DEVELOPMENTAL
BIOLOGY
Cell interactions
RUBEN Znstituto
50,
(1976)
and the Regulation of Cholinergic Neural Differentiation in Vitro
ADLER,
de Biologia
48-57
GLADYS
Celular,
TEITELMAN,
Facultad Accepted
de Medicina, November
AND ANGELA Paraguay
2155,
Enzymes during
MARIA Buenos
SUBURO Aires,
Argentina
20,1975
Seven-day-old chick embryo neural retina (NR), telencephalon (T), optic lobe (OL), and rombencephalon (Ro) were dissociated, and the resulting cell suspensions were allowed to reaggregate in vitro during 3 days either independently or in different binary combinations. Interactions could be detected by the comparison of the activity of the enzymes of the cholinergic system, choline acetyltransferase (CAT) and acetylcholinesterase (ACE), in “pure” and “combined” aggregates. The results clearly show that the activity of both enzymes in embryonic neural cells can be modified selectively by interactions between different cell populations. Thus, combined NR-OL aggregates show an increase in CAT without changes in ACE, NR-T an increase in CAT and a decrease in ACE, T-Ro a decrease in both CAT and ACE, and OL-T no changes at all. Experiments in which NR and OL cells were combined in different proportions indicate that the interactions require the presence of defined numbers of cells from each kind. Isochronous and heterochronous combinations of 7- and lo-day-old NR and OL cells show that the interactive capacities of the cells change with development.
to answer this question using an experimental approach briefly described in a preThe emergence of different cell types vious paper (Adler and Teitelman, 1974). from a common ancestor during develop- Different chick embryo neural organs are ment is considered to be the result of a dissociated and the resulting cell suspenchain of events in which various signals sions are allowed to reaggregate in vitro, trigger and/or control the switching “on” either alone or in different binary combiand “off” of genes, as well as the synthesis nations. Interactions can be detected by and assembly of macromolecules. The cen- the comparison of the activities of the chotral nervous system of vertebrates is so linergic enzymes choline acetyltransferase complex that attempts to explain the dif- and acetylcholinesterase in both “pure” ferentiation of neural cells in these terms and “combined” aggregates. could seem highly hypothetical at the Whether the interactions that were present time. However, it is accepted that found can be interpreted within the most cell interactions might play a role in the orthodox concepts of “cell differentiation” differentiation of the various types of is something which is not a subject for neural and glial cells from their common speculation at the present time. However, ancestor, the neuroepithelial cells (see, for we believe that these experiments show example, Weiss, 1955; Sperry, 1965; Adler, that embryonic neural cells can interact in 1970, 1973; Morris and Moscona, 1971; a rather selective way, and that they are Garber and Moscona, 1972). thus able to determine modifications in It remains to be ascertained to what ex- some enzymatic activities related to the tent the early embryonic neural cells are specialized functions of nerve cells. actually able to interact with each other, MATERIALS AND METHODS determining the appearance of quantitaAll the experiments were done with 7- or tive and/or qualitative changes in their lo-day-old chick embryos. Telencephalon respective properties. This work attempts INTRODUCTION
43 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
ADLER,
TEITELMAN,
AND
SLJBURO
(T), optic lobe (OL), rombencephalon (Ro), and neural retina (NR) were dissected individually and freed from their surrounding tissues. Preparation of cell suspensions. Dissociation procedures were similar to those described by Adler (1970, 1971). Small pieces of tissue were placed in a mixture of 1% trypsin (Difco, 1:250) and 1% Pancreatin (Difco, 3~) in Puck’s “A” solution. After a 40-min incubation, the enzymes were replaced by complete medium (medium 199 (Difco) plus 2.8 g/liter glucose, supplemented with 10% chick serum and 10% g-day-old chick embryo extract) and the tissues were dissociated mechanically. Cells were resuspended in fresh medium and counted with an hemocytometer. Dye exclusion (Cahn et al., 1967) was always greater than 90%. “Pure” and “combined” reaggregation cultures. Three milliliters of a cell suspension containing lo-20 x 10” cells/ml were placed in 25ml Erlenmeyer flasks and cultured during 3 days in a gyratory water bath (New Brunswick Co.) at 37°C and 90 