Hemoglobin synthesis in isolated erythroid colonies from the chick embryo

DEVELOPMENTAL

BIOLOGY

49, 556-562

Hemoglobin

(1976)

Synthesis in Isolated Erythroid from the Chick Embryo’ KAY S. PINES AND ALLAN J.

The Biological and

Colonies

TOBIN~

Laboratories, Harvard University, Cambridge, Massachusetts 02138, and Biology Department Molecular Biology Institute, University of California, Los Angeles, California 90024 Accepted

November

19, 1975

Primary cultures derived from mechanically dissociated definitive streak chick blastoderms were grown in a warm air stream on the stage of inverted phase microscope, through which in vitro erythroid development could be observed. Proerythroid cells divide three or four times in 48 hr to give rise to erythroid colonies ranging from 10 to 1000 cells, depending on the size of the blastoderm fragments from which they were derived. Erythroid cell development follows a similar course in cultures grown in a carbon dioxide incubator. Colonies consisting of about 50 cells, derived from blastoderm fragments containing 5 to 10 cells, were isolated and labeled with [3H]leucine, and their labeled hemoglobins were analyzed by isoelectric focusing. Both early hemoglobins (E,M,P,P’, and P”) and late hemoglobins (A and D) are made in colonies derived from single blastoderm fragments. The ratio of late to early hemoglobins is about 1.7 in all colonies analyzed. The implications of this finding for the clonal model of erythroid development are discussed. INTRODUCTION

Most vertebrate species exhibit changes in the pattern of hemoglobin synthesis during development. These changes are often accompanied by simultaneous changes in red cell morphology, in the site of red cell production, or both (20). An example of this is the switch from embryonic to adult hemoglobin synthesis in the mouse embryo (5, 9). On the other hand, the switch from fetal to adult hemoglobin in human infants occurs within the same cell population, with individual cells containing varying proportions of the two hemoglobins (1, 12). In metamorphosing IThis research was supported by United States Public Health Service Research Grant No. AM15885. “Support by a United States Public Health Service Training Grant in Developmental Biology NO. 5-Ol-HD00415-02 to Harvard University and by United States Public Health Service Postdoctoral Fellowship No. l-F03-HD54898-01. Present address: Department of Biology, Tufts University, Medford, Massachusetts. 3From whom reprints may be requested. Present address: Department of Biology, University of California, Los Angeles, California 90024.

Rana catesbiana, tadpole and frog hemoglobins are contained in distinct cell populations, but in Xenopus laevis, both types of hemoglobin are detected in single cells (10, 11). In the early chick embryo, the change in the pattern of hemoglobin synthesis is associated with the appearance of a new cell population (2, 3). Ingram has proposed a clonal model of erythroid development, in which early precursor cells are capable only of producing erythrocytes containing early hemoglobins, while the late hemoglobins are made by clones of cells arising from a later set of precursors (9). This model, however, does not easily account for the finding of two sets of hemoglobins within the same cell. As Maniatis and Ingram have pointed out, the model disturbingly suggests a fundamental difference in the ontogeny of erythroid development in Rana and Xenopus (11). We here suggest an alternative model, in which the restriction of synthesis to one or another set of hemoglobins occurs later than, and independently of, the restriction of pluripotent mesenchymal cells to erythroid differentiation. In this 556

Copyright All rights

0 1976 by Academic press, Inc. of reproduction in any form reserved.

