Aggregation of Cells from Early Chick Blastoderms E. J. SANDERS' and S. E. ZALIK' Departments of Physiology' and Zoologyz, University of Alberta, Edmonton, Alberta, Canada T6G 2E9
Received July 1975
The aggregation of dissociated cells from chick blastoderms at Hamburger and Hamilton stages 1-5 was studied. Aggregation was measured during the first 4 h of culture by determination of the proportion of single cells in the medium. No difference in aggregation was found when cells dissociated by either trypsin or EDTA were studied. Similarly the presence or absence of serum in the medium had no appreciable effect on early phases of aggregation, although at 24 h and thereafter, aggregate size was reduced in serum-free cultures. It was found that at all of the stages studied, cells aggregate and sort out into two groups. One group forms a continuous phase of loosely associated cells while the other segregates into several localised areas of closely associated cells within the aggregate. Examination of aggregates up to 7 days in culture showed progressive differentiation within each phase and several identifiable cell types were observed. Basal laminae were present at the boundary between the compact phase and the loose phase.
Introduction Early embryogenesis in the chick involves a complex series of cellular interactions which ultimately lead to the formation of the three-layered embryo [ 1-71. These interactions presumably depend to some extent on the surface properties of the cells present in the embryo. There is an extensive literature concerning studies on the adhesive behaviour of embryonic cells at relatively advanced stages of development [8-121. Studies on early embryonic cells have been performed on sea urchin, amphibian and fish embryos [11-151 but relatively little information is available on surface properties of chick embryonic cells prior to, and during gastrulation. Some of these surface characteristics have been defined using cell electrophoresis and electron microscopy 16- 191. Preliminary studies in our laboratory [201 have shown that in aggregates obtained from dissociated pre-streak blastoderm cells, some cells possess surface characteristics which allow them to sort out into distinct groups and continue further differentiation. Two populations of cells were distinguishable and it was our contention that these corresponded to cells of epiblast and hypoblast. These results have been substantiated by studies conducted elsewhere [211. The present report represents an attempt to study the aggregative behaviour of dissociated cells from Differentiation 6, 1-11 (1976)
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0 by Springer-Verlag 1976
pre-streak and primitive streak blastoderms. We have studied the kinetics of aggregation under a variety of conditions during the first 4 h of culture. In addition we have examined the cellular reorganisation and differentiation of the aggregates during the first 7 days of culture.
Methods In staging embryos, the criteria and numbering of Hamburger and Hamilton [221 was used. Blastoderms were removed under sterile conditions from embryos at stages 1 (pre-streak) to 4-5 (head process). Blastoderms were dissected frec from their vitelline membranes and stored overnight at room temperature or at 37" C in Panet and Compton saline containing 0.2% glucose, 300 U/ml penicillin and 300 pg/ml streptomycin (GIBCO). Dissociation of blastoderms was carried out as previously described 1161 in calcium-magnesium-free Panet and Compton saline (CMFPC), pH 7.8. containing 0.002 M EDTA at room temperature. Alternatively, blastoderms were placed in CMFPC with 0.1 % trypsin ( 1 : 250. Difco), incubated at 37" C for 10 min and dissociated as reported previously. For the aggregation experiments, cells were suspended in 2 ml Leibowitz L-15 medium (GIBCO), with or without 10% foetal calf serum, at a cell concentration varying from 0.5 to 1.0 x lo6 cells per ml. Cell suspensions were placed in 10 ml flasks and cultured at 37" C in a gyratory shaker at 70 rpm.
E. J. Sanders and S. E. Zalik:
2 Attempts were made to evaluate the viability of blastoderm cells by employing a variety of vital dyes in dye-exclusion tests. However for the same cell suspension, some dyes stained all the cells, while others did not penetrate any of them; this last instance occurred even under a variety of adverse conditions. To the present time we have not found a vital dye which stains blastoderm cells differentially. Cells. however, adhered to each other and formed aggregates in which cell division and differentiation occurred, and little sign of cell degeneration was evident when aggregates were examined with light and electron microscopes. The degree of aggregation was evaluated by determining the rate of disappearance of single cells in the culture medium. Samples were removed at defined time intervals from the culture flasks, di luted to a constant volume with saline, and the number of single cells was determined with the use of a hemocytometer. Cell counts were performed during the first 4 h of incubation. Hemocytometer counts were performed 10 times on aliquots of the diluted sample and an average of these readings was obtained at each time-interval studied. Under our conditions a decline in the number of single cells coincided with the appearance of cell aggregates. Due to the variation in size encountered in blastoderm cells, and the presence of yolk granules, also of varying size. the electronic particle counter could not be used in these experiments. After cell counts were performed, flasks with cultured aggregates were further incubated for 1, 2, 3, 5 and 7 days at 37" C and 100 rpm. During these time intervals one half of the original culture medium was removed and replaced by fresh medium. This procedure was repeated at 48 h intervals. Light microscopy was carried out on Bouin's-fixed and wax-embedded aggregates. Sections were stained using haemotoxylin and eosin. For electron microscopy, aggregates were fixed using 3?6 glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, followed by 2% osmium tetroxide in the same buffer. The material was dehydrated using graded ethanol solutions and sections were stained with uranyl acetate and lead citrate.
Results A first series of experiments was performed in order to determine the aggregative behaviour of EDTA-dissociated cells in the presence of medium containing 10% foetal calf serum. This was performed for stage 1 and stage 4-5 blastoderms. The results from these experiments are shown in Fig. 1, in which percent adhesion versus time is presented. It can be observed that by 30 min in culture, adhesion has increased to an average of 35% for experiments using blastoderrn cells at both stages. There is a time-dependent increase in aggregation, so that by 2 10 min in culture, adhesions had increased to an average value of 64 and 74% for blastoderm cells at stages 1 and 4-5 respectively. The actual extent of the time-dependent increase in aggregation tended to vary considerably between experiments, therefore no significance is attached to the variations in time-dependent aggregation found between cells of stage 1 and stage 4-5 blastoderms. Aggregation was temperature-dependent and decreased significantly when cultures were kept at 2 2 O
'O 800 1
'""1
544-5
I
60
120
I
180
240
Time (rnin)
Fig. 1. Rate of aggregation (%) of cells from dissociated blastoderms at stages 1 and stages 4-5. Blastoderms were dissociated in ealcium-magnesium free saline containing 0.002 M EDTA. Each point represents the mean and standard error obtained from four experiments. The method for assessing the degree of aggregation is presented in Table 1
C . Under the experimental conditions described in this paper, the proportion of single cells remains constant after 3y2-4 h in culture and the increase in size of aggregates which is observed further in time is, together with cell division, a reflection of adhesion between aggregates, since the latter decrease in number after 4 h in culture. In an effort to assess further the parameters affecting aggregation in this system, experiments were conducted in which blastoderm cells, dissociated in the presence of EDTA. were cultured in the presence or absence of serum. The decrease in the proportion of single cells was calculated as percent adhesion and the experiments were evaluated using a paired "t"-test. The results of these experiments are presented in Tables 1 and 2. It can be observed that under the conditions used in these experiments, the presence of serum does not have an appreciable effect on the aggregation of stage 1 blastoderm cells; the differences found were not consistent and fall between the values of experimental error. When cells of blastoderms at stage 4-5 were examined, the presence of serum tended to increase aggregation in cell suspen-
Aggregation of Cells from Early Chick Blastoderms Table 1. Effects of serum on aggregation of stage I blastoderm cells
Time (min)
Experiment I1
Experimcnt I With serum
Without serum
30 60
28.6 33.3
120
33.3 58.0
62.0 65.0
64.3 76.7
Without serum
50.9 62.3
46.6 53.0
61.6 68.2
48.1" 54.1
64.4 71.3
57.5 67.3
79.6 78.6
65.0 70.3
46.0 45.0
210 240
With serum
24.3 55.0
150 180
Time (min)
21.6 30.0
90
Table 2. Effects of serum on the aggregation o f stages 4-5 blastoderm cells
43.2a 60.0
Blastodcrms were dissociated in the presence of EDTA and subsequently cultured at 37" C in a gyratory shaker at 70 rpm. The degree of aggregation in percent was calculated as: No. single cells at zero time - No. single cells at specified time interval x 100 No. single cells at zero time When values of experiments were compared by a "t" test the probability was < 0.05%. Where probability values are missing the differences were not signifi cant.
