DEVELOPMENTAL
BIOLOGY
60, 153-162 (1977)
Temporal Changes in Embryonic Nerve Cell Recognition: Correlate with Cholinergic Development in Aggregate Cultures GALO RAMIREZ*,
l AND NICHOLAS
W. SEEDst
* Centro de Biologia Molecular (CSIC-UAMI, Velazquez 144, Madrid 6, Spain, and t Department Biophysics and Genetics, University of Colorado Medical Center, Denver, Colorado 80262 Received December 28,1976;
of
accepted in revised form June 3,1977
Chick embryo retina and optic tectum cells can be dissociated into single cells and then reaggregated in suspension cultures to give highly organized and differentiated aggregates. These aggregates show a degree of cholinergic differentiation that is characteristic of each cell type; the low activity of choline acetyltransferase in the optic tectum aggregates probably reflects the condition of natural deafferentation inherent in the culture situation. It is possible, in this respect, to study the retina-tectum interaction in vitro by preparing coaggregates including both types of cells. When coaggregates are prepared from tectum and retina cells of the same developmental age, the activity of choline acetyltransferase measured in the coaggregates is consistently higher than would be expected from the simple addition of the activities of the component cells, pointing to some kind of metabolic synergism between retinal and tectal cells. As for acetylcholinesterase, this synergism occurs only under special circumstances, and it is generally less marked. No synergism was observed when retina and tectum cells of different developmental age were coaggregated, suggesting the existence of a temporal control of neuronal interaction specificity. On the other hand, the synergism is only observed between neuronal systems that are known to establish synaptic connections during normal in viva development: No interaction could be detected when either retinal or tectal cells were combined with telencephalon, cerebellum, or liver cells. Experimental evidence is presented suggesting that the retina-tectum interaction depends on intimate cell-cell contact, and it is not mediated by freely diffusible molecules. Neurotransmission-related metabolic studies in coaggregates seem to offer a promising tool to study recognition-interaction phenomena in groups of neurons establishing synaptic links during development. INTRODUCTION
The establishment of specific cell connections during development is a major key to the complex functional organization characteristic of the mature nervous system. Much work has been done in the last 20 years to ascertain whether the amazingly accurate pattern of nerve cell connections (synaptic and other) is based on an orderly sequence of cellular and environmental events which bring into contact the right cells at the right time of development, or whether it is based on the presence of specific recognition-mediating signals in the cell surfaces responsible for the establishment of temporary or permanent ’ To whom dressed.
all
correspondence
should
Copyright 0 1977by Academic Press, Inc. All rights of reproduction in any form reserved.
be ad-
independent of the time interactions, when such contact occurs during development (Gaze, 1970; Jacobson, 1970). The visual system, in both invertebrates and vertebrates, has provided a convenient model to study growing nerve fiber-target cell recognition, and, so far, experimental evidence has been found to support the existence in living organisms of both the interaction mechanisms outlined above (Attardi and Sperry, 1963; Crossland et al., 1974; Gaze, 1970; Jacobson, 1970; Lopresti et al., 1973). Recently, the problem of the retino-tectal recognition has been approached in two different laboratories by means of in vitro techniques (Barbera et al., 1973; Barbera, 1975; Gottlieb et al., 1974). These studies 153 ISSN 0012-1606
154
DEVELOPMENTAL BIOLOGY
have demonstrated cell surface adhesive specificity between retina and optic tectum cells. Gottlieb et al. (1974) found a differential behavior of tectum and retina cell surfaces as a function of developmental age and suggested that temporal factors were a potential mechanism to achieve control of contact specificity during normal maturation in Go. Using a different methodology to measure retino-tectal specificity, Barbera et al. (1973, 1975) did not find substantial differences between tissue of various ages; however, they suggested that topographic factors were most important in controlling the specificity of cell interaction. We have proceeded with the rationale that cell recognition, to be specific and meaningful, should be able to trigger some metabolic events reflecting the establishment of functional ties between the interacting cells. Synapse formation is the most obvious and possibly the most specific form of cell-cell interaction, although there are probably many less obvious interactions between specific cell types. Since full cholinergic development of the chick optic tecturn has been shown to be contingent upon the arrival of retinal cell fibers (Filogamo, 1960; Marchisio, 1969), the influence of different patterns of interaction between tecturn and retina cells on the specific activities of two enzymes related to cholinergic neurotransmission has been examined. MATERIALS
AND
METHODS
Preparation of aggregates. Tectum and retina cell aggregates were prepared essentially as described before (Seeds, 1971; Ramirez, 1977a). Tecta and retinas (whole retinas, including the pigment epithelium) were removed from 7- or lo-day chick embryos (White Leghorn) and were cut into small (1 mm31 pieces. The tissue.was dissociated in 0.25% trypsin (Difco) in saline at 37°C for 20 min, and was pipet-triturated at 5min intervals. The resulting cell suspension was passed through a nylon screen (180 pm) and was inoculated into 25-ml
VOLUME 60, 1977
Erlenmeyer flasks containing 3 ml of basal Eagle’s medium (Gibco), supplemented with 0.4% glucose and 10% fetal calf serum, at a final cell concentration of 2-5 x lo6 cells/ml. In coaggregation experiments the flasks received aliquots of both retina and tectum cell suspensions, but the total cell concentration in the culture flask was kept within the same range as for simple aggregates. The flasks were gassed with CO,:air (5:95) and were incubated in a gyrotory shaker bath (New Brunswick) at 37°C and 70 rpm. The next day the cultures were transferred to 50-ml Erlenmeyer flasks, and the volume of medium was increased to -10 ml. Half the volume of medium was changed every other day. Enzymatic assays. Acetylcholinesterase (AChEI and choline acetyltransferase (ChATI were assayed as described (Seeds, 1971; Ramirez, 1977a). True acetylcholinesterase was distinguished from total cholinesterase activity by adding to the reaction mixture the specific inhibitor of true AChE, BW 284 C51 dibromide (Wellcome Reagents, Ltd., Beckenham, Kent, England), to a final concentration of low5 M. In the ChAT assay, blanks without added choline were routinely run to measure acetylation of endogenous substrates. This amounted to less than 2% of the cholinedependent acetylation for the different classes of nerve cell aggregates used in the present work. However, in liver cell aggregates, the acetylation of endogenous substrates accounted for practically all the observed activity. Experimental setup and treatment of data. The different retina-tectum interaction experiments presented in Figs. 3-9 employed a total of 30 flasks (Figs. 3-71, or 24 flasks (Figs. 8,9) (one to three aggregates per flask) for a time-course run. For each time point, six flasks (two retina, two 2 Abbreviations used: AChE, acetylcholinesterase; ChAT, choline acetyltransferase; BW284 C51 dibromide, 1,5-bis-(4-allyldimethylammoniumphenyl) pentane-3-one dibromide; ACh, acetylcholine.
