DEVELOPMENTAL BIOLOGY 60, 278-286 (1977)

Differentiation

of Lens and Pigment Cells in Cultures Cells of Early Chick Embryos

of Neural Retinal

MASASUKE ARAKI AND T. S. OKADA' Laboratory

for Cell Differentiation and Morphogenesis, Institute for Biophysics, University of Kyoto, Kyoto 606, Japan Received March 18,1977;

Faculty

of Science,

accepted in revised form June 8,1977

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 af?er inoculation and formed a confluent sheet within which neuroblast-like cells with long cytoplasmic processes were differentiated by 6 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 8-crystallin content, as measured by quantitative immunoelectrophoresis assay using rabbit antiserum against 6crystallin, was consistent with the increase in the number of lentoid bodies in cultures. The amount of a-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 g-day-old embryonic neural retinas.

INTRODUCTION

Previous studies have shown that the cells of neural retinas of 8- to g-day-old chick embryos differentiate in vitro into both lens-like structures and pigment cells (Itoh et al., 1975; Okada et al., 1975; Redfern et al., 1976; Okada, 1977). Another example of such a switch in differentiation is found in the differentiation of the lens from the pigmented retina (tapeturn) of 8to g-day-old chick embryos (Eguchi and Okada, 1973) and of adult newts (Eguchi et al ., 1974). There seems to be little doubt that studies on the mechanism of such a phenomenon are advantageous to our understanding of problems in the determination of the stability of the differentiated state (Eguchi, 1976; Okada, 1976). An aim of the present study is to examine the differentiation in vitro of neural retinal cells ’ To whom reprint requests should be addressed.

taken at a much earlier stage in chick embryonic development. We were interested in determining whether or not immature neural retinal cells of earlier embryos can achieve “foreign” differentiations as do cultures of cells from the same tissue from 8- to g-day-old embryos. MATERIALS

AND METHODS

Isolation of “issues to be Cultured Whole :yeballs were removed from chick embryos at 3.5 days of incubation (stages 20-21; Hamburger and Hamilton, 1951) and placed in Ca- and Mg-free Hanks’ saline with 1% fetal calf serum (CMF-S). An incision was made around the circumference of the periphery of the iris. After the anterior part comprising the lens and iris was taken off, the rest of the eyeball was left in CMF-S for 30 min (Fig. 1A). Then, the neural retinal layer was

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ISSN 0012-1606

ARAKI

AND

OKADA

Differentiation

FIG. 1. (A) The wall of eye of 3.5day-old embryo, from which the neural retina (NR) was taken for cell culture. PT, tapetum. x 140. (B) A piece of the isolated neural retina. x 140. Histological sections were stained with hematoxylin and eosin.

easily separated from the adjacent layer of the tapetum, perhaps owing to the absence of interdigitations between these two layers in these early embryos (Coulombre, 1965). The isolated neural retinal pieces were carefully examined under highpower magnification with a dissecting microscope, and any portions suspected to be contaminated by pigment cells were carefully removed (Fig. 1B; cf. also Okada et al., 1975). Cell Suspension

Cleaned pieces of the neural retinas were rinsed three times with CMF-S and once with 0.08% EDTA in CMF-S and were incubated at 37°C in the latter solution. After 40 min of incubation, the dissociation medium was removed without disrupting the softened tissue pieces. Then, fresh culture medium was added, and a cell suspension was obtained by gentle pipetting. Cell Culture

In primary cultures, cells dissociated from two eyes were planted into each Fal-

of Neural

Retinal

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Cells

con culture dish (3 cm in diameter) with 2 ml of the culture medium. Although an accurate cell count was impossible due to the presence in the suspension of a number of small cell clusters in addition to single cells, each plate contained approximately 2.0-2.5 x lo5 cells. Crude collagen was prepared according to Konigsberg (Konigsberg, 1971) with some modifications. The procedures for coating the whole culture substrate with collagen were the same as given by Konigsberg. Culture medium used in this study was Eagle’s minimum essential medium supplemented with 8% fetal calf serum, 2 mM glutamine, 26 mM sodium bicarbonate, 0.2 n-&f ascorbic acid (Itoh, 1976), and 6.8 mA4 sodium pyruvate (Rein and Rubin, 1971). The medium was changed every 2 days throughout the whole culture period. The procedure for subculturing was the same as given previously (Okada et al., 1971). Measurement

