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

DEVELOPMENTAL BIOLOGY 59, 174-182 (1977)

The Control of &CrystaIIin Gene Expression during Lens Cell Development: Dissociation of Cell Elongation, Cell Division, 6Crystallin Synthesis, and &CrystaIIin mRNA Acdumulation C. BEEBE~ AND JORAM PIATIGORSKY

DAVID Section on Cellular Differentiation, Human Development,

Laboratory of Molecular Genetics, National Institute National Institutes of Health, Bethesda, Maryland

Received March 2,1977;

accepted April

of Child Health and 20014

25,1977

Previous studies have shown that freshly explanted 6-day-old embryonic chick lens epithelial cells elongate, differentially increase their synthesis of d-crystallin, and accumulate 6crystallin mRNA when cultured with fetal calf serum; in contrast, precultured serum-starved g-day-old and freshly explanted 1%day-old embryonic epithelial cells divide when treated with fetal calf serum. We have explored whether the stimulation of d-crystallin gene expression (as measured by &crystallin synthesis and 8-crystallin mRNA accumulation) is affected by inhibiting lens cell elongation with colchicine, and whether F-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 b-crystallin gene expression does not require elongation of the cultured lens cells. Second, a decreased proportion of S-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 8-crystallin mRNA sequences per cell. Finally, serum stimulation of lens cell division does not increase the proportion of S-crystallin synthesis, but can promote the accumulation of &crystallin mRNA. Thus, the relative proportion of 6-crystallin synthesized during chick lens development is not solely a function of the number

of Scrystallin

mRNA

sequences in the lens cells.

INTRODUCTION old embryonic lens epithelial differentiate Cellular differentiation is characterized synchronously if the culture medium conby the simultaneous occurrence of differ- tains fetal calf serum (Philpott and Couent morphological and biochemical phe- lombre, 1965; Piatigorsky et al., 1972a). nomena. In view of the complexity of dif- This differentiation involves marked cell ferentiation it is difficult to know which elongation (Piatigorsky et al., 1973; Mcevents are interdependent and which ones Lean and Finnegan, 19741,decreased cell are independently controlled. This infor- division (Philpott, 1970; Piatigorsky and mation is important in order to permit a Rothschild, 1972), differential stimulation more detailed investigation of the regula- of &crystallin synthesis (Piatigorsky et al., 1972a; Milstone and Piatigorsky, tion of cell differentiation. 1975), and accumulation of &crystallin We have investigated the relationships mRNA (Milstone et al., 1976). 6Crystallin between diverse processes during differenis the principal protein of the embryonic tiation in embryonic chick lens cells, since chick lens (Rabaey, 1962; Zwaan and this system can be experimentally manipIkeda, 1968; Genis-Galvez et al., 1968) and ulated in vitro (reviewed by Piatigorsky, represents 60-80% of the soluble protein 1975). Cells of primary explants of 6-daysynthesized by differentiating lens fiber cells (Yoshida and Katoh, 1971; Piatigor1 Present address: Department of Anatomy, Uniformed Services University of the Health Sciences, sky et al., 1972a). Bethesda, Md. 20014. When 6-day-old embryonic lens epithe174 Copyright 0 1977 by Academic Press, Inc. AI1 rights of reproduction in any form resewed.

ISSN 0012-1606

BEEBE AND PIATIGORSKY

lia are precultured for 12-24 hr in serumfree medium they do not elongate during subsequent exposure to fetal calf serum; instead, the starved cells show increased division when confronted with fetal calf serum (Philpott, 1970). Furthermore, fetal calf serum initiates cell division rather than cell elongation, even without prior serum starvation, in lens epithelia derived from Is-day-old chick embryos (Piatigorsky and Rothschild, 1972). These studies did not indicate whether lens epithelial cells stimulated to divide increase &crystallin synthesis and accumulate 6crystallin mRNA, as do elongating cells differentiating in response to fetal calf serum. In the present study, we have examined the relationship between embryonic chick lens epithelial cell elongation and F-crystallin gene expression in two different kinds of experiments. In the first, we compared 6-crystallin gene expression in elongating cultured lens epithelial cells of 6day-old embryos with gene expression in those cells in which elongation had been inhibited by colchicine (Piatigorsky et al., 197213).In the second type of experiment, we compared S-crystallin gene expression in elongating cells with gene expression in nonelongating, dividing lens epithelial cells in primary embryonic explants. Gene expression was tested by determining the synthesis of 6-crystallin and the amounts of &crystallin mRNA. The latter was quantitated by molecular hybridization using a 13HlDNA probe complementary to purified b-crystallin mRNA (Zelenka and Piatigorsky, 1976; Milstone et al., 1976). Our earlier studies with &crystallin 13HlcDNA demonstrated that this probe provides an accurate measure of mRNA sequences coding for &crystallin. The results show that the regulation of&crystallin gene expression is not dependent upon cell elongation, and that increased &crystallin mRNA accumulation is not sufficient for differential stimulation of &crystallin synthesis in the cultured lens epi-