RAMIREZ AND SEEDE
Cholinergic
tectum, and two retina plus tectum) were independently processed and assayed in duplicate, so that four independent enzyme activity values would define any point in the curves. In this way the retinatectum interaction was measured against retinal and tectal basic activities from the same original batch of dissociated cells. The retinal and tectal (separate aggregates) enzyme activities measured in a given time point were used to calculate an “expected enzyme activity” for the coaggregate, taking into account the proportion (in terms of protein) between retinal and tectal cells at the time of mixing, since it would not be possible to distinguish the actual contributions of retina and tectum in a preformed coaggregate. The validity of this approach is supported by the fact that, although the protein mass of individual retina and tectum cell aggregates varied during the 4 weeks elapsed in a timecourse run, the proportion between the total protein recovered in retina and tectum cell aggregates, for any time point, remained reasonably constant (the mean +SD of this recovered protein proportion for the five or four points of a time course is given in the legend of each figure). Furthermore, the coaggregation process did not preferentially discriminate against retinal or tectal cells, since the consolidated coaggregates consistently displayed the same ratio of retinal to tectal cells as the input populations (innoculum) (Table 1). All other coaggregation experiments described in this report were handled in a similar manner, except for the specific details given in the relevant portions of the text. RESULTS
AND
DISCUSSION
Cholinergic Development Tectum Cell Aggregates
in Retina
and
Both chick retina and optic tectum show large increases in choline acetyltransferase and acetylcholinesterase specific activities from fetal Day 7 to 10 days after
Development
in Aggregate
Cultures
155
hatching (McGeer et al., 1974; Ramirez, 1977b). The specific activity of acetylcholinesterase in aggregate cultures prepared from ‘I-day chick embryo retina and optic tectum increases considerably during 4 weeks of culture (Fig. 1). Studies by Peterson et al. (1974) have also shown an increase in acetylcholinesterase activity in aggregate cultures of chick optic tectum. In retina cell aggregates, the choline acetyltransferase activity increases three- to fourfold during the same period of time (Fig. 1). In all these cases, the different enzyme activity values given in the figure vary between 40 and 90% of the activities measured in viva for the same tissue type and developmental age (Ramirez, 1977b). In contrast, optic tectum cell aggregates show an increase in choline acetyltransferase activity only during the first week of culture (Fig. 11, followed by a slow decrease to below 8% of the corresponding values obtained in vivo (Ramirez, 1977b). When aggregates are prepared from loday chick embryos (Fig. 21, the overall results are quite similar except for a significant impairment in the development of
FIG. 1. Developmental profiles of choline acetyltransferase (left) and acetylcholinesterase (right) in chick optic tectum and retina cell aggregates prepared from ‘I-day chick embryos. The values given in the curves were obtained from the enzyme assays on retina and tectum separate cell aggregates used in the different coaggregation experiments described in the present paper and give an idea of the general pattern of cholinergic development of these aggregate cultures under our experimental conditions. Signs and bars represent mean ? standard deviation. (W--B) Retina cell aggregate activities; (A-A) tectum cell aggregate activities.
156
F&G. 2. transferase chick optic pared from tails as in activities; ties.
DEVELOPMENTAL
BIOLOGY
Developmental profiles of choline acetyl(left) and acetylcholinesterase (right) in tectum and retina cell aggregates prelo-day chick embryos. Experimental deFig. 1. (H-m) Retina cell aggregate (A-A) tectum cell aggregate activi-
acetylcholinesterase activity in tectum aggregates. Thus, tectal cell aggregates show only a limited development of cholinergic enzymes and mimic to some extent the behavior of an optic tectum deprived of retinal innervation (Filogamo, 1960; Marchisio, 1969). Interaction of Retinal and Tectal Cells in Coaggregation Cultures At this point we were ready to explore whether a functional cell-cell interaction between retina and tectum could be established in aggregate cultures, and whether such interactions would indeed result in measurable changes in the cholinergic metabolism of the system. To give a quantitative estimate of the interaction, the actual results of the assays for enzyme activities in the coaggregates (aggregates formed by a mixture of retinal and tectal cells) are compared with the expected results calculated from the activities observed in aggregates of separate tectum and retina cells of the same original lot of dissociated cells, taking into account the relative proportion of the retinal and tectal components in the coaggregate. Furthermore, by coaggregating retinal and tectal cells of identical or different developmental ages, it is possible to study the influence of retino-tectal synchronization on the recognition events me-
VOLUME
60, 1977
diating metabolic interaction. Figure 3 shows the actual and expected developmental profiles of choline acetyltransferase and acetylcholinesterase in coaggregates of tectum and retina cells prepared from the same lot of 7-day chick embryos. The proportion between retina and tectum, in terms of protein, at the time of mixing the two cell populations was 85:15 for this experiment. As seen in the figure, the use of this strategy of synchronic interaction, comparable to the experimental conditions of Gottlieb et al. (19741, results in a sustained and considerable boost of choline acetyltransferase, together with a less dramatic stimulation of acetylcholinesterase. A similar experiment is presented in Fig. 4 where only the porportion between retina and tectum in the coaggregate (40:60, in terms of protein) is changed. Retino-tectal synergism is still evident in the choline acetyltransferase profile, although the interaction process seems to take more time before such synergism is measurable. However, in the case of acetylcholinesterase, the measured and expected values are practically the same, except for some “negative” interaction in the last TABLE INTEGRATION
1
OF RETINA AND TECTUM THE COAGGREGATES”
14CPH input ratio 0.63 f 0.1
“CPH
CELLS
coaggregate
INTO
ratio
0.87 2 0.08
a The relative efficiency of integration of retinal and tectal cells into the coaggregates was checked as follows: Freshly dissociated retina and tectum cells were labeled for 30 min with [Wlthymidine and [3Hlthymidine, respectively (New England Nuclear, 54.7 mCi/mmole; and The Radiochemical Centre, 29 Ci/mmole, both at a final concentration of 1 &i/ml). Aliquots from both labeled cell suspensions were either mixed and immediately acid-precipitated or were coaggregated in the same retina-tectum proportion. After 48 hr of coaggregation (assumed consolidation period), the coaggregates were in turn precipitated with 10% trichloroacetic acid, washed, dissolved, and counted along with the input mixture samples. Values given are mean 2 SD for four different samples or flasks.
RAMIREZ AND SEEDS
Cholinergic
FIG. 3. Developmental profiles of choline acetyltransferase (left) and acetylcholinesterase (right) in coaggregates of chick retina and optic tectum cells of the same developmental age (synchronic interaction paradigm). Tecta and retinas from a common lot of ‘I-day chick embryos were independently dissociated into single cells and were reaggregated in suspension cultures containing either retina cells alone, tectum cells alone, or a mixture of retina and tectum cells, as described under Materials and Methods. The measured initial ratio, in terms of protein, between retina and tectum in the coaggregate was 8515 [the ratio of recovered protein in retina and tectum separate cell aggregates, along the time course, was 86.9(*3.5):13.11. At the times shown in the figure, two retina cell aggregate flasks, two tectum cell aggregate flasks, and two retina-tectum coaggregate flasks were assayed independently for enzyme activities. An “expected activity” for the retina-tectum coaggregate was calculated from the activity values obtained for retina and tectum aggregates independently, taking into account the mentioned proportion between retina and tectum in the coaggregates, in terms of protein. (0) Retina cell aggregate activities; (A) tectum cell aggregate activities; (0- - -0) calculated (expected) retina-tecturn coaggregate activities; (0-O) measured retina-tectum coaggregate activities. Each point represents the mean of four determinations (duplicate assays on two independent homogenates for each aggregate type). The bars represent the mean + standard deviation and are reflective of all subsequent coaggregate values. A positive interaction or synergism between retina and tectum is assumed to take place when the measured activity in the coaggregate significantly exceeds the expected activity calculated as described.
time point. Thus, dissociated retinal and tectal cells of the same developmental age are capable, upon coaggregation and culture, of interacting in such a way that the choline acetyltransferase activity measured in the coaggregated system is con-
Development
in Aggregate
157
Cukures
sistently higher than the value calculated by simply adding up the expected contributions of the component cells. This is consistent with the results reported by Adler and Teitelman (19741, under comparable experimental conditions. To ascertain the importance of temporal factors in this developmental interaction, coaggregates were prepared in which the retinal and tectal cells were at different developmental stages, namely, lo-day tectal cells coaggregated with 7-day retinal cells and 7-day tectal cells coaggregated with lo-day retinal cells. However, the validity of such experiments required demonstrating that lo-day retina cells are still capable of interacting with lo-day tectum cells. This is illustrated in Fig. 5, where it is shown that choline acetyltransferase activity in the coaggregate is consistently higher than the expected activity, even after only 24 hr of coaggregation. As before, the synergism is less marked in the case of acetylcholinesterase. Figures 6 and 7 show the failure of retina and tectum cells of different developmental ages to interact meaningfully un12
,
0
c
/
10
20
300
10
20
30
Days in culture
FIG. 4. Developmental profiles of choline acetyltransferase (left) and acetylcholinesterase (right) in coaggregates of chick retina and optic tectum cells of the same developmental age (synchronic interaction paradigm). The experimental details are as for Fig. 3, but an initial retina:tectum protein ratio of 40:60 [recovered protein ratio, 41.9(?6.3):58.1] was measured in these coaggregates. (0) Retina cell aggregate activities; (A) tectum cell aggregate activities; (0- - -0) calculated (expected) retina-tectum coaggregate activities; (0-O) measured retina-tecturn coaggregate activities.