BRIEF NOTES

model, determined erythroid cells of the lday chick embryo remain dipotent with respect to the pattern of hemoglobin synthesis of their descendants. Hagopian, Lippke, and Ingram have devised a culture system of erythroid precursors from early chick embryos (8). In their cultures early and late hemoglobins are produced simultaneously, during a time which, in the intact embryo or in organ cultures of deembryonated blastoderms, only the early hemoglobins would have appeared (2, 3, 7). If clones could be obtained from single erythroid precursors, the hemoglobin synthesized by each clone would indicate the degree of restriction of the precursor. Tobin and Ingram (unpublished results), however, found that when blastoderms were dissociated into single cells, only very few cells subsequently made hemoglobin. We report here the successful growth of erythroid colonies from small fragments of early blastoderms and the pattern of their hemoglobin synthesis. MATERIALS

AND

METHODS

557

essential amino acid mixture, 1.0 j&i [:lH]leucine/bl (20-30 CUmmole), and the other additives listed above. Labeling medium was conditioned by growing in it cells from 24-hr blastoderms for 48 hr and then removing all cells by centrifugation. Collection of cells and preparation of hemoglobin. Microwells containing healthy-looking cells after 24 hr of labeling (usually only about 10% of the original wells) were collected, combined with 10 ~1 of a washed cell suspension from g-day-old chick embryo blood, washed, and lysed with 3 to 5 vol of TKM (0.01 M Tris, 0.03 M KCl, 0.002 M MgCl,, pH 7.5). The lysate was extracted with CCl,:Toluene:2:1; the red aqueous layer was removed after centrifugation and stored in liquid nitrogen (18). Analysis of hemoglobins. Isoelectric focusing was performed in 5% polyacrylamide gels in pH 7-9 Ampholine (LKB) as described by Tobin et al. (18). Electrophoresis was performed as described by Bruns and Ingram (3). Analyses of chicken hemoglobins by electrophoresis and by isoelectric focusing are shown in Fig. 1. Microscope stage cultures. A cell suspension was prepared as described and equilibrated with 5% CO,. The culture dish was then sealed with vasoline and placed on the stage of a Wild inverted phase contrast microscope and maintained at 37°C by an air stream stage incubator (Nicholson Precision Instruments). The culture dish was prepared by scoring a fine grid on the inner surface with a sterile wire screen. Selected areas of the surface could be located by their position on the grid and photographed repeatedly during development of the cells.

Culture conditions. Our culture system was adapted from the method of Hagopian et al. (8), but using the following growth medium (GM): Dulbecco’s modified Eagle medium supplemented with penicillin, fungizone, and streptomycin, 10% dialyzed fetal calf serum (GIBCO), 1% rooster serum, 0.002 kg/ml CuSO,, 0.9 pg/ml ZnCl,, and 0.8 Fg/ml FeSO,. Embryos and blastoderms were dissociated (3 ml GM/blastoderm) into small fragments by sucking in and out of a Pasteur pipet several times. [3H]Leucine-labeling of colonies. After 48 hr of culture, cohesive colonies of about 50 immature erythroid cells could be removed from the culture under the dissecting microscope, using a specially preparea RESULTS micropipet. Each colony was placed in a 10 ~1 well of a Falcon multitest plate in 10 ~1 Erythroid Differentiation in Culture of labeling medium. This medium conOur standing cultures of mechanically sisted of 20% Dulbecco’s modified Eagle disrupted definitive streak embryos show medium, 80% minimum essential medium characteristics similar to those described without leucine, supplemented with non- by Hagopian et al. (8) and by Chan and

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H ‘A ‘0

FIG. 1. Analysis of hemoglobins prepared from chick embryos of various ing; (n-6) pH 8.9 stacking gels. (a), (f) 4.8-day embryo; (b), (g) b-day embryo; 17-day embryo; (e), cj) adult rooster.

