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Identification of hemoglobin types contained in single chicken erythrocytes by fluorescent antibody technique
DEVELOPMENTAL
BIOLOGY
48,
317-326
(1976)
Identification of Hemoglobin Types Contained in Single Erythrocytes by Fluorescent Antibody Technique KOJI Department
of Morphology,
Institute
SHIMIZU
of Developmental 480-03, Accepted
Chicken
Research, Japan
September
Aichi
Prefecture
Colony,
Kasugai.
Aichi
17, 1975
It has been suggested that the switch in hemoglobin (Hb) types (from embryonic to adult) during chicken embryonic development is associated with the substitution of one erythroid cell line (“primitive”) for another (“definitive”). For the detection of two Hb types inside single erythroid cells, rabbit antibodies specific for embryonic and adult Hbs were prepared. Rabbit antibody specific for embryonic Hb cross-reacted only with embryonic major Hb components, while antibody specific for adult Hb did solely with adult minor Hb component. The antibodies were conjugated with fluorescein isothiocyanate. The conjugated antibodies were used for the fluorescent staining of blood smears of developing chicken embryos at different ages. Direct fluorescent antibody technique demonstrated that the major components of embryonic Hb and the minor component of adult Hb were not present within the same erythrocyte during chicken ontogenesis. It strongly suggested that embryonic-type Hb and adult-type Hb do not coexist within the same cell.
During the course of vertebrate development, successive changes occur in the erythroid cell populations, in the sites of erythropoiesis, and in the hemoglobin (Hb) types produced. It would be of basic importance to know whether two or more Hbs coexist within a single cell or whether ontogenie changes in Hbs are due to the appearance of a new population of erythroid cells with exclusively new Hb type. In the human, it was found that both fetal and adult Hbs are present within the same erythrocyte at the time of a switch in Hb types (Betke and Kleihauer, 1958; Tomoda, 1964; Hosoi, 1965; Kleihauer et al., 1967; Dan and Hagiwara, 1967; Gitlin et al., 1968). Similarly, in metamorphosing Xenopus laevis, it was demonstrated that erythrocytes in the circulation contain both tadpole and adult-type Hbs (Jurd and Maclean, 1970). On the other hand, in metamorphosing Rana cates beiana, it was shown that circulating erythrocytes of the tadpoles contain either tadpole or frog Hb but not both (Rosenberg, 1970; Maniatis and Ingram, 1971). In developing mice, Fantoni et al. (1969) suggested that the switch in the
types of Hb from embryonic to adult is associated with the substitution of one erythroid cell line for another. In chickens, many of the reports presented up to this time suggested that circulating erythroid cells of the embryos contain either embryonic or adult-type Hb, but not both (Wilt, 1967; Shimizu, 1972a,b; Shimizu and Hagiwara, 1973; Bruns and Ingram, 1973a,b; Keane et al., 1974). There are many conflicting reports concerning subunit compositions of chicken Hbs belonging to embryonic and adult types (Manwell et al., 1966; D’Amelio, 1966; Schalekamp et al., 1972; Shimizu, 1972a; Brown and Ingram, 1974; Moss and Hamilton, 1974). Most of them, however, demonstrated that two adult Hb components share one polypeptide chain, which embryonic Hb components do not possess, in common, and that only one or two subunits are common between two types of chicken Hb. It must be expected that antibodies specific to embryonic or adult-type Hb components are obtainable. Shimizu and Hagiwara (1973) showed that antiserum against Hb from 5-day chicken embryos, after being absorbed with Hb from 317
Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
318
DEVELOPMENTAL
BIOLOGY
adults, reacted exclusively with embryonic type Hb components on agar gel immunodiffusion tests. Now that antibodies specific to the major components of embryonic type Hb and to the minor component of adult-type Hb were obtained recently, the present work was planned to clear the question of a suggestive correlation between the populations of erythroid cells and the Hb molecules produced by fluorescent antibody technique.
