Delta-crystallin mRNA in chick lens cells: mRNA accumulates during differential stimulation of delta-crystallin synthesis in cultured cells.

Increasing specialization for δ-crystallin synthesis is a prominent feature of the differentiation of chick lens epithelial cells into lens fiber cells and can be studied in cultured embryonic lens epithelia. Quantitation of δ-crystallin mRNA by molecular hybridizaton to a [³H]DNA complementary to δ-crystallin mRNA demonstrates that differentiation, both in ovo and in tissue culture, is associated with the accumulation of δ-crystallin mRNA. In the cultures, there is an overall stimulation of protein synthesis, including δ-crystallin mRNA during the first 5 hr in vitro. Between 5 and 24 hr in vitro there is a differential stimulation of δ-crystallin synthesis and an accumulation of δ-crystallin mRNA that can quantitatively account for this stimulation.     

Differentiation of lens and pigment cells in cultures of neural retinal cells of early chick embryos

Dissociated cells of neural retinas of 3.5-day-old chick embryos (stages 20–21) were cultured as a monolayer in order to examine their differentiation in vitro. These cells started to grow actively soon after inoculation and formed a confluent sheet within which neuroblast-like cells with long cytoplasmic processes were differentiated by 8 days. At about 16 days the differentiation of both lentoid bodies and foci of pigment cells was observed, while neuronal structure disappeared. The numbers of lentoid bodies and foci of pigmented cells continued to increase up to 30 days, when primary cultures were terminated. The increase in δ-crystallin content, as measured by quantitative immunoelectrophoresis assay using rabbit antiserum against δ-crystallin, was consistent with the increase in the number of lentoid bodies in cultures. The amount of α-crystallin per culture, estimated by the same technique as above, reached a maximum at 16 days and decreased slightly during further culture. The differentiation of both lentoid bodies and pigment cells was observed also in cultures of the second generation. The results demonstrate that cells of the undifferentiated neuroepithelium of 3.5-day-old embryonic retinas can achieve at least three differentiations, neuronal, lens, and pigment cells, in vitro. We discuss several differences between the present results and the previous ones from in vitro cultures of 8- to 9-day-old embryonic neural retinas.     

COMPARISON OF NEURONAL AND LENS PHENOTYPE EXPRESSION IN THE TRANSDIFFERENTIATING CULTURES OF NUERAL RETINA WITH DIFFERENT CULTURE MEDIA*

The effects of three different culture media (Eagle's MEM, F-12 and L-15) on the transdifferentiation of 8-day chick embryonic neural retina into lens cells, were examined with respect to the expression of two phenotypes. One type referred to neuronal specificity (as represented by the level of cholineacetyl-transferase, CAT, activity) and the other to lens specificity (as represented by content of α-and δ-crystallin). In 7-day cell cultures before the visible differentiation of lentoid bodies, CAT activity was detected in all media. But, its level was about 9 times higher in cultures with L-15 than in those with MEM and 3 times higher than in F-12. In 26-day cultures, CAT activity was practically undetectable. The production of α-and δ-crystallin was detected in cultures at 26 days. There were quantitative differences in the crystallin content with different media, and it was highest in cultures with L-15. The results indicate that conditions most favourable to the maintenance of the neuronal specificity in cell cultures of neural retina, can also support the most extensive transdifferentiation. The possibility of direct transdifferentiation of once neuronally specified cells into lens cells in cultures with L-15 has been suggested to explain the present results.     

Rates of protein synthesis in explanted embryonic chick lens epithelia: differential stimulation of delta-crystallin synthesis.

Cells in the central region of 6-day-old embryonic chick lens epithelia display morphological and biochemical changes, when cultured in medium supplemented with fetal calf serum, comparable to those of lens fiber cells differentiating in vivo. In the present study the rates of synthesis of total protein and of δ-crystallin were quantitated during the first day of culture by measuring (1) ³H-valine incorporation into bulk proteins and into δ-crystallin (isolated by quantitative immunoprecipitation), (2) the specific radioactivity of picomolar amounts of intracellular valine (determined by analysis of the ¹⁴C-dansyl-derivative of ³H-valine), (3) the amount of protein degradation occurring during the labeling period (estimated by “pulse-chase” experiments with cycloheximide), and (4) the number of cells in the explants (counted following dispersal with trypsin-EDTA). The results showed that total protein synthesis increased 1.7-fold per cell during the first 24 hrs in vitro. In contrast, δ-crystallin synthesis increased 2.8-fold per cell during this time. These experiments establish that δ-crystallin synthesis is differentially stimulated in epithelia cultured in serum-supplemented medium, and provide the basis for quantitative analysis of the mechanism controlling differential protein synthesis during lens fiber differentiation in vitro.     

