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Age-dependent changes in the capacity of transdifferentiation of retinal pigment cells as revealed in clonal cell culture
Cell Differentiation, 10 (1981) 3--11 © Elsevier/North-Holland Biomedical Press
3
AGE-DEPENDENT CHANGES IN THE CAPACITY OF T R A N S D I F F E R E N T I A T I O N OF R E T I N A L PIGMENT CELLS AS R E V E A L E D IN CLONAL CELL C U L T U R E KUNIO YASUDA, GORO EGUCHI * and T.S. OKADA
Department of Biophysics, Faculty of Science, University of Kyoto, Kyoto 606, Japan (Accepted 13 July 1980)
Changes in the capacity of transdifferentiation of retinal pigment cells into lens cells with developmental age were examined by culturing cells obtained from chicks at various developmental stages ranging from 5-day-old embryos to 1-year-old adults. Secondary clonal cultures were prepared from pigment cells harvested from primary cultures of the cells dissociated from the material at different ages. The percentage of colony formation to the total number of inoculated cells, of colonies with lens differentiation, and of colonies with the differentiation of pigment cells were obtained in cultures of the material derived from the donors at different ages. The total clonal efficiency did not alter with age. Colonies with lens cells appeared only in cultures started from embryos of less than 15 days. The percentage in the formation of pigmented colonies decreased with the age of the donor. Cells of post-hatching chicks produced only unidentifiable colonies without lens and pigment cells. The results indicate that determination of retinal cells alters with age. transdifferentiation
pigment cell culture
1. Introduction
Alteration of the specificity of differentiated cells, which m a y be called transdifferentiation or cellular metaplasia, occurs extensively in tissues of the vertebrate eye (Eguchi, 1976; Okada, 1976, 1980). The classical example of this p h e n o m e n o n is the Wolffian regeneration of lens from the pigment cells of the iris epithelium in several urodelen species (Yamada, 1977). In cell culture in vitro, a number of examples of transdifferentiation have also been known in avians and mammalians, which cannot regenerate the lost parts of eyes in vivo (Okada, 1976). In these in vitro cell culture experiments, embryonic material has mainly been used. In the case of avians, embryos ranging from 3.5 to 10 days of incubation have been employed * Present address: Research Institute for Molecular Biology, Faculty of Science, University of Nagoya, Nagoya 464, Japan.
to demonstrate the occurrence of transdifferentiation in vitro as well as to study factors controlling this process. It is very likely that the capacity of transdifferentiation, which may reflect the instability in the state of determination in Cell differentiation, m a y alter with developmental stages. In fact, the transdifferentiation of chick neural retina (NR) into lens and/or pigm e n t cells occurs less extensively with the progress of embryonic age and this capacity disappears completely in NR of newly hatched chicks (De Pomerai et al., 1978; Nomura and Okada, 1979). During chick embryonic life, several different cell types are differentiated in NR in situ, and this tissue, in respect to its cellular composition, becomes complex with development. The disappearance of the capacity of transdifferentiation of NR in in vitro culture experiments could be related to the process of cell differentiation of N R in situ, as discussed by Nomura and Okada (1979).
Pigmented cells (PC) of the tapetum of chick embryos at 8--10 days of incubation can also transdifferentiate into lens under the conditions of cell culture (Eguchi and Okada, 1973; Yasuda, 1979). Once pigmented, the tapetum in chick embryos in situ remains a homogeneous cell population of only pigmented epithelial cells throughout embryonic life, while there is increasing complexity in the cell t y p e composition in NR with embryonic development. Thus, we investigated whether the capacity of transdifferentiation o f PC of the tapetum into lens in vitro is stage related. In order to obtain semi-quantitative results at the cellular level, a clonal cell culture technique was adopted. This method may permit a rough estimation of the percentage of 'transdifferentiable' cells, if any, in a given population of PC of the tapetum in the embryos at different stages.
plastic petri dishes, each with a diameter of 3.0 cm. The percentage of cells attached to the culture substrate within 24 h after inoculation decreased with developmental stages. Therefore, it was necessary to inoculate a larger number of cells to start cultures at an equal density of attached cells in all the material taken from different ages. Primary cultures were maintained for 18 days, when they became confluent. From these primary cultures, foci with heavily pigmented cells were trimmed, collected and dissociated with trypsin (Eguchi and Okada, 1973). A b o u t 500 pigmented cells thus prepared were transferred into Falcon culture dishes with a diameter of 5.5 cm each. These secondary cultures at a low cell density of clonal level were maintained for a b o u t 40 days.
2.3. Scoring the results 2. Materials and methods
White Leghorn embryos at 5, 8, 9, 10, 11, 15 and 20 days of incubation, newly hatched chicks and chicks of a b o u t 1 year old were used t o obtain PC of the tapetum for cell culture. EDTA was used to separate the pigmented epithelium of the tapeta from unpigmented neighbouring cells (Trinkaus and Lentz, 1964; Eguchi and Okada, 1973). The separation of the clean pigmented pieces was easy in most cases. However, some portions of the tapetum from embryos older than 20 days adhered so tightly to the adjacent tissue layers that some pigmented cells were left attached to the tissue.
