Localization of delta-crystallin RNA during lens morphogenesis and differentiation in the normal and talpid3 chick embryo.

Embryonic lens fiber cell differentiation in the chick is marked by the accumulation of delta-crystallin protein. The levels of delta-crystallin RNA are shown here to rise dramatically in the cells of the posterior lens pit prior to their elongation and differentiation as lens fibers. This increase correlates with regional proximity to the underlying optic cup (future retina). This accumulation of delta-crystallin RNA during lens induction operates selectively on the delta 1-crystallin transcripts whereas delta 2-crystallin/argininsosuccinate lyase RNA is detectable at lower levels in all developing ocular tissues throughout this period. The talpid3 mutant forms a flat "bridge" of thickened placode-like cells in the head epithelium between the two lens placodes, and this bridge also accumulates delta 1-crystallin RNA, suggesting that the selective increase in delta 1-crystallin RNA levels over those of delta 2-crystallin represents an early event in cellular commitment to lens fiber differentiation in the chick. The significance of the sequence of temporal changes in inductive sources for lens fiber formation is discussed, and we propose that the role of the optic cup is to provide, bound to its extra-cellular material (ECM), a high local concentration of the same growth factors which act as fiber inducers in the older eye.

THE INFLUENCE OF GROWTH-INHIBITING AND GROWTH-PROMOTING MEDIUM CONDITIONS ON CRYSTALLIN ACCUMULATION IN TRANSDIFFERENTIATING CULTURES OF EMBRYONIC CHICK NEURAL RETINA.

The effects of media containing undialysed serum (controls) or dialysed serum with or without ascorbic acid, were compared during the second half of the 41-day culture period in embryonic chick neural retina cultures, which had all been grown in control medium prior to 19 days. Conditions permitting greatest culture growth (controls) showed earlier and more extensive development of lentoids, greater accumulation of total crystallin and a higher proportion of δ relative to α+ß crystallins. Conditions allowing least culture growth (dialysed serum) gave converse results throughout. Thus changes in culture growth rate apparently affect δ crystallin production more than α or ß crystallin production. Insulin promotes growth in neural retina cultures, whether present throughout the culture period (in this case 31 days), or only from 18 days onwards. The frequency and survival of putative neuronal cell aggregates are both increased by insulin during the first 18 days of culture. Delta crystallin production during subsequent transdifferentiation is selectively promoted by insulin when present throughout, but this effect is largely obviated when insulin is present only from 18 days onwards. This anomaly could arise through percursor cell selection during the earlier phases of culture, since it is possible that some (not all) lentoids may be derived from aggregates of neuronal-like cells in neural retina cultures. Thus precursor cell selection as well as culture growth rate may influence the pattern of crystallin production during transdifferentiation.

COMPARISON OF THE GROWTH AND DIFFERENTIATION OF EPITHELIAL CELLS FROM NORMAL AND HYPERPLASTIC LENSES OF THE CHICK: STUDIES OF IN VITRO CELL CULTURES.

Epithelial cells from hyperplastic lenses of a strain of chicks (Hy-1) selected for high growth rate were dissociated and cultured in vitro and compared with lens epithelial cells from a normal strain (N) in similar conditions. The hyperplastic lens cells showed remarkable motility and adhesiveness after dissociation and formed cell aggregates of various sizes before attaching to the substrate, giving a rather low plating efficiency. The lens structures (lentoid bodies) developed in partially confluent cultures of Hy-1 cells at least three days earlier than those in the cultures from normal control cells, in which the lens structures developed only after the cultures reached confluence. The results of culture at low cell density showed that the Hy-1 cell population consisted of at least two cell types different from each other in growth capacity. These striking differences in in vitro behaviour of dissociated cells from normal and hyperplastic lens epithelia and the results of clonal culture are discussed in relation to the possible mechanisms of abnormal morphogenesis and growth which are likely to be involved in the development of the hyperplastic lens in situ.

DIFFERENTIATION AND TRANSDIFFERENTIATION IN VITRO OF NEURAL RETINA FROM MUTANT CHICKENS WITH HYPERPLASIC LENS EPITHELIUM1).