rpm. “Pure” cultures were made from the cell suspension of one of the mentioned organs, whereas in “combined” cultures two different cell suspensions were mixed before culturing. Unless otherwise specified in the text, the combined cultures contained equal numbers of both cell populations. Each experiment which included a combined culture also included the two corresponding pure ones. The different experiments were repeated at least three times. Biochemical assays. Dissociated cells or cultured aggregates collected by centrifugation, as well as freshly dissected organs, were homogenized by means of a motor driven Elvehjem homogenizer, fitted with a Teflon pestle, at 4°C in double distilled water. When not immediately assayed, homogenates were kept frozen at -70°C. DNA concentration was determined with the technique of Burton (1956). Choline acetyltransferase (CAT) was de-
Cell Interactions
in Neural
Development
4Y
termined by the micromethod of McCaman and Hunt (1965) using as a substrate synthetic [14Clacetyl CoA (New England Nuclear, sp act 58 mCi/mmole). All assays with appropriate blanks were run in triplicate and the radioactivity of [14C]acetylcholine, the product of the enzymatic reaction, was measured. Counting efficiency was monitored by the use of internal standards. Specific activity was expressed as micromoles of acetylcholine synthesized per hour per milligram DNA. Acetylcholinesterase (ACE) activity was assayed with the calorimetric method of Ellman et al. (1961) using acetylthyocholine (AThC) as a substrate. Specific activity was expressed as micromoles of AThC hydrolyzed per minute per milligram of DNA. Analysis of the data. As mentioned in the Introduction, the aim of this study was to determine whether different embryonic neural cell populations can interact with each other and thus modify their enzymatic activities. In consequence, the different experimental groups were formed by two pure cultures (from cell populations a and b), and the corresponding combined one Cab). If the a and b cells maintain the same enzymatic activity in the combined aggregates that they have in the pure cultures, it is then possible to predict the activity that should be present in the combined aggregates by the following calculation: Pa,,
= PA
+ qB
where P,, is the predicted activity of combined aggregates formed by cell populations a and b; A and B are the activities shown by a and b in pure cultures; and p and q indicate the proportions in which the two populations were mixed before culturing. Within each experimental group, enzymatic activities of the combined aggregates were compared with the corresponding predicted activity (null hypothesis) using Fisher’s modification of the two-tailed
50
DEVELOPMENTAL
BIOLOGY
Student’s t test (Mills, 1955). Significant differences indicate that an interaction has taken place.
VOLUME
50, 1976
ACE. At both stages, the higher specific activities are found in the more caudal regions (OL and Ro).
RESULTS
I. CAT and ACE Activities
In Ovo
The specific activity of CAT and ACE was determined in 7- and lo-day-old chick embryo neural retina (NR), telencephalon (T), optic lobe (OL), and rombencephalon (Ro) (see Table 1). These enzymatic activities can already be detected in the abovementioned organs from ‘I-day-old embryos. Comparison of these with the data from older embryos shows that the specific activity of both enzymes increases with developmental age and that this increase is much more pronounced for CAT than for TABLE CHOLINE
ACETYLTRANSFERASE
Activities
1 IN VIVO,
CAT Specific
activity”
in Cell Sus-
To determine the effect of dissociation procedures on CAT and ACE activities, enzymatic assays were carried out in cell suspensions immediately after their obtention. The dissociated cells, suspended in fresh culture medium, were collected by centrifugation and the resulting pellet was homogenized as described in Materials and Methods. Results shown in Table 1 indicate that the dissociation procedures have different
(CAT) AND ACETYLCHOLINESTERASE (ACE) ACTIVITIES SUSPENSIONS AND IN PURE AGGREGATES
Material
Neural retina (NR) ‘I-day-old embryos In viva Cell suspensions Cell aggregates lo-day-old embryos Telencephalon (T) 7-day-old embryos In vivo Cell suspensions Cell aggregates lo-day-old embryos Optic lobes (OL) 7-day-old embryos In vivo Cell suspensions Cell aggregates lo-day-old embryos Rombencephalon (Ro) 7-day-old embryos In vivo Cell suspensions Cell aggregates lo-day-old embryos
II. CAT and ACE pensions
IN CELL
ACE Pb
Specific
activity’
P
in viuo
0.12 k 0.02 (4)” 0.20 + 0.03 (4) 0.85 + 0.10 (22) 0.95 f 0.09 (3)