Degradation of δ-crystallin mRNA in the lens fiber cells of the chicken

δ-Crystallin is the principal protein synthesized in the embryonic chicken lens. After hatching δ-crystallin synthesis decreases and eventually ceases. We have determined when the δ-crystallin messenger RNA (mRNA) disappears from the lens fiber cells during the first year of age by cell-free translation of lens RNA in a reticulocyte lysate, RNA blot (Northern) hybridization, and in situ hybridization. The hybridization was performed with a nick-translated, cloned δ-crystallin cDNA (pδCr2). δ-Crystallin mRNA was present in the lens until 3 months of age and disappeared between the third and fifth month after hatching. The in situ hybridization experiments indicated that the δ-crystallin mRNA was present throughout the lens fiber mass until 1 month after hatching and was greatly reduced in the cortical fiber cells thereafter. In contrast to earlier stages, then, the cortical fiber cells differentiating at the lens equator after about 1 month of age do not accumulate δ-crystallin mRNA. The data also indicate that the maximal half-life of functional δ-crystallin mRNA in the posthatched chicken lens is about 2 months.     

Differentiation of lens in cultures of neural retinal cells of chick embryos

When dissociated cells of neural retinae of 8-day-old chick embryos were cultured, monolayer sheets of epithelial cells were obtained. These cells proliferated actively. After about 30 days of culture, both lentoid bodies and pigment cells were differentiated in all plates. In the second and the third generation cultures, both differentiations were also observed. Lentoid bodies showed positive immunofluorescence for fluorescein-isothiocyanate-conjugated antiserum against δ-crystallin. Molecular constituents of lentoid bodies were very similar to those of lenses developing in situ, as revealed by immunodiffusion tests. Several lines of evidence for the “neural retinal” origin of lentoid bodies, as opposed to their being derived from lens cells inadvertently included in the original culture inocula are given. Some implications of the present results for the problem of “determination” are discussed.     

The control of δ-crystallin gene expression during lens cell development: Dissociation of cell elongation,cell division, δ-crystallin synthesis,and δ-crystallin mRNA accumulation

Previous studies have shown that freshly explanted 6-day-old embryonic chick lens epithelial cells elongate, differentially increase their synthesis of δ-crystallin, and accumulate δ-crystallin mRNA when cultured with fetal calf serum; in contrast, precultured serum-starved 6-day-old and freshly explanted 19-day-old embryonic epithelial cells divide when treated with fetal calf serum. We have explored whether the stimulation of δ-crystallin gene expression (as measured by δ-crystallin synthesis and δ-crystallin mRNA accumulation) is affected by inhibiting lens cell elongation with colchicine, and whether δ-crystallin gene expression is increased in lens epithelial cells stimulated to divide by treatment with fetal calf serum, as it is in those stimulated to elongate by treatment with serum. Three new findings were made in this study. First, the stimulation of δ-crystallin gene expression does not require elongation of the cultured lens cells. Second, a decreased proportion of δ-crystallin synthesis is observed in lens epithelial cells during normal development and during serum starvation; in neither case is this decrease associated with a reduction in the number of δ-crystallin mRNA sequences per cell. Finally, serum stimulation of lens cell division does not increase the proportion of δ-crystallin synthesis, but can promote the accumulation of δ-crystallin mRNA. Thus, the relative proportion of δ-crystallin synthesized during chick lens development is not solely a function of the number of δ-crystallin mRNA sequences in the lens cells.     

