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An attempt to assay the state of determination by using transfected genes as probes in transdifferentiation of neural retina into lens
Cell Differentiation, 20 (1987) 203-207 Elsevier Scientific Publishers Ireland, Ltd.
203
CDF 00424
An attempt to assay the state of determination by using transfected genes as probes in transdifferentiation of neural retina into lens * H. K o n d o h 1, y . U e d a x, S. H a y a s h i a, K. Okazaki 1, K. Y a s u d a 1 a n d T.S. O k a d a 2 l Department of Biophysics, Faculty of Science, University of Kyoto, Kyoto 606, Japan and e National Institute for Basic Biology, Okazaki 444, Japan (Accepted 5 November 1986)
Hybrid genes coding for chloramphenicol acetyitransferase (CAT) with a non-specific retroviral, lensspecific 8-crystailin or lens-specific a-crystallin promoters were constructed to transfect the transdifferentiating (lentoidogenic) and non-transdifferentiating (non-lentoidogenic) cultures of chicken embryonic neural retina for assaying the state of determination towards lens differentiation. The expression occurred only when CAT genes with lens-specific promoters were transfected to the cultures maintained in the conditions permissive to lentoidogenesis. The expression of these exogenous, lens-specific CAT genes began at stages of culturing that were earlier than the expression of endogenous crystallin. Presumably, there are two steps in the transdifferentiation of neural retina into lens; acquisition of capacity to express crystallin genes and derepression of the endogenous crystallin genes. Embryonic neural retina; Lens differentiation; Recombinant CAT genes; Transfection; Transdifferentiation; Commitment in cell differentiation
Transdifferentiation of neural retina into lens Embryonic neural retina (NR) of vertebrates, avians in particular, gives rise to lens cells in appropriate culture conditions. This is one example of 'transdifferentiation' (for reviews, see Okada, 1980, 1983), in which the cells belonging to a neural tissue give rise to a totally different cell type. * Presented at the Symposium on Biology of Retinal Differentiation In Vitro, 7th International Congress of Research, September 1986, Nagoya, Japan. Correspondence address: Dr. H. Kondoh, Department of physics, Faculty of Science, University of Kyoto, Kyoto Japan.
Cell Eye Bio606,
When N R of 8.5-day-old chicken embryos are dissociated and cultured in spreading condition at the initial cell density of about 2.5 × 105 cells per cm 2 of the culture substrate, the neuronal cells form small clumps which lie on a layer of glial cells. The glial cells spread and multiply until they fill the space between the neuronal cell clumps. The neuronal cells stay on the glial cells usually for two weeks, during which time some neuronal phenotypes are detected (cf. Pessac, 1987), then they gradually detach. After three weeks, lens crystallins begin to accumulate in cultures, and lentoids, which are transparent masses of elongated cells filled with crystallins, emerge. The cells in the lentoids are furnished with every feature of
0045-6039/87/$03.50 v. 1987 Elsevier Scientific Publishers Ireland, Ltd.
204
A
Fig. 1. (A) A phase-contrast micrograph of a 40-day culture of NR from 8.5-day-oldchicken embryos in lentoidogeniccondition. Lentoids are pointed by arrowheads. The bar indicates 25 /Lm. (B) Comparison of soluble proteins of (a) lens of day-old chicken and (b) 40-day culture of NR shown in (A). Soluble proteins of 5 /zg each were electrophoresed in SDS-polyacrylamide gel and stained with Coomassie brilliant blue. Positions of a-. r- and 8-crystallinsare indicated.
authentic lens cells. Fig. 1 shows typical lentoids observed in a N R culture and the protein pattern of the total cell culture at 40 days.
