gels were fixed in 7 % (v/v) acetic acid for 5 min, then stained with benzidine- ... solution [0-2% (w/v) benzidine in 0-5 M acetic acid] was added immediately.
/ . Embryol. exp. Morph. Vol. 58, pp. 209-216,1980 Printed in Great Britain © Company of Biologists Limited 1980
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Erythroid cell differentiation in unincubated chick blastoderm in culture By NIKOLAS ZAGRIS 1 From the Tissue Culture Laboratory, University of Patras, Greece
SUMMARY Morphologically distinct erythroid cell types characteristic of the primitive and the definitive erythroid cell lines, and embryonic and adult haemoglobins are produced when the unincubated chick blastoderm is cultured ventral side down on afilterraft to inhibit morphogenetic movements and subsequent primitive-streak formation mechanically in serum-free minimal essential medium. The primitive and definitive erythroid cell populations appear consecutively in culture even though there is no axis formation nor apparent morphogenesis. The information to produce both the early and late haemoglobins and erythroid cell types is independent of axis formation and of specific extra-embryonic influences, such as progressive induction exerted by the yolk mass. INTRODUCTION
Erythropoiesis in the chick is far from simple and the sequential appearance in early chick embryos of morphologically distinct erythrocytes and their haemoglobins (Hb) has been thoroughly described (Sabin, 1920; Fraser, 1961; Lucas & Jamroz. 1961; Wilt, 1967; Schalekamp, Schalekamp, Van Goor & Slingerland, 1972; Bruns & Ingram, 1973; Brown & Ingram, 1974; Wainwright, & Wainwright, 1974; Cirotto, Scotto Di Telia & Geraci, 1975; Zagris & Melton, 1978; Chapman & Tobin, 1979). The presumptive erythropoietic area of primitive streak and older chick embryos has been maintained successfully in tissue culture (Murray, 1932; Settle, 1954; O'Brien, 1960; Spratt & Haas, 1960; Hell, 1964; Levere & Granick, 1967; Wilt, 1967; Hagopian & Ingram, 1971; Wenk, 1971) and in suspension culture (Hagopian, Lippke & Ingram, 1972; Chan & Ingram, 1973; Pine & Tobin, 1976). In all cases potential erythropoietic cells continue to differentiate into mature erythrocytes during the culture period. Whereas previous work on erythroid blood cells and Hb biosynthesis in chick embryos concentrated generally on embryos from the primitive streak stage and older, we showed recently (Zagris, 1979) that unincubated blastoderm can be maintained in a simple, serum-free chemically-defined medium in culture for a number of days during which it forms haemoglobinized blood islands which 1 Author's address: Tissue Culture Laboratory, School of Sciences, University of Patras, Patras, Greece.
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anastomose to form a network. Even though there is no primitive streak formation and the unincubated blastoderm is deprived of the usual flow of materials from the yolk, it forms Hb and this is inhibited when 5-bromodeoxyuridine is applied during the first two days of culture (Zagris, 1979). It seemed of interest to see whether the unincubated blastoderm, cultured ventral side down on filter rafts, to inhibit morphogenetic movements and subsequent primitive-streak formation mechanically, in a protein-free medium, contained also instructions to produce the early and late blood cells and Hbs without participation of the embryo proper, and deprived of possible extra-embryonic influences exerted by the yolk mass. MATERIALS AND METHODS
Culture Freshly-laid fertilized eggs of White Leghorn chickens were used on the day that they were obtained from a local hatchery. The explanted unincubated blastoderms were checked individually under a dissecting microscope and staged accurately (Eyal-Giladi & Kochav, 1976) so that only blastoderms at stage X were used for experiments. Blastoderms, washed free of the vitelline membrane and any adhering yolk, were attached on filter rafts, and were cultured in Petri dishes with plain Dulbecco's modified Eagle Medium (MEM) in the absence of serum as described elsewhere (Zagris, 1979). In experiments involving labelling of haemoglobins, [U-14C]protein hydrolysate (56 mCi/mAtom, Amersham) mixture was added to the culture medium to a final concentration of 6-25 /tCi/ml. Blastoderms, media and glassware were handled with sterile precautions. Collection of blood cells and preparation of Hb Erythroid cells were harvested from blastoderms under a dissecting microscope using a specially prepared fine-tipped Pasteur micropipette. By exercising gentle pressure in one direction with a fine-tipped Pasteur pipette, the anastomosed blood islands were directed to form a single large blood pool. The transparent tissue surrounding the blood pool (Zagris, 1979) was punctured gently and blood cell clusters started streaming into the micropipette opening. This method gave very good yields of red cells in that the area that the blood pool occupied originally appeared colourless after harvesting. Blood cells from five blastoderms were suspended and washed three times in a tenfold volume of ice-cold chick Ringer's solution. The packed cells, combined with 20 /A carrier Hb from 8-day-old chick embryo blood cells (packed cells: H 2 O, 1:2), were lysed in 30-50 /