GCrystallin

175

Gene Expression

thelia. A preliminary report of some of these data has been made elsewhere (Piatigorsky et al., 1976). MATERIALS

Culture

AND

and labeling

METHODS

of lens epithelia.

Fertile eggs of White Leghorn chickens (obtained from Truslow Farms, Inc., Chester-town, Md.) were maintained in a humidified forced-draft incubator at 37°C for 6 or 19 days. Lenses were surgically excised and placed into 2 ml of Ham’s F-10 medium (Ham, 1963) with or without 15% (v/v) fetal calf serum (Grand Island Biological Corp., virus-screened) as specified, contained in a sterile plastic tissue culture dish (Falcon Plastics, 10 x 35 mm). A central square, approximately 1 mm* from the 6-day-old and 2 mm2 from the 19-day-old embryonic lens epithelia, was cut out with a scalpel and was attached to the bottom of the dish, as described in detail elsewhere (Piatigorsky et al., 1972a). Four (19day-old) or 16 (6-day-old) explants were cultured per dish at 37°C in a water-saturated environment regulated to 5% CO,95% air for the times indicated. Antibiotics were not used, and occassional contaminated cultures were discarded. After the specified times of culture, the explants were washed in saline G (Puck et al., 1958), placed into 5-ml sterile plastic tubes (Falcon Plastics), and labeled in the tissue culture incubator for 60 min with 200 &i/ml of [35S]methionine (New England Nuclear Corp., 400-500 Ci/mmole) in 0.25 ml of Ham’s F-10 medium. The explants were washed twice with Ham’s F-10 medium and twice with ice-cold acetone before analysis by electrophoresis. Electrophoresis. Epithelial cell proteins were dissolved in 50 ~1 of 1% sodium dodecyl sulfate (SDS), 0.6% Tris-HCl, pH 6.8, 1% /3-mercaptoethanol, 10% (v/v) glycerol and a trace of phenol red, heated to 100°C for 3 min, and electrophoresed in a discontinuous 7.5-30% SDS-polyacrylamide gel gradient made in a Bio-Rad Model No. 221 dual vertical slab cell, as described else-

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DEVELOPMENTAL BIOLOGY

where (Beebe and Piatigorsky, 1976). The gels were stained with Coomassie brilliant blue R and were autoradiographed on Xray film (Kodak SB-5) after being dried under vacuum. Alternatively, the &crystallin band was cut from the gel, dissolved overnight in 0.1 ml of 30% H,O, at 6o”C, and assayed for radioactivity in a toluenebased scintillation fluid containing 3.7% Spectrofluor (Amersham/Searle Corp.) and 8.7% BBS 3 (Beckman Instruments). The total radioactivity applied to the gel was determined before electrophoresis by assaying a 2-~1 aliquot. Synthesis and hybridization tallin L3H]cDNA. F-Crystallin

