R.M. Clayton

Chapter 5 Problems of Differentiation in the Vertebrate Lens

Publisher Summary This chapter concentrates on the changing protein composition of the developing lens and the problems of control mechanisms which arise. Crystallin have no enzymic function and are regarded as wholly structural proteins. All available evidence shows that the assemblages synthesized and the quantities present differ according to the age of the lens. The problems of interest would seem to be the factors, both external and internal, which determine the presence or absence of a particular crystallin and the mechanism of their quantitative regulation. The relationship between capsule and crystallin synthesis requires further investigation and firm information is required in several other areas. These include the ultrastructural location of identified subunits, and their quantity per cell. Information is also required on the parameters of the RNA associated with polysomes synthesizing a given subunit at different ages, on the rate of synthesis of a subunit at different times, and on the relationship between the rate of synthesis of a peptide and the rate of turnover of the RNA associated with it.

Biochemical and immunological studies of lentoid formation in cultures of embryonic chick neural retina and day-old chick lens epithelium

Abstract During long-term cell culture of 8-day embryonic chick neural retina, lentoid bodies containing lens crystallins are developed. Although very low levels of crystallin can be detected in the embryonic neural retina, gross synthesis of each major crystallin class (α, anodal β, cathodal β, and δ) begins only after 12–16 days in culture. This occurs at least 10 days before lentoid bodies can be distinguished by eye. The concentration of each crystallin class was determined during lentoid development in cultures of both neural retina and lens epithelium. The proportions of crystallins in lentoid-containing cultures do not resemble those of embryonic lens fibres. Comparisons between two chick strains (N and Hy-1) differing in their growth rates revealed several differences in the crystallin compositions of lentoid bodies. These differences imply independent quantitative regulation for most or all of the crystallins.

A re-examination of the organ specificity of lens antigens

The cross-reacting antigenic determinants between lens proteins and other tissues of Xenopus laevis and the chick have been studied by immunodiffusion, immunoelectrophoresis and the Osserman technique, using antisera detceting minor as well as major antigenic determinants of the lens proteins. All tissues tested contained some antigenic determinants similar to those found in lens proteins and it was demonstrated that all of the major classes of lens proteins contained such cross-reacting groups. The pattern of cross-reactivity varied both qualitatively and quantitatively from tissue to tissue and there were no lens antigens which were found in all of the other tissues tested. The cross-reacting material from extralenticular tissue is not always identical in molecular form to that obtained from the lens. The results of the Osserman tests have been interpreted as indicating heterogeneity in a population of protein molecules which may be based either on a heteropolymer structure or on the selective binding of cross-reacting antigenic moieties. The results described are taken to mean that much tissue specificity is the result of a unique selection of antigens genetically available, any one of which may be found in other tissues, rather than being due exclusively to the possession of some specific protein restricted to that tissue.

Analysis of individual cataract patients and their lenses: A progress report

We are engaged in a continuing study of cataract patients and a control population matched for age and sex, in which epidemiological, ophthalmological, medical and other data is being collated and statistically processed by S.P.S.S. (Statistical Package for the Social Sciences). We report some confirmation of several significant differences found previously between our cataract and control populations, including the levels of several plasma constituents and the use of certain drugs. We also report some new data, including an increased risk of cataract to individuals without clinical diabetes whose fasting blood glucose is elevated above the normal range, and also an increased risk to individuals whose blood pressure is elevated above the normal range. Protein profiles from individual cataractous lenses are compared after grouping the lenses according to the full slit lamp description and other features, and we also present a further report on some biochemical differences observed between cataracts of similar appearance but different aetiology.

βB2-crystallin in the mammalian retina

β-crystallins are abundant lens proteins in most, if not all vertebrate species. We have previously reported the presence of low levels of β-crystallins in chick non-lens tissues, both ocular and extra-ocular, including the expression of βB2-crystallin in the retina. Here we report that extralenticular β-crystallin expression is also found in mammals. βB2-crystallin is expressed in mouse and cat neural and pigmented retinas and in cat iris. Although present at levels lower than those found in the lens, the appearance and accumulation of βB2-crystallin in the neural retina coincides with the functional maturation of this tissue.

