S.K. Brahma

Immunofluorescence Studies of Chick Lens FISC and α-Crystallin Antigens during Lens Morphogenesis and Development

Indirect staining method of Weller and Coons [23] was applied to detect the presence of FISC and α-crystallin lens antigens in the embryonic and post-embryonic chick lenses. Specific antiserum against

Ontogeny and localization of the α, β, and γ 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.

Isoelectric focusing of embryonic cow lens crystallins

Soluble lens crystallins from several embryonic stages and also from adult cow lens cortex are compared by thin layer isoelectric focusing, immunoelectrophoresis, immuno-osmophoresis and antigen/antibody crossed electrophoresis. A gradual change in crystallin composition is found between the lens proteins of 1·5–7 month embryos. Thin layer isoelectric focusing studies of various embryonic stages from 1·5 to 7 months show a decrease of γ-crystallins along with an increase of β-crystallins and a slight increase of α-crystallins. When compared with the adult lens nucleus, the cortex shows a sharp decrease in concentration of γ-crystallin components. In addition, the composition of γ-crystallin of embryonic lens is similar to that of the adult lens nucleus, but different from that of the adult lens cortex. This difference appears due to certain components of γ-crystallin that are absent or present in lower amounts in the adult lens cortex compared with the embryonic lens. The same number of α-, β- and γ-crystallin antigens are present, they differ only in the relative concentration of the protein components. In all embryonic stages two pre-α-crystallins could be detected. These pre-α-crystallins have a high anodic mobility and appear to have a molecular weight lower than α-crystallin itself; the pre-α-crystallin is found to be eluted with the low molecular weight β-crystallins in Sepharose gel chromatography. By gel chromatography procedure, crystallins of cow lens cortex could be separated into five fractions; α1-crystallins of a molecular weight > 2,000,000, α2-crystallins, two β-crystallin fractions and γ-crystallin. Immuno-osmophoresis analysis shows that α1-crystallin is present in higher amounts in embryonic lens, and in adult lens nucleus it is higher than in adult lens cortex, while the α2-crystallin is present both in cortex and nucleus.

Study of the soluble lens proteins from different amphibian species

Soluble lens proteins from five species of amphibia have been studied by zone electrophoresis and other immunochemical methods. Their patterns, as revealed by electrophoresis, do not differ markedly though the numbers of bands and subunits vary. The γ-crystallin appears to be the predominant lens protein in all the species. Immunodiffusion tests showed a reaction of complete identity of lens antigens of different species with respect to Xenopus laevis lens antiserum; while X. laevis, Triturus crislatus, Ambystoma mexicanum lens antigens revealed partial identity when tested against Rana esculenta and Bufo bufo lens antisera.

Thin layer isoelectric focusing of the soluble lens extracts from larval stages and adult Xenopus laevis

Soluble lens extracts from different larval stages and also from adult Xenopus laevis were studied by thin layer isoelectric focusing. These lens extracts were also tested against antiserum to adult X. laevis total lens proteins by two-dimensional antigen-antibody crossed electrophoresis. By isoelectric focusing the lens crystallins were resolved into 13 major components. In the adult lens some components of the γ-crystallins were lost, the β-crystallins formed the major fraction and the α-crystallin appeared to be the weakest of the crystallin fractions.

α-, β- and γ- Crystallins in the regenerating lens of Notophthalmus viridescens

Abstract Removal of the lens from the eye of adult Notophthalmus viridescens, the Eastern spotted newt, is followed by regeneration of another lens from the dorsal iris. This cell-type conversion of iris epithelial cells into lens cells is accompanied by the subsequent synthesis of α-, β- and γ-crystallins, proteins specific for the normal vertebrate lens. The immunofluorescence technique was employed to determine the ontogeny and localization of the crystallin classes in the regenerating lens rudiments. Antibodies specific for α-, β- and γ-crystallins were applied to tissue sections through regenerate Stages III–XI Yamada 1967. The first positive reaction for α-crystallins did not occur until Stage VII, in a few cells of the developing lens fiber region; and in the external layer (presumptive lens epithelium) at Stage VIII, at which time secondary lens fibers have begun to form. β-Crystallins were first detectable in the thickening internal layer of the Stage V lens (vesicle) and in the external layer at Stage VIII. The γ-crystallins also first appeared, albeit erratically, in a few cells of the internal layer of the Stage V regenerate, and were not detectable in the external layer/lens epithelium at any time. Thus the crystallins are not present in the earliest stages, but are indicative of cellular differentiation in succeeding stages of lens regeneration.

Ontogeny and localization of γ-crystallin antigen in the developing pigeon (Columba livia) lens

Ontogeny and localization of the lens γ-crystallin antigen were investigated in the embryonic and post-embryonic pigeon lenses by the indirect immunofluorescence with antiserum from rabbit immunized with isolated pigeon lens γ-crystallin. The results show that γ-crystallin appears for the first time in a 4-day embryonic lens and is distributed uniformly over the entire fibre cells. In a late and also post-embryonic lens γ-crystallin was also detected in the epithelium and annular pad areas and the intensities of immunofluorescent reaction gradually increased from embryonic to post-embryonic lens.

Immunochemical studies of chick iris

Immunochemical properties of chick iris were investigated by various methods. It appears from these studies that chick iris contains antigens which are immunologically identical to all the antigens of the adult lens.

Ontogeny of αA and αB crystallin polypeptides during Rana temporaria lens development

The ontogeny and localization of αA and αB polypeptide chains of α-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. αA and αB 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 αA cDNA of rat and mouse does not hybridize to αB mRNA ( Dodemont et al., 1981 ; King and Piatigorsky, 1983 ). Antigenically too, αA and αB polypeptides have been shown to be different. These two polypeptides were isolated from mouse lens native α-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, αA appears earlier than αB, 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 and localization of the a-, β-, and δ-crystallin antigens during lens development in mallard embryos

The ontogeny of α-, β-, and δ-crystallin antigens in the developing lens of mallard (Anaa platyrhynchos) was investigated by indirect immunofluorescence using specific antibodies to mallard α-, β-, and δ-crystallins raised in rabbits.The IgG fraction from each antiserum was isolated by affinity chromatography usign Protein A Sepharose CL-4.B. Fluoresceine (FITC) or rhodamine (TRITC) conjugated goat anti rabbit (GAR) γ-globulin was used as the secondary antibody.The results show that α-, β-, and δ-crystallins appear simultaneously in the developing mallard lens and are detectable from 66 hr (stage 15/16). This situation is different from the chick where δ-is known to appear first followed by β-, and α-. This could be due to species variation.In the epithelium, however, as in the chick, δ-emerges first followed by β-, and α-but rather late when compared with the chick and this seems probably due to a difference in the incubation time between the two species.Results from immunofluorescence and Tris-SDS gel e...