of Melanin

Content

Photometric determination of melanin content was performed according to Oikawa and Nakayasu (1975). Cells were harvested mechanically from cultures at appropriate intervals and were completely dissolved in an appropriate volume of “Soluene 100” (0.5 N solution of dimethyln-dodecyl ammonium hydroxide in toluene, Packard Instrument Co., Inc.) by incubation for 3 hr in a water bath at 40°C. Each sample, dissolved with 4 ml of “Soluene 100,” contains about 1.8 x 10’ cells of 6-day, and 6.0 x lo6 of ll-, 16-, 24-, and 30day cultures, respectively. For use as the O-day material, about 1.0 x 10’ cells from freshly dissociated neural retinas were dissolved with 2 ml of “Soluene 100.” The relative amounts of melanin were estimated spectrnphotometrically from the absorbance of e;.ch solution at 400 nm. Measurement

of Crystallin

Content

The antisera against purified CY-and 6crystallins and the saline extract of chick lens fibers were prepared by the proce-

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dures described previously (Okada et al., 1973; 1975). The specificity of these antisera was checked by immunoelectrophoresis (Fig. 2). The quantitative estimation of antigen by Laurell’s method was performed by electrophoresing test antigens into an antibody-containing agarose gel (Laurell, 1966). Cultured cells were harvested at appropriate intervals by scraping cell layers with rubber policemen and were then homogenized in Hanks’ saline. The number of culture plates used was varied according to the stages of culturing, and each saline homogenate used for measuring the antigen content was prepared from about 1.0 x lo7 cells of 6-day cultures in 0.2 ml of saline, from 1.3 X lo7 cells of ll-day cultures, or from 6.5 x lo6 cells of 16-, 24-, or 30-day cultures in 0.5 ml of saline, respectively. As the O-day material, the homogenate of freshly dissociated neural retinas, comprising about 5.0 x lo6 cells, was used. In a 50 n-&f barbital buffer, pH 8.6, containing 2 n-&f calcium lactate, 1% Agarose was melted on a boiling water bath and cooled to 45°C. To 10 ml of fluid agarose, 0.2 ml of the antiserum was added. This Agarose-antiserum mixture was poured into a mold and left overnight at 4°C. Thereafter, holes (3 mm in diameter) were punched on the immunoplate and filled with exactly 5 ml of one of the samples. The electrophoresis was run in a cold room for 6 hr at 10 V/cm in the same barbital buffer used with the Agarose gels, The distance travelled by the immunoprecipitate was measured.

VOLUME 60, 1977

A

FIG. 2. Precipitation lines appearing in immunoelectrophoresis in Agarose. L, the saline extract of homogenate of lenses of go-day-old embryos. (A) Anti-6 in the top well and anti-lens fiber extracts in the bottom well. (B) Anti-a in the top well and antilens fiber extracts in the bottom well. Note anti-a and anti-8 react only with their homologous antigens.

min after inoculation, and the cells spread as a monolayer on the following day (Fig. 3A). Many singly dissociated cells formed small aggregates while in suspension and then adhered to the substrate. The total cell number started to increase soon after plating (Fig. 4). This is in contrast to results of culturing neural retinal cells of older embryos, in which the total cell number decreased for about 1 week after inoculation and then started to increase (Okada et al., 19751. A nearly confluent cell sheet was established within 4 days. At about 8 days characteristic foci (F,) consisting of small-sized cells appeared (Fig. 3B). They were apparently similar to the rosettes reconstituted RESULTS from dissociated neural retinal cells (FuAppearance of Lentoid Bodies and Pig- jisawa, 1973). These foci were interconment Cells in Vitro nected by long cytoplasmic processes (perhaps axons) of neuronal cells (Fig. 3B, arPrimary culture. The cells from neural retinas at the stages used in this study are rows). In cultures at this stage, some small piles of transparent and swollen cells apof rather uniform size and larger than comparable cells from 8- to g-day-old em- peared (Fig. 3C, arrow). The possibility bryos. Inoculated cell suspensions con- that these piles may be the first indication of the differentiation of lentoid bodies was tained a number of small clusters consistchecked later by following the change of ing of about two to 10 cells. These clusters attached to the culture substrate within 30 selected piles during longer culturing pe-