DIFFERENTIATION AND DEDIFFERENTIATION OF RAT LENS EPITHELIAL CELLS IN SHORT- AND LONG-TERM CULTURES

The crystallin synthesis of rat lens cells in cell culture systems was studied in relevance to their terminal differentiation into lens fibers. SDS-gel electrophoresis combined with several immunological techniques showed that γ-crystallin is a fiber-specific lens protein and is not localized in the epithelium of either newborn or adult lenses. When lens epithelial cells of newborn rats were cultured in vitro , α-crystaIlin was detected in many, but not all, of cells cultured for 10 days. Cells with α-crystallin gradually changed their shape into a flattened filmy form and finally differentiated into lentoid bodies. The differentiation of lentoid bodies was also found in cultures of epithelial cells obtained from adult lenses. The molecular constitution of lentoid bodies was the same as that of lens fibers in situ . The differentiation of lentoid bodies occurred successively for 5 months in cultures of lens epithelial cells. Most of the proliferating cells, however, lost α-crystallin during the culture period. Thereafter, they did not show any sign of further differentiation into lens fibers. Four clonal lines were established from these cells. One protein which is specific to the lens epithelium and the neural retina in situ (tentatively named as β_u-crystallin) was maintained in all lines, suggesting that some specific properties of ocular cells remain in the lined cells.     

Age-dependent differences in the ratio of synthesis of delta-crystallin polypeptides in cultured vitreous-free lenses of chick embryos

We have shown previously that the synthesis of the lower molecular weight polypeptides of δ-crystallin is differentially reduced and the intracellular $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$ ratio is markedly increased in the 15-day-old embryonic chick lens cultured for 3 hr without the vitreous body or in the presence of ouabain. Here we demonstrate that neither δ-crystallin synthesis nor cation concentration is affected in the cultured, vitreous-free 6-day-old embryonic chick lens unless it is treated with ouabain. These results show that the alteration in δ-crystallin synthesis promoted by removing the vitreous body of the embryonic cultured lens is a stage-specific phenomenon, and are consistent with our previous correlation between the ratio of synthesis of the δ-crystallin polypeptides and the intracellular concentration of electrolytes.     

Dissociated limb bud cells of chick embryos can express lens specificity when reaggregated and cultured in vitro

Limb bud cells of chick embryos (stages 23–24) were dissociated into single cells, reaggregated, and cultured in vitro for about a week. δ-Crystallin, generally thought to be a lens-specific protein in the chick, was detected in the aggregates by indirect immunofluorescent staining, double immunodiffusion test, and immunoelectrophoresis with specific antiserum against δ-crystallin. Cells containing δ-crystallin were distributed in epidermal cell clusters and also in mesenchymal tissues surrounding cartilage nodules in the aggregates. Those cells in mesenchymal tissues were shown to have originated from the mesoderm of the limb bud, and those in epidermal cell clusters probably originated from the ectoderm. The possible cellular origin of this appearance of δ-crystallin was discussed.     

The combined effect of acetylation and glycation on the chaperone and anti-apoptotic functions of human α-crystallin

N^ε-acetylation occurs on select lysine residues in α-crystallin of the human lens and alters its chaperone function. In this study, we investigated the effect of N^ε-acetylation on advanced glycation end product (AGE) formation and consequences of the combined N^ε-acetylation and AGE formation on the function of α-crystallin. Immunoprecipitation experiments revealed that N^ε-acetylation of lysine residues and AGE formation co-occurs in both αA- and αB-crystallin of the human lens. Prior acetylation of αA- and αB-crystallin with acetic anhydride (Ac₂O) before glycation with methylglyoxal (MGO) resulted in significant inhibition of the synthesis of two AGEs, hydroimidazolone (HI) and argpyrimidine. Similarly, synthesis of ascorbate-derived AGEs, pentosidine and N^ε-carboxymethyl lysine (CML), was inhibited in both proteins by prior acetylation. In all cases, inhibition of AGE synthesis was positively related to the degree of acetylation. While prior acetylation further increased the chaperone activity of MGO-glycated αA-crystallin, it inhibited the loss of chaperone activity by ascorbate-glycation in both proteins. BioPORTER-mediated transfer of αA- and αB-crystallin into CHO cells resulted in significant protection against hyperthermia-induced apoptosis. This effect was enhanced in acetylated and MGO-modified αA- and αB-crystallin. Caspase-3 activity was reduced in α-crystallin transferred cells. Glycation of acetylated proteins with either MGO or ascorbate produced no significant change in the anti-apoptotic function. Collectively, these data demonstrate that lysine acetylation and AGE formation can occur concurrently in α-crystallin of human lens, and that lysine acetylation improves anti-apoptotic function of α-crystallin and prevents ascorbate-mediated loss of chaperone function.     