158
DEVELOPMENTAL BIOLOGY
c.
,
0
K)
E 20
330 10 Days in cuI+ure
20
20
FIG. 5. Developmental profiles of choline acetyltransferase (left) and acetylcholinesterase (right) in coaggregates of chick retina and optic tectum cells of the same developmental age (synchronic interaction paradigm). In this case, both tectum and retina cells were dissociated, from a common lot of lo-day chick embryos (see legend to Fig. 3). The initial retina:tectum protein ratio in these coaggre5545 [recovered protein ratio, gates was 55.4(+6.2):44.6]. (0) Retina cell aggregate activities; (A) tectum cell aggregate activities; (0- - -0) calculated (expected) retina-tectum coaggregate activities; (0-O) measured retina-&turn coaggregate activities.
{
02
E c 0 0
10
20
50
0
10
20
30
Days in culture
FIG. 6. Developmental profiles of choline acetyltransferase (left) and acetylcholinesterase (right) in coaggregates of chick retina and optic tectum cells of different developmental age (delayed or heterochronic interaction paradigm). Tecta from lo-day chick embryos and retinas from ‘I-day embryos were independently dissociated into single cells and were reaggregated in suspension cultures containing retina cells alone, tectum cells alone, or a mixture of retina and tectum cells (see legend to Fig. 3). The initial retina-tectum protein ratio in the coaggregates was 70:30 [recovered protein ratio, 71.2(?4.6):28.8]. (Cl) Retina cell aggregate activities; (A) tectum cell aggregate activities; (O- - -0) calculated (expected) retina-tectum coaggregate activities; (0-O) measured retina-tectum coaggregate activities.
VOLUME 60, 1977
Days I” culture
FIG. 7. Developmental profiles of choline acetyltransferase (left) and acetylcholinesterase (right) in coaggregates of chick retina and optic tectum cells of different developmental age (delayed or heterochronic interaction paradigm). Tecta from 7-day chick embryos and retinas from IO-day embryos were independently dissociated into single cells and were reaggregated in suspension cultures containing retina cells alone, tectum cells alone, or a mixture of retina and tectum cells (see legend to Fig. 3). The initial retina-tectum protein ratio in the coaggregates was 55:45 [recovered protein ratio, 56.5(*7.4):43.51. (0) Retina cell aggregate activities; (A) tectum cell aggregate activities; (O- - -0) calculated (expected) retina-&turn coaggregate activities; (0-O) measured retina-&turn coaggregate activities.
der our experimental conditions. The calculated enzymatic activities are either equal or more often higher than the measured ones, pointing to some kind of metabolic interference between the different cells. Interestingly, in these cases of delayed or heterochronic coaggregation of retinal and tectal cells, comparable to the experimental setup of Barbera et al. (1973, 1975), the coaggregation proceeded at the same rate as in the synchronic experiments without any readily apparent changes in the adhesive properties of the cells. Since tectum cell aggregates display only limited choline acetyltransferase development (Figs. 1 and 21, similar to deafferented tecta in viva (Marchisio, 19691, it is tempting to conclude that the observed stimulation of choline acetyltransferase activity in the retina-tectum coaggregates (Figs. 3-5) reflects a partial normalization of the development of tectal cells induced
RAMIREZ
AND SEEM
Cholinergic
Development
in Aggregate
Cultures
159
by the neighboring retina cells within the coaggregate. Furthermore, the interaction between retina and tectum is very similar in timing and effect to the one inferred from these ablation experiments in viva (Marchisio, 1969), occurring about the time when optic nerve fibers spread over the tectum to eventually make synaptic contacts with tectal neurons (Crossland et al., 1975). Mechanism of the Retina-Tectum Znteraction in Coaggregate Cultures To determine whether this cooperative phenomenon is mediated by a readily diffusible molecule or whether it requires an intimate contact between the retinal and tectal cells, two types of experiments have been conducted concerning the mechanism of induction. First, the quality of the contact between tectal and retinal cells has been reduced by coaggregating retinal cells with preformed tectal aggregates. Figure 8 shows the results of one such experiment in which freshly dissociated loday retina cells have been added to preformed aggregates of 7-day tectal cells aggregated independently for 3 days. This combination corresponds to the synchronic interaction paradigm, since the total developmental age (in uiuo plus in vitro) of both cell classes is 10 days at the time of mixing (actually they originate from the same batch of incubated eggs). However, at least initially, the contact between retina and tectum cells is limited to the outer surface of the tectal aggregate. As seen in Fig. 8, only a very slight stimulation in the choline acetyltransferase activity is seen as late as Day 24 of culture. These results, though not definitive, would favor the hypothesis of the close contact over that of a diffusible factor as being responsible for the metabolic interaction. In addition, Fig. 9 shows that, within this modality of complementary cell adhesion onto preformed aggregates, even this minor stimulation of choline acetyltransferase (Fig. 8) is not seen when both elements have different developmental ages.
FIG. 8. Developmental profiles of choline acetyltransferase (left) and acetylcholinesterase (right) in coaggregatesprepared by adding a suspension of free retina cells, freshly dissociated from lo-day chick embryos, to culture flasks containing aggregates prepared from ‘I-day chick embryo optic tecta and kept in culture for 3 days (retina and tectum cells from the original dissociated cell populations were also aggregated independently as a reference; see legend to Fig. 3). Note that the total developmental age of both tectum and retina cells used in this experiment is the same (10 days) at mixing if we take into account the time spent in culture by tectal cells. At the time of mixing the protein ratio between retina cells and tectum aggregates was 6535 [recovered protein ratio, 63.5(?8.7):36.5]. Retina cells were seen to adhere immediately to the preformed tectum aggregates. The culture time in the graph is counted from the moment of adding the retina cells. (0) Retina cell aggregate activities; (A) tectum cell aggregate activities; (0- - -0) calculated (expected) retina-tectum coaggregate activities; (0-O) measured retina-tectum coaggregate activities.
The potential role of diffusible inducing factors was further checked as follows. Aggregates of tectum or retina cells (separate) were prepared and divided into two groups. One group of tectum aggregates and one group of retina cell aggregates were maintained under the standard conditions of culture; the remaining groups of tectum and retina cell aggregates were regularly fed culture medium conditioned by the complementary kind of aggregate of the first group (i.e., retina and tectum, respectively). The concentration of conditioned medium was kept at 25% and was renewed every other day. The activities of choline acetyltransferase and acetylcholinesterase were measured weekly for a 4week period in all four lots of aggregates.
