DEVELOPMENTAL
BIOLOGY
60,
305-309
(1977)
Lens Differentiation
in Cultures of Neural Retinal Fetuses
T. S. OKADA,*
YASUDA,* MICHIKO HAYASHI,~ AND GORO EGUCHI” *
Institute
for Biophysics,*
KUNIO Faculty
Cells of Human
YOSHIO HAMADA,*
of Science and Department of Ophthalmology,t University of Kyoto, Kyoto 606, Japan
Received March 18,1977;
Faculty
of Medicine,
accepted in revised form June 8,1977
Neural retinal cells of human fetuses at approximately 9 and 15 weeks after conception were cultured in vitro. In early stages of culturing (up to about 10 days), a number of neuronal cells with axon-like processes were formed. Within about 20 days, many neuronal cells started to degenerate, while a number of “lentoid bodies” were identified by immunoelectrophoresis and immunofluorescent techniques using anti-rat lens serum which cross-reacted with human crystallins. Ultrastructural observations also revealed that cells of “lentoid bodies” represent typical profiles of lens fibers. INTRODUCTION
Several examples of a switch of once specialized cells into altered directions of differentiation have been found in cultures of cells from vertebrate eye tissues (Eguchi and Okada, 1973; Okada et al ., 1975; Itoh et al., 1975; Redfern et al., 1976; Okada, 1977). There seems to be no doubt that the ability of cells to transdifferentiate in such a way in vitro is associated with the high regenerative ability of lost parts in newt eyes in. situ (Eguchi et al., 1974; Eguchi, 1976). The occurrence of transdifferentiation in vitro, however, is not limited to newts, but is found also in several eye tissues of 8- to g-day-old chick embryos, which do not regenerate lost parts in situ (Van Deth, 1940). Thus, we were interested in examining whether or not transdifferentiation in vitro might occur in mammalian materials, in which regenerative abilities are minimal (Hay, 1966). In this communication, we now give evidence that neural retinal cells of human fetuses can switch by differentiation into lens cells 1 Present address: Research Institute for Molecular Biology, Faculty of Science, Nagoya University, Nagoya, Japan.
in vitro,
in the same manner as that shown in avian and amphibian cells. MATERIALS
AND METHODS
Aborted human fetuses with normal appearance were provided by a local gynecologist in cold sterile Hanks’ saline. Upon receipt, the eyeballs were removed and stored at 4°C in cold Eagle’s MEM for not more than 15 hr before culture experiments were conducted. The present study concerned two culture inoculations: one from a fetus approximately 9 weeks after conception at Witchi’s stage 34 (H-9 cells), and another from a fetus approximately 15 weeks after conception at stage 35 (H-15 cells) (cf. Altman and Dittmer, 1962). Clean pieces of the neural retina with negligible contamination by cells from other eye tissues were dissociated with 0.01 M EDTA in Ca- and Mg-free Hanks’ saline. Approximately 5 x lo5 cells were planted in each 30-mm plastic culture dish (Falcon) with 3 ml of Eagle’s MEM supplemented with 10% fetal calf serum, 0.03% glutamine, and 0.01% ascorbic acid. Before use, the culture substrate was coated with 305
Copyright 0 1977by Academic Press,Inc. All rights of reproduction in any form reserved.