Expression of transfected crystallin gene derivatives in transdifferentiating neural retina In the course of transdifferentiation, crystallin genes in the retinal glial cells should experience transition from unexpressible to expressible states (Thomson et al., 1979; Yasuda et al., 1983). To probe this transition, we undertook transfection of N R cultures with crystallin gene derivatives. This attempt had its basis on the previous observation that exogenously introduced crystallin genes have broader specificities than endogenous crystallin genes, as they are expressed at a high level in cell types closely related to lens (Kondoh and Okada, 1986; Kondoh et al., 1986). Therefore, exogenous
crystallin genes or their derivatives may be expressed prior to endogenous crystallin genes, as the differentiated state of the lens precursors in N R approaches the transitory state to transdifferentiate into lens. We have previously shown that the promotercontaining regions of 8- and a-crystallin genes of the chicken account for a large part of lens specificity of the genes (Hayashi et al., 1985; Okazaki et al., 1985) and generate lens specificity in the expression of artificially joined coding sequences (K. Okazaki, PhD. thesis, Kyoto University, 1986; Y. Ueda et al., in preparation). We thus joined these sequences to bacterial chloramphenicol acetyltransferase (CAT) gene to construct t$CAT and a-CAT genes which receive crystallinspecific gene regulation, but are clearly distinguished from endogenous crystallin genes in the chicken. In addition, we constructed a gen¢ MoCAT which has a non-specific promoter sequence in Moloney murine leukaemia virus to drive CAT coding sequence. Thus two lens-specific genes and one non-specific gene which code CAT enzyme were prepared (Fig. 2). N R cultures from 8.5-day-old embryos contain two basically different cell types, neuronal and glial cells, but only glial cells have been well confirmed to be precursors of lens cells (Moscona and Degenstein, 1981). To examine which cell type is transfected by our standard protocol, we made use of bacterial fl-galactosidase gene driven by each of the three promoters described above. Transfected N R cultures were histochemically stained for fl-galactosidase to localize actually transfected ceils. In every case, the expression was restricted to the glial cells (Y. Ueda et al., in preparation). Thus, the glial cells are likely to be the major and practically only cell type to be transfected with and to express transfected genes. We set up two different types of cultures from the same 8.5-day-old embryonic NR, lentoidogenic cultures with an appropriate initial cell density, and non-lentoidogenic cultures with an extremely high initial cell density to maintain characteristics of the neural tissue (Takagi et al., 1983) (Fig. 2). At each time point of culturing, we transfected the cultures with the lens-specific and nonspecific CAT genes to see whether there is any
205
Mo-CAT ~ g-CAT
Ce-CAT~
Lentoidogenic
CAT I~
I
,
CAT
~'1
CAT
~l
Non-lentoidogenic
Fig. 2. Three CAT gene constructs (upper) and two different types of cultures (lower). Structures of the CAT-coding genes are schematically shown. All are derived from pSVO-CAT (Gorman et al., 1982) and carry the same CAT-coding sequence (the boxes marked with CAT) and SV40-derived poly (A) addition signal (unmarked boxes). Promoter regions were taken from Mo-MuLV LTR (positions from 7113 to 8213 of Shinnick et al., 1981), from chicken 8-crystallin gene (positions from - 4 5 9 to + 57 of Hayashi et al., 1985) and from chicken a-crystallin gene (from - 373 to + 10 of Okazaki et al., 1985). Lentoidogenic cultures were prepared by inoculating 2.5 x 105 cells of 8.5-day NR per cm 2, whereas non-lentoidogenic cultures were prepared inoculating 4 x 1 0 6 cells per cm 2. The photographs were taken at day 5 of culturing.
correlation of CAT gene expression with lens differentiation. In both culture conditions, the non-specific Mo-CAT gene was expressed highly when transfected at an early culture period, and the expression declined with the age of cultures. This decline is a general trend of primary cultures of various cell types and presumably reflects a decrease in the transfection efficiency. Thus, with Mo-CAT gene, no essential difference was observed between lentoidogenic and non-lentoidogenic cultures.