The secondary clonal cultures were fixed with Bouin's fixative. The number of all colonies (colonies with a diameter larger than 2 mm) and of colonies with PC and of lentoid bodies (LB) (Okada et al., 1971, 1973; see Fig. 7F) were counted in each plate (Okada et al., 1979). The plating efficiency, (the number of colonies)/(the number of inoculated cells) × 100, the efficiency of PC differentiation, (the number of colonies with PC)/ (the number of total colonies) × 100 and the efficiency of LB differentiation, (the number of colonies with LB)/(the number of total colonies) × 100, were obtained. In all cultures, Eagle's minimum essential medium supplemented with 15% fetal bovine serum of one particular batch (Microbiological Inc. L o t no. 88818) was used.
2.2. Cell culture
2.4. SDS-polyacrylamide gel electrophoresis
Cell suspension was prepared from the isolated pieces of the t a p e t u m by EDTA and trypsin treatment as described previously (Eguchi and Okada, 1973). A b o u t 4 X 104 to 1.5 × l 0 s cells were inoculated into Falcon
Cultures at different stages were washed several times with Hanks' solution and each representative colony was peeled off with tweezers and solubilized in 50--200 /A of SDS-sample buffer. Aliquots containing a b o u t
2.1. Preparation o f cells to be cultured
30 pg proteins were analyzed on SDS-polyacrylamide slab gels according to the procedure of Laemmli (1970).
7
10 qb
(,3
2.5. Immunoelectrophoresis
.S
lo
Cultures at different stages were washed several times with Hanks' solution and homogenized in a small volume of the same saline. The homogenate was centrifuged at 10,000 g for 5 min to remove cell debris and the supernatant was frozen at --70°C. Aliquots of the supernatant were analyzed by immunoelectrophoresis using rabbit antiserum against the total extract of chick lenses (Yasuda, 1979).
qb
x
I
0
I
I
I
10 20 30 Days in Culture
I
40
Fig. 1. Typical growth curves of PC from various developmental stages in primary cultures. Each value represents an average of duplicate hemocytometer counts of the calls harvested from two plates starting from a single inoculum, o, 5-day embryos; A, 15day embryos; x, Adult chicks.
Fig. 2. Phase-contrast photomicrographs of pigmented epithelial cells growing in primary cultures on day 7 after inoculation. A) 5-day embryos. B) 8-day embryos. C) 1-day-old chicks. D) Adult chicks. X100.
3. Resuits 3.1. Primary cultures Many of the PC attached to the culture substrate started to grow within a b o u t 48 h of plating. PC from all the developmental stages grew equally well in primary cultures (Fig. 1). The average size of each PC differs with age; it
is usually smaller in younger materials (Fig. 2). Within about 10 days' outgrowth, however, all cultured cells of older materials became smaller reaching the size of younger materials. During the outgrowth, PC lost their pigm e n t granules by dilution with cell divisions and by discharge of the granules from PC into the medium (Whittaker, 1963; Yasuda, 1979}.
Fig. 3. Phase-contrast photomicrographs of PC at the confluent stage of the primary cultures on day 18. The heavily pigmented epithelial cells were harvested with trypsin and transferred into the secondary clonal cell culture. A) 5-day embryos. B) 8-day embryos. C) 15-day embryos. D) 1-day-old chicks. E) Adult chicks, epithelial portion. F) Adult chicks, fibroblastic portion. × 100.
PC in cultures from embryos younger than 11 days became non-pigmented within a few days of culture (Fig. 2A, B). Depigmented cells grew rapidly. When cultures reached a confluence, a number of foci of tightly packed polygonal cells appeared (Fig. 3). Cells of these foci became heavily repigmented. PC in cultures from embryonic material older than 15 days became less pigmented with growth without however completely losing the pigment granules. Practically all cells in confluent cultures at about 18 days were pigmented, although the degree of pigmentation was very different in individual cells in a single culture dish (Fig. 3). After confluence, cells from younger embryonic material appeared more heavily pigmented than those from older ones. In cultures of material from embryos older than 20 days spindle-shaped or dendritic PC appeared, in addition to pigmented epithelial cells (Fig. 3F). The spindle-shaped and dendritic PC did not attach to each other completely, while the pigmented epithelial cells came into close contact to establish a typical epithelial sheet.
~
20
o 10 "6
.,,j
0 I
I
I
5
10
15
O 0
0
I I t .;t 20 chick
t adult
Days after Incubation Age of Donors
Fig. 5. Decrease in the n u m b e r of colonies with LB in 40-day secondary clonal cultures with the development o f d o n o r age. Colonies with LB were identified and counted according to Okada et al. (1979). The data are expressed a s t h e average n u m b e r o f colonies with LB (+S.D.) o f 10 culture plates.