co.01
BIOLOGY
Cell interactions
RUBEN Znstituto
50,
(1976)
and the Regulation of Cholinergic Neural Differentiation in Vitro
ADLER,
de Biologia
48-57
GLADYS
Celular,
TEITELMAN,
Facultad Accepted
de Medicina, November
AND ANGELA Paraguay
2155,
Enzymes during
MARIA Buenos
SUBURO Aires,
Argentina
20,1975
Seven-day-old chick embryo neural retina (NR), telencephalon (T), optic lobe (OL), and rombencephalon (Ro) were dissociated, and the resulting cell suspensions were allowed to reaggregate in vitro during 3 days either independently or in different binary combinations. Interactions could be detected by the comparison of the activity of the enzymes of the cholinergic system, choline acetyltransferase (CAT) and acetylcholinesterase (ACE), in “pure” and “combined” aggregates. The results clearly show that the activity of both enzymes in embryonic neural cells can be modified selectively by interactions between different cell populations. Thus, combined NR-OL aggregates show an increase in CAT without changes in ACE, NR-T an increase in CAT and a decrease in ACE, T-Ro a decrease in both CAT and ACE, and OL-T no changes at all. Experiments in which NR and OL cells were combined in different proportions indicate that the interactions require the presence of defined numbers of cells from each kind. Isochronous and heterochronous combinations of 7- and lo-day-old NR and OL cells show that the interactive capacities of the cells change with development.
to answer this question using an experimental approach briefly described in a preThe emergence of different cell types vious paper (Adler and Teitelman, 1974). from a common ancestor during develop- Different chick embryo neural organs are ment is considered to be the result of a dissociated and the resulting cell suspenchain of events in which various signals sions are allowed to reaggregate in vitro, trigger and/or control the switching “on” either alone or in different binary combiand “off” of genes, as well as the synthesis nations. Interactions can be detected by and assembly of macromolecules. The cen- the comparison of the activities of the chotral nervous system of vertebrates is so linergic enzymes choline acetyltransferase complex that attempts to explain the dif- and acetylcholinesterase in both “pure” ferentiation of neural cells in these terms and “combined” aggregates. could seem highly hypothetical at the Whether the interactions that were present time. However, it is accepted that found can be interpreted within the most cell interactions might play a role in the orthodox concepts of “cell differentiation” differentiation of the various types of is something which is not a subject for neural and glial cells from their common speculation at the present time. However, ancestor, the neuroepithelial cells (see, for we believe that these experiments show example, Weiss, 1955; Sperry, 1965; Adler, that embryonic neural cells can interact in 1970, 1973; Morris and Moscona, 1971; a rather selective way, and that they are Garber and Moscona, 1972). thus able to determine modifications in It remains to be ascertained to what ex- some enzymatic activities related to the tent the early embryonic neural cells are specialized functions of nerve cells. actually able to interact with each other, MATERIALS AND METHODS determining the appearance of quantitaAll the experiments were done with 7- or tive and/or qualitative changes in their lo-day-old chick embryos. Telencephalon respective properties. This work attempts INTRODUCTION
43 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
ADLER,
TEITELMAN,
AND
SLJBURO
(T), optic lobe (OL), rombencephalon (Ro), and neural retina (NR) were dissected individually and freed from their surrounding tissues. Preparation of cell suspensions. Dissociation procedures were similar to those described by Adler (1970, 1971). Small pieces of tissue were placed in a mixture of 1% trypsin (Difco, 1:250) and 1% Pancreatin (Difco, 3~) in Puck’s “A” solution. After a 40-min incubation, the enzymes were replaced by complete medium (medium 199 (Difco) plus 2.8 g/liter glucose, supplemented with 10% chick serum and 10% g-day-old chick embryo extract) and the tissues were dissociated mechanically. Cells were resuspended in fresh medium and counted with an hemocytometer. Dye exclusion (Cahn et al., 1967) was always greater than 90%. “Pure” and “combined” reaggregation cultures. Three milliliters of a cell suspension containing lo-20 x 10” cells/ml were placed in 25ml Erlenmeyer flasks and cultured during 3 days in a gyratory water bath (New Brunswick Co.) at 37°C and 90 rpm. “Pure” cultures