ISSN 0012-1606
306
DEVELOPMENTAL BIOLOGY
collagen (Hauschka and Konigsberg, 1966). The procedure for subculturing was the same as that utilized in cultures of chick cells (Okada et al., 1971). For immunological identification of the lens nature of the cultured cells, antiserum against rat lens was prepared in rabbits. This antiserum cross-reacted with most of the crystallins of human lens, while it was highly lens-specific and without recognizable cross-reaction with any of the saline homogenates of non-lens tissues tested (Hamada and Okada, unpublished observations). An immunofluorescent study by the indirect method, using fluorescent goat antibody against rabbit y-globulin (Okada et al., 19731, and immunoelectrophoresis in Agarose gel containing the antiserum (Laurell, 1966) were conducted. Test antigens for the latter test were saline homogenates of cells harvested mechanically from primary cultures. Electron microscopic observations of cultured cells were made according to the procedures given previously (Eguchi and Okada, 1971). RESULTS
Primary
Culture
H-15 cells. After inoculation, most dissociated cells formed small aggregates, which adhered to the culture substrate within 24 hr. Then, aggregates started to spread, while they became interconnected with axon-like processes. Thereafter, a number of cells appeared flattened. Their number increased, and they covered the whole culture substrate. Simultaneously, many thick elongated fibers, probably nerve fibers, were differentiated overlaying the flattened cells. Cultures became confluent by 15 days. About this time, there appeared foci consisting,of epithelial cells which were smaller and more refractile than the flattened cells which were initially recognized. It is notable that these foci were observed in the location
VOLUME 60, 1977
where the development of neuronal structure had been predominant (Figs. 1 and 2). By 16 days, swollen and transparent cells were found within the foci- (Fig. 3). With further culturing, the number of these cells was increased. Sometimes they were transformed into “bottle cells”, the presence of which was a reliable marker for identifying lens differentiation in cell cultures of chick eye tissues (Okada et al., 1971). Often, several transparent cells were assembled to form the structure called “lentoid bodies” (Okada et al., 1971, 1973; see Fig. 4). Other single swollen cells became highly vacuolated and sometimes degenerated leaving only a rim of cytoplasmic materials. By 30 days, many of the neuronal structures and large flattened cells started to degenerate and to be lost from the cultures. In immunofluorescence observations, all cells of 12-day cultures were negative. On the other hand, a number of fluorescencepositive cells were recognized in the cell populations of 2%day cultures. Positive fluorescence was recognized in cells of “lentoid bodies,” Itbottle cells,” and single swollen cells with different grades of vacuolization (Fig. 5). Small epithelial cells in the foci were also weakly positive. The saline homogenate of 25day cultures reacted positively with anti-rat lens in Laurell’s test in Agarose, while that of lo-day cultures did not (Fig. 6). Electron-microscopic observation of the ultra-thin sections of “lentoid bodies” shows that these structures are composed of very elongated cells nearly free of mitochondria and endoplasmic reticulum but with a number of polysomes (Fig. 7). These profiles probably permit us to consider these elongated cells as lens fibers (Okada et al., 1973). H-9 cells. Within 10 days after inoculation, a confluent layer of flattened cells was formed. Neuronal cells with long cytoplasmic processes were differentiated by about 10 days. Apparently the number
307
BRIEF NOTES
of neuronal cells was much less here than in cultures of H-15 cells. They appeared only in several restricted areas. By 16 days, adjacent to these neuronal areas, foci of small epithelial cells were seen. Lentoid differentiation was initiated by about 30 days in the same manner as that described for cultures of H-15 cells.
Subcultures Cells harvested from a lo-day primary culture of H-15 cells were transferred to secondary cultures. Typical foci of smaller epithelial cells appeared within 7 days, and lentoid differentiation was first recognized by 13 days. Differentiation of neuronal structures was very poor in secondary cultures. DISCUSSION
Cells of vertebrate eye tissues have the very characteristic property of transdifferentiation, a property of the switching of once specialized cells into an altered direction (for reviews, see Eguchi, 1976; Okada, 1976). So far, examples of transdifferentiation have been found in avian embryos and adult newts. In the present study, we have presented evidence that the neural retina of the human fetus can alter its differentiation and form a lens. The authentic lens nature of lens-like cells and of “lentoid bodies” appearing in cultures was identified by both immunological and ultrastructural methods. It can be remarked that, in the case of the neural retinas of human fetuses, altered differentiation into a lens in vitro occurred more rapidly than it did in chick FIG. 1. Appearance of a focus of small epithelial cells encircled with dotted line. Note the presence of a nerve fiber-like structure(N) adjacent to the focus; lo-day culture of H-15 cells. x 100. FIG. 2. Epithelial cell sheet derived from the focus shown in Fig.1; 13-day culture of H-15 cells. x 100. FIG. 3. Appearance of transparent and swollen cells (indicated by arrows) in the epithelial-celled focus; 17-day culture of H-15 cells. x 100.
308
DEVELOPMENTALBIOLOGY
VOLUME 60, 1977
FIG. 4. Appearance of a “lentoid body” (L) and a “bottle cells” (B) in a focus of epithelial cells; 25-day culture of H-15 cells. x 100. FIG. 5. An immunofluorescence micrograph of a focus of epithelial cells in a 2%day culture of H-15 cells. Note the presence of strongly fluorescent cells. x 100.