3.2. Clonal cultures
Cells harvested from heavily pigmented foci of epithelial sheets of primary cultures were used for clonal cultures. Fig. 4 shows the results after a b o u t 40 days' culturing. It is im-
20 ¢
.2
100
~o
'a .Q
E o I
i
I
I
0
5
10
15
II|
//
20 chick
t adult
Days after Incubation Age of Donors
Fig. 4. The total n u m b e r of colonies 40-day secondary clonal cell cultures developmental stages. 500 single cells primary cultures were inoculated into data are expressed as the average total onies (-+S.D.) o f 1O culture plates.
"~
0
0 I
I
I
0
5
10
15
t // 20 chick
t adult
Days after Incubation
f o r m e d in the f r o m different taken from the each plate. The n u m b e r o f col-
Age of Donors
Fig. 6. Decrease in the n u m b e r of colonies with PC in 40-day secondary clonal cultures with the development of d o n o r age. The data are expressed as the average n u m b e r o f colonies (+S.D.) o f 10 culture plates.
~
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[3
Fig. 7. Typical macrophotographs of culture plates of 40-day secondary clonal cultures taken from donors at different developmental stages. A) 5-day embryos. B) 8-day embryos. C) 15-day embryos. D) 1-day-old chicks. E) Adult chicks. F) Lentoid bodies (arrow, LB) appeared in cultures of 5-day embryos. × 150.
mediately noticeable that the plating efficiency, which indicates the percentage of cells to be potent for colony formation ('clonable' cells) contained in a given inoculate, is almost the same regardless of the developmental stage. On the other hand, colonies with LB appeared only in cells derived from embryos younger than 15 days of incubation (Fig. 5). The percentage of colonies with PC also decreased gradually with the age of cultured material: no pigmented colonies were formed in cultures of cells from the adult tapetum (Fig. 6; see also Fig. 7). In clonal cultures prepared from cells of primary cultures from embryos older than 15 days, PC, if any, did not establish an epithelial sheet; they were of the spindle or dendritic form and produced colonies with very dispersed cells. The appearance of pigmented colonies occurred in two ways: 1) by repigmentation of once completely depigmented cells in the early stages of clonal outgrowth, or 2) by clonal outgrowth of cells with pigmented granules throughout all stages or the early stage of culturing. In the first case, all colonies invariably consisted of pigmented and non-pigmented cell populations, while in the second, some colonies consisted of only pigmented cells and others included a few non-pigmented cells in the peripheral portion, leaving the inner portion heavily pigmented. Pure colonies with only PC appeared in cultures derived from PC of 5--6 day old embryos. There are several 'mixed' colonies, in which both LB and PC were differentiated. In secondary clonal cultures prepared from primary cultures of embryos older than 15 days, many colonies contained neither PC nor LB. In these colonies, cellsdid not establish a coherent sheet, but were very dispersed.
3.3. Protein composition of each colony An identification of the presence of crystallins in clonal cultures was made in the culture homogenates by means of immunoelectrophoresis. In secondary clonal cultures from
primary cultures of PC from embryos younger than 15 days three classes of crystallins, ~-, ~and ~-crystallins, were detected, whereas they were not found in cultures derived from older material (Fig. 8). The homogenates prepared from each single colony were respectively' subjected to SDS--gel-electrophoresis for protein analysis. Electrophoretic patterns of several representative colonies with LB, PC and unidentifiable colonies without any identification phenotypes are shown in Fig. 9. Bands which can well be identified as crystaUins by co-migration of the latter proteins (cf. also Araki et el.,
5
=~i~i~~
Fig. 8. Immunoelectrophoretic patterns of the homogenates of 40-day secondary clonal cultures of PC taken from the donors at different developmental stages. Troughs and wells contain respectively the rabbit antiserum against total lens extract and the supernatants of the homogenates of 1) intact 1-day-old chick lens (control) sample, 2) clonal cultures of PC from 5-day embryos, 3) from 8-day embryos, 4) from l l - d a y embryos, 5) from 1-day-old chicks, and 6) from adult chicks. In all positive cases (1, 2, 3 and 4) all of the three arcs corresponding to ol-, ~- and -crystallins are detectable.
10
1
2
3
4
5
6
7
5~
i~~
Fig. 9. SDS-gel electrophoretic patterns of the homogenates of respective colony of 40-day secondary clonal cultures from different developmental stages: 1) the homogenate of 1-day-old intact chick lenses (control sample), 2) a colony with LB derived from 5day embryos, 3) a colony with LB derived from 8-day embryos, 4) a mixed colony with both LB and PC derived from 5-day embryos, 5 ) a colony with PC derived from 5-day~ld embryos, 6) an unidentifiable colony derived from 8-day chicks, 7) an unidentifiable colony derived from 1-day-old chicks. ~, ~, and 5 represent ~-, major ~-, and 6-crystallin, respectively. The fact that bands indicated by arrows (~-*), the mobility of which is very close to that of ~-crystallin, are non-crystallin proteins was confirmed by the separate tests using the two-dimensional immunoelectrophoresis. A band corresponding to ~-crystallin migrates between these two bands.
