Fibroblast growth factor receptors and regeneration of the eye lens.

If the eye lens of the adult newt, Notophthalmus viridescens, is removed, a new lens will regenerate and only from the dorsal, not the ventral, iris. The source, pigmented epithelial cells, would normally no longer divide, but upon lentectomy they do re-enter the cell cycle and form lens. The cause for this capability is unknown, but the mitogenic Fibroblast Growth Factors and their receptors may be involved. We have demonstrated that FGF receptors are present and operative in lens regeneration, since receptor-directed mitotoxins inhibit regeneration; heterogeneity and differential density in FGF-binding and receptor localization in iris sectors is also present. We propose that the spatial distribution of FGF receptors, especially the amphibian homolog of FGFR-3, is important in initiation of regeneration of eye lens.

Presence and foveal enrichment of rod opsin in the "all cone" retina of the American chameleon.

The retinal photoreceptors of the eye of the American chameleon, Anolis carolinensis, have been considered to be exclusively cones. Its retina is unusual for possessing two foveas (areas associated with heightened visual acuity), with the major, central fovea deeply incised and very densely packed with photoreceptors. Immunoblotting and light- and electron microscopic-immunocytochemistry, using several opsin monoclonal antibodies previously found specific for rods, demonstrated the presence and localization of this protein in the Anolis retina. This visual pigment appears sparsely in a subpopulation of photoreceptors in the periphery but overwhelmingly in the central fovea. Complementary results with cone-specific antibody and lectin binding corroborated this spatial organization. These results, as well as those with geckos, suggest that photoreceptor morphology is not an accurate guide among the lacertilians to visual pigment content, and that this phylogenetic grouping may constitute a crossroads in vertebrate photoreceptor evolution.

Eye lens regeneration and the crystallins in the adult newt, Notophthalmus viridescens.

Upon lens removal, the adult Eastern Spotted newt, Notophthalmus viridescens, has the capacity to regenerate an ocular lens. Crystallins, proteins characteristic of the vertebrate lens, were studied from normal and 3-month regenerated adult newt lenses. When separated by high-performance liquid chromatography (HPLC) or Sephadex G-200SF column chromatography, the crystallins from normal and regenerated lenses were fractionated into what appear to be the classical four groups: alpha, beta High, beta Low, and gamma. Upon further examination by immunoelectrophoresis, the first peak contains both alpha and beta crystallins. This study provides evidence that most of the crystallins from the regenerated lenses share biochemical properties with those of the normal lens crystallins based on their native molecular weight, isoelectric point, and the molecular wt of their constituent polypeptides, indicating that the fidelity of gene expression in reactivated iris tissue is high. Some differences are found between normal and regenerated lens crystallins and are most obvious in the beta-crystallin region: the proportion of beta crystallins is decreased in regenerated lenses when the total proteins are fractionated by column chromatography and some of the beta-crystallin polypeptide chains found in normal lenses are missing from regenerated lenses. Iris epithelial cells are normally withdrawn from the cell cycle and are synthesizing a tissue-specific product, melanin. After lentectomy these cells dedifferentiate, redifferentiate into lens cells, and their progeny then synthesize different tissue-specific proteins, crystallins. Little is known about the specific mechanism(s) for the activation of gene expression in eukaryotes, but the regenerating lens suggests itself as a good model in which to study this biological problem.

Ontogeny of alpha A and alpha B crystallin polypeptides during Rana temporaria lens development.

The ontogeny and localization of alpha A and alpha B polypeptide chains of alpha-crystallin were investigated in the developing lens of Rana temporaria, an anuran amphibian, using the indirect immunofluorescence staining method with heterologous antibodies directed against these two polypeptides. alpha A and alpha B crystallins are primary gene products and are translated by different mRNAs in mammals. Although they show about 6000 amino-acid sequence homology (Bloemendal, 1977), the alpha A cDNA of rat and mouse does not hybridize to alpha B mRNA (Dodemont et al., 1981; King and Piatigorsky, 1983). Antigenically too, alpha A and alpha B polypeptides have been shown to be different. These two polypeptides were isolated from mouse lens native alpha-crystallin by SDS-gel electrophoresis and were injected into young rabbits to raise antibodies. These antibodies were tested by immunoblotting against R. temporaria total lens soluble proteins before their use in the present investigation. Results presented here show that in the developing lens of R. temporaria, alpha A appears earlier than alpha B, suggesting a differential gene activation. In addition, these two polypeptides could not be detected either in the developing lens epithelium or in the epithelium of young froglets (2-3 weeks post-metamorphosis).