160
DEVELOPMENTAL BIOL~CY
The enzyme developmental profiles of tecturn aggregates and retina-conditioned tectum aggregates were indistinguishable; the same was true for retina and tectumconditioned retina aggregates. Again, these results fail to support the notion of factor(s)-mediated induction. Although the understanding of the actual mechanism of the cooperation between retina and tectum cells in coaggregate cultures will require additional study, these preliminary results suggest that the interaction phenomenon observed in our studies requires the intimate physical contact between tectum and retina cells, and such contact facilitates an exchange of information leading to an increase in enzyme activity. This increased enzyme activity may reflect, at the molecular level, an increased rate of enzyme synthesis, a decreased rate of enzyme degradation, an
FIG. 9. Developmental profiles of choline acetyltransferase (left) and acetylcholinesterase (right) in coaggregates prepared by adding a suspension of free retina cells, freshly dissociated from ‘I-day chick embryos, to culture flasks containing aggregates prepared from ‘I-day chick embryo optic tecta and kept in culture for 3 days (retina and tectum cells from the original dissociated cell populations were also aggregated independently as a reference; see legend to Fig. 3). Note that in this experiment the total developmental age of tectum and retina cells is different. At the time of mixing the protein ratio between retina cells and tectum aggregates was 7930 [recovered protein ratio, 71.0 (t3.5k29.01. (0) Retina cell aggregate activities; (A) tectum cell aggregate activities; (0- - -0) calculated (expected) retina-tectum coaggregate activities; (0-O) measured retina-tectum coaggregate activites. The culture time in the graph is counted from the moment of adding the retina cells.
VOLUME
60. 1977
activation of existing enzyme molecules, or, at the cellular level, a proliferation of cholinergic cell types, the differentiation of existing cholinergic cells, or their differential survival in the coaggregate culture. Future studies should elucidate the nature of this phenomenon. Specificity of the Retina-Tectum Interaction Finally, the cell-type specificity of this coaggregate interaction was demonstrated using cells from other tissues or brain regions that lack or display only limited contact in vivo. Coaggregates were prepared from the following combinations: tectum and telencephalon (7-day embryos), tecturn and cerebellum (lo-day embryos), retina and cerebellum (lo-day embryos), tecturn and liver (7-day embryos), and retina and liver (7-day embryos). This series of coaggregates was studied as usual for cholinergic enzyme activities during a 4-week period, and the results after 22 days of culture are given in Table 2 as an example. True coaggregation was observed only when both cellular types were of neural origin, with liver cells aggregating independently from the tectal or retinal cells. In all combinations studied, the activities of choline acetyltransferase and acetylcholinesterase measured in the coaggregates (or in the pooled aggregates from a culture flask when liver was involved) were consistently lower than the activities predicted from the composition of the coaggregate. Thus, if anything, there was interference between the different cell combinations studied. These results underline the relevance of regional or topographic factors in the expression of interaction specificites. CONCLUSIONS
Since initial submission of this work, a recent paper by Adler et al. (1976) has appeared in which some of the tectumretina coaggregation experiments reported here were also performed. Although we agree on many points, there are several
RAMIREZ
AND SEEDS
Cholinergic TABLE
MEASURED
AND EXPECTED
CHOLINERCIC CELL
ENZYME
COMBINATIONS
Development
in Aggregate
2
ACTIVITIES
IN COAGGREGATES
AFTER 22 DAYS
Tectum-7:telencephalon-7 (70:30) Tectum-lO:cerebellum-10 (53:47) Retina-lO:cerebellum-10 (80:20) Tectum-7:liver-7 (70:30) Retina-7:liver-7 (74:26)
Measured 0.26 0.43 0.90 0.13 0.65
2 0.05 +O.lO ” 0.14 ” 0.02 k 0.08
Expected 0.29 0.44 1.01 0.21 0.75
MADE
UP OF DIFFERENT
IN CULTURE=
Choline acetyltransferase (nmole of ACh formed/min mg of protein) Coaggregate
161
Cultures
k ? * ” k
0.11 0.14 0.12 0.09 0.17
Acetylcholinesterase acetate formed/min
(nmole of mg of pro-
tein) Measured
Expected
157 * 21+ 59 + 101 * 68 +
193 85 128 129 84
13 4 18 11 10
* k r 2 2
28 18 34 29 14
u Numbers following tissue type indicate the age of embryo in days when the aggregates were prepared. The figures in parentheses, to the right of each combination of tissues, give the proportion between the two components, in terms of protein mass, at the start of the coaggregation. Enzyme values, expressed as mean -+ SD, were obtained from a total of four determinations on two different homogenates. Expected enzyme activities were calculated as described in Materials and Methods and the legend to Fig. 3. Aggregates of telencephalon, cerebellum, and liver cells alone were also prepared to determine the enzyme specific activities characteristic of each cell type, as was needed for the above calculations. In liver cell aggregates, only the choline-dependent ChAT was taken into consideration.
discrepancies between our results; however, these are difficult to interpret at present since different cell dissociation and culture conditions were used. Furthermore, the results of Adler et al. (1976) reflect coaggregation phenomena on only the third day of culture, a time when our aggregates are still undergoing cell sorting and ,fiber outgrowth and are just beginning to express biochemical development. Previous studies on retina-tectum cell recognition in vitro (Barbera et al., 1973; Barbera, 1975; Gottlieb, et al., 1974) have used cell adhesiveness as a criterion to measure specific interaction. Although we did not formally attempt to estimate rates of adhesion, we could not detect any gross differences in the cell reassociation processes between the synchronic and heterochronic coaggregation paradigms studied. Nevertheless, enzyme measurements revealed dramatic differences between the two experimental approaches. This apparent contradiction seems to suggest that rates and specificity of adhesion may not necessarily be related to functional interaction. In the case of the retino-tectal relationship we have been able to show that, while topographic or regional factors are
essential for recognition (each cell type only interacting synergistically with cells that are a natural contact-target during development), nerve cell properties change as development proceeds, and, therefore, an adequate synchronization of the two cell types is necessary to achieve a permanent functional linkage. Coaggregation appears to be a valid method to study the establishment of temporary and permanent connections between groups of cells, together with the immediate and long-term functional consequences of such connections. Thus, the study of long-term metabolic effects of cell couplings may, in addition to adhesion studies, provide information on the importance of temporal factors in recognitioninteraction processes. These studies were initially supported by USPHS Grants NS-09818 and CA-15549 to N.W.S. and by a grant to G.R. from the Program of Cultural Cooperation between Spain and the United States of America; they have been completed with the help of the Fundacion Juan March, Spain. The assistance of Mrs. Ana Barat and Mr. Gary Ludi is gratefully acknowledged. REFERENCES R., ANY TEITELMAN, G. (1974). Aggregates formed by mixtures of embryonic neural cells:
ADLER,
162
DEVELOPMENTAL
BIOLOGY
Activity of enzymes of the cholinergic system. Develop. Biol. 39, 317-321. ADLER, R., TEITELMAN, G., AND SVBURO, A. M. (1976). Cell interaction and the regulation of cholinergic enzymes during neural differentiation in vitro. Develop. Biol. 50, 48-57. ATTARDI, D. G., AND SPERRY, R. W. (1963). Preferential selection of central pathways by regenerating optic fibers. Exp. Neural. 7, 46-64. BARBERA, A. J., MARCHASE, R. B., AND ROTH, S. (1973). Adhesive recognition and retinotectal specificity. Proc. Nat. Acad. Sci. USA 70, 24822486. BARBERA, A. J. (1975). Adhesive recognition between developing retinal cells and the optic tecta of the chick embryo. Develop. Biol. 46,167-191. CROSSLAND, W. J., COWAN, W. M., ROGERS, L. A., AND KELLY, J. P. (1974). The specification of the retino-tectal projection in the chick. J. Comp. Neural. 155, 127-164. CROSSLAND, W. J., COWAN, W. M., AND ROGERS, L. A. (1975). Studies on the development of the chick optic tectum. IV. An autoradiographic study of the development of retino-tectal connections. Brain Res. 91, l-23. FIL~GAMO, G. (1960). Recherches experimentales sur l’activite des cholinesterases specifique et non specifique, dans le developpement du lobe optique du poulet. Arch. Biol. (Liege) 71, 159-198. GAZE, R. M. (1970). “The Formation of Nerve Connections,” pp. 118-215. Academic Press, London. G~ITLIEB, D. I., MERRELL, R., AND GLASER, L. (1974).