FIG. 6. Immunelectrophoresis by Laurell’s method in an Agarose gel containing antiserum against rat lens: a, the homogenate of a IO-day culture of H-15 cells; b, the homogenate of a 25-day culture of H-15 cells; c, the homogenate of the retina of a 15-week-old fetus; e, the homogenate of the lens of a 15-week-old fetus.
embryos. It took about 20 days here, while taking 60 days in the neural retinas of 8- to g-day-old chick embryos (Okada et al., 1975). Another characteristic feature in the human cells was very extensive formation of neuronal structures in the early stages of cultures. As a rule, the lens dif-
ferentiation occurred adjacent to the areas where elongated axon-like processes had been particularly abundant. The results suggest that the neural retinas of human fetuses contain some bipotent cells, each of which can differentiate in either the neural or the lens direction. Also, the possibility that there are nonneural cells in the retina which, under the proper conditions, could form a lens, is not excluded. Factors responsible for eliciting lens formation from the neural retina remain to be elucidated. The present study showed that the instability in the state of differentiation of the cells of eye tissues is not found exclusively in species with high regenerative ability but may be a general property in all vertebrates from amphibians up to mammals. At least in fetuses, some cells of well-developed mammalian eye tissues still have the potential to alter their state of differentiation. Perhaps in the eyes of higher vertebrates, an expression of “foreign” differentiation is strictly repressed in the in situ ocular environment, and the number of cells capable of forming lens is too small to regenerate a lost lens. The instability in differentiation of pigmented retinal cells of human fetuses will be communicated in a subsequent paper (Yasuda et al., unpublished manuscript).
BRIEF NOTES
FIG. 7. An electron micrograph of an ultra-thin section of a “lentoid body” in a 27-day culture cells. Note that the cytoplasm is nearly empty except for many polysomes. x 15,000. We thank Mr. M. Araki for help in some parts of the experiments, and Miss A. Ishihara and R. Rakayasu for help in the preparation of the manuscript. The work was supported by a grant for Basic Cancer Research from the Japan Ministry of Education, Culture, and Science. REFERENCES ALTMAN, P. I., and DITTMER, D. S. (eds.) (1962). Prenatal vertebrate development. In “Biological Handbook,” pp. 275-297. Federation of American Societies for Experimental Biology, Washington, D.C. EGUCHI, 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. Nat. Acad. Sci. USA 71, 5052-5056. EGUCHI, G., and OKADA, T. S. (1971). Ultrastructure of the differentiated cell colony derived from a singly isolated chondrocyte in in vitro culture. Develop. Growth Diff. 12, 297-312. EGUCHI, G., and OKADA, T. S. (1973). Differentiation of lens tissue from the progeny of chick retinal pigment cells cultured in uitro: A demonstration of a switch of cell types in clonal cell culture. Proc. Nat. Acad. Sci. USA 70, 1495-1499. HAUSHKA, S. D., and KONIGSBERG, I. R. (1966). The influence of collagen on the development of muscle clones. Proc. Nut. Acad. Sci. USA 55, 119-126. HAY, E. D. (1966). “Regeneration.” Holt, Rinehart & Winston, New York.
309
of H-15
ITOH, Y., OKADA, T. S., IDE, H., and EGUCHI, G. (19751. The differentiation of pigment cells in cultures of chick embryonic neural retina. Deuelop. Growth Differ. 17, 39-50. LAURELL, C. -B. (1966). Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal. Biochem. 15, 45-52. 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., ~~~TAKEICHI, M. (1973). The retention of differentiated properties by lens epithelial cells in clonal cell culture. Deuelop. Biol. 34, 321-333. OKADA, T. S., ITOH, Y., WATANABE, K., andEcvc~r, G. (1975). Differentiation of lens in cultures of neural retinal cells of chick embryos. Develop. Biol. 45, 318-329. REDFERN N., ISRAEL, P., 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. VAN DETH, J. H. M. G. (1940). Induction et rogcncration du cristallin chez l’embryon de la poule. Actu Neer. Morphol. 3, 151-169.