Ontogeny of the 35K epsilon crystallin during Rana temporaria lens development.

Epsilon is a recently described eye lens protein from Rana temporaria, an anuran amphibian. It is oligomeric with a subunit M.W. of 35K. The cDNA coding for 35K E- in frog lens does not show any homology with cDNA's coding for alpha-, beta-, gamma-, and delta-crystallins. Immunologically, it also does not react with antibodies directed against alpha-, beta-, and gamma-crystallins. The ontogeny of this 35K E-protein has been investigated in R. temporaria lens development by the indirect immunofluorescence staining method with an antibody specific for the 35K E-protein. The purity of the isolated 35K protein and the specificity of the antibody were controlled by Tris-SDS gel electrophoresis and immuno-blotting, respectively. The first positive immunofluorescence reaction was observed in the inner cell wall of a stage V lens. In the external layer/epithelium the reaction was first detected in a single cell of a stage VII lens. Additional positive cells in the external layer/epithelium were detected at an early state VIII and the reaction appeared to be patchy. This type of patchy reaction was also observed in the epithelium of froglet (sub-adult) eye lens.

Conversion of iris epithelial cells as a model of differentiation control.

It has been shown that, upon lentectomy or in culture, iris epithelial cells (IECs) of adult newts become converted into lens cells, and this conversion is the basic event of lens regeneration in newts. Whether in situ or in cell culture, the conversion requires the passage of a specific number of cell cycles. The progeny of IECs which fails to traverse this cell-cycle number redifferentiates as IECs in situ. The passage through cell cycles of IECs is associated with progressive alterations of cytoplasm and cell surface, during which the original state of differentiation disappears (dedifferentiation). It is speculated that the altered state of cells caused by proliferation leads to the appearance of factors which interact with the genome and switch the gene activation pattern to that of the lens cell. In this model, developmental controls are geared to the cell-cycle progression and not directly to the activation of lens-characteristic genes. A number of points are raised which speak against the long-held idea that a factor from neural retina induces lens differentiation in IECs. It is proposed that the retinal factor plays the role of growth factor which is essential in the conversion in situ, but not required in the conversion in cell culture. The proposed model is compared with reprogramming of differentiation of some cell lines by cytidine analogs and with ontogenic systems of differentiation control.

Ontogeny and localization of the alpha, beta, and gamma crystallins in newt eye lens development.

The alpha-, beta-, and gamma-crystallins, proteins characteristic for the vertebrate eye lens, have been localized in the developing lens of Notophthalmus viridescens, the eastern spotted newt. Using the immunofluorescence technique, antibodies to the alpha-, beta-, and gamma-crystallin classes were applied to tissue sections through the eye region of developing N. viridescens embryos, Harrison (external) Stages 30 to 46+. beta-Crystallins were the first of the crystallins to appear in a few cells of the lens vesicle even before the lengthening of the prospective primary fiber cells. gamma-Crystallins were first detectable at a slightly more advanced stage in the prospective primary fibers, and alpha-crystallins in a few cells of the beginning primary fiber area. The external layer/epithelium was negative for beta-crystallins until late in lens morphogenesis, and alpha- and gamma-crystallins could not be detected in these cells at any time. This, the first use in amphibia of homologous antibodies specific for the crystallin classes, makes clear that phylogenetic differences exist as to the primacy and relevance of specific crystallins to events during morphogenesis of the eye lens.

Crystallin expression in the TVI cell line.