FIG. 3. Photographs showing characteristic changes in a primary culture. (A) Spreading of clusters or aggregates attached to the culture substrate; 1 day after inoculation. (B) The appearance of small-celled foci (F,) encircled with dotted line. Note interconnection between foci by axon-like processes indicated by arrows; 8 days after inoculation. (C) The appearance of transparent and highly swollen cells (indicated by arrow); 8 days after inoculation. (D) The appearance of foci consisting of small epithelial cells (F,) encircled with dotted iine; 10 days after inoculation. -X 125.

riods. A different type of foci (FJ consisting of densely packed small epithelial cells was also observed (Fig. 3D). Cells in F, seemed to grow faster than other cells. At about 16 days, cultures reached confluent?, and visible differentiation of a number of lentoid bodies (Okada et al., 1971; 1973) and pigment cells was conspicuous. The differentiation of pigment cells was due mostly to the pigmentation of cells in F, (Fig. 5A). The overall state of cultures at this stage was equivalent to that obtained in about a 40-day culture of neural retinal cells of older embryos (Okada et al., 1975). The cell number continued to increase up to 30 days, when primary cultures were terminated. At this stage, cultures were composed mostly of lentoid bodies and pigment cells (Fig. 5B). Neuronal cells and

rosette-like foci had disappeared. So far, five experiments have been conducted, each using a different inoculum. We found the results described above to be quite reproducible under present culture conditions. Subcdtures. Cells were transferred into secondary cultures at three different stages of primary culture, 6, 17, and 30 days after primary inoculation, as indicated in Fig. 4. When cells of g-day primary cultures were transferred, they spread on the substrate within 24 hr, and further growth and differentiation were essentially similar to those in primary cultures, except for the absence of both neuronal cells and rosette-like foci (F,). Cells transferred from 17-day primary cultures also grew rapidly after plating, and by 20 days the secondary cultures

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were composed mostly of a pigmented epithelial sheet and lentoid bodies, though the number of the latter structure was less than that in the secondary cultures derived from g-day primary cultures. In the secondary cultures of the 17-day cells, only a few lentoid bodies appeared within a few days after inoculation. Perhaps these lentoid bodies were derived from cells that were already lens-committed and ready to express visible differentiation in primary cultures. The results of secondary culture

L---d 0

10

20

30

ON!5IN CUT&E FIG. 4. A typical growth curve of neural retinal cells in primary cultures starting from a single inoculum. Each circle represents an average of duplicate hemocytometer counts of cells harvested from two plates. T, stages when secondary cultures were prepared.

VOLUME 60, 1977

of 30-day cells were essentially the same as those for secondary cultures of cells from 17-day primary cultures, though lentoid bodies were even fewer here, and they disappeared after about 30 days in the secondary cultures. Changes in Melanin Cultures

Content

in Primary

The relative amount of melanin in cultures was optically determined by using the homogenates prepared from cells harvested from primary cultures at different stages. Starting at 11 days after inoculation, optical density units began to increase rapidly and at a nearly constant rate until 30 days (Fig. 6A). Also, simultaneously, the number of pigmented foci in the living cultures increased (Fig. 6B). The first appearance of pigmented foci was recognized microscopically in some of the 11-day and in all of the 14-day primary cultures. This is about 10 days faster than in cultures of neural retinal cells of 8- to 9day-old chick embryos (Itoh et al., 1975). Changes in CY-and B-Crystallin in Primary Cultures

Contents

For calibration, purified &crystallin from chick lenses was run at several different concentrations (Fig. 7). Under fixed electrophoretic conditions, the relationship between the distance travelled by the immunoprecipitate and the antigen con-

FIG. 5. (A) The appearance of deeply pigmented cells in F, (P) and of lentoid bodies (L); 16 days after inoculation. (B) The differentiation of pigment cells and lentoid bodies 24 days after inoculation. x 125.

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Differentiation

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of Neural Retinal Cells

ot, 0

20

10

30

0

10

20

30

DAYS IN CULTURE

DAYS IN CULTURE

FIG. 6. (A) The change in the relative amount of melanin during the culture period determined by optical absorbance per 6 x lo6 cells. (B) The increase in the number of pigmented foci counted from a single living culture plate throughout the culture period.