However, with lens-specific &CAT and a-CAT genes, there was a marked difference between the two culture conditions. When these genes were transfected to non-lentoidogenic cultures, they were expressed very poorly (&CAT) or not at all (a-CAT) during the culture period. By contrast, when the same genes were transfected to lentoidogenic cultures, they were expressed at late stages of culture period. At day 3, there was almost no expression, but at day 8, a significant level of expression was observed, and thereafter the expression continued to increase although transfection efficiency itself declined (Fig. 3). Thus, being driven by lens-specific gene promoters only and only in the culture conditions supporting lens differentiation, the expression of transfected CAT genes became progressively high with culture periods. However, in both cases, the expression of exogenous lens-specific genes began much earlier than the expression of endogenous crystallin genes reported previously (Thomson et al., 1979; Nomura et al., 1980). We can interpret the results in the following way. Between 5 and 10 days in culture, cells acquire the capacity to express crystallin gene, as indicated by the transfection experiments with exogenous &CAT and a-CAT genes. However, the expression of endogenous crystallin genes is probably repressed by certain unknown mechanisms for another 10-15 days. Thus, there are two steps in the process of transdifferentiation of lens from NR: the acquisition of capacity to express crystallin genes and the derepression of the endogenous crystallin genes. In the first step, the cells may already be committed to lens differentiation. There is evidence that around this time the cells determine their future pathway of differentiation. Okada et al. (1983) examined the effect of changing the culture regime after spreading culture to aggregation culture for various periods. If NR cells are cultured in the former condition throughout the period, extensive lens transdifferentiation occurs, whereas if the same cells are cultured in the latter condition, the transdifferentiation does not take place. When NR cells are cultured for a period shorter than 5 days in spreading cultures and then cultured as aggregates, no lens differentiation occurs.
206
1000
Mo- CAT
500
~ - "-n'
0 I
"u---
-'~'n
10
20
t.-
.g
20(:]
shifting the future fate of NR cells into lens. By comparing the results based on these culture experiments with the present study on transfection experiments, the first step (defined here as an acquisition of the capacity to express exogenous genes) may coincide with the stage of determination to lens. The two steps presently assumed in this transdifferentiation system may be involved in other cases of cell differentiation. We consider that the use of exogenous gene expression as the probe for cell commitment is one of the most straightforward approaches to the molecular mechanisms of cell differentiation.
.-
.>_ 100
I,-
o
0
O
~
--0I
10
Acknowledgement !
20 /
2000
1000
This work was supported by a Cooperative Research Program of the National Institute for Basic Biology ( # 86-107) and by grant # 60790035 to S.H., grants for Basic Cancer Research #61010061 and #61010075 to H.K. and T.S.O. from the Ministry of Education, Science and Culture of Japan.
References 10
20
Days of c u l t u r e
Fig. 3. The expression of transfected CAT genes in lentoidogenie and non-lentoidogenic NR cultures. At each time point of culturing, 0.5 mg each of DNAs were transfected to cultures of 2.5 cm dishes using a calcium-phosphate technique. After 4 h, the cultures were fed with the fresh medium and after 24 h, CAT enzyme activity was measured. One unit (U) enzyme activity is to mono-acetylate [u4C]chloramphenicol at the rate 10 fmol/h. Results in lentoidogenic (©) and in non-lentoidogenic cultures (O) are compared.
On the other hand, when this change in culture regime is done after 10 days of spreading culture, then an extensive lens transdifferentiation is observed in aggregates. However, there are no obvious changes to indicate the initial expression of lens phenotypes in spreading cultures between 5 and 10 days, which period must be critical for
Gorman, C., L. Moffat and B. Haward: Recombinant genes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. Biol. 2, 1044-1051 (1982). Hayashi, S., H. Kondoh, K. Yasuda, G. Soma, Y. Ikawa and T.S. Okada: Tissue-specific regulation of the chicken 8crystallin gene in mouse cells: involvement of the 5' end region. EMBO J. 4, 2201-2207 (1985). Kondoh, H. and T.S. Okada: Dual regulation of expression of exogenous 8-crystallin gene in mammalian cells: a search for molecular background of instability in differentiation. Curr. Top. Dev. Biol. 20, 153-164 (1986). Kondoh, H., S. Hayashi, Y. Takahashi and T.S. Okada: Regulation of 8-crystallin expression: an investigation by transfer of the chicken gene into mouse cells. Cell Differ. 19, 151-160 (1986). Moscona, A.A. and L. Degenstein: Lentoids in aggregates of embryonic neural retina cells. Cell Differ. 10, 39-46 (1981). Nomura, K., S. Takagi and T.S. Okada: Expression of neural specificities in "transdifferentiating" cultures of neural retina. Differentiation 16, 141-147 (1980). Okada, T.S.: Cellular metaplasia or transdifferentiation as a model for retinal cell differentiation. Curr. Top. Dev. Biol. 16, 349-380 (1980).