of Gcrys-

[3H]cDNA was synthesized upon purified &crystallin mRNA isolated from 15-day-old embryonic lens fiber cells (Zelenka and Piatigorsky, 1974), utilizing 13H]deoxyguanosine triphosphate as given elsewhere (Zelenka and Piatigorsky, 1976). After purification by alkaline sucrose density gradient centrifugation, the 13H]cDNA was about 1100 nucleotides long and had a specific activity of 3 x lo7 cpm/pg, assuming that deoxyguanosine represents 25% of the nucleosides. RNA was extracted from thirty-two 6day-old and eight 19-day-old embryonic lens epithelia by dissolving the epithelia in 0.3 ml of 1% SDS, 0.14 M NaCl, and 0.01 M Tris-HCl, pH 7.6 and extracting with an equal volume of buffer-saturated phenol: chloroform: isoamyl alcohol (50:50:1). The aqueous phase was brought to 2% with potassium acetate, pH 5.0, reextracted with 0.3 ml of chloroform:isoamyl alcohol (50:1), and centrifuged at 10,000 rpm for 1 min at room temperature. The RNA was precipitated from the aqueous phase overnight at -20°C by addition of 2.5 vol of absolute ethanol. The precipitate was collected by centrifugation at 12,000g for 20 min at 4”C, washed with ethanol:ether (1:l) by centrifugation as above, dried under vacuum, and dissolved in either 20 (6-day-old) or 40 ~1 (19-day-old) of sterile deionized water. For hybridization, either 5-~1 aliquots or

VOLUME 59. 1977

1:l serial dilutions of 5-~1 aliquots of RNA were dried under vacuum in 1.5ml conical polypropylene tubes and were dissolved in 5 ~1 of 0.6 M NaCl, 0.2 n-&f Na2EDTA, 10 mM Tris-HCl, pH 7.5, and 0.5 pg of singlestranded salmon sperm DNA (Sigma Chemical Co.) containing 700 cpm of 6crystallin 13H]cDNA. The mixtures were incubated under a layer of paraffin oil at 75°C for 2 hr. Hybridization was stopped with 0.5 ml of 30 mM Na acetate, pH 4.5, 40 mM NaCl, 0.125 m&f ZnSO,, and 10 pgl ml of single-stranded DNA, and 5 pg of S, nuclease was added. After incubation at 45°C for 1 hr, the RNA:13HlcDNA hybrids were precipitated with ice-cold 10% trichloroacetic acid, collected on Whatman GF/C glass-fiber filters, and washed three times with cold 5% TCA. Filters were dried, and the percentage of hybridization was calculated from the radioactivity in these samples and the total amount of radioactivity added to the tubes after subtracting the amount of S,-resistant radioactivity in the 13HlcDNA preparation. Less than 3% of the [“HlcDNA in these tests was resistant to S, digestion. In order to determine the amount of &crystallin present in these samples, hybridization reactions containing different concentrations of purified S-crystallin mRNA were performed as above. Filters were assayed for radioactivity by scintillation counting. DNA determinations. DNA was quantitated from aliquots of the phenol extracts by the diaminobenzoic acid method of Kissane and Robins (1958) utilizing an Aminco-Bowman spectroflurometer with calf thymus DNA (Sigma Chemical Co.) as a reference. The aliquots used represented an equivalent of one to two explants. RESULTS

Stimulation of Cell Elongation or Cell Division in Cultured Embryonic Lens Epithelia

Previous investigations showed that serum-starved 6-day-old embryonic lens epi-

BEEBE AND PIATIGORSKY

thelial cells are stimulated to divide without cell elongation after 2 days in serumcontaining medium (Philpott, 19701, and that freshly explanted 19-day-old embryonic lens epithelial cells behave similarly after 3 days of culture in fetal calf serum (Piatigorsky and Rothschild, 1972). In the present study, we have standardized our conditions to 24 hr of culture in fetal calf serum. We confirmed, by scintillation counting and autoradiography of histological sections using 13Hlthymidine, that DNA synthesis is stimulated, without promotion of cell elongation, in both the serum-starved 6-day-old and the freshly explanted 19-day-old embryonic lens epithelial cells after 24 hr of culture in the presence of fetal calf serum. In contrast, the freshly explanted 6-day-old embryonic lens epithelial cells elongated and decreased DNA synthesis after 24 hr in medium supplemented with fetal calf serum. In the 6day-old embryonic explants after 48 hr of serum starvation, r3Hlthymidine incorporation was stimulated at least 30-fold after 24 hr of culture with serum, as measured by scintillation counting and percentage of labeled cells. In the freshly explanted 19lens day-old embryonic explants, [3Hlthymidine incorporation was stimulated threefold by both measurements after 24 hr of culture with serum. In both the starved 6-day-old and 19-day-old embryonic lens cells, there was an approximate 50% increase in the amount of DNA per explant after 24 hr of culture in the presence of fetal calf serum. On the other hand, both the amount of 13H]thymidine incorporation into DNA and the percentage of labeled cells decreased by approximately half when freshly explanted 6-dayold embryonic lens explants were cultured for 24 hr in the presence of fetal calf serum. Stimulation of &Crystallin Gene Expression without Cell Elongation in Cultured Embryonic Lens Epithelia