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Temporal changes in embryonal cell surface recognition. Proc. Nat. Acad. Sci. USA 17, 18001802. JACOBSON, M. (1970) “Developmental Neurobiology,” pp. 294-344. Holt, Rinehart and Winston, New York. LOPRESTI, B., MACAGNO, E. R., AND LEVINTHAL, C. (1973). Structure and development of neural connections in isogenic organisms: Cellular interactions in the development of the optic lamina of Daphnia. Proc. Nat. Acad. Sci. USA 70, 433-437. MARCHISIO, P. C. (1969) Choline acetyltransferase activity in developing chick optic centres and the effects of monolateral removal of retina at an early embryonic stage and at hatching. J. Neurothem. 16, 665-671. MCGEER, E. G., MALER, L., AND FITZSIMMONS, R. C. (1974). Comparative enzymatic development in chick embryonic brain areas. Develop. Biol. 38, 165-174. PETERSON, G., FISHER, P., AND BURKHALTER, A. (1974). Rotation cultures from different regions of embryonic chick brain. Neurobiology 4,210-221. RAMIREZ, G. (1977a) Cholinergic development in chick brain reaggregated cell cultures. Neurothem. Res. 2, 417-425. RAMIREZ, G. (1977b). Cholinergic development in chick optic tectum and retina reaggregated cell cultures. Neurochem. Res. 2, 427-438. SEEDS, N. W. (1971). Biochemical differentiation in reaggregating brain cell cultures. Proc. Nat. Acad. Sci. USA 68, 1858-1861.
BIOLOGY
60, 153-162 (1977)
Temporal Changes in Embryonic Nerve Cell Recognition: Correlate with Cholinergic Development in Aggregate Cultures GALO RAMIREZ*,
l AND NICHOLAS
W. SEEDst
* Centro de Biologia Molecular (CSIC-UAMI, Velazquez 144, Madrid 6, Spain, and t Department Biophysics and Genetics, University of Colorado Medical Center, Denver, Colorado 80262 Received December 28,1976;
of
accepted in revised form June 3,1977
Chick embryo retina and optic tectum cells can be dissociated into single cells and then reaggregated in suspension cultures to give highly organized and differentiated aggregates. These aggregates show a degree of cholinergic differentiation that is characteristic of each cell type; the low activity of choline acetyltransferase in the optic tectum aggregates probably reflects the condition of natural deafferentation inherent in the culture situation. It is possible, in this respect, to study the retina-tectum interaction in vitro by preparing coaggregates including both types of cells. When coaggregates are prepared from tectum and retina cells of the same developmental age, the activity of choline acetyltransferase measured in the coaggregates is consistently higher than would be expected from the simple addition of the activities of the component cells, pointing to some kind of metabolic synergism between retinal and tectal cells. As for acetylcholinesterase, this synergism occurs only under special circumstances, and it is generally less marked. No synergism was observed when retina and tectum cells of different developmental age were coaggregated, suggesting the existence of a temporal control of neuronal interaction specificity. On the other hand, the synergism is only observed between neuronal systems that are known to establish synaptic connections during normal in viva development: No interaction could be detected when either retinal or tectal cells were combined with telencephalon, cerebellum, or liver cells. Experimental evidence is presented suggesting that the retina-tectum interaction depends on intimate cell-cell contact, and it is not mediated by freely diffusible molecules. Neurotransmission-related metabolic studies in coaggregates seem to offer a promising tool to study recognition-interaction phenomena in groups of neurons establishing synaptic links during development. INTRODUCTION
The establishment of specific cell connections during development is a major key to the complex functional organization characteristic of the mature nervous system. Much work has been done in the last 20 years to ascertain whether the amazingly accurate pattern of nerve cell connections (synaptic and other) is based on an orderly sequence of cellular and environmental events which bring into contact the right cells at the right time of development, or whether it is based on the presence of specific recognition-mediating signals in the cell surfaces responsible for the establishment of temporary or permanent ’ To whom dressed.
all
correspondence
should
Copyright 0 1977by Academic Press, Inc. All rights of reproduction in any form reserved.
be ad-
independent of the time interactions, when such contact occurs during development (Gaze, 1970; Jacobson, 1970). The visual system, in both invertebrates and vertebrates, has provided a convenient model to study growing nerve fiber-target cell recognition, and, so far, experimental evidence has been found to support the existence in living organisms of both the interaction mechanisms outlined above (Attardi and Sperry, 1963; Crossland et al., 1974; Gaze, 1970; Jacobson, 1970; Lopresti et al., 1973). Recently, the problem of the retino-tectal recognition has been approached in two different laboratories by means of in vitro techniques (Barbera et al., 1973; Barbera, 1975; Gottlieb et al., 1974). These studies 153 ISSN 0012-1606
154
DEVELOPMENTAL BIOLOGY
have demonstrated cell surface adhesive specificity between retina and optic tectum cells. Gottlieb et al. (1974) found a differential behavior of tectum and retina cell surfaces as a function of developmental age and suggested that temporal factors were a potential mechanism to achieve control of contact specificity during normal maturation in Go. Using a different methodology to measure retino-tectal specificity, Barbera et al. (1973, 1975) did not find substantial differences between tissue of various ages; however, they suggested that topographic factors were most important in controlling the specificity of cell interaction. We have proceeded with the rationale that cell recognition, to be specific and meaningful, should be able to trigger some metabolic events reflecting the establishment of functional ties between the interacting cells. Synapse formation is the most obvious and possibly the most specific form of cell-cell interaction, although there are probably many less obvious interactions between specific cell types. Since full cholinergic development of the chick optic tecturn has been shown to be contingent upon the arrival of retinal cell fibers (Filogamo, 1960; Marchisio, 1969), the influence of different patterns of interaction between tecturn and retina cells on the specific activities of two enzymes related to cholinergic neurotransmission has been examined. MATERIALS
AND
METHODS
Preparation of aggregates. Tectum and retina cell aggregates were prepared essentially as described before (Seeds, 1971; Ramirez, 1977a). Tecta and retinas (whole retinas, including the pigment epithelium) were removed from 7- or lo-day chick embryos (White Leghorn) and were cut into small (1 mm31 pieces. The tissue.was dissociated in 0.25% trypsin (Difco) in saline at 37°C for 20 min, and was pipet-triturated at 5min intervals. The resulting cell suspension was passed through a nylon screen (180 pm) and was inoculated into 25-ml
VOLUME 60, 1977
Erlenmeyer flasks containing 3 ml of basal Eagle’s medium (Gibco), supplemented with 0.4% glucose and 10% fetal calf serum, at a final cell concentration of 2-5 x lo6 cells/ml. In coaggregation experiments the flasks received aliquots of both retina and tectum cell suspensions, but the total cell concentration in the culture flask was kept within the same range as for simple aggregates. The flasks were gassed with CO,:air (5:95) and were incubated in a gyrotory shaker bath (New Brunswick) at 37°C and 70 rpm. The next day the cultures were transferred to 50-ml Erlenmeyer flasks, and the volume of medium was increased to -10 ml. Half the volume of medium was changed every other day. Enzymatic assays. Acetylcholinesterase (AChEI and choline acetyltransferase (ChATI were assayed as described (Seeds, 1971; Ramirez, 1977a). True acetylcholinesterase was distinguished from total cholinesterase activity by adding to the reaction mixture the specific inhibitor of true AChE, BW 284 C51 dibromide (Wellcome Reagents, Ltd., Beckenham, Kent, England), to a final concentration of low5 M. In the ChAT assay, blanks without added choline were routinely run to measure acetylation of endogenous substrates. This amounted to less than 2% of the cholinedependent acetylation for the different classes of nerve cell aggregates used in the present work. However, in liver cell aggregates, the acetylation of endogenous substrates accounted for practically all the observed activity. Experimental setup and treatment of data. The different retina-tectum interaction experiments presented in Figs. 3-9 employed a total of 30 flasks (Figs. 3-71, or 24 flasks (Figs. 8,9) (one to three aggregates per flask) for a time-course run. For each time point, six flasks (two retina, two 2 Abbreviations used: AChE, acetylcholinesterase; ChAT, choline acetyltransferase; BW284 C51 dibromide, 1,5-bis-(4-allyldimethylammoniumphenyl) pentane-3-one dibromide; ACh, acetylcholine.