VOLUME
48, 1976
phate buffer (pH 7.1) and 0.01% sodium merthiolate. The results were obtained after incubation at 20°C for 6 days. The precipitate lines were stained with o-dianisidine for Hb. Zmmunofluorescent staining. Both antisera were made specific for the homologous antigens by absorbing antiserum to Hb from 5-day embryos with Hb from adults (anti-embryonic-Hb), and antiserum to Hb from adults with Hb from 5-day embryos (anti-adult-Hb). Absorption was carried out at 37°C for 2 hr, and subseMATERIALS AND METHODS quently at 4°C for 2 days. Absorbed antiAnimal source. Fresh fertilized eggs and sera were first fractionated in 50% ammoadult hens of White Leghorn chickens for nium sulfate saturation at 4”C, then globucommercial source were obtained from the lin fraction was obtained by three cycles of Yatsurughi Agricultural Cooperative Asso- precipitation with 33% saturated ammociation near Nagoya. Eggs were immedinium sulfate (Kawamura, 1969). Conjugaately incubated blunt end up to 38°C in an tion with fluorescein isothiocyanate electric cabinet incubator. (FIT0 was carried out according to the Hemoglobin and erythrocyte preparaprocedure given by Kawamura (1969). Contions. Early embryos were bled from the jugated antibodies were absorbed before heart and the blood vessels on the yolk sac. use with both mouse liver powder and defatted chicken yolk powder at 20°C for 1 hr. Late embryos were bled from the umbilical vessels, while adult chickens were bled Fluorescent antibodies were employed in from a jugular vein. Erythrocytes were pre- the direct technique on blood smears prepared by the procedure of Dan and Hagipared by conventional methods (Wilt, 1962). Hemoglobin was prepared by the wara (19671, but after fixation the slides method of Drabkin (19491, and cyanmetwere treated with 3% Tween-80 (Sigma) in hemoglobin was prepared in the cold from the saline according to the method of Mancrystallized oxyhemoglobin according to iatis and Ingram (1971). The preparations the procedure of Allen, Schroeder, and were examined by fluorescence microscopy Balog (1958). with a Nikon FL. Zmmunization. Male New Zealand RESULTS White rabbits were immunized subcutaneously with cyanmethemoglobins from 5 Properties of antibodies. Anti-embryday chicken embryos or adults by the proce- onic-Hb reacted with Hb from 5-day dure mentioned previously (Shimizu and chicken embryos by forming precipitate lines positive for o-dianisidine staining on Hagiwara, 1973). Complement inhibition of the antisera obtained was carried out at agar gel immunodiffusion test, but did not 56°C for 30 min. The maximum dilution of with Hb from adults (Fig. 1A). Anti-adultthe antisera to give positive reaction in the Hb produced a clear precipitin line in the inter-facial ring test against cyanmethemoreaction with Hb from adult chickens, globins from &day embryos or adults was while its reaction with Hb from 5-day embryos was extremely feeble (Fig. 1B). The 64. Agar gel immunodiffusion tests. Agar reaction of the antibodies obtained to Hbs gel immunodiffusion tests were carried out from lo- and 15-day embryos was variable in 1% agarose gel plate with 0.15 M phos- in each test. Sometimes no visible lines
Ko.n
SHIMIZU
Identification
appeared (Figs. 2A and 2B), while in other tests very weak reactions were discernible. The reasons why no clear precipitate lines appeared in the tests were obscure. Anti-embryonic-Hb reacted with the major components of embryonic Hb (E-I and E-II), isolated from Hb of &day embryos on starch gel electrophoresis, by forming three precipitate lines positive for o-dianisidine staining, while it did not with the minor component of embryonic Hb (E-III), isolated from Hb of &day embryos, and both components of adult Hb (A-I and A-II) isolated from adult chicken Hb (Fig. 3A). Anti-adult-Hb reacted with the minor adult Hb (A-I), by forming a very weak precipitate line on immunodiffusion test, but it did not with the major adult Hb (AII) and any components of embryonic Hb (Fig. 3B).