We have utilized colchicine to test whether cell elongation is required for the differential stimulation of 6-crystallin syn-

GCrystallin

Gene Expression

177

thesis and the corresponding accumulation of 6-crystallin mRNA which take place in g-day-old embryonic chick lens epithelia cultured in the presence of fetal calf serum. Colchicine has been shown to inhibit lens cell elongation in vitro (Piatigorsky et al., 1972b). &Crystallin synthesis was measured in lens explants labeled with [35S]methionine and fractionated by SDSpolyacrylamide gel electrophoresis. Inspection of the autoradiograms of these gels shows, as usual, an increase in the incorporation of labeled amino acid into 6crystallin by elongating lens cells during the first 24 hr of culture under these conditions (Milstone and Piatigorsky, 1975; Beebe and Piatigorsky, 1976) (Fig. 1). Earlier studies of cultured lens explants have established that this increase in labeling is indeed due to enhanced 6-crystallin synthesis and not to increases in the specific activity of the amino acid precursor pool or to decreased turnover of newly synthesized &crystallin (Milstone and Piatigorsky, 1975; Beebe and Piatigorsky, 1976). Figure 1 also shows that colchicine did not inhibit the increased incorporation of label into 6crystallin by the explants. Moreover, a molecular hybridization experiment utilizing &crystallin 13H]cDNA revealed that colchicine-treated lens epithelia contained as much S-crystallin mRNA as epithelia cultured without colchicine (Fig. 2). In this experiment, both sets of epithelia cultured for 24 hr contained 4.3 times more Gcrystallin mRNA than at the time of explantation. This represents a 3. l-fold elevation in the average &crystallin mRNA content per cell in the epithelia cultured without colchicine, after adjustment for the l.Cfold increase in cell number which occurs during cultivation (Milstone and Piatigorsky, 1975), and an even larger increase in the average amount of 6-crystallin mRNA per cell in the colchicine-treated epithelia, since cell division is prevented by this alkaloid. Cell elongation, then, is not required for either the increase in a-crystallin synthesis or the accumulation of &crystallin mRNA that occurs during fiber cell

178

DEVELOPMENTAL

0

5

BIOLOGY

VOLUME

59, 1977

24 24+C

s

NUMBER

OF EPITHEUA

x HR

FIG. 2. Hybridization of d-crystallin 13H]cDNA to RNA extracts from g-day-old embryonic chick lens epithelia (Al or to purified &crystallin mRNA (Bl. Immediately after explantation (0); after 24 hr of culture in fetal calf serum (A); after 24 hr of culture in fetal calf serum supplemented with 2 x 10m5M colchicine (0). The abscissa in A is the number of epithelia x hours, rather than R,t, because the actual amounts of RNA in the lens extracts were too small to determine by A,,,, absorption. The amount of Gcrystallin mRNA in the extracts can be determined by reference to the standard curve (B). There were approximately 0.075 and 0.32 ng of Scrystallin mRNA per explant immediately after explantation and after 24 hr of culture, respectively, in this experiment. Note the similarity of F-crystallin mRNA content in the epithelia cultured in the presence and absence of colchicine.

P

B A

FIG. 1. Autoradiograms of SDS-polyacrylamide gels of proteins labeled with [Vlmethionine from 6day-old embryonic lens explants cultured for 0, 5, or 24 hr in the presence of fetal calf serum alone, or for 24 hr in the presence of fetal calf serum plus 2 x 10e5 M colchicine. Identification of the 6-, p-, and (Ycrystallin bands is from Beebe and Piatigorsky (1976).

differentiation in cultured embryonic lens epithelia. We repeatedly observed three noteworthy alterations in the synthesis of non+ crystallin proteins in explants cultured with colchicine. Explants treated with colchicine for 24 hr showed a diminished incorporation of [35Slmethionine into the /3 and crB bands denoted in Fig. 1 and an elevated incorporation into the band immediately above that indicated by aB in Fig. 1. These induced changes in incorporation suggest that the synthesis of specific polypeptides may be coordinately reg-

