Alterations in pH and Serum Concentration have Contrasting Effects on Normal and "Foreign" Pathways of Differentiation in Cultures of Embryonic Chick Neuroretinal Cells

Nine-day chicken embryo neuroretinal cells transdifferentiate into both lens and pigment cells after 3–4 weeks when cultured in MEM medium containing 10% foetal calf serum at pH 7.4. At pH 6.8. the appearance of lens crystallins is retarded and cholineacetyltransferase (CAT) activity persists for longer, whereas at pH 8.0 crystallins appear earlier and CAT activity declines more rapidly. Cell survival and culture growth are about 10% lower at pH 6.8 than at pH 8.0. If the concentration of foetal calf serum (FCS) is increased from 10% to 25% (at pH 7.4), cell survival and growth are both promoted, crystallins appear slightly earlier and CAT activity declines more rapidly. Converse effects are observed with 5 % serum, accumulation of crystallins being greatly inhibited and CAT activity prolonged. Crystallin production in cultures with 10% or 25% chicken serum (CS) is much less extensive than in similar FCS cultures, but in cultures with 5 % CS, crystallins appear more rapidly, reaching higher levels than in 5 % FCS cultures. However, the pattern of CAT activity in response to different serum levels is similar for both CS and FCS. This might imply the presence of some factor(s) able to stimulate transdifferentiation in FCS, whereas CS can apparently inhibit this process.     

Stress Protein and Crystallin Synthesis During Heat Shock and Transdifferentiation of Embryonic Chick Neural Retina Cells

Embryonic chick neural retina responds to heat shock by the synthesis of "stress" polypeptides with molecular weights of 85 and 70 kd. Both stress proteins are synthesised from newly-transcribed messenger RNA. Sodium arsenite induces an additional stress protein of MW 25 kd. The heat shock response does not change during culture and subsequent transdifferentiation, and crystallin synthesis is not coinducible with the heat-shock proteins. We have also examined the pattern of protein synthesis at various stages of culture in both monolayer and aggregate systems; although changes in the protein synthetic profine are evident, there is no stress protein induction above basal levels at any time. Whilst mammalian α crystallin (B₂ chain) exhibits considerable homology to four small Drosophila heat-shock proteins, no significant antigenic similarity is apparent between δ crystallin and the major avian heat shock proteins. Thus during transdifferentiation, (a) the crystallin proteins do not behave in a manner analogous to stress proteins; moreover (b) crystallin production is not mediated by stress proteins resulting from a culture-induced stress response.     

Pathways of Differentiation in Chick Embryo Neuroretinal Cultures

Markers of neuronal cell differentiation (GABA accumulation, choline acetyltransferase activity) are shown to increase initially and then decline sharply in monolayer cultures of 9 day embryo neuroretinal (NR) cells. A glial marker (glutamine synthetase, GSase) is precociously inducible by hydrocortisone (HC) in dense'monolayer' NR cultures (containing aggregates of neuronal cells overlying the glial sheet) as well as in chick embryo retinal explants. The induced level of GSase activity is not maintained in the continued presence of HC, but rather declines by 20 days in vitro. Choline acetyltransferase (CAT) activity is higher in HC-treated cultures than in controls only during the period when induced GSase activity is detectable. Furthermore, the subsequent transdifferentiation of lens cells (monitored as δ crystallin content) in these cultures is delayed by 10 days and much reduced in extent when HC is present throughout the culture period.
We suggest a simple model to account for these results, on the basis of recent evidence that lens cells are derived mainly from the retinal epithelial cells (immature Müller glia) of 9-day embryonic NR, and that transdifferentiation results from a change in cell determination during the early stages of'monolayer' culture. In outline, our model proposes that early dedetermination of the retinal glia is associated with a decline of neuronal cell markers (dedifferentiation) followed eventually by loss of the neuronal cells. Hydrocortisone, by inducing transient glial cell differentiation (GSase activity), both prolongs the expression of a neuronal marker (CAT) and also reduces later transdifferentiation into lens.     