Erythroid cell differentiation in unincubated chick blastoderm 211 24 h after preparation, and after conversion of Hbs to the cyanomethemoglobin form with ferricyanide. To study cell morphology, collected blood cells were placed in one drop of chick Ringer's solution on a slide. After drying, cells were stained with Harris' haematoxylin overnight. Analysis of haemoglobin Electrophoresis was performed in 10% polyacrylamide gels according to Barker (1968) as modified by Zagris & Melton (1978). At the end of the run, the gels were fixed in 7 % (v/v) acetic acid for 5 min, then stained with benzidineperoxide solution for 1 h. Hydrogen peroxide (30 %) at 0-2 % (v/v) in benzidine solution [0-2% (w/v) benzidine in 0-5 M acetic acid] was added immediately before use. Colour developed within the first few minutes after application of the staining solution, and gels were scanned at 510 nm in a Gilford 240 spectrophotometer. After scanning, gels were cut in 1-5 cm slices which were digested overnight in 0-2 ml of 30 % H 2 O 2 each in scintillation vials at 37 °C. The sample in each vial was counted for radioactivity in a toluenefluor [0-5 % PPO, 0-03 % POPOP in toluene (w/v)]-Triton X-100 (4:1, v/v) scintillation cocktail.
RESULTS
Unincubated blastoderms produce blood islands which appear after 2-5-3-0 days in culture as dense bright red clusters visible to the naked eye, and on subsequent days as a tissue which is heavily haemoglobinized. The first erythroblasts appear in similar structures in ovo, but with the blood island networks being much more reticulate. After pricking the haemoglobinized tissue with a glass microneedle, the blood cells stream in groups (clusters) indicating that the blood island is constructed by tight grouping of erythroid cells. This observation is in accord with the view of Miura & Wilt (1970) on the blood island cytoarchitecture. Microscopic examination of erythroblasts showed morphological differences in the erythroid cells of the blastoderms. There seem to be at least four distinct erythroid cell populations appearing during the 10-0-day culture. On day 3-8 of culture the erythroid cell population that appears to develop from precursor cells as a cohort is uniform, roughly circular in shape, and granulate in appearance, with most cells in mitosis. These cells, which, according to the terminology of Lucas & Jamroz (1961), correspond to early primary ('primitive') erythrocytes, constitute the only cell type (Fig. 1 A). On day 6-0 of culture there are three erythroid cell types present. The cells described previously as early primary erythrocytes seem to be at a resting phase with only an occasional cell in mitosis. These cells display delicate extensions of cytoplasm as if to facilitate their anchoring within the blood island, or to begin a
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Fig. 1. Blood cells collected from unincubated blastoderms which were cultured for (A) 3-8, (B) 60, (C) 80 and (D) 100 days in serum-free MEM. Cells stained with Harris' haematoxylin. Scale bar = 10 /tm. ep, Early primary erythrocyte; Id, late polychromatic definitive erythrocyte; md, mid-polychromatic definitive erythrocyte; mp, mid-polychromatic primary erythrocyte; pc, pycnotic cell.
degenerative pycnotic process. The most numerous population consists of small, pycnotic cells with fragmented nuclei. These could be early primary erythrocytes in the process of pycnosis and degeneration. The larger cells with the irregular shape and sharper nuclear contour could be mid-polychromatic primary erythrocytes. An occasional semi-elliptical cell with a well-defined nucleus is also observed (Fig. I B ) . . On day 8-0 of culture, blood consists primarily of cells semi-elliptical in form with a well-defined roughly oval nucleus. These cells resemble morphologically the mid-polychromatic definitive erythrocytes characteristic of 6-5- to 8-5-day in ovo developing chick embryos. A few pycnotic cells are still present. An occasional erythroid cell, ellipsoid in general and nuclear shape, is evident. There is absence of cell divisions in the blood cell population (Fig. 1C). By day 10 of culture, blood consists primarily of mid-polychromatic definitive erythrocytes, most of which seem to be in the process of degeneration. The rest of the blood population consists of erythrocytes, elliptical in general and nuclear shape, with a low nuclear-to-cytoplasmic ratio. These cells resemble morphologically late polychromatic definitive erythrocytes characteristic of older than 9-0-day embryos in ovo, and of the adult chicken (Fig. 1D).
