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Two different populations of primitive erythroid cells in the chick embryo
DEVELOPMENTAL
BIOLOGY
Two Different
61, 384-387 (1977)
Populations
of Primitive Embryo
C. CIROTTO,* F. PANARA,” * Zstituto di Zstologia ed Embriologia, Embryology, CNR,
Erythroid
AND
G.
Cells in the Chick
GERACI~
Universitci di Perugia, Perugia, and t Laboratory Via Toiano, 2-80072 Arco Felice, Naples, Italy
Received July 5,1977;
of Molecular
accepted July 19,1977
Antibodies prepared against the two hemoglobins of the adult chick cross react with the two minor hemoglobins and do not react with the two major hemoglobins isolated from lysates of primitive erythroid cells of the I-day-old embryo. The different immunological reactivities of the two primitive hemoglobin pairs have permitted us to discriminate, in smears of primitive erythroid cells, two populations on the basis of their hemoglobin contents. INTRODUCTION
We have demonstrated that four hemoglobins are contained in the primitive erythroid cells of the chick embryo (Cirotto et al., 1975). The four hemoglobins, isolated by column chromatography, were called El, E2, E3, and E4, according to their order of elution from the CM-cellulose column. Each hemoglobin was discriminated from the others by gel electrophoresis of the component globins. These results confirmed those of Schalekamp et al. (1972) who detected, by column chromatography, four hemoglobins in the primitive erythroid cells. We have already shown the presence of four hemoglobins in primitive erythroid cells in a study of the oxygen equilibrium properties of the two major components isolated in pure form from 5day-old embryos (Cirotto and Geraci, 1975). We have also shown that erythrocytes of the definitive type, found in the blood circulation at 7 days of development, can be isolated on a Ficoll gradient and contain exclusively the two adult-type hemoglobins (Cirotto et al., 1975). These were called Al and A2 according to their order of elution from the CM-cellulose column, as suggested by Matsuda and Takei (1963) for adult chick hemoglobins. Becently, this result has been confirmed by
analyzing definitive erythrocytes isolated from &day-old embryos on a BSA gradient (Mahoney et al., 1977). We have suggested the possibility that the two major and two minor primitive hemoglobins, like the adult ones, are contained in two different primitive erythroid cell types (Cirotto et al., 1975). This suggestion has been advanced because the time courses of the component hemoglobins, during embryonic development are distinctly different. In addition, only four hemoglobins can be observed, that are composed of four different a-like chain types and at least of two p-like chain types. Our argument was that if all those chains were synthesized in the same cell, one would expect eight different hemoglobins deriving from their random assortment. Attempts at discriminating the primitive erythroid cells on the basis of their morphology have been unsuccessful. Thus, it was decided to try and discriminate them on the basis of their hemoglobin contents. We describe here the results of such studies based on immunofluorescence labeling of specific hemoglobin types. MATERIALS
AND
METHODS
Four-day-old White Leghorn chick embryos were obtained from a local poultry farm. The erythrocytes were isolated from
384 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0012-1606
BRIEF NOTES
385
the embryos and, the four primitive embryonic hemoglobins were prepared as previously described (Cirotto et al., 1975). Preparation of antibodies. The adult chick hemoglobins were isolated on a CMcellulose column as previously described (Cirotto and Geraci, 1974), mixed stoichiometrically, and used for antibody formation in rabbits as described by Kabat (1968). The isolated r-globulins were coupled to fluorescein isothiocyanate (Serval) according to Maniatis and Ingram (1971) and were stored at -20°C in a lyophilized form. Antibody specificity was controlled by using the double-diffusion method in agar. Immunofluorescence labeling of primitive erythrocyte smears. Erythrocyte
smears were prepared and labeled with fluorescent antibodies as reported by Maniatis and Ingram (1971), except that the slides were fixed in methanol for 20 min. A Zeiss fluorescence microscope was used for observations and photography. RESULTS
AND
DISCUSSION
Figure 1 shows the specificity of the antibodies prepared against the two adult chick hemoglobins. It is clear that the antibodies do not discriminate between the two adult hemoglobins Al and A2 and the two minor primitive embryonic hemoglobins E3 and E4. In contrast, these antibodies do not react appreciably with the two major primitive embryonic hemoglobins El and E2. Such a result is not in contrast with those reported by Wilt (1962) who found hemoglobins cross-reacting with antiadult hemoglobin antibodies since the earliest appearance of hemoglobins in the very young embryo. Indeed, by globin chain analysis, we have found and identified E3 and E4 in the embryo from the earliest stages at which detection of hemoglobins was possible (Cirotto et al., 1975). The different antibody specificity of El, E2 and of E3, E4 is not surprising since the two major and two minor embryonic hemo-
FIG. 1. Specificity of antibodies against the two adult hemoglobins, Al and A2, as determined by double diffusion in agar. The y-globulin from the immune serum is placed in the central well. Hemoglobins are placed in the peripheral wells as shown in the figure. The antibodies do not discriminate between Al, A2 and the minor primitive embryonic hemoglobins E3, E4. In contrast, they do not react with the two major primitive embryonic hemoglobins El, E2.