RAMIREZ AND SEEDE
Cholinergic
tectum, and two retina plus tectum) were independently processed and assayed in duplicate, so that four independent enzyme activity values would define any point in the curves. In this way the retinatectum interaction was measured against retinal and tectal basic activities from the same original batch of dissociated cells. The retinal and tectal (separate aggregates) enzyme activities measured in a given time point were used to calculate an “expected enzyme activity” for the coaggregate, taking into account the proportion (in terms of protein) between retinal and tectal cells at the time of mixing, since it would not be possible to distinguish the actual contributions of retina and tectum in a preformed coaggregate. The validity of this approach is supported by the fact that, although the protein mass of individual retina and tectum cell aggregates varied during the 4 weeks elapsed in a timecourse run, the proportion between the total protein recovered in retina and tectum cell aggregates, for any time point, remained reasonably constant (the mean +SD of this recovered protein proportion for the five or four points of a time course is given in the legend of each figure). Furthermore, the coaggregation process did not preferentially discriminate against retinal or tectal cells, since the consolidated coaggregates consistently displayed the same ratio of retinal to tectal cells as the input populations (innoculum) (Table 1). All other coaggregation experiments described in this report were handled in a similar manner, except for the specific details given in the relevant portions of the text. RESULTS
AND
DISCUSSION
Cholinergic Development Tectum Cell Aggregates
in Retina
and
Both chick retina and optic tectum show large increases in choline acetyltransferase and acetylcholinesterase specific activities from fetal Day 7 to 10 days after
Development
in Aggregate
Cultures
155
hatching (McGeer et al., 1974; Ramirez, 1977b). The specific activity of acetylcholinesterase in aggregate cultures prepared from ‘I-day chick embryo retina and optic tectum increases considerably during 4 weeks of culture (Fig. 1). Studies by Peterson et al. (1974) have also shown an increase in acetylcholinesterase activity in aggregate cultures of chick optic tectum. In retina cell aggregates, the choline acetyltransferase activity increases three- to fourfold during the same period of time (Fig. 1). In all these cases, the different enzyme activity values given in the figure vary between 40 and 90% of the activities measured in viva for the same tissue type and developmental age (Ramirez, 1977b). In contrast, optic tectum cell aggregates show an increase in choline acetyltransferase activity only during the first week of culture (Fig. 11, followed by a slow decrease to below 8% of the corresponding values obtained in vivo (Ramirez, 1977b). When aggregates are prepared from loday chick embryos (Fig. 21, the overall results are quite similar except for a significant impairment in the development of
FIG. 1. Developmental profiles of choline acetyltransferase (left) and acetylcholinesterase (right) in chick optic tectum and retina cell aggregates prepared from ‘I-day chick embryos. The values given in the curves were obtained from the enzyme assays on retina and tectum separate cell aggregates used in the different coaggregation experiments described in the present paper and give an idea of the general pattern of cholinergic development of these aggregate cultures under our experimental conditions. Signs and bars represent mean ? standard deviation. (W--B) Retina cell aggregate activities; (A-A) tectum cell aggregate activities.
156
F&G. 2. transferase chick optic pared from tails as in activities; ties.
DEVELOPMENTAL
BIOLOGY
Developmental profiles of choline acetyl(left) and acetylcholinesterase (right) in tectum and retina cell aggregates prelo-day chick embryos. Experimental deFig. 1. (H-m) Retina cell aggregate (A-A) tectum cell aggregate activi-
acetylcholinesterase activity in tectum aggregates. Thus, tectal cell aggregates show only a limited development of cholinergic enzymes and mimic to some extent the behavior of an optic tectum deprived of retinal innervation (Filogamo, 1960; Marchisio, 1969). Interaction of Retinal and Tectal Cells in Coaggregation Cultures At this point we were ready to explore whether a functional cell-cell interaction between retina and tectum could be established in aggregate cultures, and whether such interactions would indeed result in measurable changes in the cholinergic metabolism of the system. To give a quantitative estimate of the interaction, the actual results of the assays for enzyme activities in the coaggregates (aggregates formed by a mixture of retinal and tectal cells) are compared with the expected results calculated from the activities observed in aggregates of separate tectum and retina cells of the same original lot of dissociated cells, taking into account the relative proportion of the retinal and tectal components in the coaggregate. Furthermore, by coaggregating retinal and tectal cells of identical or different developmental ages, it is possible to study the influence of retino-tectal synchronization on the recognition events me-
VOLUME
60, 1977
diating metabolic interaction. Figure 3 shows the actual and expected developmental profiles of choline acetyltransferase and acetylcholinesterase in coaggregates of tectum and retina cells prepared from the same lot of 7-day chick embryos. The proportion between retina and tectum, in terms of protein, at the time of mixing the two cell populations was 85:15 for this experiment. As seen in the figure, the use of this strategy of synchronic interaction, comparable to the experimental conditions of Gottlieb et al. (19741, results in a sustained and considerable boost of choline acetyltransferase, together with a less dramatic stimulation of acetylcholinesterase. A similar experiment is presented in Fig. 4 where only the porportion between retina and tectum in the coaggregate (40:60, in terms of protein) is changed. Retino-tectal synergism is still evident in the choline acetyltransferase profile, although the interaction process seems to take more time before such synergism is measurable. However, in the case of acetylcholinesterase, the measured and expected values are practically the same, except for some “negative” interaction in the last TABLE INTEGRATION
1
OF RETINA AND TECTUM THE COAGGREGATES”
14CPH input ratio 0.63 f 0.1
“CPH
CELLS
coaggregate
INTO
ratio
0.87 2 0.08
a The relative efficiency of integration of retinal and tectal cells into the coaggregates was checked as follows: Freshly dissociated retina and tectum cells were labeled for 30 min with [Wlthymidine and [3Hlthymidine, respectively (New England Nuclear, 54.7 mCi/mmole; and The Radiochemical Centre, 29 Ci/mmole, both at a final concentration of 1 &i/ml). Aliquots from both labeled cell suspensions were either mixed and immediately acid-precipitated or were coaggregated in the same retina-tectum proportion. After 48 hr of coaggregation (assumed consolidation period), the coaggregates were in turn precipitated with 10% trichloroacetic acid, washed, dissolved, and counted along with the input mixture samples. Values given are mean 2 SD for four different samples or flasks.