of Hemoglobin
Types
319
ZmmunofZuorescent staining of erythrocytes. Figure 4 shows smears of erythroid cells in the circulation from 5day embryos, which possess almost embryonic type Hb, when it is said that erythroblasts of the second, or definitive, cell line appear in their circulation (Lemez, 1964). In Fig. 4A, all cells were stained with FITC-conjugated anti-embryonic-Hb (anti-embryonicHb-FITC), while in Fig. 4B no cells stained with FITC-conjugated anti-adult-Hb (antiadult-Hb-FITC). More than 70% of the circulating erythroid cells from 6-day embryos, which have definitive cells as well as primitive cells, were stained with antiembryonic-Hb-FITC, while less than 15% of the cells stained with anti-adult-HbFITC. Figure 5 shows smears of the circulating erythroid cells from 7-day embryos. More than 35% of the cells were stained
FIG. 1. Specificity of absorbed antibodies examined by agar gel immunodiffusion tests. A, Hb prepared from adult chickens; 5, Hb prepared from 5-day chicken embryos. Each outer well contains 2-4 cog of proteins. (A) Anti-embryonic-Hb in the center well. (B) Anti-adult-Hb in the center well. Each center well contains 2-4 mg of proteins.
FIG. 2. The time of disappearance or appearance of embryonic type Hb or adult-type Hb demonstrated by immunodiffusion tests on agar gel plate with specific antibodies. 5, Hb from 5day embryos; 10, Hb from IOday embryos. 15, Hb from 15-day embryos; A, Hb from adult chickens; S, adult chicken whole serum. L, mouse liver powder. Each outer well contains 2-4 cog of proteins. (A) Anti-embryonic-Hb in the center well. rB) Anti-adult-Hb in the center well. Each center well contains 2-4 mg of proteins.
320
DEVELOPMENTAL
0Eli
BIOLOGY
VOLUME
48. 1976
\ 0 II
@>II
FIG. 3. Specificity of each absorbed antibody examined further with isolated Hb components by immunodiffusion tests. A-I, adult minor Hb; A-II, adult major Hb; E-I, embryonic major Hb; E-II, embryonic major Hb; E-III, embryonic minor Hb. Each Hb component was isolated from starch gel after electrophoresis at pH 6.8 for 16 hr. Each outer well contains 2 pg of Hb. (A) Anti-embryonic-Hb in the center well. (B) Anti-adultHb in the center well. Each center well contains 2-4 mg of proteins. Anti-embryonic-Hb reacted exclusively with embryonic major Hb components. The reactions of anti-embryonic-Hb with Hb components E-I and E-II were those of identity. Anti-adult-Hb reacted solely with adult minor Hb component by forming a very weak precipitate line.
FIG. 4. Direct fluorescent staining chicken embryos with almost embryonic with anti-adult-Hb-FITC. x 900.
of circulating erythroid type Hb. (A) Staining
cells, almost primitive with anti-embryonic-Hb-FITC.
cells,
from 5-day (B) Staining
KOJI
SHIMIZU
Identification
of
Hemoglobin
Types
321
with anti-embryonic-Hb-FITC, while less FITC (Fig. 6B). Figure 7 represents smears of erythroid cells from 11-day emthan 50% of the cells stained with antiadult-Hb-FITC. In the circulation of g-day bryos. Less than 10% of erythroid cells embryos, less than 20% of erythroid cells were stained with anti-embryonic-HbFITC,while more than 75% of erythroid were stained with anti-embryonic-HbFITC (Fig. 6A), while more than 60% of cells stained with anti-adult-Hb-FITC. In erythroid cells stained with anti-adult-Hbthe circulating erythrocytes of 15-day em-
FIG. 5. Direct fluorescent staining embryonic type Hb and 75% adult-type anti-adult-Hb-FITC. x 900.