BEEBE AND PIATICORSKY

GCrystallin

mated with lens cell elongation. Alternatively, these alterations in incorporation may represent specific secondary effects caused by colchicine. Relationship between &Crystallin Gene Expression and Stimulation of Cell Division in Cultured Embryonic Lens Epithelia

We examined the relationship between 6-crystallin gene expression in nonelongating epithelial lens cells which were stimulated to divide by fetal calf serum in explants derived from 6- and 19-day-old embryos. &Crystallin synthesis was assayed in these experiments by determining the percentage of label incorporated into &crystallin relative to the total incorporation into protein. This allowed a measure of specialization for &crystallin synthesis which obviated possible differences in the intracellular specific activity of the radioactively labeled amino acid used for the incorporation tests. The data given in Table 1 show that &crystallin represents a considerably smaller proportion of protein synthesized by both 6-day-old embryonic lens epithelia deprived of fetal calf serum for 48 hr and 19-day-old embryonic lens epithelial than was synthesized by freshly TABLE RELATIVE PROPORTION OF KRYSTALLIN Treatment

explantation hr with serum hr without serum hr with serum

explanted lens epithelia from 6-day-old embryos. Thus, both serum-starvation in vitro and aging of the embryonic lens epithelium in ovo cause a decrease in the specialization, or percentage of Scrystallin synthesis. The gradual loss of specialization for Fj-crystallin synthesis in the epithelial cells during development has been observed by numerous investigators (Zwaan and Ikeda, 1968; Genis-Galvez et al., 1968; Yoshida and Katoh, 1971). When lens cell division was stimulated by addition of fetal calf serum to either serum-starved 6-day-old or freshly explanted 19-day-old embryonic lens epithelia, the relative proportion of &crystallin synthesized after 24 hr was, at best, slightly increased in the former and not increased in the latter (Table 1). In contrast, when cell elongation was stimulated in freshly explanted 6-day-old embryonic lens epithelia, there was an approximately 2.5fold increase in the relative proportion of 6-crystallin synthesized after 24 hr. The amounts of S-crystallin mRNA present in the lens explants were determined by molecular hybridization experiments using &crystallin r3HlcDNA. The amount of hybridization of RNA from the 6- and 19day-old embryonic lens explants to the 1

SYNTHESIZED IN CULTURED EMBRYONIC LENS EPITHELIA”

Percentage

of Gcrystallin

6-dayb At 24 24 24

179

Gene Expression

synt~~~Ere$tive + Precultured

6-dayc

8.6 ‘- 1.2 20.0 -t 0.9

to total protein

synthesis

19-dayd 2.3 -t 0.1 2.5 e 0.4

4.1 * 0.4 5.5 + 0.5

4 a-Crystallin was isolated from labeled homogenates by SDS-polyacrylamide gel electrophoresis. b Calculated from experiments presented in Beebe and Piatigorsky (1976) and from unpublished data. Our earlier experiments (Piatigorsky et al., 1972a; Milstone and Piatigorsky, 1975) gave higher values for the relative proportion of Gcrystallin synthesized in the explanted lens epithelium. These, however, were based on less highly resolving gels and on immunoprecipitation of Gcrystallin, which may have some nonspecific adsorption of proteins contaminating the Gcrystallin precipitate, and which are probably overestimates. ’ Values represent an average of four (without serum) or six (with serum) experiments. These explants were precultured for 48 hr without serum before culture for 24 hr with or without fetal calf serum. d Values represent an average of five (0 time in culture) or three (24 hr in culture) experiments.

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DEVELOPMENTAL BIOLOGY

[3HlcDNA was proportional to the amount of tissue extract in these tests, as exemplified in Fig. 2. This indicates that there was an excess of &crystallin mRNA in the reaction mixtures and establishes the validity of calculating the amount of S-crystallin mRNA in the extracts by reference to a standard curve. Such standard curves were performed with each experiment under identical conditions using known concentrations of purified &crystallin mRNA. To obtain sufficient data for meaningful statistics, quadruplicate determinations were made at a concentration of lens RNA which gave between 20 and 50% hybridization of the L3H]cDNA. After calculation of the number of molecules of S-crystallin mRNA from the hybridization tests, the data were normalized on the basis of the amount of DNA in the epithelia. The results are summarized in Table 2 and demonstrate that average S-crystallin mRNA content per cell does not increase significantly in the precultured serumstarved 6-day-old embryonic lens epithelia when cell division is stimulated, whereas there is an approximately 25fold increase in Ccrystallin mRNA in the freshly explanted epithelia when cell elongation is stimulated by fetal calf serum. Unexpectedly and in contrast to the results for precultured 6-day-old embryonic lens epithelia, the average amount of &crystallin TABLE BCRYSTALLIN mRNA Treatment