Ingram (41, when examined in slides of unattached cells. During the first 24 h of culture, there are few cells of any kind in the liquid medium. By 48 hr, there are large, cohesive groups of immature erythroid cells which contain hemoglobin, as judged by benzidine-peroxide staining. As many as 4% of these cells are in mitosis. At 72 hr, up to 90% of the unattached cells stained with benzidine; most are free, single cells. After 3 days of culture, the number of cells decreases. When older embryos (up to four somites) are used as a source of cells, the progression of the culture is similar, with development corresponding to the total incubation time, rather than the time in culture. We had originally assumed that the masses of cells seen on slides from 48-hr cultures were either artifacts of our slide preparation method, or were the result of the aggregation of erythroid precursors in blood islands (13). By observing cultures grown on the stage of an inverted microscope, however, we determined that the large masses originate from smaller, tightly packed cell clumps resulting from the initial disaggregation (Fig. 2A). Although these early fragments cling to the dish surface and move small distances during the first few hours, they do not form new agglomerations. Rather, it appears that the 48-hr mass (Fig. 2B) must be

ages, (a)-(e) Isoelectric (c), (h) E-day embryo;

focus(d), (i)

formed by growth of the original fragment. The number of early divisions is difficult to determine through phase-contrast observation, because the cells are so tightly packed. From 24 hr on, however, the number of cells in a colony can be estimated reasonably. During this period, the number of cells in each colony doubles in approximately 18 hr (note that the cells in Fig. 2C are more widely spaced than in Figs. 2A and B). As the cells accumulate hemoglobin, their refractive index increases and they appear darker under phase contrast. A few of these more mature cells can be seen, free from the main cell mass, at 48 hr (Fig. 2B). At 72 hr the majority of cells are free and many appear to have sufficient hemoglobin to appear dark with phase contrast optics (Fig. 20. These observations are generally consistent with We&s description of the development of chick embryo erythroid cells in plasma clot culture (19). Erythroid colonies are recognizable under phase contrast before hemoglobin synthesis can be detected. After 48 hr of culture they are clumps of cells that are not attached to the substrate (Figs. 2B and 3). Even after 1 hr of culture, we were able to identify the larger proerythroid fragments. They are more adherent to each other than to the culture dish and appear as bumps above the surface of the dish

FIG. 2. Development of erythroid colony in microscope stage culture. (A) Precursor fragment 0.5 hr after dissociation. Yolk particles and cell debris typical of first few hours of culture are abundant. (B) Colony at right is the same mass after 29 hr of culture. Individual cells are discernible. At this stage, some hemoglobin may be detectable by benzidine staining. Colony contains about 600 cells. (C) Forty-eight hr of culture. Cells at periphery have begun to break loose as they mature. Center-of-mass is still cohesive, tightly packed. Nonerythroid cells at right are the result of spreading of a nearby tissue fragment. The mark at the bottom of each micrograph is part of the grid scored on the culture dish surface for the purpose of locating the same positions repeatedly during the culture period. The colony in C contains about 2500 cells; those used in hemoglobin-labeling experiments were composed of only 20-80 cells,

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predominating. The ratio of late to early hemoglobins is about 1.7 (Table 1). This is true whether the embryo was definitive streak or as old as the four-somite stage when explanted. DISCUSSION

FIG. colony,

3. Colony of 35 cells. Typical isolated in a IO-JJ microwell.

cohesive,

48-hr

(Fig. 2A). In one culture grown on the microscope stage, we identified 30 cell groups as proerythroid fragments: nine of these actually differentiated into mature erythroid cells. Of the colonies that we identified at 48 hr of culture, more than 90% produced mature erythroid cells. In these erythroid colonies, all the cells contained hemoglobin by 72 hr of culture. The fragment from which they were derived therefore must have been composed entirely of erythroid precursors. Hemoglobin

Synthesis

Although the mature descendants of the precursor fragment break loose from each other and mix with other cells in the culture if disturbed, the cohesive cell mass of the 48-hr culture can be transferred to a lo-p1 spot culture containing [3H]leucine. Twenty-four hours later, the descendents of these cells can be harvested, and their 3H-labeled hemoglobins analyzed by isoelectric-focusing in polyacrylamide gels. Although there is a wide range of colony sizes at 48 hr, those selected for transfer were composed of from 20 to 80 cells. A typical colony, consisting of about 35 cells, is shown in Fig. 3. Twelve of these colonies were analyzed in this manner, and all were found to have the same distributions of label among the hemoglobins (Fig. 4). All hemoglobins present in the g-day embryo are synthesized, with the adult types