1979) were recognized only in colonies with the visible formation of LB. All three classes of crystallins, a, fl, and 5, were present in colonies with LB. The unidentifiable colonies did n o t contain any detectable amount of crystallins. Band patterns of electrophoresis among three different types of colonies were basically similar to each other, except for the presence or the absence of crystallins.
4. Discussion It has been demonstrated that the capacity of transdifferentiation of chick NR in culture into LB and PC progressively decreased with developmental age (De Pomerai and Clayton, 1978; Nomura and Okada, 1979). The present study regarding the capacity of PC of the tapetum to transdifferentiate into lens cells in culture confirms this progressive decrease. However, as our conclusions rest on the results of the secondary clonal cell culture, it is necessary to evaluate the limitations of this technique. Firstly, with the present method we are not able to detect the presence of cells that would differentiate into PC or lens cells without producing large colonies. These cells, if present, may disappear earlier on in clonal cultures. Therefore, what we detected here is the percentage of transdifferentiable cells only in 'clonable' cell population. Secondly, instead of the whole cell population, we transferred only the heavily pigmented cell population of primary cultures into the assay system of secondarily clonal culture, not the whole cell population. Then, with the present method it is not possible to compare directly the capacity of transdifferentiation in the tapetum of different ages, b u t the comparison is limited to the capacity of 'stable' cell population, which can maintain or recover the original differentiative trait as PC in primary cultures. Within such limitations, the present results demonstrate in a semi-quantitative manner that in vitro the capacity of transdifferentiation of PC of the tapetum into lens cells decreases progressively with the developmental age of the starting material and that the capacity is almost completely lost in the t a p e t u m from embryos older than 15--16 days. On the other hand, the plating efficiency which may indicate the percentage of 'clonable' cells remains unchanged throughout all the stages examined. N o t only the differentiation of LB but also the differentiation of PC in secondary clonal
11
culture occurred more frequently in younger materials. Thus, the capacity of both transdifferentiation and redifferentiation decreases with age. Colonies derived from 'clonable' cells of aged material remained mostly 'unidentifiable'. It still remains to be examined whether this result indicates the permanent inhibition of redifferentiation or transdifferentation into any identifiable characteristics with development. In the present study, the decrease of the capacity of transdifferentiation is shown in a semi-quantitative manner by comparing the percentage within material at different ages, of the formation of colonies with LB to the number of inoculated cells or to the number of the 'clonable' cells. The results can be interpreted to indicate that the tapetum consists of qualitatively different cell populations, the transdifferentiable and non-transdifferentiable cells, though apparently consisting of a very homogeneous population of PC. The number of transdifferentiable cells decreases with age. It may be possible that there are no such qualitative distinctions in the cell population, but, for u n k n o w n reasons, the number of transdifferentiable cells detectable under the present assay system decreases with age. It has been .repeatedly shown that an overall trend of transdifferentiation is influenced by many factors of culture conditions in both N R and PC of the tapetum (Itoh, 1976; Clayton et al., 1977; Araki and Okada, 1978; Pritchard et al., 1978). This, however, does not exclude that the state of determination of PC in the tapetum, as assayed by the capacity of transdifferentiation, changes with developmental age.
Acknowledgements The authors thank Miss Y. Katsurayama for helping with the preparation of the manu-
script. The present work was supported by a research grant for Basic Cancer Research from the Japan Ministry of Education, Science and Culture, and by a research grant from Yamada Science Foundation.
