Electron Microscopic Studies on Lens Regeneration

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141

(10,11,27,28).We have shown that the switch from pigmented retinal cells of 9-day-old check embryos into lens cells occurs in clonal cell culture without interactions with other cell types (14). The question then arises whether this approach can be extended to iris epithelial cells of newts which possess the capacity for Wolffian regeneration in vivo. This communication describes the differentiation of lens-like structures in cultures of dissociated iris epithelial cells of adult newts. MATERIALS AND METHODSTwenty-five to 50 adult newts, Cynops (Triturus) pyrrhogaster were used in each experiment. Isolated whole eyes were sterilized by three 1-min immersions in 70% ethanol. The irisrings (iris pars iridica) were then removed (11) and treated in an enzyme mixture that contained 5 parts of 0.4% (v/v) Diluted Leibovitz medium IL15 (12) was used as the culture medium and was prepared by dissolving 0.894 g of commercial Leibovitz L-15 powder (GIBCO) in 90 ml of triple-distilled water; this was supplemented with 10 ml of fetal calf serum (GIBCO, Control No. A322102), 3200 IU of penicillin, and 4 mg of streptomycin before filtration by Millipore filter.For the immunological identification of lens products in cultured cells, antisera were prepared in rabbits against the 15,000 X g supernatant of adult newt lenses in 20 mM Tris HCl buffer (pH 7.5) and against the a-, f-, and y-crystallin fractions, respectively. The separation of crystallins from the whole lens extract was made by column chromatography through Sephadex G-50 and G-200, and DEAE-Sephadex A-50 according to established methods (13). Sixteen rabbits were injected, one each with 40, 5, 3, and 1 mg of each protein fraction, respectively. Each rabbit received injections of protein with an incomplete Freund's adjuvant at intervals of two weeks. Antisera were usually obtained 10 days-after the second injection. In immunodiffusion tests (15) anti-whole lens extract and anti-a-crystallin cross-reacted faintly with extracts of heterologous organs of newts. This cross-reactivity was removed by absorbing the antisera with saline extracts of liver homogenate of adult newts. Cross-reactions of anti-acrystallin with the heterologous crystallins were also removed by absorption with 0-crystallin solution, whereas anti-acrystallin and anti-y-crystallin did not cross-react with the heterologous test-antigens (Fig. 1)

Section: Discussionmentioning

confidence: 90%

mentioning

confidence: 99%

Differentiation of Lens-Like Structures from Newt Iris Epithelial Cells In Vitro

Eguchi¹,

Abe²,

Watanabe³

1974

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141

“…Once the lens is removed the process of regeneration is initiated by dedifferentiation of the dorsal iris pigment epithelium (3)(4)(5). About 6 days later, transdifferentiation of the iris into lens cells begins.…”

mentioning

confidence: 99%

Expression of pax-6 during urodele eye development and lens regeneration.

Rio‐Tsonis

Washabaugh

Tsonis

1995

105

Regeneration of eye tissues, such as lens, seen in some urodeles involves dedifferentiation of the dorsal pigmented epithelium and subsequent differentiation to lens cells. Such spatial regulation implies possible action of genes known to be specific for particular spell lineages and/or axis.Hox genes have been the best examples of genes for such actions. We have, therefore, investigated the possibility that such genes are expressed during lens regeneration in the newt. Thepax-6 gene (a gene that contains a homeobox and a paired box) has been implicated in the development of the eye and lens determination in various species ranging from Drosophila to human and, because of these properties, could be instrumental in the regeneration of the urodele eye tissues as well. We present data showing thatpax-6 transcripts are present in the developing and the regenerating eye tissues. Furthermore, expression in eye tissues, such as in retina, declines when a urodele not capable of lens regeneration (axolotl) surpasses the embryonic stages. Such a decline is not seen in adult newts capable of lens regeneration. This might indicate a vital role of pax-6 in newt lens regeneration.

“…After lentectomy, the pigment epithelial cells of the dorsal iris dedifferentiate, by shedding their pigments, proliferate, and eventually transdifferentiate to lens cells. These lens epithelial cells can subsequently differentiate to lens fibers (2)(3)(4). These morphogenetic events during lens regeneration differ from the corresponding events that take place during lens development.…”

mentioning

confidence: 95%

Conservation of fibroblast growth factor function in lens regeneration

Rio‐Tsonis

Jung

Chiu

et al. 1997

In urodele amphibians, lens induction during development and regeneration occurs through different pathways. During development, the lens is induced from the mutual interaction of the ectoderm and the optic vesicle, whereas after lentectomy the lens is regenerated through the transdifferentiation of the iris-pigmented epithelial cells. Given the known role of fibroblast growth factors (FGFs) during lens development, we examined whether or not the expression and the effects of exogenous FGF during urodele lens regeneration were conserved. In this paper, we describe expression of FGF-1 and its receptors, FGFR-2 (KGFR and bek variants) and FGFR-3, in newts during lens regeneration. Expression of these genes was readily observed in the dedifferentiating pigmented epithelial cells, and the levels of expression were high in the lens epithelium and the differentiating fibers and lower in the retina. These patterns of expression implied involvement of FGFs in lens regeneration. To further elucidate this function, we examined the effects of exogenous FGF-1 and FGF-4 during lens regeneration. FGF-1 or FGF-4 treatment in lentectomized eyes resulted in the induction of abnormalities reminiscent to the ones induced during lens development in transgenic mice. Effects included transformation of epithelial cells to fiber cells, double lens regeneration, and lenses with abnormal polarity. These results establish that FGF molecules are key factors in fiber differentiation, polarity, and morphogenesis of the lens during regeneration even though the regenerating lens is induced by a different mechanism than in lens development. In this sense, FGF function in lens regeneration and development should be regarded as conserved. Such conservation should help elucidate the mechanisms of lens regeneration in urodeles and its absence in higher vertebrates.