II

I

0 CRVSTALLIN

0.1

I

a2

I

a3

CONCENTRATION

I

0.4 (mglml

I

a5 1

FIG. 7. The relationship between the distance travelled by the immunoprecipitate and the concentration of the test antigen, purified &crystallin.

centrations was linear below 0.35 mg of antigen/ml. As long as the migration distance is shorter than 20 mm, the concentration of the antigen contained in unknown samples can thus be calculated from the calibration unit. Figure 8 shows the results of estimation of b-crystallin content in the primary cultures. Deltacrystallin was first detected 11 days after inoculation, and it increased markedly per dish until 16 days and gradually thereafter (Fig. 9A). This pattern of change in 6-

FIG. 8. A quantitative homogenates anti-6 serum. cultures were

photograph showing the results of the estimation of S-crystallin content in of cultured cells by Laurell’s test using Cells of 0-, 6-, ll-, 17-, 24-, and 30-day tested.

crystallin content was roughly consistent with the increase in the number of lentoid bodies counted directly in living culture plates (Fig. 9B). The change in a-crystallin content per dish was different from that of b-crystallin. Although a-crystallin was also detected first at 11 days (Fig. 9B), its content reached a maximum at 16 days and then decreased slightly during further culture. The results were also confirmed by SDS-polyacrylamide gel electrophoresis (unpublished data). It was revealed by immunoelectrophoresis using anti-lens fi-

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DEVELOPMENTALBIOLOGY

VOLUME 60, 1977

A

ii 0

10 20 OAYS IN CLILNRE

30

0

10 20 OAYS IN CULTURE

30

FIG. 9. (A) The increase in the number of lentoid bodies counted from a living culture plate throughout the culture period. (B) The change of Q- (dashed line) and &crystallin (solid line) contents determined by Laurell’s test (cf. Fig. 8) in primary cultures.

ber serum that three groups of crystallins, (Y, /3, and 6, were present in 30-day cultures. DISCUSSION

It has been demonstrated previously that the neural retinal cells of 8- to g-dayold chick embryos will differentiate into lentoid bodies and pigment cells when cultured in vitro (Okada et al., 1975; Itoh et al., 1975; Okada, 1976). Such “foreign” differentiations were obtained in the present study utilizing neural retinas of 3.5dayold embryos (stages 20 and 21). The possibility that nonneural retinal differentiations in cultures might be due to a selective outgrowth of a small number of lens and retinal pigment cells inadvertently included in the original inocula is very unlikely, as discussed by Okada et al. (1975). In addition, histological observations of isolated neural retinas showed that the isolated pieces were very “pure” without any adherent cells (Fig. 1B). Thus, it can be concluded that the neural retina of 3.5day-old embryos can differentiate in cell culture into lens and pigmented cells. There are several differences between the present results and the previous in. vitro studies using neural retinal cells of 8to g-day-old embryos. (1) The increase in cell number per plate occurred soon after

plating, within 24 hr here, while it occurred only after 7 days in the cultures of the previous studies. (2) The onset of “foreign” differentiations was detected earlier here, lo-12 days, instead of 30 days as in the previous cases. (3) The appearance of neuronal cells occurred after about 4 to 6 days here and within 24 hr in the previous cases. In the present study some changes were adopted in the culture methods. For instance, cells were dissociated with EDTA only and without trypsin treatment; ascorbic acid and sodium pyruvate were added to the culture medium (Itoh, 1976), and culture plates were coated with collagen. These alterations probably do not account for the differences observed, because the neural retinal cells of &dayold chick embryos, when cultured with the same methods used here, did not differ in their growth and differentiation from cells cultured by the older technique except for the somewhat earlier differentiation of lentoid bodies (unpublished data). It should be noted here, however, that the use of trypsin should be avoided for successful culture of dissociated neural retinal cells of earlier embryos. Few of the cells from trypsin-dissociated younger retinas adhered to the substrate and grew. Probably, the surface of younger cells has properties different from that of older cells