BIOLOGY
60,
305-309
(1977)
Lens Differentiation
in Cultures of Neural Retinal Fetuses
T. S. OKADA,*
YASUDA,* MICHIKO HAYASHI,~ AND GORO EGUCHI” *
Institute
for Biophysics,*
KUNIO Faculty
Cells of Human
YOSHIO HAMADA,*
of Science and Department of Ophthalmology,t University of Kyoto, Kyoto 606, Japan
Received March 18,1977;
Faculty
of Medicine,
accepted in revised form June 8,1977
Neural retinal cells of human fetuses at approximately 9 and 15 weeks after conception were cultured in vitro. In early stages of culturing (up to about 10 days), a number of neuronal cells with axon-like processes were formed. Within about 20 days, many neuronal cells started to degenerate, while a number of “lentoid bodies” were identified by immunoelectrophoresis and immunofluorescent techniques using anti-rat lens serum which cross-reacted with human crystallins. Ultrastructural observations also revealed that cells of “lentoid bodies” represent typical profiles of lens fibers. INTRODUCTION
Several examples of a switch of once specialized cells into altered directions of differentiation have been found in cultures of cells from vertebrate eye tissues (Eguchi and Okada, 1973; Okada et al ., 1975; Itoh et al., 1975; Redfern et al., 1976; Okada, 1977). There seems to be no doubt that the ability of cells to transdifferentiate in such a way in vitro is associated with the high regenerative ability of lost parts in newt eyes in. situ (Eguchi et al., 1974; Eguchi, 1976). The occurrence of transdifferentiation in vitro, however, is not limited to newts, but is found also in several eye tissues of 8- to g-day-old chick embryos, which do not regenerate lost parts in situ (Van Deth, 1940). Thus, we were interested in examining whether or not transdifferentiation in vitro might occur in mammalian materials, in which regenerative abilities are minimal (Hay, 1966). In this communication, we now give evidence that neural retinal cells of human fetuses can switch by differentiation into lens cells 1 Present address: Research Institute for Molecular Biology, Faculty of Science, Nagoya University, Nagoya, Japan.
in vitro,
in the same manner as that shown in avian and amphibian cells. MATERIALS
AND METHODS
Aborted human fetuses with normal appearance were provided by a local gynecologist in cold sterile Hanks’ saline. Upon receipt, the eyeballs were removed and stored at 4°C in cold Eagle’s MEM for not more than 15 hr before culture experiments were conducted. The present study concerned two culture inoculations: one from a fetus approximately 9 weeks after conception at Witchi’s stage 34 (H-9 cells), and another from a fetus approximately 15 weeks after conception at stage 35 (H-15 cells) (cf. Altman and Dittmer, 1962). Clean pieces of the neural retina with negligible contamination by cells from other eye tissues were dissociated with 0.01 M EDTA in Ca- and Mg-free Hanks’ saline. Approximately 5 x lo5 cells were planted in each 30-mm plastic culture dish (Falcon) with 3 ml of Eagle’s MEM supplemented with 10% fetal calf serum, 0.03% glutamine, and 0.01% ascorbic acid. Before use, the culture substrate was coated with 305
Copyright 0 1977by Academic Press,Inc. All rights of reproduction in any form reserved.