Mutant chickens, Hy-1 and Hy-2, show abnormalities in growth and differentiation of the lens epithelium. In this study, neural retinal cells (NR cells) from 3.5-day-old embryos of these mutants were cultured, and the differentiation in vitro was compared with the cells of the normal strain. Hy-1 cells in vitro were characterized by a delay in the first appearance of neuronal cells (N-cells) and by excessive production of this cell type at later stages. By contrast, the Hy-2 cells were indistinguishable from the normal cells in the early phase of culturing. In spite of the marked difference of Hy-1 NR cells in neuronal differentiation up to about 7 days in culture, the transdifferentiation of lens and pigmented cells occurred to a similar extent and with the same time schedule as cultures of normal cells. A number of lentoid bodies were formed by about 10 days. The relative composition of the three major classes of crystallins in transdifferentiated lens cells was almost identical between normal and Hy-1 strains. The results were discussed in comparison with the previous results of cell culture of NR of 8-day embryonic mutant chickens, and it was concluded that the process of transdifferentiation in cell culture is different between NR from 3.5-day-old and 8-day-old embryos.

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 Earle's salt formulation rather than Hank's, 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.

Differential effects of prenatal exposure to phenobarbital on the behaviour and neurochemistry of CBA and C57BL/6J mice.

Pregnant C57BL/6J and CBA mice were administered 60 mg/kg phenobarbital intraperitoneally from days 10 to 16 of gestation. On day 18 of pregnancy half of the control and drug-treated mice were killed and the embryonic brains removed for cell cultures. The remaining mice were allowed to have their litter. After cross-fostering the mice were used for behavioural studies. Pups born to drug-treated CBA mice had birth-weights similar to controls, but their weights had fallen behind controls by day 18 after birth. They were slower at attaining mature responses in tests for sensory motor development and became progressively more hyperactive (three times more active at day 18) compared to controls. Drug-exposed C57 pups also had birth weights similar to controls. After cross-fostering, 19% of control and 31% of drug-exposed pups died, but the remaining drug-exposed pups showed no deficits in weight gain. In contrast to drug-treated CBA pups, drug-exposed C57 pups were slightly quicker in attaining mature responses in some tests. There was no difference in activity between them and their controls. In neurochemical analyses, uptake of neurotransmitters by cerebral cultures from CBA showed that uptake of GABA was increased by 5%, choline by 95%, dopamine 120%, serotonin 165% and noradrenaline by 160% in cultures from drug exposed embryos compared to controls. In cerebral cultures from C57, GABA uptake was reduced by 18%, choline 33%, dopamine 35% and noradrenaline by 25%. Only serotonin uptake was increased by 182% compared to controls. Differences between C57 and CBA were also apparent in the uptake of neurotransmitters by neuronal cultures from the mesencephalon.(ABSTRACT TRUNCATED AT 250 WORDS)

Some blood plasma constituents correlate with human cataract.

AIMS: To look for differences between matched pairs of patients and controls in concentrations of various plasma constituents which might indicate dysfunctions associated with cataract. METHOD: One thousand patients were taken from the cataract waiting list of a specialist eye hospital. For each patient a matched control of the same sex and half-decade of age but without cataract was taken from the patient list of the family doctor of the patient; the control was the next alphabetically after the patient on the doctor's list. The patients and controls were visited in their homes by a team of nurses who performed venepunctures and collected information for a questionnaire. Eye examinations were performed by a team of ophthalmologists. RESULTS: Significant differences were found between the cataract and control groups in 10 of the 18 examined plasma constituents. A constellation of three--bilirubin, alkaline phosphatase, and gamma glutamyl transpeptidase--was significantly higher in the cataract group, suggesting subclinical liver dysfunction as a risk factor. Steroid treatment and diabetes increased cataract risk. Endogenous basal plasma cortisol levels were raised in the cataract group, irrespective of steroid use and diabetic status. Alkaline phosphatase, calcium, glucose, and sodium were all raised in the cataract group. Given the raised total protein and albumin also found in the cataract group, the lower albumin/(total protein-albumin) ratio (an approximation for albumin/globulin ratio) may imply an increase in globulin, suggestive of possible (chronic) infection. Total cholesterol was lower in the cataract group. CONCLUSION: Human cataract in older age groups seems to be due to an accumulation of risk factors, even if individual mean concentrations are well within normal limits but, of course, differing significantly from the corresponding means in the control population.

Long-term effects of aluminium on the fetal mouse brain.