References Araki, M. and T.S. Okada: Dev. Growth Differ. 20, 71--78 (1978). Araki, M., M. Yanagida and T.S. Okada: Dev. Biol. 69, 170--181 (1979). Clayton, R.M., D.I. de Pomerai and D.J. Pritchard: Dev. Growth Differ, 19, 319--328 (1977). De Pomerai, D.I. and R.M. Clayton:J. Embryol. Exp. Morphol. 47, 179--193 (1978). Eguchi, G.: In: Embryogenesis in Mammals, Ciba Symposium 40 (Elsevier, Amsterdam) pp. 241-258 (1976). Eguchi, G. and T.S. Okada: Proc. Natl. Acad. Sci. U.S.A. 70, 1495--1499 (1973). Itoh, Y.: Dev. Biol. 54, 157--162 (1976). Laemmli, U.K.: Nature (London) 227, 680--685 (1970). Nomura, K. and T.S. Okada: Dev. Growth Differ. 21, 161--168 (1979). Okada, T.S.: In: Tests of Teratogenicity in Vitro, eds. J.D. Ebert and M. Marois (North-Holland, Amsterdam) pp. 91--105 (1976). Okada, T.S.: In: Current Topics in Developmental Biology, Vol. 16, eds. R.K. Hunt, A. Monroy and A.A. Moseona (Academic Press, New York and London) (1980) in press. Okada, T.S., G. Eguehi and M. Takeichi: Dev. Growth Differ. 13, 323--335 (1971). Okada, T.S., G. Eguehi and M. Takeiehi: Dev. Biol. 34, 321--333 (1973). Okada, T.S., K. Yasuda, M. Araki and G. Eguchi: Dev. Biol. 68, 600-617 (1979). Pritchard, D.J., R.M. Clayton and D.I. de Pomerai: J. Embryol. Exp. Morphol. 48, 1--21 (1978). Trinkaus, J.P. and J.P. Lentz: Dev. Biol. 9, 115-136 (1964). Whittaker, J.R.: Dev. Biol. 8, 99--127 (1963). Yamada, T.: Control Mechanisms in Cell-Type Conversions in Newt Lens Regeneration. Monographs in Developmental Biology, ed. Awalsky, 13, (S. Karger, Basel) (1977). Yasuda, K.: Dev. Biol. 68, 618--623 (1979).
3
AGE-DEPENDENT CHANGES IN THE CAPACITY OF T R A N S D I F F E R E N T I A T I O N OF R E T I N A L PIGMENT CELLS AS R E V E A L E D IN CLONAL CELL C U L T U R E KUNIO YASUDA, GORO EGUCHI * and T.S. OKADA
Department of Biophysics, Faculty of Science, University of Kyoto, Kyoto 606, Japan (Accepted 13 July 1980)
Changes in the capacity of transdifferentiation of retinal pigment cells into lens cells with developmental age were examined by culturing cells obtained from chicks at various developmental stages ranging from 5-day-old embryos to 1-year-old adults. Secondary clonal cultures were prepared from pigment cells harvested from primary cultures of the cells dissociated from the material at different ages. The percentage of colony formation to the total number of inoculated cells, of colonies with lens differentiation, and of colonies with the differentiation of pigment cells were obtained in cultures of the material derived from the donors at different ages. The total clonal efficiency did not alter with age. Colonies with lens cells appeared only in cultures started from embryos of less than 15 days. The percentage in the formation of pigmented colonies decreased with the age of the donor. Cells of post-hatching chicks produced only unidentifiable colonies without lens and pigment cells. The results indicate that determination of retinal cells alters with age. transdifferentiation
pigment cell culture
1. Introduction
Alteration of the specificity of differentiated cells, which m a y be called transdifferentiation or cellular metaplasia, occurs extensively in tissues of the vertebrate eye (Eguchi, 1976; Okada, 1976, 1980). The classical example of this p h e n o m e n o n is the Wolffian regeneration of lens from the pigment cells of the iris epithelium in several urodelen species (Yamada, 1977). In cell culture in vitro, a number of examples of transdifferentiation have also been known in avians and mammalians, which cannot regenerate the lost parts of eyes in vivo (Okada, 1976). In these in vitro cell culture experiments, embryonic material has mainly been used. In the case of avians, embryos ranging from 3.5 to 10 days of incubation have been employed * Present address: Research Institute for Molecular Biology, Faculty of Science, University of Nagoya, Nagoya 464, Japan.
to demonstrate the occurrence of transdifferentiation in vitro as well as to study factors controlling this process. It is very likely that the capacity of transdifferentiation, which may reflect the instability in the state of determination in Cell differentiation, m a y alter with developmental stages. In fact, the transdifferentiation of chick neural retina (NR) into lens and/or pigm e n t cells occurs less extensively with the progress of embryonic age and this capacity disappears completely in NR of newly hatched chicks (De Pomerai et al., 1978; Nomura and Okada, 1979). During chick embryonic life, several different cell types are differentiated in NR in situ, and this tissue, in respect to its cellular composition, becomes complex with development. The disappearance of the capacity of transdifferentiation of NR in in vitro culture experiments could be related to the process of cell differentiation of N R in situ, as discussed by Nomura and Okada (1979).
Pigmented cells (PC) of the tapetum of chick embryos at 8--10 days of incubation can also transdifferentiate into lens under the conditions of cell culture (Eguchi and Okada, 1973; Yasuda, 1979). Once pigmented, the tapetum in chick embryos in situ remains a homogeneous cell population of only pigmented epithelial cells throughout embryonic life, while there is increasing complexity in the cell t y p e composition in NR with embryonic development. Thus, we investigated whether the capacity of transdifferentiation o f PC of the tapetum into lens in vitro is stage related. In order to obtain semi-quantitative results at the cellular level, a clonal cell culture technique was adopted. This method may permit a rough estimation of the percentage of 'transdifferentiable' cells, if any, in a given population of PC of the tapetum in the embryos at different stages.
plastic petri dishes, each with a diameter of 3.0 cm. The percentage of cells attached to the culture substrate within 24 h after inoculation decreased with developmental stages. Therefore, it was necessary to inoculate a larger number of cells to start cultures at an equal density of attached cells in all the material taken from different ages. Primary cultures were maintained for 18 days, when they became confluent. From these primary cultures, foci with heavily pigmented cells were trimmed, collected and dissociated with trypsin (Eguchi and Okada, 1973). A b o u t 500 pigmented cells thus prepared were transferred into Falcon culture dishes with a diameter of 5.5 cm each. These secondary cultures at a low cell density of clonal level were maintained for a b o u t 40 days.