globins have globin chains which are clearly different by gel electrophoresis (Cirotto and Geraci, 1975). On the other hand, the minor primitive embryonic hemoglobin E3 and the adult hemoglobin Al and the minor primitive embryonic hemoglobin E4 and the adult hemoglobin A2 have identical (Ychains and differ by their P-chain types (Brown and Ingram, 1974; Cirotto et al., 1975; Cirotto and Geraci, 1977). The apparently complete absence of antibody cross reaction between the major primitive embryonic hemoglobins and the adult hemoglobins indicates that the differences in these hemoglobins concern more than their electrophoretic mobilities since they appear to have very little homology. This has been of advantage in the
386
DEVELOPMENTAL BIOLOGY
identification of a specific cell type, in the primitive erythroid population, containing preferentially or exclusively, the two primitive minor hemoglobins E3 and E4. As clearly shown in Fig. 2, which represents a typical field, the fluorescent antibody labeling technique demonstrates the existence of a limited number of cells containing the two minor hemoglobins. In fact, 75 erythroid cells were found to be labeled with fluorescence out of a total of 1000 cells counted. This percentage value is lower than the combined amounts of the two minor hemoglobins (Cirotto et al., 1975). This might derive from a different
VOLUME 61, 1977
concentration of the hemoglobins in the different erythrocyte types. E3 and E4 might also be present, but at much lower concentration, in the remaining 92% of the erythrocytes containing the two major primitive hemoglobins. In any case, a special population, rich in E3 and E4, is distinctly and clearly present in the primitive erythroid cells of the embryo. The presence of a special cell type in the primitive erythroid population and the compartmentalization of the definitive hemoglobins Al and A2 in the definitive erythrocyte type (Cirotto et al., 1975; Mahoney et al., 1977) suggest that each cell type might be able to produce specific hemoglobins. A shift from one hemoglobin type to another would then be the result of a shift of cell types probably deriving from different erythropoietic territories, rather than from the reordering of the activity of the individual genes in the same stem cell during embryonic development. This work was supported in part for Carlo Cirotto by Grant No. CT76. 01201.04 of the Consiglio Nazionale delle Ricerche. REFERENCES
FIG. 2. Labeling with fluorescent antibodies anti-Al, A2 of a smear of primitive erythroid cells isolated from 4-day-old embryos. The photograph was overexposed in order to show the background of unreacted cells. Note that the fluorescence appearing in the center of the cells with unreacted cytoplasm is due to nonspecific reaction of the rabbit antiserum with nuclear components of the erythrocytes. This reaction also occurs with the antisera of nonimmunized rabbits. It is important to remember that the erythrocytes are deprived of their membrane by treatment with a detergent in order to expose the molecules contained inside them; consequently, the nuclei are also exposed.