Potentially noxious substances may act as fetal teratogens at levels far lower than those required to produce detectable effects in adults, and behavioural teratogenicity may occur at levels lower than those which produce morphological teratogenesis. Aluminium (Al) is a potential neurotoxin in adults. Since pregnant women may be exposed to untoward levels of Al compounds under certain conditions, we have examined the long-term effects of treating the pregnant mouse with intraperitoneal or oral aluminium sulphate on brain biochemistry and behaviour of the offspring. The cholinergic system, as evaluated by the activity of choline acetyltransferase (ChAT), was affected differentially in different regions of the brain, and still showed significant effects in the adult. Differences between the intraperitoneal and oral series in the magnitude of effect seen in the regions of the brain probably reflect differences in the effective level of exposure. Growth rate and psychomotor maturation in the pre-weaning mouse were affected in the intraperitoneal series only, showing a marked post-natal maternal effect.

Ageing in the chick lens: in vitro studies.

In principle, ageing may be due to the interaction of several factors, including the accumulation of random changes both genomic and non-genomic, secondary changes in a tissue contingent upon the changing function of other tissues, and programmed non-random changes in the tissue-specific expression of various genes. The use of a single tissue comprising one cell type only, in which the major gene products are well defined, in which there is a well attested series of developmental and age-related changes in cell properties and gene expression and which can be studied and compared in vivo and in vitro, offers advantages for investigation of these questions. The vertebrate eye lens possesses these advantages. The crystallins (proteins expressed at super-abundant levels in the lens) are well characterised. The lens epithelial cells (LEC) grow readily and can differentiate into the lens fibre cells in vitro, and, finally, such terminally differentiated cells may also be derived, by a process of transdifferentiation, from neural retina cells (NRC) in vitro. Thus the effect on ageing changes of the tissue of origin may also be studied. This article reviews our previous studies on long-term changes in growth potential, differentiation capacity and crystallin expression of chick lens cells in ageing cultures, their overall similarity to events in vivo and the effect on ageing changes of genotypes affecting the growth rate. It presents new information on these genetic aspects, and on crystallin expression in long-term ageing cultures of transdifferentiated neural retina, and compares the behaviour of ageing chick lens cells with that reported for mammals.

The penetration of detergents into adult and infant eyes: possible hazards of additives to ophthalmic preparations.

In studies of surfactant penetration into the eye, radiolabelled detergent was found to penetrate the rabbit cornea, to accumulate readily in ocular tissues and to be released slowly. Repeated applications led to increasing binding of the detergent. Permeability, ocular and systemic uptake and retention were all greater in juvenile than in adult rabbits. Preliminary tests of the effects of surfactants on ocular cells in vitro using concentrations found in vivo, suggest that some low-level effects might occur, particularly in juveniles.

Comparative studies of normal and genetically hyperplastic lens epithelia II: lectin-induced cell agglutination and 125I-lectin uptake by cells.

The reactions of lectins with dissociated and cultured epithelial cells from hyperplastic lenses of two unrelated chick strains, Hy-1 and Hy-2, characterised by hyperplasia of the lens epithelium, and lenses of a normal genotype (N) were investigated using four different lectins. Three methods of monitoring lectin-binding to cell surfaces were employed. Each of the four lectins used showed an individual pattern of reactivity to separated membrane components. Data obtained with the three labelling methods showed the same trend viz: increased agglutinability and high affinity for binding of all four lectins by Hy-1 and Hy-2 cells. These results suggest that Hy-1 and Hy-2 lens epithelial cells are characterised by alteration in their cell surfaces.

Alterations in crystallin gene expression during subculture of chick lens cells.

We report here on the changes in crystallin gene expression during serial subculture of lens epithelial cells derived from day-old post-hatch chicks. Total cellular RNA from mass cultures were analysed by in vitro cell-free translation and by RNA blot (Northern) hybridization using a cloned delta-crystallin cDNA. Our results indicate that following subculture, lens epithelial cells which still retain the capacity for lens fibre differentiation (lentoid body formation) show a selective loss of delta-crystallin synthesis, and that this is related to the loss of delta-crystallin mRNA. The data suggest that older epithelial-cell populations give rise to lentoid bodies which in terms of crystallin gene expression closely resemble the later-formed cortical fibres of the adult chick lens. Tertiary cultures had an accelerated growth rate, formed no lentoids, contained no translatable alpha- or delta-crystallin mRNAs but still contained translatable beta-crystallin mRNAs.