RAMIREZ AND SEEDS
Cholinergic
FIG. 3. Developmental profiles of choline acetyltransferase (left) and acetylcholinesterase (right) in coaggregates of chick retina and optic tectum cells of the same developmental age (synchronic interaction paradigm). Tecta and retinas from a common lot of ‘I-day chick embryos were independently dissociated into single cells and were reaggregated in suspension cultures containing either retina cells alone, tectum cells alone, or a mixture of retina and tectum cells, as described under Materials and Methods. The measured initial ratio, in terms of protein, between retina and tectum in the coaggregate was 8515 [the ratio of recovered protein in retina and tectum separate cell aggregates, along the time course, was 86.9(*3.5):13.11. At the times shown in the figure, two retina cell aggregate flasks, two tectum cell aggregate flasks, and two retina-tectum coaggregate flasks were assayed independently for enzyme activities. An “expected activity” for the retina-tectum coaggregate was calculated from the activity values obtained for retina and tectum aggregates independently, taking into account the mentioned proportion between retina and tectum in the coaggregates, in terms of protein. (0) Retina cell aggregate activities; (A) tectum cell aggregate activities; (0- - -0) calculated (expected) retina-tecturn coaggregate activities; (0-O) measured retina-tectum coaggregate activities. Each point represents the mean of four determinations (duplicate assays on two independent homogenates for each aggregate type). The bars represent the mean + standard deviation and are reflective of all subsequent coaggregate values. A positive interaction or synergism between retina and tectum is assumed to take place when the measured activity in the coaggregate significantly exceeds the expected activity calculated as described.
time point. Thus, dissociated retinal and tectal cells of the same developmental age are capable, upon coaggregation and culture, of interacting in such a way that the choline acetyltransferase activity measured in the coaggregated system is con-
Development
in Aggregate
157
Cukures
sistently higher than the value calculated by simply adding up the expected contributions of the component cells. This is consistent with the results reported by Adler and Teitelman (19741, under comparable experimental conditions. To ascertain the importance of temporal factors in this developmental interaction, coaggregates were prepared in which the retinal and tectal cells were at different developmental stages, namely, lo-day tectal cells coaggregated with 7-day retinal cells and 7-day tectal cells coaggregated with lo-day retinal cells. However, the validity of such experiments required demonstrating that lo-day retina cells are still capable of interacting with lo-day tectum cells. This is illustrated in Fig. 5, where it is shown that choline acetyltransferase activity in the coaggregate is consistently higher than the expected activity, even after only 24 hr of coaggregation. As before, the synergism is less marked in the case of acetylcholinesterase. Figures 6 and 7 show the failure of retina and tectum cells of different developmental ages to interact meaningfully un12
,
0
c
/
10
20
300
10
20
30
Days in culture
FIG. 4. Developmental profiles of choline acetyltransferase (left) and acetylcholinesterase (right) in coaggregates of chick retina and optic tectum cells of the same developmental age (synchronic interaction paradigm). The experimental details are as for Fig. 3, but an initial retina:tectum protein ratio of 40:60 [recovered protein ratio, 41.9(?6.3):58.1] was measured in these coaggregates. (0) Retina cell aggregate activities; (A) tectum cell aggregate activities; (0- - -0) calculated (expected) retina-tectum coaggregate activities; (0-O) measured retina-tecturn coaggregate activities.
158
DEVELOPMENTAL BIOLOGY
c.
,
0
K)
E 20
330 10 Days in cuI+ure
20
20
FIG. 5. Developmental profiles of choline acetyltransferase (left) and acetylcholinesterase (right) in coaggregates of chick retina and optic tectum cells of the same developmental age (synchronic interaction paradigm). In this case, both tectum and retina cells were dissociated, from a common lot of lo-day chick embryos (see legend to Fig. 3). The initial retina:tectum protein ratio in these coaggre5545 [recovered protein ratio, gates was 55.4(+6.2):44.6]. (0) Retina cell aggregate activities; (A) tectum cell aggregate activities; (0- - -0) calculated (expected) retina-tectum coaggregate activities; (0-O) measured retina-&turn coaggregate activities.
{
02
E c 0 0
10
20
50
0
10
20
30
Days in culture
FIG. 6. Developmental profiles of choline acetyltransferase (left) and acetylcholinesterase (right) in coaggregates of chick retina and optic tectum cells of different developmental age (delayed or heterochronic interaction paradigm). Tecta from lo-day chick embryos and retinas from ‘I-day embryos were independently dissociated into single cells and were reaggregated in suspension cultures containing retina cells alone, tectum cells alone, or a mixture of retina and tectum cells (see legend to Fig. 3). The initial retina-tectum protein ratio in the coaggregates was 70:30 [recovered protein ratio, 71.2(?4.6):28.8]. (Cl) Retina cell aggregate activities; (A) tectum cell aggregate activities; (O- - -0) calculated (expected) retina-tectum coaggregate activities; (0-O) measured retina-tectum coaggregate activities.
VOLUME 60, 1977
Days I” culture
FIG. 7. Developmental profiles of choline acetyltransferase (left) and acetylcholinesterase (right) in coaggregates of chick retina and optic tectum cells of different developmental age (delayed or heterochronic interaction paradigm). Tecta from 7-day chick embryos and retinas from IO-day embryos were independently dissociated into single cells and were reaggregated in suspension cultures containing retina cells alone, tectum cells alone, or a mixture of retina and tectum cells (see legend to Fig. 3). The initial retina-tectum protein ratio in the coaggregates was 55:45 [recovered protein ratio, 56.5(*7.4):43.51. (0) Retina cell aggregate activities; (A) tectum cell aggregate activities; (O- - -0) calculated (expected) retina-&turn coaggregate activities; (0-O) measured retina-&turn coaggregate activities.
der our experimental conditions. The calculated enzymatic activities are either equal or more often higher than the measured ones, pointing to some kind of metabolic interference between the different cells. Interestingly, in these cases of delayed or heterochronic coaggregation of retinal and tectal cells, comparable to the experimental setup of Barbera et al. (1973, 1975), the coaggregation proceeded at the same rate as in the synchronic experiments without any readily apparent changes in the adhesive properties of the cells. Since tectum cell aggregates display only limited choline acetyltransferase development (Figs. 1 and 21, similar to deafferented tecta in viva (Marchisio, 19691, it is tempting to conclude that the observed stimulation of choline acetyltransferase activity in the retina-tectum coaggregates (Figs. 3-5) reflects a partial normalization of the development of tectal cells induced
RAMIREZ
AND SEEM
Cholinergic
Development
in Aggregate
Cultures
159
by the neighboring retina cells within the coaggregate. Furthermore, the interaction between retina and tectum is very similar in timing and effect to the one inferred from these ablation experiments in viva (Marchisio, 1969), occurring about the time when optic nerve fibers spread over the tectum to eventually make synaptic contacts with tectal neurons (Crossland et al., 1975). Mechanism of the Retina-Tectum Znteraction in Coaggregate Cultures To determine whether this cooperative phenomenon is mediated by a readily diffusible molecule or whether it requires an intimate contact between the retinal and tectal cells, two types of experiments have been conducted concerning the mechanism of induction. First, the quality of the contact between tectal and retinal cells has been reduced by coaggregating retinal cells with preformed tectal aggregates. Figure 8 shows the results of one such experiment in which freshly dissociated loday retina cells have been added to preformed aggregates of 7-day tectal cells aggregated independently for 3 days. This combination corresponds to the synchronic interaction paradigm, since the total developmental age (in uiuo plus in vitro) of both cell classes is 10 days at the time of mixing (actually they originate from the same batch of incubated eggs). However, at least initially, the contact between retina and tectum cells is limited to the outer surface of the tectal aggregate. As seen in Fig. 8, only a very slight stimulation in the choline acetyltransferase activity is seen as late as Day 24 of culture. These results, though not definitive, would favor the hypothesis of the close contact over that of a diffusible factor as being responsible for the metabolic interaction. In addition, Fig. 9 shows that, within this modality of complementary cell adhesion onto preformed aggregates, even this minor stimulation of choline acetyltransferase (Fig. 8) is not seen when both elements have different developmental ages.
FIG. 8. Developmental profiles of choline acetyltransferase (left) and acetylcholinesterase (right) in coaggregatesprepared by adding a suspension of free retina cells, freshly dissociated from lo-day chick embryos, to culture flasks containing aggregates prepared from ‘I-day chick embryo optic tecta and kept in culture for 3 days (retina and tectum cells from the original dissociated cell populations were also aggregated independently as a reference; see legend to Fig. 3). Note that the total developmental age of both tectum and retina cells used in this experiment is the same (10 days) at mixing if we take into account the time spent in culture by tectal cells. At the time of mixing the protein ratio between retina cells and tectum aggregates was 6535 [recovered protein ratio, 63.5(?8.7):36.5]. Retina cells were seen to adhere immediately to the preformed tectum aggregates. The culture time in the graph is counted from the moment of adding the retina cells. (0) Retina cell aggregate activities; (A) tectum cell aggregate activities; (0- - -0) calculated (expected) retina-tectum coaggregate activities; (0-O) measured retina-tectum coaggregate activities.