207 Okada, T.S.: Recent progress in studies of transdifferentiation of eye tissues in vitro. Cell Differ. 13, 177-183 (1983). Okada, T.S., K. Nomura and K. Yasuda: Commitment to transdifferentiation into lens occurs in neural retina cells after brief spreading culture of dissociated cells. Cell Differ. 12, 85-92 (1983). Okazaki, K., K. Yasuda, H. Kondoh and T.S. Okada: DNA sequences responsible for tissue-specific expression of a chicken a-crystallin gene in mouse cells. EMBO J. 4, 2589-2595 (1985). Pessac, B.: Differentiation of retrovirus infected avian neuroretina cells. Cell Differ. 20 (1987). Shinnick, T.M., R.A. Lerner and J.G. Sutcliffe: Nucleotide sequence of Moloney murine leukaemia virus. Nature 293, 543-548 (1981).
Takagi, S., H. Kondoh, K. Nomura and T,S. Okada: Lentoidogenesis from neural retina cells in culture is affected by interactive relationships between different cell types. J. Embryol. Exp. Morphol. 73, 97-109 (1983). Thomson, I., D.I. de Pomerai, J.F. Jackson and R.M. Clayton: Lens specific mRNA in transdifferentiating cultures of embryonic chicken neural retina and pigmented epithelium. Exp. Cell. Res. 122, 73-81 (1979). Yasuda, K., K. Okuyama and T.S. Okada: The accumulation of 8-crystallin mRNA in transdetermination and transdifferentiation of neural retina cells into lens. Cell Differ. 12, 177-183 (1983).
203
CDF 00424
An attempt to assay the state of determination by using transfected genes as probes in transdifferentiation of neural retina into lens * H. K o n d o h 1, y . U e d a x, S. H a y a s h i a, K. Okazaki 1, K. Y a s u d a 1 a n d T.S. O k a d a 2 l Department of Biophysics, Faculty of Science, University of Kyoto, Kyoto 606, Japan and e National Institute for Basic Biology, Okazaki 444, Japan (Accepted 5 November 1986)
Hybrid genes coding for chloramphenicol acetyitransferase (CAT) with a non-specific retroviral, lensspecific 8-crystailin or lens-specific a-crystallin promoters were constructed to transfect the transdifferentiating (lentoidogenic) and non-transdifferentiating (non-lentoidogenic) cultures of chicken embryonic neural retina for assaying the state of determination towards lens differentiation. The expression occurred only when CAT genes with lens-specific promoters were transfected to the cultures maintained in the conditions permissive to lentoidogenesis. The expression of these exogenous, lens-specific CAT genes began at stages of culturing that were earlier than the expression of endogenous crystallin. Presumably, there are two steps in the transdifferentiation of neural retina into lens; acquisition of capacity to express crystallin genes and derepression of the endogenous crystallin genes. Embryonic neural retina; Lens differentiation; Recombinant CAT genes; Transfection; Transdifferentiation; Commitment in cell differentiation
Transdifferentiation of neural retina into lens Embryonic neural retina (NR) of vertebrates, avians in particular, gives rise to lens cells in appropriate culture conditions. This is one example of 'transdifferentiation' (for reviews, see Okada, 1980, 1983), in which the cells belonging to a neural tissue give rise to a totally different cell type. * Presented at the Symposium on Biology of Retinal Differentiation In Vitro, 7th International Congress of Research, September 1986, Nagoya, Japan. Correspondence address: Dr. H. Kondoh, Department of physics, Faculty of Science, University of Kyoto, Kyoto Japan.