BROWN, J. L., and INGRAM, V. M. (1974). Structural studies on chick embryonic hemoglobins. J. Biol. Chem. 249, 3960-3972. CIROTTO, C., and GERACI, G. (1974). Exposed sulphydry1 groups of chicken haemoglobins: Globin localization and effect of oxygenation on their reactivity. J. Mol. Biol. 84, 103-114. CIROTTO, C., and GERACI, G. (1975). Embryonic chicken hemoglobins. Studies on the oxygen equilibrium of two pure components. Camp. Biochem. Physiol. 51A, 159-163. CIROTTO, C., SC~TTO DI TELLA, A., and GERACI, G. (1975). The hemoglobins of the developing chicken embryos. Fractionation and globin composition of the individual components of total erythrocytes and of a single erythrocyte type. Cell Differ. 4,8799. CIROTTO, C., and GERACI, G. (1977). The hemoglobins of the developing chicken embryo. A system for the study of the switch from fetal to adult hemoglobins. Bull. Mol. Biol. Med. 2, 59-71. KABAT, D. (1968). Organization of hemoglobin synthesis in chicken erythrocytes. J. Biol. Chem. 243, 2597-2606. MAHONEY, K. A., HYER, B. J., and CHEN, L. N. L.
BRIEF NOTES (19’77). Separation of primitive and definitive erythroid cells of the chick embryo. Deuelop. Biol. 56, 412416. MANIATIS, G. M., and INGRAM, V. M. (19711. Erythropoiesis during amphybian metamorphosis. J. Cell Biol. 49, 390-404. MATSUDA, G., and TAKEI, H. (1963). The studies on the structure of chicken hemoglobin. I. Chromatographic purification of chicken hemoglobin. J. Biochem. 54. 156-160.
387
SCHALEKAMP, M., SCHALEKAMP, M., VAN-GOOR, D., and SLINGERLAND, R. (1972). Re-evaluation of the presence of multiple haemoglobin during the ontogenesis of the chicken. Electrophoretic and chromatographic characterization, polypeptide composition and immunochemical properties. J. Embryol. Exp. Morphol. 28, 681-713. WILT, F. H. (1962). The ontogeny of chick embryo hemoglobin. Proc. Nat. Acad. Sci. USA 48, 15821590.
BIOLOGY
Two Different
61, 384-387 (1977)
Populations
of Primitive Embryo
C. CIROTTO,* F. PANARA,” * Zstituto di Zstologia ed Embriologia, Embryology, CNR,
Erythroid
AND
G.
Cells in the Chick
GERACI~
Universitci di Perugia, Perugia, and t Laboratory Via Toiano, 2-80072 Arco Felice, Naples, Italy
Received July 5,1977;
of Molecular
accepted July 19,1977
Antibodies prepared against the two hemoglobins of the adult chick cross react with the two minor hemoglobins and do not react with the two major hemoglobins isolated from lysates of primitive erythroid cells of the I-day-old embryo. The different immunological reactivities of the two primitive hemoglobin pairs have permitted us to discriminate, in smears of primitive erythroid cells, two populations on the basis of their hemoglobin contents. INTRODUCTION
We have demonstrated that four hemoglobins are contained in the primitive erythroid cells of the chick embryo (Cirotto et al., 1975). The four hemoglobins, isolated by column chromatography, were called El, E2, E3, and E4, according to their order of elution from the CM-cellulose column. Each hemoglobin was discriminated from the others by gel electrophoresis of the component globins. These results confirmed those of Schalekamp et al. (1972) who detected, by column chromatography, four hemoglobins in the primitive erythroid cells. We have already shown the presence of four hemoglobins in primitive erythroid cells in a study of the oxygen equilibrium properties of the two major components isolated in pure form from 5day-old embryos (Cirotto and Geraci, 1975). We have also shown that erythrocytes of the definitive type, found in the blood circulation at 7 days of development, can be isolated on a Ficoll gradient and contain exclusively the two adult-type hemoglobins (Cirotto et al., 1975). These were called Al and A2 according to their order of elution from the CM-cellulose column, as suggested by Matsuda and Takei (1963) for adult chick hemoglobins. Becently, this result has been confirmed by