a
sions examined at 210 min of incubation, and otherwise did not seem to have any appreciable effect. The effects of serum in the medium, however, were noticeable after 20 h of culture. Several large aggregates of a mean diameter of 1-1.5 mm were present in cultures of all blastoderm cells studied. In these cases the proportion of single cells had remained constant and we assumed the results reflected adhesion between small aggregates. The incidence of the presence of such large aggregates in media devoid of serum was very small; these aggregates when formed were healthy, and sorting out was evident upon microscopical examination of tissue sections [201. Experiments were designed to determine the effect of trypsin dissociation on aggregation of blastoderm cells. The results of these experiments are presented in Table 3. When examined at 30 min in culture, trypsin dissociation increased aggregation of cells from blastoderm stages 4-5. Trypsin dissociation had no effect on aggre-
Experiment I
Experiment I1
With serum
Without serum
With serum
Without serum
30
24.5 28.0
28.3a 20.4
32.6 41.5
31.3" 34.4
90
36.5 54.7
41.2 58.0
64.5 66.9
52.8 59.3
150
63.3 63.4
54.4 66.9
74.7 76.1
57.1 65.2
210
78.3 69.6
64.4 65.5
85.1 80.6
62.8 68.2"
Blastoderm cells were dissociated in the presence of EDTA. Culture conditions and evaluation of degree of adhesion were conducted as explained in Table 1. Whcn the values of the two experiments were compared by a "t" test the probability was < 0.05%. Where probability values are missing the differences were not significant.
gation thereafter. The effect of serum on trypsinised cells of blastoderm stages 4-5 is presented in Table 4. It can be observed that the serum hat no effect on aggregation up to 3y2 h in culture. As in experiments performed with EDTA-dissociated cells, serum effects were observed after 20 h of culture. Generally, blastoderm cells, cultured in serum containing media, formed large aggregates of 1-1.5 mm in diameter, while this was rarely the case in serum-free cultures where aggregates 0.7 mm in diameter were occasionally found. In an earlier paper [201 we described the pattern of sorting out in aggregates from stage 1 blastoderms as well as their further differentiation during the first 7 days of culture. Cells were arranged into two distinct groups: one of them was characterised by close intercellular associations, while the other displayed a loose packing of cells. The former had an internal location, while the latter formed the periphery of the aggregate and surrounded the compact cells, forming a continuous phase. We interpreted the compact phase as being composed mainly of epiblast cells, and the loosely packed cells as being of hypoblast origin. Thc events which occur when cells from stage 3 and 5 embryos reaggregate are similar, except that further differentiation has occurred in aggregates cultured for 5 and 7 days (Figs. 2-7). Sorting out into two populations is evident after 24 h culture (Fig. 2). As in the case of stage 1 aggregates, the inter-
E. J. Sanders and S. E. Zalik
4 Table 3. Effects of trypsin and EDTA on the aggregation of blastoderm cells EDTA
Trypsin
29.6 46.3
11.1 25.0
51.9 55.6
47.2 58.3
150
61.1 63.0
61.1 66.7
210
70.4 72.7
61.1 69.4
37.8 31.7 18.2 24.6
43.1" 42.1a 40. la 38.7"
68.4 68.7 40.1 53.4
67.0 58.8 64.5 52.6
150
75.7 66.4 59.2 55.6
73.2 73.0 72.9 64.0
210
75.2 75.1 63.9 66.7
74.9 67.0 59.7 67.6
Time (min) Stage I 30
90
Stage 4-5 30
90
Blastodcrm cells were dissociated with EDTA or trypsin as described in the text. Values for stage 1 blastoderm represent duplicates for one experiment. Values for stage 4-5 blastoderms represent duplicates for two experiments. Adhesion values when compared by a "t" test had probability values of < 0.05%. Wherc probability values are missing the differences were not significant.
nalised population consisted of patches of closely packed cells and these were surrounded by more loosely packed cells. After 2 days culture (Fig. 3) the closely packed population had usually aggregated into a single mass, without evidence of further differentiation. Characteristic changes had begun to occur in aggregates examined after 5 days' culture (Fig. 4). The contours of the aggregate surface showed evidence of folding, and the loosely packed cells at the periphery took on the appearance of yolk-sac endoderm. Signs of erythroid
Table 4. Effects of serum on the aggregation of trypsinised cells of stage 4-5 blastoderms Time (min)
With serum
Without serum
30
52.1 25.9 32.6 41.5
39.7 38.7 31.4 34.4
90
49.6 42.2 64.5 66.9
52.9 51.0 52.9 59.3
150
50.7 41.9 74.7 76.1
41.2 44.0 57.1 65.2
2 10
85.0 80.6 48.2 47.0
62.8 68.3 57.0 55.0
Adhesion
(Oh)
was evaluated as explained in Table 1.