Cell Eye Bio606,
When N R of 8.5-day-old chicken embryos are dissociated and cultured in spreading condition at the initial cell density of about 2.5 × 105 cells per cm 2 of the culture substrate, the neuronal cells form small clumps which lie on a layer of glial cells. The glial cells spread and multiply until they fill the space between the neuronal cell clumps. The neuronal cells stay on the glial cells usually for two weeks, during which time some neuronal phenotypes are detected (cf. Pessac, 1987), then they gradually detach. After three weeks, lens crystallins begin to accumulate in cultures, and lentoids, which are transparent masses of elongated cells filled with crystallins, emerge. The cells in the lentoids are furnished with every feature of
0045-6039/87/$03.50 v. 1987 Elsevier Scientific Publishers Ireland, Ltd.
204
A
Fig. 1. (A) A phase-contrast micrograph of a 40-day culture of NR from 8.5-day-oldchicken embryos in lentoidogeniccondition. Lentoids are pointed by arrowheads. The bar indicates 25 /Lm. (B) Comparison of soluble proteins of (a) lens of day-old chicken and (b) 40-day culture of NR shown in (A). Soluble proteins of 5 /zg each were electrophoresed in SDS-polyacrylamide gel and stained with Coomassie brilliant blue. Positions of a-. r- and 8-crystallinsare indicated.
authentic lens cells. Fig. 1 shows typical lentoids observed in a N R culture and the protein pattern of the total cell culture at 40 days.
Expression of transfected crystallin gene derivatives in transdifferentiating neural retina In the course of transdifferentiation, crystallin genes in the retinal glial cells should experience transition from unexpressible to expressible states (Thomson et al., 1979; Yasuda et al., 1983). To probe this transition, we undertook transfection of N R cultures with crystallin gene derivatives. This attempt had its basis on the previous observation that exogenously introduced crystallin genes have broader specificities than endogenous crystallin genes, as they are expressed at a high level in cell types closely related to lens (Kondoh and Okada, 1986; Kondoh et al., 1986). Therefore, exogenous
crystallin genes or their derivatives may be expressed prior to endogenous crystallin genes, as the differentiated state of the lens precursors in N R approaches the transitory state to transdifferentiate into lens. We have previously shown that the promotercontaining regions of 8- and a-crystallin genes of the chicken account for a large part of lens specificity of the genes (Hayashi et al., 1985; Okazaki et al., 1985) and generate lens specificity in the expression of artificially joined coding sequences (K. Okazaki, PhD. thesis, Kyoto University, 1986; Y. Ueda et al., in preparation). We thus joined these sequences to bacterial chloramphenicol acetyltransferase (CAT) gene to construct t$CAT and a-CAT genes which receive crystallinspecific gene regulation, but are clearly distinguished from endogenous crystallin genes in the chicken. In addition, we constructed a gen¢ MoCAT which has a non-specific promoter sequence in Moloney murine leukaemia virus to drive CAT coding sequence. Thus two lens-specific genes and one non-specific gene which code CAT enzyme were prepared (Fig. 2). N R cultures from 8.5-day-old embryos contain two basically different cell types, neuronal and glial cells, but only glial cells have been well confirmed to be precursors of lens cells (Moscona and Degenstein, 1981). To examine which cell type is transfected by our standard protocol, we made use of bacterial fl-galactosidase gene driven by each of the three promoters described above. Transfected N R cultures were histochemically stained for fl-galactosidase to localize actually transfected ceils. In every case, the expression was restricted to the glial cells (Y. Ueda et al., in preparation). Thus, the glial cells are likely to be the major and practically only cell type to be transfected with and to express transfected genes. We set up two different types of cultures from the same 8.5-day-old embryonic NR, lentoidogenic cultures with an appropriate initial cell density, and non-lentoidogenic cultures with an extremely high initial cell density to maintain characteristics of the neural tissue (Takagi et al., 1983) (Fig. 2). At each time point of culturing, we transfected the cultures with the lens-specific and nonspecific CAT genes to see whether there is any
205
Mo-CAT ~ g-CAT
Ce-CAT~
Lentoidogenic
CAT I~
I
,
CAT
~'1
CAT
~l
Non-lentoidogenic
Fig. 2. Three CAT gene constructs (upper) and two different types of cultures (lower). Structures of the CAT-coding genes are schematically shown. All are derived from pSVO-CAT (Gorman et al., 1982) and carry the same CAT-coding sequence (the boxes marked with CAT) and SV40-derived poly (A) addition signal (unmarked boxes). Promoter regions were taken from Mo-MuLV LTR (positions from 7113 to 8213 of Shinnick et al., 1981), from chicken 8-crystallin gene (positions from - 4 5 9 to + 57 of Hayashi et al., 1985) and from chicken a-crystallin gene (from - 373 to + 10 of Okazaki et al., 1985). Lentoidogenic cultures were prepared by inoculating 2.5 x 105 cells of 8.5-day NR per cm 2, whereas non-lentoidogenic cultures were prepared inoculating 4 x 1 0 6 cells per cm 2. The photographs were taken at day 5 of culturing.