Expression of Toxin Receptors on Cell Surfaces in Transdifferentiating Cultures of Neural Retina

It has often been asked which of the cell types found during the early stages of culturing embryonic chick neural retina can undergo transdifferentiation into lens in vitro. Since neuronal cell-surface toxin receptors are maintained in NR cultures for much longer than internal neuronal enzymes (e.g. choline acetyltransferase), and since the transdifferentiation process can be greatly accelerated by preparing reaggregates of neural retina cells after about 10 days of preculture as "monolayers", a direct test of this question became feasible. 7 or 9 day embryonic chick neural retina cells, precultured for 10–12 days as monolayers, were dissociated and reaggregated under continuous gyration. Reaggregates were maintained for 8 days in the presence of either tetanus toxin or FITC-conjugated α-bungarotoxin, to permit surface-bound toxins to become internalised via receptor turnover. The reaggregates were then dissociated, stained with rabbit antitoxin and FITC-conjugated anti-antibody in the case of tetanus toxin-labelled material, and restained with a rat or mouse antibody against chick δ crystallin followed by the appropriate rhodamine-conjugated anti-antibody. Although both FITC/toxin-labelled cells (putative neurones) and rhodamine/δ crystallin-labelled cells (transdifferentiated lens cells) were abundant, no examples of double-labelled cells were observed with 9 day starting material, and only a very few with 7 day starting material. We conclude that the vast majority of differentiated neuronal cells expressing surface receptors for these toxins do not transdifferentiate directly into lens cells.     

Embryonic Serum Factors Required for Transdifferentiation of Chick Embryo Neuroretinal Cells in Culture

The accumulation of δ crystallin (chick lens marker) in cultures of 9 day chick embryo neuroretinal cells is strongly promoted by chick embryo extract (CEE) or foetal calf serum (FCS), but much less so by adult sera (horse, chicken and newborn bovine serum). The "transdifferentiation-promoting" (TP) activity of FCS is absent from dialysed FCS but is largely recovered in the initial dialysis medium (F_DM). Similarly, the initial dialysis medium from CEE (E_DM) shows strong TP activity, whereas that from chicken or from horse serum does not. We conclude that the proposed TP factor(s) is (are) of relatively low molecular weight. By contrast, horse serum contains macromolecular factor(s) able to inhibit the TP activity of E_DM or F_DM. Rapid loss of neuronal cells (including those expressing choline acetyltransferase activity) is also observed in media based on F_DM, though whether this effect is mediated by the proposed TP factor(s) has not been determined. The TP activity is not directly related to growth rate or cell density, since cultures in F_DM alone grow poorly yet still accumulate δ crystallin.     

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.     

Determination of Chick Neuro-retinal Cells in Culture: Serum Factors Acting between 12 and 20 Days of Culture Influence the Extent of Subsequent Lens Cell Formation

Embryonic chick neuroretinal cells transdifferentiate into lens cells during culture in media containing foetal calf serum (F). This process is largely inhibited if horse serum plus supplementary glucose (Hg) is substituted for F. This paper explores the effect of medium changeover (from F to Hg or vice versa ) on the subsequent appearance of lens-specific δ-crystallin. If cultures are changed from Hg to F up to 12 days of culture, δ-production at 40 days is similar to that for controls maintained in F throughout. Changeovers between 14 and 17 days progressively inhibit subsequent δ production, and after 19 days in Hg, lens transdifferentiation cannot be induced by F. Conversely, if cultures are maintained in F for up to 17 days, a changeover to Hg blocks transdifferentiation, whereas similar transfers performed after 19 days give increased δ production. These results suggest that some retinal cells which will eventually form lens in vitro become so determined between the 12th and 20th days of culture. A mixture of 50% Hg and 50% F medium (FHg) does not support δ production even after 60 days, but in the absence of supplementary glucose (FH), δ appears in considerable amounts by 30 days.