differentiation were evident in both the loose and compact phases (Fig. 5); in the latter, formed blood elements were seen most frequently in the region lining a cavity within the aggregate. Groups of compact cells were differentiated into a variety of structures. At the boundary between the compact phase and the loose phase some cells of the former had flattened and elongated considerably and acquired a fibroblast-like shape (Fig. 5). Deeper within the compact phase, groups of cells appeared which were similar to notochord and early cartilage (Fig. 6). Lumina, which were apparent to a lesser degree in 1- and 2-day aggregates, were now consistently present in an enlarged form. Seven days in culture resulted in aggregates in which several distinct cell groups had differentiated (Fig. 7). The peripheral loosely packed cells showed features similar to those seen in 7-day aggregates from stage 1 embryos, and had the appearance of yolk-sac endodermal cells. The aggregate surface was highly folded. The cells derived from the closely packed population, by contrast, were more highly differentiated than those of stage 1 embryos after 7 days of culture. A number of different cavities or lumina had developed within distinct cell groups of the compact phase, some of which were closely juxtaposed forming a trabecular structure. The trabeculae, when examined by electron microscopy, were seen to be a series of cavities lined by intact cells (Fig. 8). Occasionally a large central cavity occurred as in the case of
Fig. 2. 24-h aggregate from stage 3 blastoderm cells showing the initial stages of sorting out into compact (*) and loosely packed cell populations. x 400 Fig. 3. 48-h aggregate from stage 3 blastoderm cells showing the accumulation of the compact cell populations into a single mass. x 440 Fig. 4. Aggregate from stage 3 blastoderms cultured for 5 days. Elongated cells (arrow) are present at the boundary of the compact phase. x 890 Fig. 5. 5-day aggregate showing flattened cells (arrow) between compact and loose phases and formed blood elements (B). x 1300 Fig. 6. 5 day aggregate showing the presence of notochord and cartilage-like cells within the compact phase (arrows). x 1300
Fig. 7. Aggregates from stage 5 blastoderms cultured for 7 days. Cavities or lumina are evident within the compact phase. Yolk sac-like cells are present at the periphery. x 380 Fig. 8. Electron micrograph of a region of a stage 1 aggregate cultured for 5 days. The trabecular structures are evident. x 4000 Fig. 9. A 5-day cultured aggregate from stage 1 blastoderm. The electron micrograph shows cells of the compact phase. x 4000 Fig. 10. The same aggregate as in Fig. 9. Cells of the loosely packed phase are shown. x 5300
Fig. 11. 24-h aggregate from stage 1 blastoderm cells. A basal lamina (b) is shown between closely packed (c) and loosely packed cells (1). x 23,600 Fig. 12. Electron micrograph of a 4-h aggregate from stage 4 blastoderm cells. A region of parallel membrane apposition is illustrated. x 47,800
Fig. 13. A 3-day aggregate from stage 3 blastoderm cells showing a lumen bounded by cells which have acquired a villous transformation. x 7400 Fig. 14. A 7-day aggregate from stage 1 blastoderm cells. A junctional complex is evident between cells bounding a lumen (L). x 83,500
Fig. 15. A 3-day aggregate from stage 1 blastoderms. A thin strip of cytoplasm from a single cell is surrounding a lumen (L). x 23,600 Fig. 16. 24-h aggregate of stage 1 blastoderm showing a highly differentiated desmosome between cells of the compact phase. x 75,300 Fig. 17. A 7-day aggregate from stage 1 blastoderm cells. Intercellular contacts in which the intercellular space is reduced to 30 A or less (arrows) are evident. x 89,000 Fig. 18. A 7-day aggregate from stage 1 blastoderm cells. A cell from the periphery of the aggregate with a morphology characteristic of yolksac epithelium. x 10,100 Fig. 19. A 5-day aggregate from stage 5 blastoderm. The cell illustrated has a microfilament tract (m) situated parallel to the long axis of the nucleus (n). x 17,500
9
Aggregation of Cells from Early Chick Blastoderms
stage 1 embryos [201. No difference in aggregation pattern could be detected between EDTA- and trypsin-dissociated cells. In aggregates cultured in the absence of serum, there was a reduction in aggregate size and a slight initial lag in sorting out. Electron micrographs showed that both the closely packed and loosely packed populations of cells (Figs. 9 and 10) were composed of cells of healthy appearance. The ultrastructural appearance, as well as the occurrence of frequent mitotic figures, provided evidence for the presence of healthy cells in both populations. In the early stages of reaggregation, cells of the loosely packed population possessed adhering extracellular yolk granules which became trapped within the aggregate. This is due to the presence of yolk of extracellular origin associated with the excised blastoderms at these developmental stages, which cannot be completely removed. Small aggregates examined after 4 h of incubation showed relatively undifferentiated cell contacts characterised by large areas of membrane apposition with a 200 A intercellular space (Fig. 12). The cell contours were smooth, showing little or no interdigitation. By the time aggregates of cells from embryos of all stages had been cultured for 24 h, basal laminae appeared, sharply separating the closely packed and loosely packed cells (Fig. 11). The basal lamina was present at the boundaries of the closely packed cell masses and consisted of a moderately electron-dense fibrous layer of variable thickness. No striated collagen fibrils were observed in any aggregates. The lumina, which occurred in aggregates cultured from 24 h onwards (Fig. 13), were bounded by several cells displaying a villous transformation on the luminal surface. These cells were attached to one another at their luminal ends by a terminal complex of intercellular junctions (Fig. 14) similar to those occurring at the dorsal surface of epiblast cells in situ [181. These junctions, which had developed by 24-h culture, were characterised by the presence of electron density on the cytoplasmic surface of the membrane and focal contacts between the cells. Lumina were occasionally observed entirely bounded by a thin strip of cytoplasm belonging to a single cell (Fig. 15). When 24-h aggregates were examined, considerable interdigitation was observed between some cells, although large regions of parallel apposition remained. Highly developed desmosomes (Fig. 16) were present in parallel as well as interdigital regions and by 3 days in culture, contacts in which the intercellular space was reduced to 30 A or less were present (Fig. 17). With regard to morphological differentiation within the aggregates, by 5 days in culture a number of distinct
cell types were present, a limited number of which were identifiable by their electron microscopic appearance. Some cells resembled yolk-sac epithelium (Fig. 18) which are characterised in several species by large numbers of microvilli with fibrillar cores and coated invaginations of the cell surface [23-261. The most readily distinguishable cells of the more compact phase were those displaying highly developed microfilament tracts (Fig. 19). These cells correspond in appearance to chick embryo myogenic cells which have previously been shown to differentiate within aggregates [27, 281, and in many such cells, Z material was seen, as well as contact specialisations similar to intercalated discs.