analyzing definitive erythrocytes isolated from &day-old embryos on a BSA gradient (Mahoney et al., 1977). We have suggested the possibility that the two major and two minor primitive hemoglobins, like the adult ones, are contained in two different primitive erythroid cell types (Cirotto et al., 1975). This suggestion has been advanced because the time courses of the component hemoglobins, during embryonic development are distinctly different. In addition, only four hemoglobins can be observed, that are composed of four different a-like chain types and at least of two p-like chain types. Our argument was that if all those chains were synthesized in the same cell, one would expect eight different hemoglobins deriving from their random assortment. Attempts at discriminating the primitive erythroid cells on the basis of their morphology have been unsuccessful. Thus, it was decided to try and discriminate them on the basis of their hemoglobin contents. We describe here the results of such studies based on immunofluorescence labeling of specific hemoglobin types. MATERIALS
AND
METHODS
Four-day-old White Leghorn chick embryos were obtained from a local poultry farm. The erythrocytes were isolated from
384 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0012-1606
BRIEF NOTES
385
the embryos and, the four primitive embryonic hemoglobins were prepared as previously described (Cirotto et al., 1975). Preparation of antibodies. The adult chick hemoglobins were isolated on a CMcellulose column as previously described (Cirotto and Geraci, 1974), mixed stoichiometrically, and used for antibody formation in rabbits as described by Kabat (1968). The isolated r-globulins were coupled to fluorescein isothiocyanate (Serval) according to Maniatis and Ingram (1971) and were stored at -20°C in a lyophilized form. Antibody specificity was controlled by using the double-diffusion method in agar. Immunofluorescence labeling of primitive erythrocyte smears. Erythrocyte
smears were prepared and labeled with fluorescent antibodies as reported by Maniatis and Ingram (1971), except that the slides were fixed in methanol for 20 min. A Zeiss fluorescence microscope was used for observations and photography. RESULTS
AND
DISCUSSION
Figure 1 shows the specificity of the antibodies prepared against the two adult chick hemoglobins. It is clear that the antibodies do not discriminate between the two adult hemoglobins Al and A2 and the two minor primitive embryonic hemoglobins E3 and E4. In contrast, these antibodies do not react appreciably with the two major primitive embryonic hemoglobins El and E2. Such a result is not in contrast with those reported by Wilt (1962) who found hemoglobins cross-reacting with antiadult hemoglobin antibodies since the earliest appearance of hemoglobins in the very young embryo. Indeed, by globin chain analysis, we have found and identified E3 and E4 in the embryo from the earliest stages at which detection of hemoglobins was possible (Cirotto et al., 1975). The different antibody specificity of El, E2 and of E3, E4 is not surprising since the two major and two minor embryonic hemo-
FIG. 1. Specificity of antibodies against the two adult hemoglobins, Al and A2, as determined by double diffusion in agar. The y-globulin from the immune serum is placed in the central well. Hemoglobins are placed in the peripheral wells as shown in the figure. The antibodies do not discriminate between Al, A2 and the minor primitive embryonic hemoglobins E3, E4. In contrast, they do not react with the two major primitive embryonic hemoglobins El, E2.