Characterization of chick lens soluble proteins and the control of their synthesis.

Abstract A single step column procedure for the separation of chick lens soluble proteins is described. α-, β- and δ-crystallin, which account for 80–90% of the soluble protein are separated using this technique, with little or no cross-contamination between classes, as judged by immuoelectrophoresis. Analysis of the fractions in sodium dodecylsulphate-polyacrylamide (SDS) and isoelectric focusing (IEF) gels in dissociating conditions identifies and characterizes the major α-, β- and δ-crystallin subunits. Fresh lenses from day-old chicks were labelled by culturing in medium containing radioactive amino acids and the synthesis of individual crystallin subunits analysed. In both pulse and pulse-chase labelled lenses at least 70% of the incorporated radioactivity could be assigned to characterized crystallin subunits. Differences were observed in the radioactivity profiles from pulse and pulse-chase labelled lenses when analysed on SDS and IEF gels and some polypeptides showed labelling characteristics typical of post-translational modifications of existing protein chains.

EXPERIMENTAL MANIPULATION OF ALTERNATIVE PATHWAYS OF DIFFERENTIATION IN CULTURES OF EMBRYONIC CHICK NEURAL RETINA

Embryonic chick neural retina cells can transdifferentiate during long‐term cell culture into either pigmented epithelium or lens fibres. We have found that some culture conditions influence the choice between these pathways. Pigment cell development is promoted by low initial cell densities and by the use of a medium based on Earles salt formulation rather than Hanks, while lens fibre development is encouraged by high initial cell densities and by folding the cell sheet into multilayered regions. Some differences in in vitro cell properties of neural retina are reported for two genotypes previously found to exhibit differences in in vitro cell properties of lens epithelial cells.

Properties of the crystallins of the chick in terms of their subunit composition

Chick crystallins, FISC (first important soluble crystallins, also known as β-mobility or δ-crystallins) and several longline (also known as β or γ) crystallins have been isolated from polyacrylamide gels and their subunit composition and antigenic properties investigated. Some comparisons have been made with duck and quail. All crystallins have a subunit structure and each class appears to be a population of molecules in dynamic equilibrium with their subunits. The longline crysallins are a family of heteropolymers whose mobilities are related to their subunit structure. Their antigenicity varies, and that of their subunits, only some of them showing species cross-reactivity.

Isolation and cell-free translation of chick lens crystallin mRNA during normal development and transdifferentiation of neural retina.

Abstract Messenger RNA has been isolated from day-old chick lens. Size characterization and heterologous cell-free translation demonstrate that the predominant species of mRNA present code for α-, β- and δ-crystallins. Total polysomal RNA and polysomal RNA which did not bind to oligo (dT)-cellulose translate in the cell-free system to give a crystallin profile qualitatively similar to that of poly(A)+ mRNA. RNA from postribosomal supernatant which binds to oligo(dT)-cellulose also translates to give crystallins, but the products are enriched for β-crystallins. Messenger RNAs isolated from 15-day embryo lens fiber and lens epithelium cells give products on translation which reflect the different protein compositions of these two cell types, as do mRNAs isolated from chick lenses at various developmental stages. Messenger RNAs were isolated from freshly excised 8-day embryo neural retina and from this tissue undergoing transdifferentiation into lens cells in cell culture. Cell-free translation demonstrates no detectable crystallin mRNAs in the freshly excised material, but by 42 days in cell culture, crystallin mRNAs are the most prominent species.

Antigens of the lens of Xenopus laevis.

The antigens of the lens of Xenopus laevis were investigated by elect rophoresis, immunological double-diffusion methods and immuno-electrophoresis. Twenty-two lens antigens were detected. A new nomenclature for lens proteins is suggested.