Discussion
Under the present experimental conditions, results show that regardless of the method of dissociation, the presence of serum in the culture medium has no effect on early adhesion of blastoderm cells at any of the developmental stages studied. Although some increase in adhesion was observed in EDTA-dissociated cells from stage 4-5 blastoderms, this was only evident at 3y2 h of culture. It should be pointed out that under our experimental procedures, adhesion was not evaluated during the first 30 min of culture. The extent of influence exerted by serum during this time remains to be established. A number of investigations have shown that embryonic cells are able to reaggregate in a medium which lacks serum [29-341. In many cases the aggregates are reduced in size and are less compact. The present results are in agreement with these findings. The reduction in aggregate size may reflect a decrease in adhesion between aggregates, a parameter that was not determined in our experiments. The results of the present experiments show that, under the conditions employed, early chick embryonic cells reaggregate just as well during the first 4 h after trypsin dissociation as after EDTA dissociation. There is no general agreement as to the effects of trypsin on cell-cell adhesiveness 1351. While there is certainly an initial decrease in cell-glass adhesiveness 136, 371, the effects on aggregation are unclear. Where the effects of trypsin and EDTA dissociation have previously been compared, investigators have reported that there is no difference either in reaggregative competence (3 1, 381, or reduced aggregation after EDTA [391, or an initial reduction in aggregation after trypsin [34, 40, 411. Differences may not only be due to the various cell types and developmental ages [421 but to differences in the technique of evaluation of aggregation and the time peri-
10
ods of observation. The lack of any reduction of aggregative efficiency shown in the present study after trypsin dissociation may correlate with the observation that the effects of calcium on cell-surface charge density, as well as lectin-induced agglutination, are not appreciably affected by this enzyme 119, 431. Electron microscopic studies of cell aggregation during the first few hours of culture have shown that adhering cells make contact by means of relatively smooth areas of apposed membrane with few cytoplasmic processes [ 13, 44-461. The surface projections that do occur have been suggested to have a role in promoting a subsequent closer contact between apposing cells [471. Our observations on 4-h aggregates suggest that early contacts in chick blastoderm cells also involve large areas of smooth undifferentiated contact, with no interdigitation, but with focal contacts via surface undulations; whether a similar situation occurs at earlier stages of aggregation remains to be established. When aggregates were observed at 24 h, when sorting out is actively taking place, evidence of interdigitation between cells was found, similar to that reported by Sheffield and Moscona [48], Armstrong [49, 501 and Adler [51]. Whether this phenomenon has any significance for initial stages of sorting out is uncertain. The only previous study on reaggregation of cells from early chick blastoderms was that of Zwilling 152, 531 in which embryos at the primitive streak stage and onwards were used. This investigator found that LLwhen the entire pellucid area of chick embryo at the definitive streak stage is subjected to dissociation and allowed to reaggregate spontaneously . . . embryonic structures fail to develop” [531. Our results show that cell suspensions from whole blastoderms at these stages and earlier are able to aggregate and further sort out into two phases. In both phases blood elements developed, while in the internal compact phase, structures resembling notochordal cartilage and muscle cells were found. Our evidence suggests that the compact phase arises from epiblast cells and includes nodal cells of the primitive streak. We cannot therefore comment on Zwilling’s conclusion that “the nodal cells . . . do not sort out from the non-nodal cells” [531. However our experiments suggest that epiblast cells in the proportion present in the embryo are capable of segregating from the hypoblast cells which under our conditions differentiated into yolk-sac-like cells. Furthermore, the former are capable of forming discrete masses within the aggregates which are capable of further differentiation. The conclusion of Zwilling [521 that “nodal area cells must be present in considerable numbers before they can express their full morpho-
E. J. Sanders and S . E. Zalik:
genetic potential” has not been tested by us. It should be pointed out that the experimental conditions used by this author were considerably different from our own. Electron microscope observations show that a basal lamina is laid down at the boundary of each mass of compact tissue. According to Low 1541, the epiblast is the first tissue to acquire a basal lamina in situ. Chick as well as amphibian embryos synthesise mucopolysaccharide, before the onset of gastrulation [ 5 5 , 561 and in the former this is in association with the presence of a basal lamina. As pointed out by O’Hare [55], the origin of a basal lamina cannot be inferred from its location, but an ectodermal origin is usually found to be the case. In the aggregates studied here we have a situation in which the compact population can form lumina sealed by intercellular junctions characteristic of the dorsal surface of the epiblast [ 1XI and also a basal lamina characteristic of the basal surface of the epiblast. Acknowledgements: This work was supported by the National Cancer Institute, the National Research Council and the Medical Research Council of Canada. We thank Miss Carol Ward for her technical assistance.
References 1. Spratt, N. T., Haas, H.: J. exp. 2001.144, 139, 1960 2. Spratt, N. T., Haas, H.: J. exp. 2001.144, 257, 1960 3. Spratt, N. T., Haas, H.: J. exp. 2001.158, 9, 1965 4. Vakaet. L.: J . Ernbyol. exp. Morph. 10, 38, 1962 5. Eyal-Giladi, H.: J . Embryol. exp. Morph. 23, 739. 1970 6. Bellairs, R. : Developmental Processes in Higher Vertebrates. London: Logos Academic Press 1971 7. Nicolet, G.: In: Advanced Morphogenesis. Abercrombie and Brachet (eds.), Vol. 9, p. 231. New York: Academic Press 1971 8. Moscona, A. A.: In: Cells and Tissue in Culture. Willmcr (ed.), p. 489. New York: Academic Press 1965 9. Steinberg, M. S.: In: Cellular Membranes in Development. Locke (ed.), p. 321. New York: Academic Press 1964 10. Steinberg, M. S.: J. exp. Zoof. 173, 395, 1970 11. Trinkaus, J. P.: In: Organogenesis. DeHaan and Ursprung (eds.), p. 55. New York: Holt, Rinehart and Winston 1965 12. Weiss, L.: The Cell Periphery, Metastasis and Other Contact Phenomena. Amsterdam: North Holland Pub. Co. 1967 13. Giudice, G., Mutolo. V.: In: Advanced Morphogenesis. Abercrombie and Brachet (eds.), Vol. 8. p. 11 5. New York: Academic Press 1970 14. Holtfreter, J.: Ann. N . Y. Acud. Sci. 49, 709, 1948 15. Curtis, A. S. G . : The Cell Surface: Its Molecular Role in Morphogenesis. London: Logos Academic Press 1967 16. Zalik, S. E., Sanders, E. J., Tilley, C.: J. Cell Physiol. 79, 225, 1972 17. Sanders. E. J., Zalik, S . E.: J. Cell Physiol. 79, 235, 1972 18. Sanders, F.. J.: 2. Zellforsch. 141, 459, 1973 19. Harris. H. L., Zalik, S. E.: J. Cell Physiol. 83, 359, 1974