explantation hr with serum hr without serum hr with serum

mRNA per cell doubled in 19-day-old embryonic lens explants when division was promoted by fetal calf serum, even though the relative proportion of Ccrystallin synthesized did not increase. DISCUSSION

The present experiments demonstrate that four events which occur during lens fiber cell differentiation in chick embryos, namely, cell elongation, cell division, differential synthesis of Ccrystallin, and accumulation of 6-crystallin mRNA, can be experimentally uncoupled from each other (see Table 3 for summary). Recent studies with the “aphakia” mouse mutant (Varnum and Stevens, 1968) also suggest that crystallin synthesis and lens morphogenesis are independently controlled in mammals. The “aphakia” lens is grossly disorganized and morphologically abnormal, yet, although crystallin synthesis is delayed (Zwaan, 1973), it is not prevented (Zwaan, personal communication). We cannot determine from the present data whether the small increases in both the proportion of &crystallin synthesized and the average cellular content of Ccrystallin mRNA in precultured lens epithelia after serum treatment occur uniformly in the cultured epithelial cells or are localized to a portion of the explants. The first 2

CONTENT IN CULTURED EMBRYONIC LENS EPITHELIA Molecules

of S-crystallin

6-day” At 24 24 24

VOLUME 59, 1977

mRNA per picogram Precultured

6-dayb

of DNA (* SEM) Is-day’ 1487 f 42 3019 f 226

1638 k 370 4243 k 434 1832 f 610 2417 T 402

@Data taken from Milstone et&. (1976). The conversion of molecules per cell to molecules per picogram of DNA was made by using 3.71 pg of DNA/cell. This was based on a direct comparison of cell number and DNA content in explanted epithelia (Milstone and Piatigorsky, 1977). * Values represent an average of 14 determinations from a total of 3 different experiments. These explants were precultured for 48 hr without serum before culture for 24 hr with or without fetal calf serum. c Values represent an average of 13 (0 time in culture) or 9 determinations (24 hr in culture) from 3 different experiments.

BEEBE AND PIATICORSKY

GCrystallin

181

Gene Expression

TABLE 3 CELL ELONGATION, CELL DIVISION, PROPORTIONAL SYNTHESIS OF NRYSTALLIN, AND ACCUMULATION OF NRYSTALLIN mRNA IN EMBRYONIC LENS EPITHELIA CULTURED FOR 24 Hours IN 15% FETAL CALF SERUM Embryonic age Cell elongation Cell division Approximate Approximate and treatment of relative inrelative inexplant crease in procrease in 6portion of 6 crystallin crystallin synmRNA conthesized tent ner cell 6-day 6-day, colchicine g-day, precultured without serum 19-day

Yes” No” Only periphery’

Decreasedb Inhibited’ Increased’

2-4’ 2-4 1.3

2-4d 2-4 None

No’

Increased#

None

2.0

a Philpott and Coulombre (1965); Piatigorsky et al. (1972a). * Philpott (197’0); Piatigorsky and Rothschild (19721. c Piatigorsky et al. (1972a); Milstone and Piatigorsky (1975); Beebe and Piatigorsky d Milstone et al. (1976). e Piatigorsky et al. (1972b). ’ Philpott (1970). 0 Piatigorsky and Rothschild (1972).

case would be an example of partial differential stimulation of S-crystallin gene expression in nonelongating lens cells and may represent an early event in the recovery of fiber-forming ability by the serum-starved lens explants (Philpott and Coulombre, 1968). On the other hand, the elevated proportion of S-crystallin synthesis may be confined to the peripheral cells of these explants, which generally show some elongation (Philpott, 1970). Our most surprising findings were that the 19-day-old and the serum-deprived 6day-old embryonic lens epithelial cells contained approximately the same number of S-crystallin mRNA sequences per cell as did the freshly explanted 6-day-old embryonic epithelial cells, and, yet, the relative proportion of S-crystallin was only onequarter to one-half that of the freshly explanted epithelia from 6-day-old embryos; moreover, the average content of S-crystallin mRNA per cell doubled in the cultured 19-day-old embryonic lens epithelia without a corresponding increase in the relative proportion of S-crystallin synthesized. This is an important finding and indicates that the relative proportion of S-crystallin synthesis in the lens cells is not only a