ISSN 0012-1606
306
DEVELOPMENTAL BIOLOGY
collagen (Hauschka and Konigsberg, 1966). The procedure for subculturing was the same as that utilized in cultures of chick cells (Okada et al., 1971). For immunological identification of the lens nature of the cultured cells, antiserum against rat lens was prepared in rabbits. This antiserum cross-reacted with most of the crystallins of human lens, while it was highly lens-specific and without recognizable cross-reaction with any of the saline homogenates of non-lens tissues tested (Hamada and Okada, unpublished observations). An immunofluorescent study by the indirect method, using fluorescent goat antibody against rabbit y-globulin (Okada et al., 19731, and immunoelectrophoresis in Agarose gel containing the antiserum (Laurell, 1966) were conducted. Test antigens for the latter test were saline homogenates of cells harvested mechanically from primary cultures. Electron microscopic observations of cultured cells were made according to the procedures given previously (Eguchi and Okada, 1971). RESULTS
Primary
Culture
H-15 cells. After inoculation, most dissociated cells formed small aggregates, which adhered to the culture substrate within 24 hr. Then, aggregates started to spread, while they became interconnected with axon-like processes. Thereafter, a number of cells appeared flattened. Their number increased, and they covered the whole culture substrate. Simultaneously, many thick elongated fibers, probably nerve fibers, were differentiated overlaying the flattened cells. Cultures became confluent by 15 days. About this time, there appeared foci consisting,of epithelial cells which were smaller and more refractile than the flattened cells which were initially recognized. It is notable that these foci were observed in the location
VOLUME 60, 1977
where the development of neuronal structure had been predominant (Figs. 1 and 2). By 16 days, swollen and transparent cells were found within the foci- (Fig. 3). With further culturing, the number of these cells was increased. Sometimes they were transformed into “bottle cells”, the presence of which was a reliable marker for identifying lens differentiation in cell cultures of chick eye tissues (Okada et al., 1971). Often, several transparent cells were assembled to form the structure called “lentoid bodies” (Okada et al., 1971, 1973; see Fig. 4). Other single swollen cells became highly vacuolated and sometimes degenerated leaving only a rim of cytoplasmic materials. By 30 days, many of the neuronal structures and large flattened cells started to degenerate and to be lost from the cultures. In immunofluorescence observations, all cells of 12-day cultures were negative. On the other hand, a number of fluorescencepositive cells were recognized in the cell populations of 2%day cultures. Positive fluorescence was recognized in cells of “lentoid bodies,” Itbottle cells,” and single swollen cells with different grades of vacuolization (Fig. 5). Small epithelial cells in the foci were also weakly positive. The saline homogenate of 25day cultures reacted positively with anti-rat lens in Laurell’s test in Agarose, while that of lo-day cultures did not (Fig. 6). Electron-microscopic observation of the ultra-thin sections of “lentoid bodies” shows that these structures are composed of very elongated cells nearly free of mitochondria and endoplasmic reticulum but with a number of polysomes (Fig. 7). These profiles probably permit us to consider these elongated cells as lens fibers (Okada et al., 1973). H-9 cells. Within 10 days after inoculation, a confluent layer of flattened cells was formed. Neuronal cells with long cytoplasmic processes were differentiated by about 10 days. Apparently the number
307
BRIEF NOTES
of neuronal cells was much less here than in cultures of H-15 cells. They appeared only in several restricted areas. By 16 days, adjacent to these neuronal areas, foci of small epithelial cells were seen. Lentoid differentiation was initiated by about 30 days in the same manner as that described for cultures of H-15 cells.
Subcultures Cells harvested from a lo-day primary culture of H-15 cells were transferred to secondary cultures. Typical foci of smaller epithelial cells appeared within 7 days, and lentoid differentiation was first recognized by 13 days. Differentiation of neuronal structures was very poor in secondary cultures. DISCUSSION
Cells of vertebrate eye tissues have the very characteristic property of transdifferentiation, a property of the switching of once specialized cells into an altered direction (for reviews, see Eguchi, 1976; Okada, 1976). So far, examples of transdifferentiation have been found in avian embryos and adult newts. In the present study, we have presented evidence that the neural retina of the human fetus can alter its differentiation and form a lens. The authentic lens nature of lens-like cells and of “lentoid bodies” appearing in cultures was identified by both immunological and ultrastructural methods. It can be remarked that, in the case of the neural retinas of human fetuses, altered differentiation into a lens in vitro occurred more rapidly than it did in chick FIG. 1. Appearance of a focus of small epithelial cells encircled with dotted line. Note the presence of a nerve fiber-like structure(N) adjacent to the focus; lo-day culture of H-15 cells. x 100. FIG. 2. Epithelial cell sheet derived from the focus shown in Fig.1; 13-day culture of H-15 cells. x 100. FIG. 3. Appearance of transparent and swollen cells (indicated by arrows) in the epithelial-celled focus; 17-day culture of H-15 cells. x 100.
308
DEVELOPMENTALBIOLOGY
VOLUME 60, 1977
FIG. 4. Appearance of a “lentoid body” (L) and a “bottle cells” (B) in a focus of epithelial cells; 25-day culture of H-15 cells. x 100. FIG. 5. An immunofluorescence micrograph of a focus of epithelial cells in a 2%day culture of H-15 cells. Note the presence of strongly fluorescent cells. x 100.