Developmental changes in proteins of purified membranes of chicken lenses and evidence for contamination by cytoplasmic δ-crystallin

Urea-washed membranes from embryonic chick lenses (15 days old) and from the cortical and nuclear regions of adult chicken lenses (1 year) have been prepared by repeated centrifugation through discontinuous density gradients. The protein components of the isolated membranes have been examined by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate and urea. Proteins with molecular weights of 75 000, 56 000, 54 000, 48 000, 34 000, 32 000, 25 000, and 22 000 were present in all the membrane preparations, although their proportions changed during development. One additional protein, molecular weight 70 000, was seen only in the embryonic lens membranes. The greatest developmental change was the increase in 25 000 molecular weight protein from 12% in the embryonic lens to about 45% in the adult lens. Since it has been suggested that this protein is associated with gap junctions, its increase during development may reflect a corresponding increase in the number of gap junctions in the lens.The 50 000 molecular weight protein of embryonic lens membranes and membranes of adult nuclear lens fibers consisted at least partly of δ-crystallin, since δ-crystallin peptides could be identified in tryptic pepetide maps of the isolated protein after in vitro radioiodination. Peptide maps of the 50 000 molecular weight protein of cortical lens fiber membranes contained no identifiable δ-crystallin peptides, although it is possible that modified δ-crystallin peptides may be present. The level of cytoplasmic contamination of the membrane fraction was estimated by preparing lens membranes in the presence of added δ-[³⁵S]crystallin. The results indicated that cytoplasmic contamination contributes significantly to the presence of δ-crystallin in lens membrane preparations.     

Expression of Neuronal Specificities in "Transdifferentiating" Cultures of Neural Retina

Cells dissociated from the neural retina of embryonic chick differentiate into lens and pigment cells, when cultured in vitro. Using 3.5-day-old and 8.5-day-old chick embryos, we examined whether neuronal specificities would be expressed in such transdifferentiating cultures of neural retinal cells. The synthesis of acetylcholine and γ-aminobutyric acid (GABA) and the activity of choline acetyl transferase (CAT) was searched for in these cultures. The synthesis of an appreciable amount of these two putative neurotransmitters was detected in cultures of 3.5-day-old embryonic retinas by about 15 days. The activity of CAT was maximum in 7-day cultures of the 3.5-day-old materials and in 2-day cultures of the 8.5-day-old materials, and then decreased. Concomitant with the decrease of CAT-activity, δ-crystallin became detectable and increased thereafter. CAT-activity changed in parallel with the increase in the number of small neuroblast-like cells in cultures. The results demonstrate that the neuronal specificity identified by the appearance of acetylcholine and GABA and of the enzyme for the synthesis of acetylcholine is expressed in the early period of transdifferentiating cultures, which would later differentiate into lens and pigment cells. The possible mechanisms of the transition from neuronal to non-neuroretinal specificities of the transdifferentiating cultures are discussed.     

Distinct interactions of αA-crystallin with homologous substrate proteins, δ-crystallin and argininosuccinate lyase, under thermal stress

δ-Crystallin is a taxon-specific eye lens protein that was recruited from argininosuccinate lyase (ASL) through gene sharing. ASL is a metabolic enzyme that catalyzes the reversible conversion of argininosuccinate into arginine and fumarate and shares about 70% sequence identity and similar overall topology with δ-crystallin. ASL has a lower thermal stability than δ-crystallin. In this study, we show that the small heat shock protein, αA-crystallin, functions as a molecular chaperone, and enhanced thermal stability of both δ-crystallin and ASL. The stoichiometry for efficient protection of the two substrate proteins by αA-crystallin was determined by slowly increasing the temperature. N- or C-terminal truncated mutants of δ-crystallin co-incubated with αA-crystallin showed higher thermal stability than wild-type enzyme, and the stoichiometry for efficient protection was the same. Thermal unfolding of δ-crystallin or ASL in the presence of αA-crystallin followed a similar three-state model, as determined by circular dichroism analyses. A stable intermediate which retained about 30% α-helical structure was observed. Protection from thermal denaturation by αA-crystallin was by interaction with partly unfolded ASL or δ-crystallin to form high molecular weight heteroligomers, as judged by size-exclusive chromatography and SDS-PAGE analyses. Aggregate formation of ASL was significantly reduced in the presence of αA-crystallin. The extent of protection of ASL and δ-crystallin at different ratios of αA-crystallin were described by hyperbolic and sigmoidal curves, respectively. These results suggest the preferential recognition of partly unfolded ASL by αA-crystallin. In contrast, unstable δ-crystallin might trigger a cooperative interaction by higher stoichiometries of αA-crystallin leading to fuller protection. The different interactions of αA-crystallin with the two homologous but functionally different substrate proteins show its behavior as a chaperone is variable.     