The potential role of diffusible inducing factors was further checked as follows. Aggregates of tectum or retina cells (separate) were prepared and divided into two groups. One group of tectum aggregates and one group of retina cell aggregates were maintained under the standard conditions of culture; the remaining groups of tectum and retina cell aggregates were regularly fed culture medium conditioned by the complementary kind of aggregate of the first group (i.e., retina and tectum, respectively). The concentration of conditioned medium was kept at 25% and was renewed every other day. The activities of choline acetyltransferase and acetylcholinesterase were measured weekly for a 4week period in all four lots of aggregates.
160
DEVELOPMENTAL BIOL~CY
The enzyme developmental profiles of tecturn aggregates and retina-conditioned tectum aggregates were indistinguishable; the same was true for retina and tectumconditioned retina aggregates. Again, these results fail to support the notion of factor(s)-mediated induction. Although the understanding of the actual mechanism of the cooperation between retina and tectum cells in coaggregate cultures will require additional study, these preliminary results suggest that the interaction phenomenon observed in our studies requires the intimate physical contact between tectum and retina cells, and such contact facilitates an exchange of information leading to an increase in enzyme activity. This increased enzyme activity may reflect, at the molecular level, an increased rate of enzyme synthesis, a decreased rate of enzyme degradation, an
FIG. 9. Developmental profiles of choline acetyltransferase (left) and acetylcholinesterase (right) in coaggregates prepared by adding a suspension of free retina cells, freshly dissociated from ‘I-day chick embryos, to culture flasks containing aggregates prepared from ‘I-day chick embryo optic tecta and kept in culture for 3 days (retina and tectum cells from the original dissociated cell populations were also aggregated independently as a reference; see legend to Fig. 3). Note that in this experiment the total developmental age of tectum and retina cells is different. At the time of mixing the protein ratio between retina cells and tectum aggregates was 7930 [recovered protein ratio, 71.0 (t3.5k29.01. (0) Retina cell aggregate activities; (A) tectum cell aggregate activities; (0- - -0) calculated (expected) retina-tectum coaggregate activities; (0-O) measured retina-tectum coaggregate activites. The culture time in the graph is counted from the moment of adding the retina cells.
VOLUME
60. 1977
activation of existing enzyme molecules, or, at the cellular level, a proliferation of cholinergic cell types, the differentiation of existing cholinergic cells, or their differential survival in the coaggregate culture. Future studies should elucidate the nature of this phenomenon. Specificity of the Retina-Tectum Interaction Finally, the cell-type specificity of this coaggregate interaction was demonstrated using cells from other tissues or brain regions that lack or display only limited contact in vivo. Coaggregates were prepared from the following combinations: tectum and telencephalon (7-day embryos), tecturn and cerebellum (lo-day embryos), retina and cerebellum (lo-day embryos), tecturn and liver (7-day embryos), and retina and liver (7-day embryos). This series of coaggregates was studied as usual for cholinergic enzyme activities during a 4-week period, and the results after 22 days of culture are given in Table 2 as an example. True coaggregation was observed only when both cellular types were of neural origin, with liver cells aggregating independently from the tectal or retinal cells. In all combinations studied, the activities of choline acetyltransferase and acetylcholinesterase measured in the coaggregates (or in the pooled aggregates from a culture flask when liver was involved) were consistently lower than the activities predicted from the composition of the coaggregate. Thus, if anything, there was interference between the different cell combinations studied. These results underline the relevance of regional or topographic factors in the expression of interaction specificites. CONCLUSIONS
Since initial submission of this work, a recent paper by Adler et al. (1976) has appeared in which some of the tectumretina coaggregation experiments reported here were also performed. Although we agree on many points, there are several
RAMIREZ
AND SEEDS
Cholinergic TABLE
MEASURED
AND EXPECTED
CHOLINERCIC CELL
ENZYME
COMBINATIONS
Development
in Aggregate
2
ACTIVITIES
IN COAGGREGATES
AFTER 22 DAYS
Tectum-7:telencephalon-7 (70:30) Tectum-lO:cerebellum-10 (53:47) Retina-lO:cerebellum-10 (80:20) Tectum-7:liver-7 (70:30) Retina-7:liver-7 (74:26)
Measured 0.26 0.43 0.90 0.13 0.65
2 0.05 +O.lO ” 0.14 ” 0.02 k 0.08
Expected 0.29 0.44 1.01 0.21 0.75
MADE
UP OF DIFFERENT
IN CULTURE=
Choline acetyltransferase (nmole of ACh formed/min mg of protein) Coaggregate
161
Cultures
k ? * ” k
0.11 0.14 0.12 0.09 0.17
Acetylcholinesterase acetate formed/min
(nmole of mg of pro-
tein) Measured
Expected
157 * 21+ 59 + 101 * 68 +
193 85 128 129 84
13 4 18 11 10
* k r 2 2
28 18 34 29 14
u Numbers following tissue type indicate the age of embryo in days when the aggregates were prepared. The figures in parentheses, to the right of each combination of tissues, give the proportion between the two components, in terms of protein mass, at the start of the coaggregation. Enzyme values, expressed as mean -+ SD, were obtained from a total of four determinations on two different homogenates. Expected enzyme activities were calculated as described in Materials and Methods and the legend to Fig. 3. Aggregates of telencephalon, cerebellum, and liver cells alone were also prepared to determine the enzyme specific activities characteristic of each cell type, as was needed for the above calculations. In liver cell aggregates, only the choline-dependent ChAT was taken into consideration.
discrepancies between our results; however, these are difficult to interpret at present since different cell dissociation and culture conditions were used. Furthermore, the results of Adler et al. (1976) reflect coaggregation phenomena on only the third day of culture, a time when our aggregates are still undergoing cell sorting and ,fiber outgrowth and are just beginning to express biochemical development. Previous studies on retina-tectum cell recognition in vitro (Barbera et al., 1973; Barbera, 1975; Gottlieb, et al., 1974) have used cell adhesiveness as a criterion to measure specific interaction. Although we did not formally attempt to estimate rates of adhesion, we could not detect any gross differences in the cell reassociation processes between the synchronic and heterochronic coaggregation paradigms studied. Nevertheless, enzyme measurements revealed dramatic differences between the two experimental approaches. This apparent contradiction seems to suggest that rates and specificity of adhesion may not necessarily be related to functional interaction. In the case of the retino-tectal relationship we have been able to show that, while topographic or regional factors are
essential for recognition (each cell type only interacting synergistically with cells that are a natural contact-target during development), nerve cell properties change as development proceeds, and, therefore, an adequate synchronization of the two cell types is necessary to achieve a permanent functional linkage. Coaggregation appears to be a valid method to study the establishment of temporary and permanent connections between groups of cells, together with the immediate and long-term functional consequences of such connections. Thus, the study of long-term metabolic effects of cell couplings may, in addition to adhesion studies, provide information on the importance of temporal factors in recognitioninteraction processes. These studies were initially supported by USPHS Grants NS-09818 and CA-15549 to N.W.S. and by a grant to G.R. from the Program of Cultural Cooperation between Spain and the United States of America; they have been completed with the help of the Fundacion Juan March, Spain. The assistance of Mrs. Ana Barat and Mr. Gary Ludi is gratefully acknowledged. REFERENCES R., ANY TEITELMAN, G. (1974). Aggregates formed by mixtures of embryonic neural cells:
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