globins have globin chains which are clearly different by gel electrophoresis (Cirotto and Geraci, 1975). On the other hand, the minor primitive embryonic hemoglobin E3 and the adult hemoglobin Al and the minor primitive embryonic hemoglobin E4 and the adult hemoglobin A2 have identical (Ychains and differ by their P-chain types (Brown and Ingram, 1974; Cirotto et al., 1975; Cirotto and Geraci, 1977). The apparently complete absence of antibody cross reaction between the major primitive embryonic hemoglobins and the adult hemoglobins indicates that the differences in these hemoglobins concern more than their electrophoretic mobilities since they appear to have very little homology. This has been of advantage in the
386
DEVELOPMENTAL BIOLOGY
identification of a specific cell type, in the primitive erythroid population, containing preferentially or exclusively, the two primitive minor hemoglobins E3 and E4. As clearly shown in Fig. 2, which represents a typical field, the fluorescent antibody labeling technique demonstrates the existence of a limited number of cells containing the two minor hemoglobins. In fact, 75 erythroid cells were found to be labeled with fluorescence out of a total of 1000 cells counted. This percentage value is lower than the combined amounts of the two minor hemoglobins (Cirotto et al., 1975). This might derive from a different
VOLUME 61, 1977
concentration of the hemoglobins in the different erythrocyte types. E3 and E4 might also be present, but at much lower concentration, in the remaining 92% of the erythrocytes containing the two major primitive hemoglobins. In any case, a special population, rich in E3 and E4, is distinctly and clearly present in the primitive erythroid cells of the embryo. The presence of a special cell type in the primitive erythroid population and the compartmentalization of the definitive hemoglobins Al and A2 in the definitive erythrocyte type (Cirotto et al., 1975; Mahoney et al., 1977) suggest that each cell type might be able to produce specific hemoglobins. A shift from one hemoglobin type to another would then be the result of a shift of cell types probably deriving from different erythropoietic territories, rather than from the reordering of the activity of the individual genes in the same stem cell during embryonic development. This work was supported in part for Carlo Cirotto by Grant No. CT76. 01201.04 of the Consiglio Nazionale delle Ricerche. REFERENCES
FIG. 2. Labeling with fluorescent antibodies anti-Al, A2 of a smear of primitive erythroid cells isolated from 4-day-old embryos. The photograph was overexposed in order to show the background of unreacted cells. Note that the fluorescence appearing in the center of the cells with unreacted cytoplasm is due to nonspecific reaction of the rabbit antiserum with nuclear components of the erythrocytes. This reaction also occurs with the antisera of nonimmunized rabbits. It is important to remember that the erythrocytes are deprived of their membrane by treatment with a detergent in order to expose the molecules contained inside them; consequently, the nuclei are also exposed.
BROWN, J. L., and INGRAM, V. M. (1974). Structural studies on chick embryonic hemoglobins. J. Biol. Chem. 249, 3960-3972. CIROTTO, C., and GERACI, G. (1974). Exposed sulphydry1 groups of chicken haemoglobins: Globin localization and effect of oxygenation on their reactivity. J. Mol. Biol. 84, 103-114. CIROTTO, C., and GERACI, G. (1975). Embryonic chicken hemoglobins. Studies on the oxygen equilibrium of two pure components. Camp. Biochem. Physiol. 51A, 159-163. CIROTTO, C., SC~TTO DI TELLA, A., and GERACI, G. (1975). The hemoglobins of the developing chicken embryos. Fractionation and globin composition of the individual components of total erythrocytes and of a single erythrocyte type. Cell Differ. 4,8799. CIROTTO, C., and GERACI, G. (1977). The hemoglobins of the developing chicken embryo. A system for the study of the switch from fetal to adult hemoglobins. Bull. Mol. Biol. Med. 2, 59-71. KABAT, D. (1968). Organization of hemoglobin synthesis in chicken erythrocytes. J. Biol. Chem. 243, 2597-2606. MAHONEY, K. A., HYER, B. J., and CHEN, L. N. L.
BRIEF NOTES (19’77). Separation of primitive and definitive erythroid cells of the chick embryo. Deuelop. Biol. 56, 412416. MANIATIS, G. M., and INGRAM, V. M. (19711. Erythropoiesis during amphybian metamorphosis. J. Cell Biol. 49, 390-404. MATSUDA, G., and TAKEI, H. (1963). The studies on the structure of chicken hemoglobin. I. Chromatographic purification of chicken hemoglobin. J. Biochem. 54. 156-160.
387
SCHALEKAMP, M., SCHALEKAMP, M., VAN-GOOR, D., and SLINGERLAND, R. (1972). Re-evaluation of the presence of multiple haemoglobin during the ontogenesis of the chicken. Electrophoretic and chromatographic characterization, polypeptide composition and immunochemical properties. J. Embryol. Exp. Morphol. 28, 681-713. WILT, F. H. (1962). The ontogeny of chick embryo hemoglobin. Proc. Nat. Acad. Sci. USA 48, 15821590.