Samir K. Brahma

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. Dev. Dyn. 208:220–226, 1997.

Ontogeny and localization of gamma-crystallins in Rana temporaria, Ambystoma mexicanum and Pleurodeles waltlii normal lens development

Rana pipiens lens γ-crystallin antibodies were used in the indirect immunofluorescence staining method to investigate the role of γ-crystallins in the normal lens development of the amphibians Rana temporaria, Ambystoma mexicanum and Pleurodeles waltlii Michah. In each case, the fluorescence was first localized in a number of cells in the inner wall of the lens, which was in the vesicle stage. With further differentiation, the intensity of immunofluorescence gradually increased and was restricted only to the fibre cells. These results support the concept that, though the γ-crystallins of different amphibian species studied so far show different numbers of protein components by thin layer isoelectric focusing on polyacrylamide gel, they all have similar immunological properties, and are specific for the lens fibre differentiation.

Immunohistochemical Studies of Lens Crystallins in the Dysgenetic Lens (dyl) Mutant Mice

The lens in the dyl mutant mice shows a persistent lens-ectodermal connection as well as degeneration and extrusion of lens materials after the initial differentiation of the fibres. Immunohistochemical investigation of the ontogeny of the lens crystallins in this developing mutant lens has been carried out using the indirect immunofluorescence staining method with antiserum to adult mouse lens total soluble proteins. The results have been compared with those for coisogenic normal lens used as a control. In both, the first positive reaction was detectable at identical stages of lens development. A rapid increase in the intensity of fluorescence, most marked in the elongating fibre progressing through the equatorial region to the epithelium, was recorded in the mutant as well as in the normal lens. However, the stalk leading to the lens epithelium did not show any reaction. Appearance of vacuoles in the lens nucleus and cortex marked the beginning of degeneration of fibres which otherwise showed strong fluorescence. This was followed by extrusion of lens crystallin materials through the stalk. As a result, the lens became increasingly reduced and malformed but the surviving cells making up the vestigeal lens in the adult showed positive immunofluorescence. The results demonstrate that despite a failure of lens-ectoderm separation in the mutant mice, the ontogeny of the lens crystallins and differentiation of the lens up to a certain stage of development follow an apparently normal course before the commencement of cataractous degeneration.

Ontogeny and localization of pre-α crystallin antigen in Rana temporaria lens development

The ontogeny and localization of Rana temporaria pre-α, the fastest moving crystallin were investigated during normal lens development using indirect immuno-cytochemical staining method and antibodies to R. esculenta pre-α crystallin antigen. It was found that this pre-α crystallin appears immediately after the γ-, and is also localized in the fibre cells like the γ-crystallin. The esculenta pre-α crytallin is antigenically different from the fastest moving fraction from bovine lens which is now called Fast Migrating or FM-crystallin.

Isofocusing and immunological investigations on cephalopod lens proteins

Soluble lens proteins from Octopus vulgaris, Sepia officinalis, and Loligo vulgaris were analyzed by thin-layer isoelectric focusing and compared by various immunochemical methods using antibodies directed against total soluble lens protein antigens from the said three species. The results show close similarity between Sepia and Loligo lens proteins. Antibodies made against Sepia and Loligo lens proteins did not react against Octopus total lens soluble protein antigens. Our results suggest a common ancestry for Sepia and Loligo which is different from that of Octopus.

Isofocusing and immunoelectrophoretic studies of soluble eye lens proteins from regenerated and normally developed Xenopus laevis

Abstract Soluble eye lens proteins from regenerated and normally developed 6-month-old Xenopus laevis a p a p -mutant froglets were compared by thin-layer isoelectric focusing and microimmunoelectrophoresis. Larvae belonging to the same age group were reared through metamorphosis after the removal of the left lens at different larval stages as explained in the text. The results show that regenerating and normally developing lenses of similar ages have identical profiles of α-, β- and γ-crystallins and also identical antigenic determinants suggesting that gene expression for lens crystallins follows the same pathway in a regenerating lens as in a normally developing lens.

Embryonic appearance of rod opsin in the urodele amphibian eye

Notophthalmus (Triturus) viridescens, a urodele amphibian (newt) common to the Eastern United States, is a promising subject for developmental and regeneration studies. We have available a monoclonal antibody shown to be specific in many vertebrates for rod opsin, the membrane apoprotein of the visual pigment rhodopsin. This antibody to an N-terminal epitope, by rigorous biochemical and immunological criteria, recognizes only rod photoreceptor cells of the retina in light-and electron-microscopic immunocytochemistry. To determine the ontogeny and localization of rhodopsin in developing rods as an indicator of function in the embryonic urodele retina, we have utilized this antibody in the immunofluorescence technique on sections of developing N. viridescens. It was applied to serial sections of the eye region of Harrison stage 28 (optic vesicle) through stage 43 (most adult retina histology complete) embryos, and subsequently visualized with biotinylated species antibody followed by extravidin fluorescein isothiocyanate. The first positive reaction to rhodopsin could be detected in two to four cells (total) of the stage 37 embryonic eye, in the region of the central retinal primordium where the photoreceptors will be found. Some indications of retinal outer nuclear and inner plexiform layers could be seen at this time. Later embryonic stages demonstrated increasing numbers of positive cells in the future photoreceptor outer nuclear layer and outer and inner segments, spreading even to the peripheral retina. Nevertheless, by stale 43, no positive cells could be found at the dorsal or ventral retinal margins. Thus, biochemical differentiation of a photoreceptor population in the urodele retina occurs at a stage before retinal histogenesis is complete. The total maturation of retinal rods occurs topographically over a long period until the adult distribution is achieved.