Aggregation of Cells from Early Chick Blastodcrms 20. Zalik, S. E., Sanders, E. J.: Differentiation 2,25, 1974 21. Menashi. M. K., Kochav, S., Eyal-Giladi, H.: Int. Symp. on Early Avian Development. Belgium: Rijksuniversitair Centrum Antwerpen 1975 22. Hamburger, V., Hamilton, N. L.: J. Morph. 88, 49, 1951 23. Padykula, H. A., Deren, J. J., Wilson. H. T.: Dev. Biol. 13, 311, 1966 24. King, B. F., Endcrs, A. C . : Amer. J. Anat. 127, 397, 1970 25. Lambson, R. 0.: Amer. J. Anat. 129, I , 1970 26. Sladc, B. S.: J. Anal. 107, 531, 1970 27. Shimada, Y.: Z . Anat. EntwickL-Gesch. 138, 255, 1972 28. Zacchei, A. M.. Caravita, S.: J. Embryol. exp. Morph. 28, 571, 1972 29. Ball, W. D.: Nature 210, 1075, 1966 30. Ishii, K.: Cyrologia 31, 89, 1966 31. Moscona, A. A., Moscona, M. H.: Exp. Cell Res. 41, 697, 1966 32. Kemp, R. B., Jones, B. M., Cunningham, I., James, M. C. M.: J. Cell Sci. 2, 323, 1967 33. Daday, H.: Wilhelm Roux’ Arehiv 171, 244, 1972 34. Steinberg, M. S., Armstrong, P. B., Granger, R. E.: J. Memb. Biol. 13, 91. 1973 35. Lilicn, J. E.: Curr. Top. Dev. Biol. 4, 169, 1969 36. Weiss, L., Kapes, D. L.: Exp. Cell Res. 41. 601, 1966 37. Poste, G.: Exp. Cell Res. 65, 359. 1971
11 38. Glacscr, R. M.. Richmond, J. E., Todd, P. W.: Exp. Cell Res. 52, 71, 1968 39. Moscona, A. A., Moscona, M. H.: Exp. Cell Res. 45, 239, 1967 40. McQuiddy, P., Lilien, J.: J. Cell Sci. 9, 823, 1971 41. Cassiman, J. J., Bernfield, M. R.: Exp. Cell Res. 91, 31, 1975 42. Kleinschuster, S. J., Moscona, A. A.: Exp. Cell Res. 70, 397, 1972 43. Zalik, S. E., Cook, G. M. W.: J. Cell Biol. 63, 385a, 1974 44. Shefield, J. B.: J. Morph. 132, 245, 1970 45. Shefield, J. B., Moscona, A. A.: Exp. Cell Res. 57, 462, 1969 46. Pucci-Minafra, I., Bosco, M., Giamhcrtone, L.: Exp. Cell Res. 53, 177, 1968 47. Lesseps, R. J.: J. exp. Zool. 153. 171, 1963 48. Shefield, J. B., Moscona, A. A,: Dev. Biol. 23, 36, 1970 49. Armstrong. P. B.: J. Cell Biol. 47, 197, 1970 50. Armstrong, P. B.: Wilhelm Roux’ Archiv 168, 125, 1971 51. Adlcr, R.: Exp. Cell Res. 77, 367, 1973 52. Zwilling, E.: Natl. Cancer Inst. Monogr. 2, 19, 1960 53. Zwilling, E.: Dev. Biol. 7, 642, 1963 54.Low, F. N.: Anat. Rec. 159, 231, 1967 55. O’Hare, M. J.: J. Embryol. exp. Morph. 29, 197, 1973 56. Kosher, R. A,, Searls, R. L.: Dev. Biol. 32, 50, 1973
Received July 1975
The aggregation of dissociated cells from chick blastoderms at Hamburger and Hamilton stages 1-5 was studied. Aggregation was measured during the first 4 h of culture by determination of the proportion of single cells in the medium. No difference in aggregation was found when cells dissociated by either trypsin or EDTA were studied. Similarly the presence or absence of serum in the medium had no appreciable effect on early phases of aggregation, although at 24 h and thereafter, aggregate size was reduced in serum-free cultures. It was found that at all of the stages studied, cells aggregate and sort out into two groups. One group forms a continuous phase of loosely associated cells while the other segregates into several localised areas of closely associated cells within the aggregate. Examination of aggregates up to 7 days in culture showed progressive differentiation within each phase and several identifiable cell types were observed. Basal laminae were present at the boundary between the compact phase and the loose phase.
Introduction Early embryogenesis in the chick involves a complex series of cellular interactions which ultimately lead to the formation of the three-layered embryo [ 1-71. These interactions presumably depend to some extent on the surface properties of the cells present in the embryo. There is an extensive literature concerning studies on the adhesive behaviour of embryonic cells at relatively advanced stages of development [8-121. Studies on early embryonic cells have been performed on sea urchin, amphibian and fish embryos [11-151 but relatively little information is available on surface properties of chick embryonic cells prior to, and during gastrulation. Some of these surface characteristics have been defined using cell electrophoresis and electron microscopy 16- 191. Preliminary studies in our laboratory [201 have shown that in aggregates obtained from dissociated pre-streak blastoderm cells, some cells possess surface characteristics which allow them to sort out into distinct groups and continue further differentiation. Two populations of cells were distinguishable and it was our contention that these corresponded to cells of epiblast and hypoblast. These results have been substantiated by studies conducted elsewhere [211. The present report represents an attempt to study the aggregative behaviour of dissociated cells from Differentiation 6, 1-11 (1976)
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0 by Springer-Verlag 1976
pre-streak and primitive streak blastoderms. We have studied the kinetics of aggregation under a variety of conditions during the first 4 h of culture. In addition we have examined the cellular reorganisation and differentiation of the aggregates during the first 7 days of culture.
Methods In staging embryos, the criteria and numbering of Hamburger and Hamilton [221 was used. Blastoderms were removed under sterile conditions from embryos at stages 1 (pre-streak) to 4-5 (head process). Blastoderms were dissected frec from their vitelline membranes and stored overnight at room temperature or at 37" C in Panet and Compton saline containing 0.2% glucose, 300 U/ml penicillin and 300 pg/ml streptomycin (GIBCO). Dissociation of blastoderms was carried out as previously described 1161 in calcium-magnesium-free Panet and Compton saline (CMFPC), pH 7.8. containing 0.002 M EDTA at room temperature. Alternatively, blastoderms were placed in CMFPC with 0.1 % trypsin ( 1 : 250. Difco), incubated at 37" C for 10 min and dissociated as reported previously. For the aggregation experiments, cells were suspended in 2 ml Leibowitz L-15 medium (GIBCO), with or without 10% foetal calf serum, at a cell concentration varying from 0.5 to 1.0 x lo6 cells per ml. Cell suspensions were placed in 10 ml flasks and cultured at 37" C in a gyratory shaker at 70 rpm.
