression pattern of gamma-crystallins, the most abundant crystallins of the embryonic lens in Xenopus laevis. The expression profile of five Xenopus gamma-crystallin genes mirrors the pattern of lens differentiation in X. laevis, exhibiting on average a 100-fold increase between tailbud and tadpole stages. Four of these genes are also ubiquitously expressed outside the lens at a very low level, the first demonstration of nonlens expression of any gamma-crystallin gene; expression of the remaining gene was not detected outside the head region, thus suggesting that there may be two classes of gamma-crystallin genes in X. laevis. Predictions regarding control mechanisms responsible for this dual mode of expression are discussed. This study raises the question of whether any crystallin, on stringent examination, will be found exclusively in the lens.     

delta- and beta-Crystallin mRNA levels in the embryonic and posthatched chicken lens: temporal and spatial changes during development

The levels of delta- and beta-crystallin mRNAs were examined by cDNA hybridization in the embryonic and posthatched chicken eye lens. Four different cloned beta-crystallin cDNAs were used, allowing discrimination among different members of the beta-crystallin family. Each crystallin mRNA displayed a characteristic temporal and spatial pattern in the developing lens. delta-Crystallin mRNA accumulated rapidly during early embryonic development; by contrast, the beta-crystallin mRNAs began to accumulate rapidly near the end of embryogenesis. Both delta- and beta-crystallin mRNAs increased in the lens for the first month after hatching and began to decrease 3 months after hatching. The levels of the delta- and the different beta-crystallin mRNAs were also differentially regulated in cultured embryonic lens epithelia. The most fiber cell specific crystallin gene product in the differentiating lens was the beta 35 mRNA. These experiments provide a quantitative basis for exploring the differential expression of the delta- and beta-crystallin gene families in the chicken lens.     

Crystallins in Retinal Ganglion Cell Survival and Regeneration

Crystallins are heterogeneous proteins classified into alpha, beta, and gamma families. Although crystallins were first identified as the major structural components of the ocular lens with a principal function to maintain lens transparency, further studies have demonstrated the expression of these proteins in a wide variety of tissues and cell types. Alpha crystallins (alpha A and alpha B) share significant homology with small heat shock proteins and have chaperone-like properties, including the ability to bind and prevent the precipitation of denatured proteins and to increase cellular resistance to stress-induced apoptosis. Stress-induced upregulation of crystallin expression is a commonly observed phenomenon and viewed as a cellular response mechanism against environmental and metabolic insults. However, several studies reported downregulation of crystallin gene expression in various models of glaucomatous nerodegeneration suggesting that that the decreased levels of crystallins may affect the survival properties of retinal ganglion cells (RGCs) and thus, be associated with their degeneration. This hypothesis was corroborated by increased survival of axotomized RGCs in retinas overexpressing alpha A or alpha B crystallins. In addition to RGC protective functions of alpha crystallins, beta and gamma crystallins were implicated in RGC axonal regeneration. These findings demonstrate the importance of crystallin genes in RGC survival and regeneration and further in-depth studies are necessary to better understand the mechanisms underlying the functions of these proteins in healthy RGCs as well as during glaucomatous neurodegeneration, which in turn could help in designing new therapeutic strategies to preserve or regenerate these cells.     

Collagen synthesis by long-lived mRNA in embryonic chicken lens

Lens capsule collagen synthesis by epithelial and fiber cells was examined by immunoprecipitation and collagenase digestion in embryonic and posthatch chicken eye lens. Epithelial cells and lens fibers in the process of terminal differentiation produce alpha 1 and alpha 2 type IV collagen chains. At 6 days of embryonic development in addition to the alpha 1 (IV) and alpha 2 (IV) collagen chains, lens cells produce high molecular weight collagenase-sensitive proteins not immunologically related to type IV collagen. Lens capsule collagen components have been identified in central and outer fibers isolated from 18-day embryos and from 10-day posthatch chicken eyes. At these stages, fibers which have an increasing number of picnotic nuclei still show collagen synthesis due to long-lived mRNA. Analysis of collagen synthesis by lens cells incubated with actinomycin D suggests that stabilization of collagen mRNA occurs in lens fiber cells and to a lesser extent in epithelial cells as early as 6 days of embryonic development.