ARAKI AND OKADA

Differentiation

and is more easily damaged by trypsin. The neural retina of embryos used in the present study consists of a morphologically very homogeneous cell population. In situ, the thickness and cytoarchitectural complexity of the neural retina starts to increase after 3.5 days of incubation, as a result of the rapid increase in total cell number due to high mitotic activity (Coulombre, 1955). Perhaps the immediate growth in vitro of younger cells may be ascribed to the inclusion in the innocula of a large number of cells which had mitotic potential in situ. “Transdifferentiation”, i.e., rechanneling of differentiation in already differentiated cells, occurs in the case of lens differentiation from pigment cells (Eguchi and Okada, 1973; Eguchi et al., 1974; Eguchi, 1976; Okada, 1976). The differentiation of lens and pigmented cells from older neural retinal cells also can be considered as another example of the same phenomenon with some reservations (Okada et al., 1975; Okada, 1976). In these cases the appearance of rechanneled differentiation is preceded by the process of dedifferentiation or the loss of already expressed specificity and is necessarily a long process. It is possible that the very fast appearance of lentoid bodies and pigmented cells from earlier neural retinal cells in vitro is not by “transdifferentiation,” but by “differentiation” not preceded by the step of dedifferentiation. It is very probable that the neural retina of 3.5-day-old embryos may contain quite a number of cells that are not only undifferentiated, but also uncommitted (or undetermined) with respect to their pathways in further differentiation. The delayed appearance of neuronal cells in cultures of cells from younger retinas is perhaps due to the fact that even those cells committed to differentiate along the normal pathway are still immature in the neural retina of 3.5-day-old embryos, while many of the neural retinal cells of 8- to g-day-old embryos may be fully matured and ready for neuronal differentiation in vitro immediately.

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Contents of crystallins and melanin in cultures at different stages were determined as shown in Figs. 6A ;nd 9B. ‘IOgether with the results of direct counts of the numbers of lentoid bodies and pigmented foci in living cultures (Figs. 6B and 9A), these data provide a rough picture of differentiation in terms of production of marker molecules. A comparison of the patterns of change of a-crystallin and &crystallin content during a 30-day period of culturing is of special interest. Both proteins initially appeared at about the same time (11 days) and both accumulated rapidly. The amount of a-crystallin, however, started to decrease after 16 days, while 6crystallin content still increased, though gradually, after this time. Thus, the pattern in the change of &crystallin content in cultures is correlated well with the change in the number of lentoid bodies, whereas the pattern in a-crystallin content is not. This fact is understandable, if cells with the properties of lens epithelial cells are predominant in 16-day cultures, and if these cells mature into lens fibers during prolonged culture in uitro. Very poor lens differentiation in the secondary cultures derived from 30-day primary cultures may support this interpretation, since it suggests that immature lens cells with mitotic potential (lens epithelial cells) are quite rare in older primary cultures. It is known that the differentiation of lens epithelial cells to lens fibers is accompanied by the alteration in the synthetic pattern of different crystallin molecules (Clayton, 1970; Yoshida and Katoh, 1971). Fiber cells synthesize mainly $-crystallin, and a- and /3-crystallins much less. Recently, Beebe and Piatigorsky (1976; see also Piatigorsky et al., 1973, and Milstone and Piatigorsky, 1975) reported that each protein species is synthesized at different rates at different stages of differentiation of the lens fibers in vitro. The difference in the patterns of accumulation between aand &crystallins obtained here may indicate that situations similar to those above

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may exist in the case of lens differentiation from the neural retina in vitro. The present study concerns a more complex system of multiple differentiation from neural retinal cells to lens fibers, pigment cells, and neuronal cells. The cell population in cultures is naturally not homogeneous, and the relative number of different cell types must be constantly changed during culture. The possibility that interactions between different cell types influence the final differentiation of cultures should be examined to elucidate the mechanism of the very rapid and extensive “foreign” differentiations in vitro of the neural retina of 3.5day-old embryos shown in the present study. We thank Drs. G. Eguchi, M. Yanagida, and K. Yasuda for valuable advice, Dr. J. Pilzer for reading the manuscript, and Miss A. Ishihara and Miss R. Takayasu for their help in its preparation. The work was supported by a grant for Basic Cancer Research from The Japan Ministry of Education, Culture, and Science. REFERENCES BEEBE, D. C., and PIATIGORSKY, J. (1976). Differential synthesis of crystallin and noncrystallin polypeptides during lens fiber cell differentiation in vitro. Exp. Eye Res. 22, 231-249. CLAYTON, R. M. (1970). Problems of differentiation in the vertebrate lens. Curr. Top. Develop. Biol. 5, 115-180. COULOMBRE, A. J. (1955). Correlations of structural and biochemical changes in the developing retina of chick. Amer. J. Anat. 96, 153-193. COULOMBRE, A. J. (1965). The eye. In “Organogenesis” (R. L. DeHaan and H. Ursprung, eds.), pp. 219-251. Holt, New York. ECUCHI, G. (1976). “Transdifferentiation” of vertebrate cells in cell culture. In “Embryogenesis in Mammals,” Ciba Foundation Symposium, No. 40, pp. 241-258. Elsevier, Amsterdam. EGUCHI, G., ABE, S., and WATANABE, K. (1974). Differentiation of lens-like structures from newt epithelial cells in vitro. Proc. Nut. Acad. Sci. USA 71, 5052-5056. EGUCHI, G., and OKADA, T. S. (1973). Differentiation of lens tissue from the progeny of chick retinal pigment cells cultured in vitro: A demonstration of a switch of cell type in clonal cell culture. Proc. Nat. Acad. Sci. USA 70, 14951499. FUJISAWA, H. (1973). The process of reconstruction of histological architecture from dissociated retinal cells. Wilhelm Roux’ Archiv. Entwicklungs-