2.3. Scoring the results 2. Materials and methods
White Leghorn embryos at 5, 8, 9, 10, 11, 15 and 20 days of incubation, newly hatched chicks and chicks of a b o u t 1 year old were used t o obtain PC of the tapetum for cell culture. EDTA was used to separate the pigmented epithelium of the tapeta from unpigmented neighbouring cells (Trinkaus and Lentz, 1964; Eguchi and Okada, 1973). The separation of the clean pigmented pieces was easy in most cases. However, some portions of the tapetum from embryos older than 20 days adhered so tightly to the adjacent tissue layers that some pigmented cells were left attached to the tissue.
The secondary clonal cultures were fixed with Bouin's fixative. The number of all colonies (colonies with a diameter larger than 2 mm) and of colonies with PC and of lentoid bodies (LB) (Okada et al., 1971, 1973; see Fig. 7F) were counted in each plate (Okada et al., 1979). The plating efficiency, (the number of colonies)/(the number of inoculated cells) × 100, the efficiency of PC differentiation, (the number of colonies with PC)/ (the number of total colonies) × 100 and the efficiency of LB differentiation, (the number of colonies with LB)/(the number of total colonies) × 100, were obtained. In all cultures, Eagle's minimum essential medium supplemented with 15% fetal bovine serum of one particular batch (Microbiological Inc. L o t no. 88818) was used.
2.2. Cell culture
2.4. SDS-polyacrylamide gel electrophoresis
Cell suspension was prepared from the isolated pieces of the t a p e t u m by EDTA and trypsin treatment as described previously (Eguchi and Okada, 1973). A b o u t 4 X 104 to 1.5 × l 0 s cells were inoculated into Falcon
Cultures at different stages were washed several times with Hanks' solution and each representative colony was peeled off with tweezers and solubilized in 50--200 /A of SDS-sample buffer. Aliquots containing a b o u t
2.1. Preparation o f cells to be cultured
30 pg proteins were analyzed on SDS-polyacrylamide slab gels according to the procedure of Laemmli (1970).
7
10 qb
(,3
2.5. Immunoelectrophoresis
.S
lo
Cultures at different stages were washed several times with Hanks' solution and homogenized in a small volume of the same saline. The homogenate was centrifuged at 10,000 g for 5 min to remove cell debris and the supernatant was frozen at --70°C. Aliquots of the supernatant were analyzed by immunoelectrophoresis using rabbit antiserum against the total extract of chick lenses (Yasuda, 1979).
qb
x
I
0
I
I
I
10 20 30 Days in Culture
I
40
Fig. 1. Typical growth curves of PC from various developmental stages in primary cultures. Each value represents an average of duplicate hemocytometer counts of the calls harvested from two plates starting from a single inoculum, o, 5-day embryos; A, 15day embryos; x, Adult chicks.
Fig. 2. Phase-contrast photomicrographs of pigmented epithelial cells growing in primary cultures on day 7 after inoculation. A) 5-day embryos. B) 8-day embryos. C) 1-day-old chicks. D) Adult chicks. X100.
3. Resuits 3.1. Primary cultures Many of the PC attached to the culture substrate started to grow within a b o u t 48 h of plating. PC from all the developmental stages grew equally well in primary cultures (Fig. 1). The average size of each PC differs with age; it
is usually smaller in younger materials (Fig. 2). Within about 10 days' outgrowth, however, all cultured cells of older materials became smaller reaching the size of younger materials. During the outgrowth, PC lost their pigm e n t granules by dilution with cell divisions and by discharge of the granules from PC into the medium (Whittaker, 1963; Yasuda, 1979}.
Fig. 3. Phase-contrast photomicrographs of PC at the confluent stage of the primary cultures on day 18. The heavily pigmented epithelial cells were harvested with trypsin and transferred into the secondary clonal cell culture. A) 5-day embryos. B) 8-day embryos. C) 15-day embryos. D) 1-day-old chicks. E) Adult chicks, epithelial portion. F) Adult chicks, fibroblastic portion. × 100.