The blastoderm fragments that undergo erythroid development in cultures are probably pieces of the blood island precursors which have been described in embryos of this age (16). These cells are already committed to produce red blood cells, and

FRACTION

FIG. 4. Electrofocusing of products of a single colony. Solid line, A,,,, of carrier 8-day hemoglobins; broken line,radioactivity of [3H]Leu-labeled products from single colony. TABLE SYNTHESIS

Colony

1 2 3 4 5 6 7 8 9 10 11

12

1

OF HEMOGLOBINS COLONIES

Late

BY INDIVIDUAL

Early

8 of cw in A

% of cpm in D

% of cpm in E

34 35 30 22 34 42 38 33 38 27 36 27

25 26 29 41 27 25 23 33 28 40 31 40 Mean

19 20 25 10 19 17 21 15 16 8 13 10 2 SD

Late/early % of wm in P 21 17 16 26 20 16 18 18

1.48 1.65 1.44 1.75 1.56 2.03 1.56 2.00

19

1.89

24 20 23

2.09 2.03 2.03 1.79 ~fr 0.25

BRIEF

their direction is not altered by our culture conditions. Hagopian and Ingram found that in organ cultures the yolk sac blood islands produce both sets of hemoglobins with the same time sequence as that found in ouo (7). Our cultures contain all the cells from the embryo, so that the endodermal induction demonstrated by Miura and Wilt is probably not interrupted (14). Hagopian et al. (8) and Tobin and Ingram (unpublished) found that chick erythroblasts undergo three to six cell divisions in the first 48 hr of culture. Insofar as we can determine, the cells of individual colonies divide at the same rate as those in the bulk of the culture. Indeed, most of the erythroid cells in this culture system are found in colonies for the first 48 hr. Since the colonies we selected for subculture after 48 hr consisted of 20 to 80 cells, they must have been derived from blastoderm fragments containing no more than 3 to 10 erythroblasts. The pattern of hemoglobin synthesis of the subcultured cells appears to have been altered by the physical disruption of the embryo. There are two kinds of explanations for the appearance of all hemoglobin types in each colony. A “clonal” model would postulate that each clump is composed of a mixture of both types of precursors, so that the resulting colony contains a mixture of all hemoglobins. If fragmentation of the blastoderm is at random, however, the probability of obtaining the same ratios of early to late hemoglobins in twelve separate colonies originating from 3-10 precursor cells would be extremely low (p G lo-41.4 4Since the average fraction of late hemoglobins in each colony is 0.64, the most probable fraction of “late” erythroid precursors is 0.64. If a colony is derived from 10 cells, and if the descendants of each precursor type make the same amount of hemoglobin, then each of the colonies we examined must have been derived from six or seven “late” precursors and four or three “early” precursors. Our measurements would certainly have distinguished colonies making only 50% or as much as 80% late hemoglobins. Assuming a random spatial distribution of

561

NOTES

Alternatively, it may be that in the environment of the cell culture each erythroid cell responds to the same combination of stimuli by producing the same spectrum of hemoglobins. Although we do not know whether each cell in a colony of 20-80 cells makes all hemoglobins, our data suggest that each precursor in a fragment of 3 to 10 cells is dipotent with respect to the pattern of hemoglobin synthesis. We propose the following model to account for our observations: Mesenchymal cells are first restricted to erythroid differentiation and are only later restricted to a single pattern of hemoglobin synthesis. At the time of explantation, cells from the 24hr embryo are identifiable as erythroid precursors, but they are apparently still flexible with respect to the pattern of hemoglobin synthesis. These determined erythroid cells are then receptive to two signals, one stimulating (or permitting) the synthesis of early hemoglobins and the other stimulating the synthesis of late hemoglobins. The conditions of culture stimulate (or permit) both sets of hemoglobins to be made, but the embryonic environment before 6 days of incubation stimu“late” and “early” precursors and that 64% of the erythroid precursors are “late” precursors, we can calculate the probability that a fragment of 10 cells will consist of six “late” and four “early” precursors: p(6)

=-

lo! 4! 6!