NMR spin–echo studies of hydration properties of the molecular chaperone α-crystallin in the bovine lens

The water-binding properties of bovine lens α-crystallin, collagen from calf skin and bovine serum albumin (BSA), were investigated with various techniques. The water absorptive capacity was obtained in high vacuum desorption experiments volumetrically, and also gravimetrically in controlled atmosphere experiments. NMR spin–echo technique was used to study the hydration of protein samples and to determine the spin–spin relaxation times (T₂) from the protons of water, absorbed on the proteins. Isolated bovine lenses were sectioned into 11–12 morphological layers (from anterior cortex through nucleus to posterior cortex). Crystallin profiles were obtained for each lens layer using thin-layer isoelectric focusing in polyacrylamide gel (IEF). The water content in relation to dry weight of proteins was measured in individual morphological lens layers. During the water vapor uptake P/P₀=0.75, α-crystallin did not absorb water, suggesting that hydrophobic regions of the protein are exposed to the aqueous solvent. At P/P₀=1.0, the absorption of water by α-crystallin was 17% with a single component decay character of spin–echo (T₂=3 ms). Addition of water to α-crystallin to about 50% of its w/w in the protein sample showed T₂=8 ms with only one single component decay of the spin–echo signal. The single component decay character of the spin–echo indicates at the tightly bound water by α-crystallin. Under a relative humidity P/P₀=1.0, collagen and BSA absorbed correspondingly 19.3% and 28% of water and showed a two-component decay curve with T₂ of about 5 and 40 ms. The findings demonstrate the presence of two water fractions in collagen and BSA which are separated in space. The IEF data suggest a tight binding of water with α-crystallin with similar distribution patterns in the lens layers. The IEF data demonstrate a possible chaperone-like function for α-crystallin in the nucleus and inner cortex of the lens, but not in the outer cortex. To conclude, it was found that α-crystallin can immobilize and bind water to a greater extent than other proteins such as collagen and BSA. These results shed new light on structural properties of α-crystallin and have important implications for understanding the mechanism of the chaperone-like action of this protein in the lens and non-ocular tissues.     

In vitro interactions of histones and α-crystallin

The aggregation of crystallins in lenses is associated with cataract formation. We previously reported that mutant crystallins are associated with an increased abundance of histones in knock-in and knockout mouse models. However, very little is known about the specific interactions between lens crystallins and histones. Here, we performed in vitro analyses to determine whether α-crystallin interacts with histones directly. Isothermal titration calorimetry revealed a strong histone–α-crystallin binding with a K_d of 4 × 10^?7 M, and the thermodynamic parameters suggested that the interaction was both entropy and enthalpy driven. Size-exclusion chromatography further showed that histone–α-crystallin complexes are water soluble but become water insoluble as the concentration of histones is increased. Right-angle light scattering measurements of the water-soluble fractions of histone–α-crystallin mixtures showed a decrease in the oligomeric molecular weight of α-crystallin, indicating that histones alter the oligomerization of α-crystallin. Taken together, these findings reveal for the first time that histones interact with and affect the solubility and aggregation of α-crystallin, indicating that the interaction between α-crystallin and histones in the lens is functionally important.     