The TVI cell line, derived from dorsal iris cells of adult newts (notophthalmus viridescens), was investigated for the presence of crystallins. Since there is reason to believe that iris epithelial cells are the main sources of this cell line and since iris epithelial cells are known to convert into lens cells in primary cultures, it is possible that TVI cells also possess the capacity to synthesize crystallins, those proteins characteristic of lens cells. It is also possible, however, that the large number of passages gone through by TVI cells in the past has eliminated such differentiated synthetic capacity expressed in earlier generations. Our immunoelectrophoresis studies reveal the presence of small amounts of alpha and beta crystallins, and the absence of gamma crystallins in TVI cells. Furthermore, immunofluorescence observations demonstrate that a small number of cells comparable to lens epithelial cells in crystallin composition and morphology are present in TVI cultures. In view of the fact that in the amphibian lens, epithelial cells which retain proliferative activity accumulate alpha and beta crystallins but not gamma crystallins, while fiber cells which are devoid of proliferative activity accumulate all three classes of crystallins, the present results suggest that the TVI cell line has lost the capacity to maintain lens fiber cells, which are known to be present in primary culture of iris epithelial cells.

Ontogeny and localization of the crystallins during lens development in normal and Hy-1 (hyperplastic lens epithelium) chick embryos.

A strain of chickens selected for high growth rate has been found to exhibit an anomalous eye lens morphology indicating a failure of the normal process of growth regulation. Hyperplasia of the lens epithelium and annular pad, often with fiber formation, has recently been described in day-old chicks of this strain, termed Hy-1 (Clayton, 1975). The earliest evidence of this condition in this study has been found in the 11-day embryonic Hy-1 lens. Before this time, no definitive lens abnormality could be detected histologically in the Hy-1 embryos. However, the indirect immunofluorescence technique had revealed early temporal and spatial differences with regard to the lens crystallins. Antibodies, specific for delta, cathodal beta, anodal beta and alpha crystallins, were applied to sections through the lens of 2 1/2-, 3-, 3 1/2, 4-, 5-, 8- and 16-day embryonic and 1-day post-hatch normal and Hy-1 chicks. delta crystallin appears precociously in the external layer, and alpha crystallin in the prospective fiber region, of the lens rudiment of Hy-1 embryos. Both anodal and cathodal beta crystallins are retarded, however, in their appearance in the external layer/epithelium of Hy-1 lenses. Localization of the crystallin classes within the lenses of the two strains continues to vary during lens differentiation until 1 day post-hatch, at which time and during late embryogenesis annular pad and epithelium abnormalities can be frequently be seen in the Hy-1 lens. This inability to control normal lens histogenesis thus manifests itself early as alterations in the appearance of an organ-specific gene product, the crystallins.

Embryonic appearance of alpha, beta, and gamma crystallins in the periodic albinism (ap) mutant of Xenopus laevis.

The appearance of the crystallins during lens development in the periodic albinism (ap/ap) mutant of Xenopus laevis has been studied. Using antibodies specific for total crystallins, alpha + beta crystallins, and gamma crystallins in the immunofluorescence technique, the first positive reaction for all could be demonstrated in the Nieuwkoop-Faber Stage 31 lens rudiment. The antibody to alpha + beta crystallins exhibited differences in intensity from cell to cell in the early rudiment, while the reaction to the other antibodies was uniform throughout the rudiment. As lens differentiation progressed, immunofluorescence was restricted in all cases to the lens fiber area, up to and including Nieuwkas positive, however, for total lens crystallins. These results are at variance with earlier studies on lens development and the crystallins in wildtype (+/+) X. laevis, where a positive reaction for gamma and total crystallins could be detector total lens crystallins. That this divergence in the mutant is due to a pleiotropic effect or directly to the inductive failure of the endomesoderm to initiate melanogenesis, is discussed.

Eye lens development and gamma crystallins in Discoglossus pictus (Anura).

The ontogeny and localization of the gamma crystallins in Discoglossus pictus lens development has been determined. Using antibody specific for amphibian gamma crystallins in the immunofluorescence technique, it was found that gamma crystallins first appear in primary lens fibre cells in the lens rudiment, and continue to be restricted to the fibre area as lens development progresses. Thus the role of gamma crystallins as indicators of a differentiated state remains constant in amphibian evolution, having been demonstrated in the most archaic anuran superfamily, as well as in others more recently evolved.