- Email: [email protected]
The similarity of hemoglobins in two cell populations in the chick embryo
Hemoglobins
in the chick embryo
429
REFERENCES V. H. and WAISMAN, H. A., J. Biof. Chem. 234, 304 (1959). P. and GREENGARD, O., J. Riof. Chem. 236, 153 (1961). GREENGARD, O., SMITH, M. A. and Acs, G., J. Biol. Chem. 238, 1548 (1963). KNOX, W. E., in Methods in Enzymology. Vol. 2, p. 242. S. P. COLOWICK and (eds.) Academic Press Inc., New York, 1955. KNOX, W. E. and AUERBACH, V. H., J. Biof. Chem. 214, 307 (1955). KNOX, W. E., AUERBACH, V. H. and LIN, E. C. C., Physiof. Rev. 36, 164 (1956). KNOX, W. E. and MEHLER, A. H., J. Biol. Chem. 187, 419 (1950). LORESZEX, E. J. and SMITH, S. E., J. Nufr. 33, 143 (1947). NEMETH, A. M. and NACIIMIAS, V. T., Science 128, 1085 (1958).
1. AUERBACH, 2. FEIGELSON,
3. 4. 5. 6. 7. 6. 9.
THE SIMILARITY
OF HEMOGLOBINS
POPULATIONS
IN THE
CHICK
N.
0.
RAPL.4N
IN TWO CELL EMBRYO1
R. C. FRASER Department
of Zoology
and Entomology, Knoxville, Term., Received
August
The University U.S.A.
of Tennessee,
16, 1964
THE question as to whether two or more forms of hemoglobin exist in one erythrocyte has been of concern to the worker in blood chemistry for some time. There is evidence that co-habitation of this type does exist in human fetal cells [2, 71 and in those of the adult chicken [7]. The question becomes particularly interesting to developmental biologists, when posed in relation to embryonic cells during differentiation. On the sixth day of incubation there is a remarkable change in the circulating blood of the embryonic chick, in which the primitive erythroid line begins to wane with the appearance of the definitive line [3, 61. By the thirteenth day there remains only an occasional erythrocyte of the primitive series. The changes in hemoglobin content of these cells, however, are not consistent with the rather sharp transformation in cell types. When assayed chromatographically [4] or electrophoretically [5] hemoglobin concentrations are found to shift in a gradual manner throughout embryonic development. One form of this respiratory pigment gradually increases during ontogeny, while another decreases, and a third changes only slightly [5]. The present brief study is addressed to the nature of hemoglobins in the two morphologically distinct cell types found in circulating blood of the 7-day chick embryo. The method of withdrawing blood from punctured chorio-allantois vessels, and of preparing hemoglobin samples for electrophoresis has been described previously [4, 51. Several methods of separating the red blood cells of the primitive line from the definitive line that were used were: light sonication alone, filtration through 1 Supported
by
USPHS
grant
H-7027.
Experimenfal
Cell
Research
36
Pig. 1.. -Photographs of red blood cells of 7-dav chick embryos along with clcclrophorclograms of the hemoglobins and their integrated recordings. A, Erythrocytes from whole l~lood. B, The definitive line population of erythrocytcs remaining after treatment of whole-blood cells with 0.001 per cent dodecyl sodium sulfate followed by mild sonication. Hemoglobin types and concentrations in the total ccl1 population and in the dcfinitivc line alone arc identical.
Hemoglobins
in the chick embryo
431
glass wool, selective absorption on glass paper fibers, preferential lysis with rabbit antiserum produced against the primitive cell line, and selective lysis with detergents. None of these procedures were entirely satisfactory, because of failure to separate the cells or because of non-reproducible separations. The procedure finally adopted involved the weakening of cell membranes with 0.001 per cent dodecyl sulfate, followed by mild sonication for 30 sec. The results of such treatment can be seen in the two photographs of Fig. 1. The large cells (the mature erythrocytes of the primitive cellline) were lysed preferentially. Hemoglobins prepared from wh.ole 7-day embryo blood and from the non-lysed definitive-line erythrocytes of embryos of the same age were separated electrophoretically on Oxoid cellulose acetate strips at a potential of 400 volts for 2 hr in a trisEDTA buffer at pH 8.9 [l]. The strips were then dried and stained with o-dianisidine 151. Densitometric recordings were made of the stained strips. The left-hand halves of Fig. 1 show that the recordings are similar. The average of 5 runs indicated that 85 per cent of the hemoglobin in both the total 7-day cells and the definitive-line cells alone was in the major component, with approximately 5 and 10 per cent in the two minor fractions. The persisting changes in the concentrations of hemoglobins in older embryos who have only one morphologically distinct type of erythroid cell, and the results presented here indicate clearly that all three forms of hemoglobin are present in the cells of both lines. Moreover, the changes in cellular content of the hemoglobins occur in all erythroid cells during ontogeny. I wish to acknowledge the expert technical and Mrs. Lorraine Fraser.
assistance
of Mrs. Catherine Martin
REFERENCES
1. 2. 3.
ARONSSON, RETKE, FRASER,
T. and GRGNwALL, A., Sci. Tools 5, 21 (1958). K. and KLEIHAUER, E., Blut 4, 241 (1958). R. C., J. Exptl Zool. 152, 297 (1963).
4. __ Exptl Cell Res. 25, 418 (1961). 5. __ J. Exptl Zool. 156, 185 (1964). 6. I,UCAS, A. iV. and JAMROZ, C., Atlas 7.
Washington, D.C., 1961. MATIOLI, G. and THORELL, B.,
29-641807
Blood
of Avian Hematology. U.S. Department of Agriculture, 21, 1 (1963).