E. J. Sanders and S. E. Zalik:
2 Attempts were made to evaluate the viability of blastoderm cells by employing a variety of vital dyes in dye-exclusion tests. However for the same cell suspension, some dyes stained all the cells, while others did not penetrate any of them; this last instance occurred even under a variety of adverse conditions. To the present time we have not found a vital dye which stains blastoderm cells differentially. Cells. however, adhered to each other and formed aggregates in which cell division and differentiation occurred, and little sign of cell degeneration was evident when aggregates were examined with light and electron microscopes. The degree of aggregation was evaluated by determining the rate of disappearance of single cells in the culture medium. Samples were removed at defined time intervals from the culture flasks, di luted to a constant volume with saline, and the number of single cells was determined with the use of a hemocytometer. Cell counts were performed during the first 4 h of incubation. Hemocytometer counts were performed 10 times on aliquots of the diluted sample and an average of these readings was obtained at each time-interval studied. Under our conditions a decline in the number of single cells coincided with the appearance of cell aggregates. Due to the variation in size encountered in blastoderm cells, and the presence of yolk granules, also of varying size. the electronic particle counter could not be used in these experiments. After cell counts were performed, flasks with cultured aggregates were further incubated for 1, 2, 3, 5 and 7 days at 37" C and 100 rpm. During these time intervals one half of the original culture medium was removed and replaced by fresh medium. This procedure was repeated at 48 h intervals. Light microscopy was carried out on Bouin's-fixed and wax-embedded aggregates. Sections were stained using haemotoxylin and eosin. For electron microscopy, aggregates were fixed using 3?6 glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, followed by 2% osmium tetroxide in the same buffer. The material was dehydrated using graded ethanol solutions and sections were stained with uranyl acetate and lead citrate.
Results A first series of experiments was performed in order to determine the aggregative behaviour of EDTA-dissociated cells in the presence of medium containing 10% foetal calf serum. This was performed for stage 1 and stage 4-5 blastoderms. The results from these experiments are shown in Fig. 1, in which percent adhesion versus time is presented. It can be observed that by 30 min in culture, adhesion has increased to an average of 35% for experiments using blastoderrn cells at both stages. There is a time-dependent increase in aggregation, so that by 2 10 min in culture, adhesions had increased to an average value of 64 and 74% for blastoderm cells at stages 1 and 4-5 respectively. The actual extent of the time-dependent increase in aggregation tended to vary considerably between experiments, therefore no significance is attached to the variations in time-dependent aggregation found between cells of stage 1 and stage 4-5 blastoderms. Aggregation was temperature-dependent and decreased significantly when cultures were kept at 2 2 O
'O 800 1
'""1
544-5
I
60
120
I
180
240
Time (rnin)
Fig. 1. Rate of aggregation (%) of cells from dissociated blastoderms at stages 1 and stages 4-5. Blastoderms were dissociated in ealcium-magnesium free saline containing 0.002 M EDTA. Each point represents the mean and standard error obtained from four experiments. The method for assessing the degree of aggregation is presented in Table 1
C . Under the experimental conditions described in this paper, the proportion of single cells remains constant after 3y2-4 h in culture and the increase in size of aggregates which is observed further in time is, together with cell division, a reflection of adhesion between aggregates, since the latter decrease in number after 4 h in culture. In an effort to assess further the parameters affecting aggregation in this system, experiments were conducted in which blastoderm cells, dissociated in the presence of EDTA. were cultured in the presence or absence of serum. The decrease in the proportion of single cells was calculated as percent adhesion and the experiments were evaluated using a paired "t"-test. The results of these experiments are presented in Tables 1 and 2. It can be observed that under the conditions used in these experiments, the presence of serum does not have an appreciable effect on the aggregation of stage 1 blastoderm cells; the differences found were not consistent and fall between the values of experimental error. When cells of blastoderms at stage 4-5 were examined, the presence of serum tended to increase aggregation in cell suspen-
Aggregation of Cells from Early Chick Blastoderms Table 1. Effects of serum on aggregation of stage I blastoderm cells
Time (min)
Experiment I1
Experimcnt I With serum
Without serum
30 60
28.6 33.3
120
33.3 58.0
62.0 65.0
64.3 76.7
Without serum
50.9 62.3
46.6 53.0
61.6 68.2
48.1" 54.1
64.4 71.3
57.5 67.3
79.6 78.6
65.0 70.3
46.0 45.0
210 240
With serum
24.3 55.0
150 180
Time (min)
21.6 30.0
90
Table 2. Effects of serum on the aggregation o f stages 4-5 blastoderm cells
43.2a 60.0
Blastodcrms were dissociated in the presence of EDTA and subsequently cultured at 37" C in a gyratory shaker at 70 rpm. The degree of aggregation in percent was calculated as: No. single cells at zero time - No. single cells at specified time interval x 100 No. single cells at zero time When values of experiments were compared by a "t" test the probability was < 0.05%. Where probability values are missing the differences were not signifi cant.
a
sions examined at 210 min of incubation, and otherwise did not seem to have any appreciable effect. The effects of serum in the medium, however, were noticeable after 20 h of culture. Several large aggregates of a mean diameter of 1-1.5 mm were present in cultures of all blastoderm cells studied. In these cases the proportion of single cells had remained constant and we assumed the results reflected adhesion between small aggregates. The incidence of the presence of such large aggregates in media devoid of serum was very small; these aggregates when formed were healthy, and sorting out was evident upon microscopical examination of tissue sections [201. Experiments were designed to determine the effect of trypsin dissociation on aggregation of blastoderm cells. The results of these experiments are presented in Table 3. When examined at 30 min in culture, trypsin dissociation increased aggregation of cells from blastoderm stages 4-5. Trypsin dissociation had no effect on aggre-
Experiment I
Experiment I1
With serum
Without serum
With serum
Without serum
30
24.5 28.0
28.3a 20.4
32.6 41.5
31.3" 34.4
90
36.5 54.7
41.2 58.0
64.5 66.9
52.8 59.3
150
63.3 63.4
54.4 66.9
74.7 76.1
57.1 65.2
210
78.3 69.6
64.4 65.5
85.1 80.6
62.8 68.2"
Blastoderm cells were dissociated in the presence of EDTA. Culture conditions and evaluation of degree of adhesion were conducted as explained in Table 1. Whcn the values of the two experiments were compared by a "t" test the probability was < 0.05%. Where probability values are missing the differences were not significant.