were made from the cell suspension of one of the mentioned organs, whereas in “combined” cultures two different cell suspensions were mixed before culturing. Unless otherwise specified in the text, the combined cultures contained equal numbers of both cell populations. Each experiment which included a combined culture also included the two corresponding pure ones. The different experiments were repeated at least three times. Biochemical assays. Dissociated cells or cultured aggregates collected by centrifugation, as well as freshly dissected organs, were homogenized by means of a motor driven Elvehjem homogenizer, fitted with a Teflon pestle, at 4°C in double distilled water. When not immediately assayed, homogenates were kept frozen at -70°C. DNA concentration was determined with the technique of Burton (1956). Choline acetyltransferase (CAT) was de-
Cell Interactions
in Neural
Development
4Y
termined by the micromethod of McCaman and Hunt (1965) using as a substrate synthetic [14Clacetyl CoA (New England Nuclear, sp act 58 mCi/mmole). All assays with appropriate blanks were run in triplicate and the radioactivity of [14C]acetylcholine, the product of the enzymatic reaction, was measured. Counting efficiency was monitored by the use of internal standards. Specific activity was expressed as micromoles of acetylcholine synthesized per hour per milligram DNA. Acetylcholinesterase (ACE) activity was assayed with the calorimetric method of Ellman et al. (1961) using acetylthyocholine (AThC) as a substrate. Specific activity was expressed as micromoles of AThC hydrolyzed per minute per milligram of DNA. Analysis of the data. As mentioned in the Introduction, the aim of this study was to determine whether different embryonic neural cell populations can interact with each other and thus modify their enzymatic activities. In consequence, the different experimental groups were formed by two pure cultures (from cell populations a and b), and the corresponding combined one Cab). If the a and b cells maintain the same enzymatic activity in the combined aggregates that they have in the pure cultures, it is then possible to predict the activity that should be present in the combined aggregates by the following calculation: Pa,,
= PA
+ qB
where P,, is the predicted activity of combined aggregates formed by cell populations a and b; A and B are the activities shown by a and b in pure cultures; and p and q indicate the proportions in which the two populations were mixed before culturing. Within each experimental group, enzymatic activities of the combined aggregates were compared with the corresponding predicted activity (null hypothesis) using Fisher’s modification of the two-tailed
50
DEVELOPMENTAL
BIOLOGY
Student’s t test (Mills, 1955). Significant differences indicate that an interaction has taken place.
VOLUME
50, 1976
ACE. At both stages, the higher specific activities are found in the more caudal regions (OL and Ro).
RESULTS
I. CAT and ACE Activities
In Ovo
The specific activity of CAT and ACE was determined in 7- and lo-day-old chick embryo neural retina (NR), telencephalon (T), optic lobe (OL), and rombencephalon (Ro) (see Table 1). These enzymatic activities can already be detected in the abovementioned organs from ‘I-day-old embryos. Comparison of these with the data from older embryos shows that the specific activity of both enzymes increases with developmental age and that this increase is much more pronounced for CAT than for TABLE CHOLINE
ACETYLTRANSFERASE
Activities
1 IN VIVO,
CAT Specific
activity”
in Cell Sus-
To determine the effect of dissociation procedures on CAT and ACE activities, enzymatic assays were carried out in cell suspensions immediately after their obtention. The dissociated cells, suspended in fresh culture medium, were collected by centrifugation and the resulting pellet was homogenized as described in Materials and Methods. Results shown in Table 1 indicate that the dissociation procedures have different
(CAT) AND ACETYLCHOLINESTERASE (ACE) ACTIVITIES SUSPENSIONS AND IN PURE AGGREGATES
Material
Neural retina (NR) ‘I-day-old embryos In viva Cell suspensions Cell aggregates lo-day-old embryos Telencephalon (T) 7-day-old embryos In vivo Cell suspensions Cell aggregates lo-day-old embryos Optic lobes (OL) 7-day-old embryos In vivo Cell suspensions Cell aggregates lo-day-old embryos Rombencephalon (Ro) 7-day-old embryos In vivo Cell suspensions Cell aggregates lo-day-old embryos
II. CAT and ACE pensions
IN CELL
ACE Pb
Specific
activity’
P
in viuo
0.12 k 0.02 (4)” 0.20 + 0.03 (4) 0.85 + 0.10 (22) 0.95 f 0.09 (3)
co.01