correlation of CAT gene expression with lens differentiation. In both culture conditions, the non-specific Mo-CAT gene was expressed highly when transfected at an early culture period, and the expression declined with the age of cultures. This decline is a general trend of primary cultures of various cell types and presumably reflects a decrease in the transfection efficiency. Thus, with Mo-CAT gene, no essential difference was observed between lentoidogenic and non-lentoidogenic cultures.
However, with lens-specific &CAT and a-CAT genes, there was a marked difference between the two culture conditions. When these genes were transfected to non-lentoidogenic cultures, they were expressed very poorly (&CAT) or not at all (a-CAT) during the culture period. By contrast, when the same genes were transfected to lentoidogenic cultures, they were expressed at late stages of culture period. At day 3, there was almost no expression, but at day 8, a significant level of expression was observed, and thereafter the expression continued to increase although transfection efficiency itself declined (Fig. 3). Thus, being driven by lens-specific gene promoters only and only in the culture conditions supporting lens differentiation, the expression of transfected CAT genes became progressively high with culture periods. However, in both cases, the expression of exogenous lens-specific genes began much earlier than the expression of endogenous crystallin genes reported previously (Thomson et al., 1979; Nomura et al., 1980). We can interpret the results in the following way. Between 5 and 10 days in culture, cells acquire the capacity to express crystallin gene, as indicated by the transfection experiments with exogenous &CAT and a-CAT genes. However, the expression of endogenous crystallin genes is probably repressed by certain unknown mechanisms for another 10-15 days. Thus, there are two steps in the process of transdifferentiation of lens from NR: the acquisition of capacity to express crystallin genes and the derepression of the endogenous crystallin genes. In the first step, the cells may already be committed to lens differentiation. There is evidence that around this time the cells determine their future pathway of differentiation. Okada et al. (1983) examined the effect of changing the culture regime after spreading culture to aggregation culture for various periods. If NR cells are cultured in the former condition throughout the period, extensive lens transdifferentiation occurs, whereas if the same cells are cultured in the latter condition, the transdifferentiation does not take place. When NR cells are cultured for a period shorter than 5 days in spreading cultures and then cultured as aggregates, no lens differentiation occurs.
206
1000
Mo- CAT
500
~ - "-n'
0 I
"u---
-'~'n
10
20
t.-
.g
20(:]
shifting the future fate of NR cells into lens. By comparing the results based on these culture experiments with the present study on transfection experiments, the first step (defined here as an acquisition of the capacity to express exogenous genes) may coincide with the stage of determination to lens. The two steps presently assumed in this transdifferentiation system may be involved in other cases of cell differentiation. We consider that the use of exogenous gene expression as the probe for cell commitment is one of the most straightforward approaches to the molecular mechanisms of cell differentiation.
.-
.>_ 100
I,-
o
0
O
~
--0I
10
Acknowledgement !