(19761.

function of the amount of S-crystallin sequences present, but is also controlled either by specific post-transcriptional or translational regulation of S-crystallin n-RNA or by the relative activity or amount of non-S-crystallin mRNAs in the cells. In view of the present results, then, one might speculate that the high proportion of S-crystallin synthesis obtained during lens fiber cell differentiation (7040% of protein synthesis) is not controlled strictly by the cellular levels of S-crystallin mRNA, but may also require a specific regulation of the synthesis of non+crystallin proteins. We have previously shown such a differential regulation of protein synthesis during lens fiber cell differentiation in vitro, which involves a decrease in the rate of synthesis of noncrystallin polypeptides (Beebe and Piatigorsky, 1976). This view of the mechanism of specialization for S-crystallin gene expression during lens fiber cell differentiation agrees well with a recent model for the specialization of globin gene expression during erythrocyte differentiation, in which destabilization of nonglobin mRNA sequences was required in order to achieve the high

182

DEVELOPMENTAL BIOLOGY

proportion of globin mRNA present in the mature cells (Aviv et al., 1976).

VOLUME 59, 1977

AVIV, H., VOLOCH, Z., BASTOS, R., and LEVY, S.

STONE,L. M., AND SHINOHARA,T. (1976). Regulation of &crystallin gene expression during development of the embryonic chick lens. Les Colloques de L’Institute de la Sante et de la Recherche Medicale 60, 85-104. PIATIGORSKY, J., and ROTHSCHILD, S. S. (1972). Loss during development of the ability of chick embryonic lens cells to elongate in culture: Inverse relationship between cell division and elongation. De-

(1976). Biosynthesis and stability of globin mRNA in cultured erythroleukemic Friend cells. Cell 8,

velop. Biol. 28, 382-389. PIATIGORSKY, J., ROTHSCHILD, S. S., and MILSTONE,

We thank Dr. Peggy Zelenka for critically reading this manuscript and Ms. Catherine Kunkle for expert typing. REFERENCES

495-503. BEEBE, D. C., and PIATIGORSKY, J. (1976). Differen-

tial synthesis of crystallin and noncrystallin polypeptides during lens fiber cell differentiation in vitro. Exp. Eye Res. 22, 237-249. GENIS-GALVEZ, J. M., MAISEL, H., and CASTRO, J.

(1968). Changes in chick lens proteins with aging. Exp. Eye Res. 7, 593-602. HAM, R. G. (1963). An improved nutrient

solution for diploid Chinese hamster and human cell lines. Exp. Cell Res. 29, 515-526. KISSANE, J. M., and ROBINS, E. (1958). The fluorometric measurement of deoxyribonucleic acid in animal tissues with special reference to the central nervous system. J. Biol. Chem. 233, 184-188. MCLEAN, B. G., and FINNEGAN, C. V. (1974). Observations on chick-embryo lens hi&genesis in vivo and in vitro. Canad. J. Zool. 52, 345-352. MILSTONE, L. M., and PIATIGORSKY, J. (1975). Rates of protein synthesis in explanted chick lens epithelia: Differential stimulation of S-crystallin synthesis. Develop. Biol. 43, 91-100. MILSTONE, L. M., and PIATIGORSKY, J. (1977). 6Crystallin gene expression in embryonic chick lens epithelia cultured in the presence of insulin. Exp. Cell Res. 105, 9-14. MILLSTONE, L. M., ZELENKA, P., and PIATIGORSKY,J. (1976). &Crystallin mRNA in chick lens cells: mRNA accumulates during differential stimulation of &crystallin synthesis in cultured cells. Develop. Biol. 48, 197-204. PHILPOTT, G. W. (1970). Growth and cytodifferentiation of embryonic chick lens epithelial cells in vitro. Exp. Cell Res. 59, 57-68.

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