PC in cultures from embryos younger than 11 days became non-pigmented within a few days of culture (Fig. 2A, B). Depigmented cells grew rapidly. When cultures reached a confluence, a number of foci of tightly packed polygonal cells appeared (Fig. 3). Cells of these foci became heavily repigmented. PC in cultures from embryonic material older than 15 days became less pigmented with growth without however completely losing the pigment granules. Practically all cells in confluent cultures at about 18 days were pigmented, although the degree of pigmentation was very different in individual cells in a single culture dish (Fig. 3). After confluence, cells from younger embryonic material appeared more heavily pigmented than those from older ones. In cultures of material from embryos older than 20 days spindle-shaped or dendritic PC appeared, in addition to pigmented epithelial cells (Fig. 3F). The spindle-shaped and dendritic PC did not attach to each other completely, while the pigmented epithelial cells came into close contact to establish a typical epithelial sheet.
~
20
o 10 "6
.,,j
0 I
I
I
5
10
15
O 0
0
I I t .;t 20 chick
t adult
Days after Incubation Age of Donors
Fig. 5. Decrease in the n u m b e r of colonies with LB in 40-day secondary clonal cultures with the development o f d o n o r age. Colonies with LB were identified and counted according to Okada et al. (1979). The data are expressed a s t h e average n u m b e r o f colonies with LB (+S.D.) o f 10 culture plates.
3.2. Clonal cultures
Cells harvested from heavily pigmented foci of epithelial sheets of primary cultures were used for clonal cultures. Fig. 4 shows the results after a b o u t 40 days' culturing. It is im-
20 ¢
.2
100
~o
'a .Q
E o I
i
I
I
0
5
10
15
II|
//
20 chick
t adult
Days after Incubation Age of Donors
Fig. 4. The total n u m b e r of colonies 40-day secondary clonal cell cultures developmental stages. 500 single cells primary cultures were inoculated into data are expressed as the average total onies (-+S.D.) o f 1O culture plates.
"~
0
0 I
I
I
0
5
10
15
t // 20 chick
t adult
Days after Incubation
f o r m e d in the f r o m different taken from the each plate. The n u m b e r o f col-
Age of Donors
Fig. 6. Decrease in the n u m b e r of colonies with PC in 40-day secondary clonal cultures with the development of d o n o r age. The data are expressed as the average n u m b e r o f colonies (+S.D.) o f 10 culture plates.
~
i ii~iiii! i~
[3
Fig. 7. Typical macrophotographs of culture plates of 40-day secondary clonal cultures taken from donors at different developmental stages. A) 5-day embryos. B) 8-day embryos. C) 15-day embryos. D) 1-day-old chicks. E) Adult chicks. F) Lentoid bodies (arrow, LB) appeared in cultures of 5-day embryos. × 150.
mediately noticeable that the plating efficiency, which indicates the percentage of cells to be potent for colony formation ('clonable' cells) contained in a given inoculate, is almost the same regardless of the developmental stage. On the other hand, colonies with LB appeared only in cells derived from embryos younger than 15 days of incubation (Fig. 5). The percentage of colonies with PC also decreased gradually with the age of cultured material: no pigmented colonies were formed in cultures of cells from the adult tapetum (Fig. 6; see also Fig. 7). In clonal cultures prepared from cells of primary cultures from embryos older than 15 days, PC, if any, did not establish an epithelial sheet; they were of the spindle or dendritic form and produced colonies with very dispersed cells. The appearance of pigmented colonies occurred in two ways: 1) by repigmentation of once completely depigmented cells in the early stages of clonal outgrowth, or 2) by clonal outgrowth of cells with pigmented granules throughout all stages or the early stage of culturing. In the first case, all colonies invariably consisted of pigmented and non-pigmented cell populations, while in the second, some colonies consisted of only pigmented cells and others included a few non-pigmented cells in the peripheral portion, leaving the inner portion heavily pigmented. Pure colonies with only PC appeared in cultures derived from PC of 5--6 day old embryos. There are several 'mixed' colonies, in which both LB and PC were differentiated. In secondary clonal cultures prepared from primary cultures of embryos older than 15 days, many colonies contained neither PC nor LB. In these colonies, cellsdid not establish a coherent sheet, but were very dispersed.
3.3. Protein composition of each colony An identification of the presence of crystallins in clonal cultures was made in the culture homogenates by means of immunoelectrophoresis. In secondary clonal cultures from
primary cultures of PC from embryos younger than 15 days three classes of crystallins, ~-, ~and ~-crystallins, were detected, whereas they were not found in cultures derived from older material (Fig. 8). The homogenates prepared from each single colony were respectively' subjected to SDS--gel-electrophoresis for protein analysis. Electrophoretic patterns of several representative colonies with LB, PC and unidentifiable colonies without any identification phenotypes are shown in Fig. 9. Bands which can well be identified as crystaUins by co-migration of the latter proteins (cf. also Araki et el.,
5
=~i~i~~
Fig. 8. Immunoelectrophoretic patterns of the homogenates of 40-day secondary clonal cultures of PC taken from the donors at different developmental stages. Troughs and wells contain respectively the rabbit antiserum against total lens extract and the supernatants of the homogenates of 1) intact 1-day-old chick lens (control) sample, 2) clonal cultures of PC from 5-day embryos, 3) from 8-day embryos, 4) from l l - d a y embryos, 5) from 1-day-old chicks, and 6) from adult chicks. In all positive cases (1, 2, 3 and 4) all of the three arcs corresponding to ol-, ~- and -crystallins are detectable.