VOLUME 60, 1977 mech. Orgunismen 171, 312-330. HAMBURGER, V., and HAMILTON, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49-92. ITOH, Y., OKADA, T. S., IDE, H., and EGUCHI, G. (1975). The differentiation of pigment cells in cultures of chick embryonic neural retinae. Develop. Growth Differ. 17, 39-50. ITOH, Y. (1976). Enhancement of differentiation of lens and pigment cells by ascorbic acid in cultures of neural retinal cells of chick embryos. Develop. Biol. 54, 157-162. KONIGSBERG, I. R. (1971). Diffusion-mediated control of myoblast fusion. Develop. Biol. 26, 133-152. LAURELL, C. -B. (1966). Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal. Biochem. 15, 45-52. MILSTONE, L. M., and PIATIGORSKY, J. (1975). Rates of protein synthesis in explanted embryonic chick lens epithelia: Differential stimulation of &crystallin synthesis. Develop. Biol. 43, 91-100. OIKAWA, A., and NAKAYASU, M. (1975). Solution of eumelanin showing no light scattering. Anal. Biothem. 63, 634-637. OKADA, T. S. (1976). “Transdifferentiation” of cells of specialized eye tissues in cell culture. In “Tests of Teratogenicity In Vitro,” pp. 91-105. North Holland, Amsterdam. OKADA, T. S. (1977). A demonstration of lens forming-cells in neural retina in clonal cell culture. Develop. Growth Differ. 19, 47-55. OKADA, T. S., EGUCHI, G., and TAKEICHI, M., (1971). The expression of differentiation by chicken lens epithelium in vitro cell culture. Develop. Growth Differ. 13, 323-335. OKADA, T. S., EGUCHI, G., and TAKEICHI, M. (1975). The retention of differentiated properties by lens epithelial cells in clonal cell culture. Develop. Biol. 45, 318-329. OKADA, T. S., ITOH, I., WATANABE, K., and EGUCHI, G. (1975). Differentiation of lens in cultures of neural retinal cells of chick embryos. Develop. Biol. 45, 318-329. PIATIGORSKY, J., ROTHCHILD, S. S., and MILSTONE, L. M. (1973). Differentiation of lens fibers in explanted embryonic chick lens epithelia. Develop. Biol. 34, 334-345. REDFERN, N., ISRAEL, P., BERGSMA, D., ROBISON, W. G., JR., WHIKEHART, D., and CHADER, G. (1976). Neural retinal and pigment epithelial cells in culture: Patterns of differentiation and effects of prostaglandins and cyclic-AMP on pigmentation. Exp. Eye Res. 22, 559-568. REIN, A., and RUBIN, H. (1971). On the survival of chick embryo cells at low concentrations in culture. Exp. Cell Res. 65, 209-214. YOSHIDA, K., and KATOH, A. (1971). Crystallin synthesis by chick lens. II. Changes in synthetic activities of epithelial and fiber cells during embryonic development. Exp. Eye Res. 11, 184-194.

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

DEVELOPMENTAL BIOLOGY 60, 278-286 (1977) Differentiation of Lens and Pigment Cells in Cultures Cells of Early Chick Embryos of Neural Retinal MASA...
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