Experimental
Cell Research
36
in the chick embryo
429
REFERENCES V. H. and WAISMAN, H. A., J. Biof. Chem. 234, 304 (1959). P. and GREENGARD, O., J. Riof. Chem. 236, 153 (1961). GREENGARD, O., SMITH, M. A. and Acs, G., J. Biol. Chem. 238, 1548 (1963). KNOX, W. E., in Methods in Enzymology. Vol. 2, p. 242. S. P. COLOWICK and (eds.) Academic Press Inc., New York, 1955. KNOX, W. E. and AUERBACH, V. H., J. Biof. Chem. 214, 307 (1955). KNOX, W. E., AUERBACH, V. H. and LIN, E. C. C., Physiof. Rev. 36, 164 (1956). KNOX, W. E. and MEHLER, A. H., J. Biol. Chem. 187, 419 (1950). LORESZEX, E. J. and SMITH, S. E., J. Nufr. 33, 143 (1947). NEMETH, A. M. and NACIIMIAS, V. T., Science 128, 1085 (1958).
1. AUERBACH, 2. FEIGELSON,
3. 4. 5. 6. 7. 6. 9.
THE SIMILARITY
OF HEMOGLOBINS
POPULATIONS
IN THE
CHICK
N.
0.
RAPL.4N
IN TWO CELL EMBRYO1
R. C. FRASER Department
of Zoology
and Entomology, Knoxville, Term., Received
August
The University U.S.A.
of Tennessee,
16, 1964
THE question as to whether two or more forms of hemoglobin exist in one erythrocyte has been of concern to the worker in blood chemistry for some time. There is evidence that co-habitation of this type does exist in human fetal cells [2, 71 and in those of the adult chicken [7]. The question becomes particularly interesting to developmental biologists, when posed in relation to embryonic cells during differentiation. On the sixth day of incubation there is a remarkable change in the circulating blood of the embryonic chick, in which the primitive erythroid line begins to wane with the appearance of the definitive line [3, 61. By the thirteenth day there remains only an occasional erythrocyte of the primitive series. The changes in hemoglobin content of these cells, however, are not consistent with the rather sharp transformation in cell types. When assayed chromatographically [4] or electrophoretically [5] hemoglobin concentrations are found to shift in a gradual manner throughout embryonic development. One form of this respiratory pigment gradually increases during ontogeny, while another decreases, and a third changes only slightly [5]. The present brief study is addressed to the nature of hemoglobins in the two morphologically distinct cell types found in circulating blood of the 7-day chick embryo. The method of withdrawing blood from punctured chorio-allantois vessels, and of preparing hemoglobin samples for electrophoresis has been described previously [4, 51. Several methods of separating the red blood cells of the primitive line from the definitive line that were used were: light sonication alone, filtration through 1 Supported
by
USPHS
grant
H-7027.
Experimenfal
Cell
Research
36
Pig. 1.. -Photographs of red blood cells of 7-dav chick embryos along with clcclrophorclograms of the hemoglobins and their integrated recordings. A, Erythrocytes from whole l~lood. B, The definitive line population of erythrocytcs remaining after treatment of whole-blood cells with 0.001 per cent dodecyl sodium sulfate followed by mild sonication. Hemoglobin types and concentrations in the total ccl1 population and in the dcfinitivc line alone arc identical.
Hemoglobins
in the chick embryo
431
glass wool, selective absorption on glass paper fibers, preferential lysis with rabbit antiserum produced against the primitive cell line, and selective lysis with detergents. None of these procedures were entirely satisfactory, because of failure to separate the cells or because of non-reproducible separations. The procedure finally adopted involved the weakening of cell membranes with 0.001 per cent dodecyl sulfate, followed by mild sonication for 30 sec. The results of such treatment can be seen in the two photographs of Fig. 1. The large cells (the mature erythrocytes of the primitive cellline) were lysed preferentially. Hemoglobins prepared from wh.ole 7-day embryo blood and from the non-lysed definitive-line erythrocytes of embryos of the same age were separated electrophoretically on Oxoid cellulose acetate strips at a potential of 400 volts for 2 hr in a trisEDTA buffer at pH 8.9 [l]. The strips were then dried and stained with o-dianisidine 151. Densitometric recordings were made of the stained strips. The left-hand halves of Fig. 1 show that the recordings are similar. The average of 5 runs indicated that 85 per cent of the hemoglobin in both the total 7-day cells and the definitive-line cells alone was in the major component, with approximately 5 and 10 per cent in the two minor fractions. The persisting changes in the concentrations of hemoglobins in older embryos who have only one morphologically distinct type of erythroid cell, and the results presented here indicate clearly that all three forms of hemoglobin are present in the cells of both lines. Moreover, the changes in cellular content of the hemoglobins occur in all erythroid cells during ontogeny. I wish to acknowledge the expert technical and Mrs. Lorraine Fraser.
assistance
of Mrs. Catherine Martin
REFERENCES
1. 2. 3.
ARONSSON, RETKE, FRASER,
T. and GRGNwALL, A., Sci. Tools 5, 21 (1958). K. and KLEIHAUER, E., Blut 4, 241 (1958). R. C., J. Exptl Zool. 152, 297 (1963).
4. __ Exptl Cell Res. 25, 418 (1961). 5. __ J. Exptl Zool. 156, 185 (1964). 6. I,UCAS, A. iV. and JAMROZ, C., Atlas 7.
Washington, D.C., 1961. MATIOLI, G. and THORELL, B.,
29-641807
Blood
of Avian Hematology. U.S. Department of Agriculture, 21, 1 (1963).
Experimental
Cell Research
36