gation thereafter. The effect of serum on trypsinised cells of blastoderm stages 4-5 is presented in Table 4. It can be observed that the serum hat no effect on aggregation up to 3y2 h in culture. As in experiments performed with EDTA-dissociated cells, serum effects were observed after 20 h of culture. Generally, blastoderm cells, cultured in serum containing media, formed large aggregates of 1-1.5 mm in diameter, while this was rarely the case in serum-free cultures where aggregates 0.7 mm in diameter were occasionally found. In an earlier paper [201 we described the pattern of sorting out in aggregates from stage 1 blastoderms as well as their further differentiation during the first 7 days of culture. Cells were arranged into two distinct groups: one of them was characterised by close intercellular associations, while the other displayed a loose packing of cells. The former had an internal location, while the latter formed the periphery of the aggregate and surrounded the compact cells, forming a continuous phase. We interpreted the compact phase as being composed mainly of epiblast cells, and the loosely packed cells as being of hypoblast origin. Thc events which occur when cells from stage 3 and 5 embryos reaggregate are similar, except that further differentiation has occurred in aggregates cultured for 5 and 7 days (Figs. 2-7). Sorting out into two populations is evident after 24 h culture (Fig. 2). As in the case of stage 1 aggregates, the inter-
E. J. Sanders and S. E. Zalik
4 Table 3. Effects of trypsin and EDTA on the aggregation of blastoderm cells EDTA
Trypsin
29.6 46.3
11.1 25.0
51.9 55.6
47.2 58.3
150
61.1 63.0
61.1 66.7
210
70.4 72.7
61.1 69.4
37.8 31.7 18.2 24.6
43.1" 42.1a 40. la 38.7"
68.4 68.7 40.1 53.4
67.0 58.8 64.5 52.6
150
75.7 66.4 59.2 55.6
73.2 73.0 72.9 64.0
210
75.2 75.1 63.9 66.7
74.9 67.0 59.7 67.6
Time (min) Stage I 30
90
Stage 4-5 30
90
Blastodcrm cells were dissociated with EDTA or trypsin as described in the text. Values for stage 1 blastoderm represent duplicates for one experiment. Values for stage 4-5 blastoderms represent duplicates for two experiments. Adhesion values when compared by a "t" test had probability values of < 0.05%. Wherc probability values are missing the differences were not significant.
nalised population consisted of patches of closely packed cells and these were surrounded by more loosely packed cells. After 2 days culture (Fig. 3) the closely packed population had usually aggregated into a single mass, without evidence of further differentiation. Characteristic changes had begun to occur in aggregates examined after 5 days' culture (Fig. 4). The contours of the aggregate surface showed evidence of folding, and the loosely packed cells at the periphery took on the appearance of yolk-sac endoderm. Signs of erythroid
Table 4. Effects of serum on the aggregation of trypsinised cells of stage 4-5 blastoderms Time (min)
With serum
Without serum
30
52.1 25.9 32.6 41.5
39.7 38.7 31.4 34.4
90
49.6 42.2 64.5 66.9
52.9 51.0 52.9 59.3
150
50.7 41.9 74.7 76.1
41.2 44.0 57.1 65.2
2 10
85.0 80.6 48.2 47.0
62.8 68.3 57.0 55.0
Adhesion
(Oh)
was evaluated as explained in Table 1.
differentiation were evident in both the loose and compact phases (Fig. 5); in the latter, formed blood elements were seen most frequently in the region lining a cavity within the aggregate. Groups of compact cells were differentiated into a variety of structures. At the boundary between the compact phase and the loose phase some cells of the former had flattened and elongated considerably and acquired a fibroblast-like shape (Fig. 5). Deeper within the compact phase, groups of cells appeared which were similar to notochord and early cartilage (Fig. 6). Lumina, which were apparent to a lesser degree in 1- and 2-day aggregates, were now consistently present in an enlarged form. Seven days in culture resulted in aggregates in which several distinct cell groups had differentiated (Fig. 7). The peripheral loosely packed cells showed features similar to those seen in 7-day aggregates from stage 1 embryos, and had the appearance of yolk-sac endodermal cells. The aggregate surface was highly folded. The cells derived from the closely packed population, by contrast, were more highly differentiated than those of stage 1 embryos after 7 days of culture. A number of different cavities or lumina had developed within distinct cell groups of the compact phase, some of which were closely juxtaposed forming a trabecular structure. The trabeculae, when examined by electron microscopy, were seen to be a series of cavities lined by intact cells (Fig. 8). Occasionally a large central cavity occurred as in the case of
Fig. 2. 24-h aggregate from stage 3 blastoderm cells showing the initial stages of sorting out into compact (*) and loosely packed cell populations. x 400 Fig. 3. 48-h aggregate from stage 3 blastoderm cells showing the accumulation of the compact cell populations into a single mass. x 440 Fig. 4. Aggregate from stage 3 blastoderms cultured for 5 days. Elongated cells (arrow) are present at the boundary of the compact phase. x 890 Fig. 5. 5-day aggregate showing flattened cells (arrow) between compact and loose phases and formed blood elements (B). x 1300 Fig. 6. 5 day aggregate showing the presence of notochord and cartilage-like cells within the compact phase (arrows). x 1300
Fig. 7. Aggregates from stage 5 blastoderms cultured for 7 days. Cavities or lumina are evident within the compact phase. Yolk sac-like cells are present at the periphery. x 380 Fig. 8. Electron micrograph of a region of a stage 1 aggregate cultured for 5 days. The trabecular structures are evident. x 4000 Fig. 9. A 5-day cultured aggregate from stage 1 blastoderm. The electron micrograph shows cells of the compact phase. x 4000 Fig. 10. The same aggregate as in Fig. 9. Cells of the loosely packed phase are shown. x 5300
Fig. 11. 24-h aggregate from stage 1 blastoderm cells. A basal lamina (b) is shown between closely packed (c) and loosely packed cells (1). x 23,600 Fig. 12. Electron micrograph of a 4-h aggregate from stage 4 blastoderm cells. A region of parallel membrane apposition is illustrated. x 47,800
Fig. 13. A 3-day aggregate from stage 3 blastoderm cells showing a lumen bounded by cells which have acquired a villous transformation. x 7400 Fig. 14. A 7-day aggregate from stage 1 blastoderm cells. A junctional complex is evident between cells bounding a lumen (L). x 83,500
Fig. 15. A 3-day aggregate from stage 1 blastoderms. A thin strip of cytoplasm from a single cell is surrounding a lumen (L). x 23,600 Fig. 16. 24-h aggregate of stage 1 blastoderm showing a highly differentiated desmosome between cells of the compact phase. x 75,300 Fig. 17. A 7-day aggregate from stage 1 blastoderm cells. Intercellular contacts in which the intercellular space is reduced to 30 A or less (arrows) are evident. x 89,000 Fig. 18. A 7-day aggregate from stage 1 blastoderm cells. A cell from the periphery of the aggregate with a morphology characteristic of yolksac epithelium. x 10,100 Fig. 19. A 5-day aggregate from stage 5 blastoderm. The cell illustrated has a microfilament tract (m) situated parallel to the long axis of the nucleus (n). x 17,500
9
Aggregation of Cells from Early Chick Blastoderms