20 /
2000
1000
This work was supported by a Cooperative Research Program of the National Institute for Basic Biology ( # 86-107) and by grant # 60790035 to S.H., grants for Basic Cancer Research #61010061 and #61010075 to H.K. and T.S.O. from the Ministry of Education, Science and Culture of Japan.
References 10
20
Days of c u l t u r e
Fig. 3. The expression of transfected CAT genes in lentoidogenie and non-lentoidogenic NR cultures. At each time point of culturing, 0.5 mg each of DNAs were transfected to cultures of 2.5 cm dishes using a calcium-phosphate technique. After 4 h, the cultures were fed with the fresh medium and after 24 h, CAT enzyme activity was measured. One unit (U) enzyme activity is to mono-acetylate [u4C]chloramphenicol at the rate 10 fmol/h. Results in lentoidogenic (©) and in non-lentoidogenic cultures (O) are compared.
On the other hand, when this change in culture regime is done after 10 days of spreading culture, then an extensive lens transdifferentiation is observed in aggregates. However, there are no obvious changes to indicate the initial expression of lens phenotypes in spreading cultures between 5 and 10 days, which period must be critical for
Gorman, C., L. Moffat and B. Haward: Recombinant genes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. Biol. 2, 1044-1051 (1982). Hayashi, S., H. Kondoh, K. Yasuda, G. Soma, Y. Ikawa and T.S. Okada: Tissue-specific regulation of the chicken 8crystallin gene in mouse cells: involvement of the 5' end region. EMBO J. 4, 2201-2207 (1985). Kondoh, H. and T.S. Okada: Dual regulation of expression of exogenous 8-crystallin gene in mammalian cells: a search for molecular background of instability in differentiation. Curr. Top. Dev. Biol. 20, 153-164 (1986). Kondoh, H., S. Hayashi, Y. Takahashi and T.S. Okada: Regulation of 8-crystallin expression: an investigation by transfer of the chicken gene into mouse cells. Cell Differ. 19, 151-160 (1986). Moscona, A.A. and L. Degenstein: Lentoids in aggregates of embryonic neural retina cells. Cell Differ. 10, 39-46 (1981). Nomura, K., S. Takagi and T.S. Okada: Expression of neural specificities in "transdifferentiating" cultures of neural retina. Differentiation 16, 141-147 (1980). Okada, T.S.: Cellular metaplasia or transdifferentiation as a model for retinal cell differentiation. Curr. Top. Dev. Biol. 16, 349-380 (1980).
207 Okada, T.S.: Recent progress in studies of transdifferentiation of eye tissues in vitro. Cell Differ. 13, 177-183 (1983). Okada, T.S., K. Nomura and K. Yasuda: Commitment to transdifferentiation into lens occurs in neural retina cells after brief spreading culture of dissociated cells. Cell Differ. 12, 85-92 (1983). Okazaki, K., K. Yasuda, H. Kondoh and T.S. Okada: DNA sequences responsible for tissue-specific expression of a chicken a-crystallin gene in mouse cells. EMBO J. 4, 2589-2595 (1985). Pessac, B.: Differentiation of retrovirus infected avian neuroretina cells. Cell Differ. 20 (1987). Shinnick, T.M., R.A. Lerner and J.G. Sutcliffe: Nucleotide sequence of Moloney murine leukaemia virus. Nature 293, 543-548 (1981).
Takagi, S., H. Kondoh, K. Nomura and T,S. Okada: Lentoidogenesis from neural retina cells in culture is affected by interactive relationships between different cell types. J. Embryol. Exp. Morphol. 73, 97-109 (1983). Thomson, I., D.I. de Pomerai, J.F. Jackson and R.M. Clayton: Lens specific mRNA in transdifferentiating cultures of embryonic chicken neural retina and pigmented epithelium. Exp. Cell. Res. 122, 73-81 (1979). Yasuda, K., K. Okuyama and T.S. Okada: The accumulation of 8-crystallin mRNA in transdetermination and transdifferentiation of neural retina cells into lens. Cell Differ. 12, 177-183 (1983).