FIG. 6. Immunelectrophoresis by Laurell’s method in an Agarose gel containing antiserum against rat lens: a, the homogenate of a IO-day culture of H-15 cells; b, the homogenate of a 25-day culture of H-15 cells; c, the homogenate of the retina of a 15-week-old fetus; e, the homogenate of the lens of a 15-week-old fetus.
embryos. It took about 20 days here, while taking 60 days in the neural retinas of 8- to g-day-old chick embryos (Okada et al., 1975). Another characteristic feature in the human cells was very extensive formation of neuronal structures in the early stages of cultures. As a rule, the lens dif-
ferentiation occurred adjacent to the areas where elongated axon-like processes had been particularly abundant. The results suggest that the neural retinas of human fetuses contain some bipotent cells, each of which can differentiate in either the neural or the lens direction. Also, the possibility that there are nonneural cells in the retina which, under the proper conditions, could form a lens, is not excluded. Factors responsible for eliciting lens formation from the neural retina remain to be elucidated. The present study showed that the instability in the state of differentiation of the cells of eye tissues is not found exclusively in species with high regenerative ability but may be a general property in all vertebrates from amphibians up to mammals. At least in fetuses, some cells of well-developed mammalian eye tissues still have the potential to alter their state of differentiation. Perhaps in the eyes of higher vertebrates, an expression of “foreign” differentiation is strictly repressed in the in situ ocular environment, and the number of cells capable of forming lens is too small to regenerate a lost lens. The instability in differentiation of pigmented retinal cells of human fetuses will be communicated in a subsequent paper (Yasuda et al., unpublished manuscript).
BRIEF NOTES
FIG. 7. An electron micrograph of an ultra-thin section of a “lentoid body” in a 27-day culture cells. Note that the cytoplasm is nearly empty except for many polysomes. x 15,000. We thank Mr. M. Araki for help in some parts of the experiments, and Miss A. Ishihara and R. Rakayasu for help in the preparation of the manuscript. The work was supported by a grant for Basic Cancer Research from the Japan Ministry of Education, Culture, and Science. REFERENCES ALTMAN, P. I., and DITTMER, D. S. (eds.) (1962). Prenatal vertebrate development. In “Biological Handbook,” pp. 275-297. Federation of American Societies for Experimental Biology, Washington, D.C. EGUCHI, 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. Nat. Acad. Sci. USA 71, 5052-5056. EGUCHI, G., and OKADA, T. S. (1971). Ultrastructure of the differentiated cell colony derived from a singly isolated chondrocyte in in vitro culture. Develop. Growth Diff. 12, 297-312. EGUCHI, G., and OKADA, T. S. (1973). Differentiation of lens tissue from the progeny of chick retinal pigment cells cultured in uitro: A demonstration of a switch of cell types in clonal cell culture. Proc. Nat. Acad. Sci. USA 70, 1495-1499. HAUSHKA, S. D., and KONIGSBERG, I. R. (1966). The influence of collagen on the development of muscle clones. Proc. Nut. Acad. Sci. USA 55, 119-126. HAY, E. D. (1966). “Regeneration.” Holt, Rinehart & Winston, New York.
309
of H-15
ITOH, Y., OKADA, T. S., IDE, H., and EGUCHI, G. (19751. The differentiation of pigment cells in cultures of chick embryonic neural retina. Deuelop. Growth Differ. 17, 39-50. LAURELL, C. -B. (1966). Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal. Biochem. 15, 45-52. 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., ~~~TAKEICHI, M. (1973). The retention of differentiated properties by lens epithelial cells in clonal cell culture. Deuelop. Biol. 34, 321-333. OKADA, T. S., ITOH, Y., WATANABE, K., andEcvc~r, G. (1975). Differentiation of lens in cultures of neural retinal cells of chick embryos. Develop. Biol. 45, 318-329. REDFERN N., ISRAEL, P., 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. VAN DETH, J. H. M. G. (1940). Induction et rogcncration du cristallin chez l’embryon de la poule. Actu Neer. Morphol. 3, 151-169.