α-Crystallin: molecular chaperone and protein surfactant

Bovine lens α-crystallin has recently been shown to function as a molecular chaperone by stabilizing proteins against heat denaturation (Horwitz, J. (1992) Proc. Natl. Acad. Sci. USA, 89, 10449–10453). An investigation, using a variety of physico-chemical methods, is presented into the mechanism of stabilization. α-Crystallin exhibits properties of a surfactant. Firstly, a plot of conductivity of α-crystallin versus concentration shows a distinct inflection in its profile, i.e., a critical micelle concentration (cmc), over a concentration range from 0.15 to 0.17 mM. Gel chromatographic and ¹H-NMR spectroscopic studies spanning the cmc indicate no change in the aggregated state of α-crystallin implying that a change in conformation of the aggregate occurs at the cmc. Secondly, spectrophotometric studies of the rate of heat-induced aggregation and precipitation of alcohol dehydrogenase (ADH), β_L- and γ-crystallin in the presence of α-crystallin and a variety of synthetic surfactants show that stabilization against precipitation results from hydrophobic interactions with α-crystallin and monomeric anionic surfactants. Per mole of subunit or monomer, α-crystallin is the most efficient at stabilization. α-Crystallin, however, does not preserve the activity of ADH after heating. After heat inactivation, gel permeation HPLC indicates that ADH and α-crystallin form a high molecular weight aggregate. Similar results are obtained following incubation of β_L- and γ-crystallin with α-crystallin. ¹H-NMR spectroscopy of mixtures of α- and β_L-crystallin, in their native states, reveals that the C-terminus of βB2-crystallin is involved in interaction with α-crystallin. In the case of γ- and α-crystallin mixtures, a specific interaction occurs between α-crystallin and the C-terminal region of γ_B-crystallin, an area which is known from the crystal structure to be relatively hydrophobic and to be involved in intermolecular interactions. The short, flexible C-terminal extensions of α-crystallin are not involved in specific interactions with these proteins. It is concluded that α-crystallin interacts with native proteins in a weak manner. Once a protein has become denatured, however, the soluble complex with α-crystallin cannot be readily dissociated. In the aging lens this finding may have relevance to the formation of high molecular weight crystallin aggregates.     

Partially Folded Aggregation Intermediates of Human γD-, γC-, and γS-Crystallin Are Recognized and Bound by Human αB-Crystallin Chaperone

Human γ-crystallins are long-lived, unusually stable proteins of the eye lens exhibiting duplicated, double Greek key domains. The lens also contains high concentrations of the small heat shock chaperone α-crystallin, which suppresses aggregation of model substrates in vitro. Mature-onset cataract is believed to represent an aggregated state of partially unfolded and covalently damaged crystallins. Nonetheless, the lack of cell or tissue culture for anucleate lens fibers and the insoluble state of cataract proteins have made it difficult to identify the conformation of the human γ-crystallin substrate species recognized by human α-crystallin. The three major human lens monomeric γ-crystallins, γD, γC, and γS, all refold in vitro in the absence of chaperones, on dilution from denaturant into buffer. However, off-pathway aggregation of the partially folded intermediates competes with productive refolding. Incubation with human αB-crystallin chaperone during refolding suppressed the aggregation pathways of the three human γ-crystallin proteins. The chaperone did not dissociate or refold the aggregated chains under these conditions. The αB-crystallin oligomers formed long-lived stable complexes with their γD-crystallin substrates. Using α-crystallin chaperone variants lacking tryptophans, we obtained fluorescence spectra of the chaperone-substrate complex. Binding of substrate γ-crystallins with two or three of the four buried tryptophans replaced by phenylalanines showed that the bound substrate remained in a partially folded state with neither domain native-like. These in vitro results provide support for protein unfolding/protein aggregation models for cataract, with α-crystallin suppressing aggregation of damaged or unfolded proteins through early adulthood but becoming saturated with advancing age.     

A switch for transdifferentiation in culture: Effects of glucose on cell determination in chick embryo neuroretinal cultures

Chick embryo neuroretinal cells accumulate lens-specific δ-crystallin when cultured in Eagle's minimal essential medium containing 5% foetal calf serum and 5% horse serum (FH), but fail to do so if supplementary glucose is present (FHG). Culture growth rates are similar in the two media, but choline acetyltransferase activity is maintained for longer in FHG medium. By transferring cultures from FHG into FH medium and vice versa, we show that potential lens precursor cells survive for at least 21 days in FHG medium, while some retinal cells become irreversibly committed to crystallin production after 18 days in FH medium.     

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

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.