Erythroid cell differentiation in unincubated chick blastoderm 213 100 80 60 40
1
20
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60 80 Time in culture (days)
100
Fig. 2. Alterations in the blood cell population of unincubated blastoderm during 100-day culture in terms of percentage of the cells morphologically classified as early primary erythrocytes (| 1), pycnotic cells (H^>mid-polychromatic primary erythrocytes ([JTflD, mid-polychromatic definitive erythrocytes (1—|) and late-polychromatic definitive erythrocytes ( ^ | ) . The time scale refers to the day blood cells were collected.
Slides with the fixed blood cells were scanned microscopically, and percentages of the various cell populations were calculated from about 500 cells per culture point. The results were reproducible in three separate experiments. Figure 2 shows histograms of the frequencies of erythroid cell populations at 3-8, 6-0, 8-0 and 10-0 days of unincubated blastoderm in culture. The entire erythroid cell population consists of early primary erythrocytes on day 3-8 of culture. On day 6-0 the proportion of early primary erythrocytes has dropped to 20% with the pycnotic cells being the predominant cell type, while the midpolychromatic primitive erythrocytes comprise 22 % of the total erythroid cell population. On day 8-0 of culture the proportion of mid-polychromatic definitive erythrocytes has risen to about 97 % of the entire cell population with 2 % mid-polychromatic primitive erythrocytes and 1 % pycnotic cells present. By day 10-0 of culture the cells defined as mid-polychromatic definitive erythrocytes constitute about 65% of the total blood cell population, while the late polychromatic definitive erythrocytes comprise the rest of the erythroid cell population. Electrophoresis on polyacrylamide gels of the Hbs produced by the unincubated chick blastoderm after 9 days of culture shows the presence of all four Hbs (Fig. 3). The electrophoretic profile of Hb was reproducible in two separate experiments (one electrophoretic run per experiment) performed. In keeping with the nomenclature advocated by Bruns & Ingram (1973), these are Hbs E, A, P, D, the embryonic type being E and P, with A and D being the adult Hbs. According to Hagopian & Ingram (1973), the peak of radioactivity which is present between Hbs P and D is a breakdown product from Hb A as a result of overnight storage in the refrigerator.
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-0-4
-0-3
-0-2
-01
00
10
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Fig. 3. Polyacrylamide gel electrophoretic profile of Hbs extracted from unincubated blastoderms cultured in serum-free MEM for 90 days. On the last 2 days of culture, the medium contained in addition 6-25 /*Ci/ml [U-14C]protein hydrolysate (56 mCi/ mAtom). Haemoglobin preparation for electrophoresis and counting of radioactivity as described in Materials and Methods. Direction of migration from left to right. , Absorbance at 510 nm; # — # , radioactivity of [14C]amino acid-labelled products. DISCUSSION
The results show that unincubated chick blastoderm which, cultured in a serum-free medium, supports differentiation of precursor cells to the erythroid cell line (Zagris, 1979), contained also the information to produce the early and late Hbs and erythroid cell types. This shows that the formation of morphologically distinct cell populations and Hbs is independent of specific extra-embryonic influences, such as progressive induction exerted by the yolk mass. The numerous cells in mitosis give evidence that blood cells are the haemopoietic tissue around day 3-5 of culture. Later in culture, there is an occasional blood cell observed in mitosis, and by day 8-0 of culture there is a conspicuous absence of mitotic figures. The small pyenotic cells which comprise the largest cell population on day 6-0 of culture seem to be early primary erythrocytes in the process of degeneration, fragments of degenerated cells, and/or these could be the innermost blood island cells which according to Sabin (1920) disintegrate to form plasma. In a thorough study of chick haemopoiesis in ovo, Lucas & Jamroz (1961) consider similar elements to be embryonic thrombocytes that clump accompanied by degeneration as shown by loss of cytoplasm and by pycnosis of the nucleus.