10
1
2
3
4
5
6
7
5~
i~~
Fig. 9. SDS-gel electrophoretic patterns of the homogenates of respective colony of 40-day secondary clonal cultures from different developmental stages: 1) the homogenate of 1-day-old intact chick lenses (control sample), 2) a colony with LB derived from 5day embryos, 3) a colony with LB derived from 8-day embryos, 4) a mixed colony with both LB and PC derived from 5-day embryos, 5 ) a colony with PC derived from 5-day~ld embryos, 6) an unidentifiable colony derived from 8-day chicks, 7) an unidentifiable colony derived from 1-day-old chicks. ~, ~, and 5 represent ~-, major ~-, and 6-crystallin, respectively. The fact that bands indicated by arrows (~-*), the mobility of which is very close to that of ~-crystallin, are non-crystallin proteins was confirmed by the separate tests using the two-dimensional immunoelectrophoresis. A band corresponding to ~-crystallin migrates between these two bands.
1979) were recognized only in colonies with the visible formation of LB. All three classes of crystallins, a, fl, and 5, were present in colonies with LB. The unidentifiable colonies did n o t contain any detectable amount of crystallins. Band patterns of electrophoresis among three different types of colonies were basically similar to each other, except for the presence or the absence of crystallins.
4. Discussion It has been demonstrated that the capacity of transdifferentiation of chick NR in culture into LB and PC progressively decreased with developmental age (De Pomerai and Clayton, 1978; Nomura and Okada, 1979). The present study regarding the capacity of PC of the tapetum to transdifferentiate into lens cells in culture confirms this progressive decrease. However, as our conclusions rest on the results of the secondary clonal cell culture, it is necessary to evaluate the limitations of this technique. Firstly, with the present method we are not able to detect the presence of cells that would differentiate into PC or lens cells without producing large colonies. These cells, if present, may disappear earlier on in clonal cultures. Therefore, what we detected here is the percentage of transdifferentiable cells only in 'clonable' cell population. Secondly, instead of the whole cell population, we transferred only the heavily pigmented cell population of primary cultures into the assay system of secondarily clonal culture, not the whole cell population. Then, with the present method it is not possible to compare directly the capacity of transdifferentiation in the tapetum of different ages, b u t the comparison is limited to the capacity of 'stable' cell population, which can maintain or recover the original differentiative trait as PC in primary cultures. Within such limitations, the present results demonstrate in a semi-quantitative manner that in vitro the capacity of transdifferentiation of PC of the tapetum into lens cells decreases progressively with the developmental age of the starting material and that the capacity is almost completely lost in the t a p e t u m from embryos older than 15--16 days. On the other hand, the plating efficiency which may indicate the percentage of 'clonable' cells remains unchanged throughout all the stages examined. N o t only the differentiation of LB but also the differentiation of PC in secondary clonal
11
culture occurred more frequently in younger materials. Thus, the capacity of both transdifferentiation and redifferentiation decreases with age. Colonies derived from 'clonable' cells of aged material remained mostly 'unidentifiable'. It still remains to be examined whether this result indicates the permanent inhibition of redifferentiation or transdifferentation into any identifiable characteristics with development. In the present study, the decrease of the capacity of transdifferentiation is shown in a semi-quantitative manner by comparing the percentage within material at different ages, of the formation of colonies with LB to the number of inoculated cells or to the number of the 'clonable' cells. The results can be interpreted to indicate that the tapetum consists of qualitatively different cell populations, the transdifferentiable and non-transdifferentiable cells, though apparently consisting of a very homogeneous population of PC. The number of transdifferentiable cells decreases with age. It may be possible that there are no such qualitative distinctions in the cell population, but, for u n k n o w n reasons, the number of transdifferentiable cells detectable under the present assay system decreases with age. It has been .repeatedly shown that an overall trend of transdifferentiation is influenced by many factors of culture conditions in both N R and PC of the tapetum (Itoh, 1976; Clayton et al., 1977; Araki and Okada, 1978; Pritchard et al., 1978). This, however, does not exclude that the state of determination of PC in the tapetum, as assayed by the capacity of transdifferentiation, changes with developmental age.
Acknowledgements The authors thank Miss Y. Katsurayama for helping with the preparation of the manu-
script. The present work was supported by a research grant for Basic Cancer Research from the Japan Ministry of Education, Science and Culture, and by a research grant from Yamada Science Foundation.
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