(0.64)6

(0.36).'

= 0.242.

(1)

Similarly, the probability that such a fragment will consist of seven “late” and three “early” precursors is 0.246. Therefore, the probability that a given colony will have a hemoglobin distribution within the limits found in Table 1 (after tailoring all assumptions for maximum consistency with the clonal model) is 0.488. The probability that all 12 colonies will be within these limits, however, is (0.488)” = 1.8 x 10m4. If colonies are derived from blastoderm fragments consisting of less than 10 cells, the probability of such a clonal explanation is correspondingly reduced. Our data can be explained by a clonal model only if there is close physical association of “late” and “early” precursors and if our culture and subculture conditions allowed the growth only of those colonies containing precursors of both types.

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lates (or permits) the synthesis early hemoglobins.

BIOLOGY

of only the

AJT began this work as a postodoctoral fellow in the laboratory of Dr. Vernon M. Ingram, where he was supported by a United States Public Health Service Postdoctoral Fellowship and a United States Public Health Service Research Grant to VMI. We are grateful to Drs. Vernon Ingram, Michael Brenner, Raymond Kaempfer, and John Fessler, and Ms. Hildur Colot for many helpful discussions. REFERENCES 1. BETKE, VON K., and KLEIHAUER, E. (1958). Blut 4, 241-249. 2. BRUNS, G. A. P., and INGRAM, V. M. (1973). Develop. Biol. 30, 455-459. 3. BRUNS, G. A. P and INGRAM, V. M. (1973). Philos. Trans. Royal Sot. (London) 266, 22% 305. 4. CHAN, L., and INGRAM, V. M. (1973). J. Cell Biol. 56, 861-865. 5. FANTONI, A., BANK, A., and MARKS, P. (1967). Science 157, 1327-1329. 6. GARRICK, M. D., MANNING, R. F., REICHLIN, M., and MATTIOLI, M. (1973). In “Biochemistry of Gene Expression in Higher Organisms,” (J. Pollak and J. W. Lee, eds.), Reidel, Dordrecht.

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7. HAGOPIAN, H. K., and INGRAM, V. M. (1971) J. Cell Biol. 51, 440-451. 8. HAGOPIAN, H. K., LIPPKE, J. A., and INGRAM, V. M. (1972) J. Cell Biol. 54, 98-106. 9. INGRAM, V. M. (1972). Nature (London) 235,338339. 10. JURD, R. D., and MACLEAN, N. (1970). J. Embryol. Exp. Morph. 23, 299-309. 11. MANIATIS, G. M., and Ingram, V. M. (1971) J. Cell Biol. 49, 390-404. 12. MATIOLI, G., and THORELL, B. (1963). Blood 21, 13. MIURA, Y., and WILT, F. H. (1969). Develop. Biol. 19, 201-211. 14. MIURA, Y., and WILT, F. H. (1970). Exp. Cell Res. 59, 217-226. 15. NIENHUIS, A. W., and BUNN, H. F. (19741. Science 185, 946-948. 16. ROSENBERG, M. (1970). Proc. Nat. Acad. Sci. USA 67, 32-36. 17. SABIN, F. R. (1920). Contributions to Embryology 9, 213. 18. TOBIN, A. J., COLOT, H. V., KAO, J., PINE, K. S., PORTNOFF, S. R., ZAGRIS, N., and ZARIN, N. (in press). In Eukaryotes at the Subcellular Level: Development and Differentiation (J. Last, ed.), Dekker, New York. 19. WENK, M. L. (1971). Anat. Rec. 169, 453. 20. WILT, F. H. (1967). Adv. Morphogenesis 6, 89125.