stage 1 embryos [201. No difference in aggregation pattern could be detected between EDTA- and trypsin-dissociated cells. In aggregates cultured in the absence of serum, there was a reduction in aggregate size and a slight initial lag in sorting out. Electron micrographs showed that both the closely packed and loosely packed populations of cells (Figs. 9 and 10) were composed of cells of healthy appearance. The ultrastructural appearance, as well as the occurrence of frequent mitotic figures, provided evidence for the presence of healthy cells in both populations. In the early stages of reaggregation, cells of the loosely packed population possessed adhering extracellular yolk granules which became trapped within the aggregate. This is due to the presence of yolk of extracellular origin associated with the excised blastoderms at these developmental stages, which cannot be completely removed. Small aggregates examined after 4 h of incubation showed relatively undifferentiated cell contacts characterised by large areas of membrane apposition with a 200 A intercellular space (Fig. 12). The cell contours were smooth, showing little or no interdigitation. By the time aggregates of cells from embryos of all stages had been cultured for 24 h, basal laminae appeared, sharply separating the closely packed and loosely packed cells (Fig. 11). The basal lamina was present at the boundaries of the closely packed cell masses and consisted of a moderately electron-dense fibrous layer of variable thickness. No striated collagen fibrils were observed in any aggregates. The lumina, which occurred in aggregates cultured from 24 h onwards (Fig. 13), were bounded by several cells displaying a villous transformation on the luminal surface. These cells were attached to one another at their luminal ends by a terminal complex of intercellular junctions (Fig. 14) similar to those occurring at the dorsal surface of epiblast cells in situ [181. These junctions, which had developed by 24-h culture, were characterised by the presence of electron density on the cytoplasmic surface of the membrane and focal contacts between the cells. Lumina were occasionally observed entirely bounded by a thin strip of cytoplasm belonging to a single cell (Fig. 15). When 24-h aggregates were examined, considerable interdigitation was observed between some cells, although large regions of parallel apposition remained. Highly developed desmosomes (Fig. 16) were present in parallel as well as interdigital regions and by 3 days in culture, contacts in which the intercellular space was reduced to 30 A or less were present (Fig. 17). With regard to morphological differentiation within the aggregates, by 5 days in culture a number of distinct
cell types were present, a limited number of which were identifiable by their electron microscopic appearance. Some cells resembled yolk-sac epithelium (Fig. 18) which are characterised in several species by large numbers of microvilli with fibrillar cores and coated invaginations of the cell surface [23-261. The most readily distinguishable cells of the more compact phase were those displaying highly developed microfilament tracts (Fig. 19). These cells correspond in appearance to chick embryo myogenic cells which have previously been shown to differentiate within aggregates [27, 281, and in many such cells, Z material was seen, as well as contact specialisations similar to intercalated discs.
Discussion
Under the present experimental conditions, results show that regardless of the method of dissociation, the presence of serum in the culture medium has no effect on early adhesion of blastoderm cells at any of the developmental stages studied. Although some increase in adhesion was observed in EDTA-dissociated cells from stage 4-5 blastoderms, this was only evident at 3y2 h of culture. It should be pointed out that under our experimental procedures, adhesion was not evaluated during the first 30 min of culture. The extent of influence exerted by serum during this time remains to be established. A number of investigations have shown that embryonic cells are able to reaggregate in a medium which lacks serum [29-341. In many cases the aggregates are reduced in size and are less compact. The present results are in agreement with these findings. The reduction in aggregate size may reflect a decrease in adhesion between aggregates, a parameter that was not determined in our experiments. The results of the present experiments show that, under the conditions employed, early chick embryonic cells reaggregate just as well during the first 4 h after trypsin dissociation as after EDTA dissociation. There is no general agreement as to the effects of trypsin on cell-cell adhesiveness 1351. While there is certainly an initial decrease in cell-glass adhesiveness 136, 371, the effects on aggregation are unclear. Where the effects of trypsin and EDTA dissociation have previously been compared, investigators have reported that there is no difference either in reaggregative competence (3 1, 381, or reduced aggregation after EDTA [391, or an initial reduction in aggregation after trypsin [34, 40, 411. Differences may not only be due to the various cell types and developmental ages [421 but to differences in the technique of evaluation of aggregation and the time peri-
10
ods of observation. The lack of any reduction of aggregative efficiency shown in the present study after trypsin dissociation may correlate with the observation that the effects of calcium on cell-surface charge density, as well as lectin-induced agglutination, are not appreciably affected by this enzyme 119, 431. Electron microscopic studies of cell aggregation during the first few hours of culture have shown that adhering cells make contact by means of relatively smooth areas of apposed membrane with few cytoplasmic processes [ 13, 44-461. The surface projections that do occur have been suggested to have a role in promoting a subsequent closer contact between apposing cells [471. Our observations on 4-h aggregates suggest that early contacts in chick blastoderm cells also involve large areas of smooth undifferentiated contact, with no interdigitation, but with focal contacts via surface undulations; whether a similar situation occurs at earlier stages of aggregation remains to be established. When aggregates were observed at 24 h, when sorting out is actively taking place, evidence of interdigitation between cells was found, similar to that reported by Sheffield and Moscona [48], Armstrong [49, 501 and Adler [51]. Whether this phenomenon has any significance for initial stages of sorting out is uncertain. The only previous study on reaggregation of cells from early chick blastoderms was that of Zwilling 152, 531 in which embryos at the primitive streak stage and onwards were used. This investigator found that LLwhen the entire pellucid area of chick embryo at the definitive streak stage is subjected to dissociation and allowed to reaggregate spontaneously . . . embryonic structures fail to develop” [531. Our results show that cell suspensions from whole blastoderms at these stages and earlier are able to aggregate and further sort out into two phases. In both phases blood elements developed, while in the internal compact phase, structures resembling notochordal cartilage and muscle cells were found. Our evidence suggests that the compact phase arises from epiblast cells and includes nodal cells of the primitive streak. We cannot therefore comment on Zwilling’s conclusion that “the nodal cells . . . do not sort out from the non-nodal cells” [531. However our experiments suggest that epiblast cells in the proportion present in the embryo are capable of segregating from the hypoblast cells which under our conditions differentiated into yolk-sac-like cells. Furthermore, the former are capable of forming discrete masses within the aggregates which are capable of further differentiation. The conclusion of Zwilling [521 that “nodal area cells must be present in considerable numbers before they can express their full morpho-
E. J. Sanders and S . E. Zalik:
genetic potential” has not been tested by us. It should be pointed out that the experimental conditions used by this author were considerably different from our own. Electron microscope observations show that a basal lamina is laid down at the boundary of each mass of compact tissue. According to Low 1541, the epiblast is the first tissue to acquire a basal lamina in situ. Chick as well as amphibian embryos synthesise mucopolysaccharide, before the onset of gastrulation [ 5 5 , 561 and in the former this is in association with the presence of a basal lamina. As pointed out by O’Hare [55], the origin of a basal lamina cannot be inferred from its location, but an ectodermal origin is usually found to be the case. In the aggregates studied here we have a situation in which the compact population can form lumina sealed by intercellular junctions characteristic of the dorsal surface of the epiblast [ 1XI and also a basal lamina characteristic of the basal surface of the epiblast. Acknowledgements: This work was supported by the National Cancer Institute, the National Research Council and the Medical Research Council of Canada. We thank Miss Carol Ward for her technical assistance.
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