Erythroid cell differentiation in unincubated chick blastoderm 215 The electrophoretic pattern of Hb from the blood of unincubated blastoderm after 9 days in culture is comparable to that of the 8-day chick embryo in ovo (Bruns & Ingram, 1973; Brown & Ingram, 1974). The electrophoretic profile of Hb is also comparable to that observed with primitive streak and older blastoderms in tissue culture (Hagopian & Ingram, 1971), and in suspension culture (Hagopian et al. 1972; Chan & Ingram, 1973; Pine & Tobin, 1976) in a serumenriched defined medium. Perhaps the most important aspect of our results is that, even though there is neither axis formation nor apparent morphogenesis (Zargis, 1979), the morphologically distinct erythroid cells of the primitive and the definitive series appear consecutively in culture as is the case with in ovo development. Because blastoderms are cultured ventral side down on filter rafts, morphogenetic cell movements are inhibited mechanically, and the primitive streak is not formed. This shows that the interacting components for the erythroid cell formation in this system need not invaginate through a primitive streak. It is possible that the endodermal component is the primary hypoblast which is formed from the epiblast by a process of polyinvagination (Vakaet, 1962), while the mesodermal component is the dispersed cells which are found between the epiblast and the hypoblast even in the absence of a primitive streak (Azar & Eyal-Giladi, 1979). The appearance of morphologically distinct erythrocytes consecutively in culture shows that the embryo proper is not essential for the formation of the early and late erythroid eelte. However, k is possible that some of the erythroid cell populations, such as those of the definitive series, originate inside the embryo in normal development. I wish to thank Professor Hefzibah Eyal-Giladi (Hebrew Univeristy of Jerusalem, Israel) for the critical reading of the manuscript. REFERENCES Y. & EYAL-GILADI, H. (1979). Marginal zone cells-the primitive streak-inducing component of the primary hypoblast in the chick. /. Embryol. exp. Morph. 52, 79-88. BARKER, J. (1968). Development of the mouse haematopoietic system. I. Types of hemoglobin produced in embryonic yolk sac and liver. Devi Biol. 18, 14-29. BROWN, J. L. & INGRAM, V. M. (1974). Structural studies on chick embryonic hemoglobins. /. biol. Chem. 249, 3960-3972. BRUNS, G. A. P. & INGRAM, V. M. (1973). The erythroid cells and haemoglobins of the chick embryo. Proc. Trans. R. Soc. B 265, 225-305. CHAN, L. L. & INGRAM, V. M. (1973). Culture of erythroid cells from chick blastoderms. /. Cell Biol. 56, 861-865. CHAPMAN, B. S. & TOBIN, A. J. (1979). Distribution of developmentally regulated hemoglobins in embryonic erythroid populations. Devi Biol. 69, 375-387. CIROTTO, C., SCOTTO, Di TELLA A. & GERACI, jG. (1975). The hemoglobins of the developing chicken embryos. Fractionation and globin composition of the individual component of total erythrocytes and of a single erythrocyte type. Cell Differ. 4, 87-99. EYAL-GILADI, H. & KOCHAV, S. (1976). From cleavage to primitive streak formation: a complementary normal table and a new look at the first stages of the development of the chick. I. General morphology. Devi Biol. 49, 321-337. AZAR,
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ation of the presence of multiple haemoglobins during the ontogenesis of the chicken: electrophoretic and chromatographic characterization, polypeptide composition and immunochemical properties. /. Embryol. exp. Morph. 28, 681-713. SETTLE, G. W. (1954). Localization of the erythrocyte-forming areas in the early chick blastoderm cultivated in vitro. Contr. Embryol. 241, 223-237. SPRATT, N. T. & HAAS, H. (1960). Morphogenetic movements in the lower surface of the unincubated and early chick blastoderm. / . exp. Zool. 144, 139-157. TOBIN, A. J., COLOT, H. V., KAO, J., PINE, S. K., PORTNOFF, S., ZAGRIS, N. & ZARIN,N. (1976). Analysis of erythroid development. In Eukaryotes at the Subcellular Level: Development and Differentiation (ed. J. Last), pp. 211-255. New York, Basel: Marcel Dekker. VAKAET, L. (1962). Some new data concerning the formation of the definitive endoblast in the chick embryo. / . Embryol. exp. Morph. 10, 38-57. WAINWRIGHT, S. D. & WAINWRIGHT, L. K. (1974). Isolation of two erythrocyte cell populations from the early chick blastodisc and the further resolution of one into two essential sub-populations. Expl Cell Res. 88, 143-152. WENK, M. (1971). Effect of BUdR on initiation of hemoglobin synthesis by erythroid precursor cell. Anat. Rec. 169, 453. WILT, F. H. (1967). The control of embryonic hemoglobin synthesis. In Advances in Morphogenesis (ed. M. Abercrombie & J. Brachet), pp. 89-125. New York, London: Academic Press. ZAGRIS, N. & MELTON, C. G. (1978). Hemoglobins in single chick erythrocytes as determined by a differential elution procedure Z. Naturf. 33 c, 330-336. ZAGRIS, N. (1979). Differentiation capacity of unincubated chick blastoderm in culture. /. Embryol. exp. Morph. 50, 47-55.
{Received 26 November 1979, revised 16 January 1980)