FIG. 6. Direct definitive erythroid (B) Staining with
of circulating erythroid Hb. (A) Staining with
fluorescent staining of circulating erythroid cells prevails over primitive erythroid cells. anti-adult-Hb-FITC. x 900.
cells from ‘I-day chicken anti-embryonic-Hb-FITC.
cells from (A) Staining
embryos with 25q (B’I Staining with
g-day embryos, at which with anti-embryonic-Hb-FITC.
time
322
DEVELOPMENTAL
BIOLOGY
bryos, when erythropoietic function begins in the bone marrow, cells stained with anti-embryonic-Hb-FITC were scarcely detected. Figure 8 shows smears of erythrocytes of 17-day embryos, that is, definitive cells, which contain solely adult type Hb.
VOLUME
48, 1976
In Fig. 8A none of the cells were stained with anti-embryonic-Hb-FITC, while in Fig. 8B all of the cells stained with antiadult-Hb-FITC. Similar results were obtained with erythrocytes in the circulation of a 6-month old hen (Fig. 9).
FE. 7. Direct fluorescent staining of circulating erythrocytes from cells and 87% definitive cells. (A) Staining with anti-embryonic-Hb-FITC. FITC. x 900.
FIG. 8. Direct fluorescent ryos with almost adult-type :b-FITC. x 900.
staining of circulating Hb. (A) Staining with
almost ei rythrocytes, an ti-embryonic-Hb-FITC.
U-day embryos (B) Staining
with with
definitive cells, (B) Staining
13% primitive anti-adult-Hb-
from with
17-day emanti-adult-
KOJI
SHIMIZU
FIG. 9. Direct fluorescent staining ens with exclusively adult-type Hb. adult-Hb-FITC.
Identification
of circulating (A) Staining
of Hemoglobin
erythrocytes, definitive with anti-embryonic-Hb-FITC.
DISCUSSION
Previously, Shimizu and Hagiwara (1973) were successful in obtaining rabbit antiserum specific to chicken embryonic type Hb, while not in obtaining antiserum specific to adult-type Hb. In the present work, antibody specific to the minor component of adult-type Hb (anti-adult-Hb), as well as that to the major components of embryonic type Hb (anti-embryonic-Hb), was successfully obtained. It was, however, very difficult to obtain completely absorbed antibodies with sufficiently high titer. Fig. 2A shows that Hbs from lo- and l&day embryos did not react with antiembryonic-Hb, although immunologically embryonic type Hb was clearly detectable in lo-day chicken embryos with absorbed antiserum on agar gel immunodiffusion tests (Shimizu and Hagiwara, 1973). Fig. 2B also represents that Hbs from lo- and 15-day embryos did not react with antiadult-Hb, though adult-type Hb components are sufficiently detectable in those embryos by electrophoresis or chromatography. The reasons why Hbs from lo- and 15-day embryos did not react clearly with
Types
erythrocytes, from (B) Staining
323
adult chickwith anti-
absorbed antibodies were not proved in the present work. The agar gel immunodiffusion test given in Fig. 3A shows that embryonic major Hb components (E-I and E-11) were apparently distinguished from embryonic minor (EIII) and both adult Hb components (A-I and A-II), implying that embryonic major Hb components possess unique globin chain (or chains), which other Hb components do not have. The reasons why antiembryonic-Hb produced three precipitin lines in the reaction with embryonic major Hb components were not clear. Further studies were expected to make clear the chemical properties of embryonic type Hb components. In the previous work, Shimizu and Hagiwara (1973) failed to obtain the antiserum that enables us to distinguish adult major Hb from adult minor Hb. Brown and Ingram (1974) reported, however, that antigenie determinants of Hb D (adult minor Hb or A-I) and Hb A (adult major Hb or AII) are similar in the reaction with antiHb-D serum, but anti-Hb-A serum contains antibodies for such antigenic determi-
324
DEVELOPMENTALBIOLOGY
nants on Hb A that are not present in Hb D. Figure 3B shows that only adult minor Hb (A-1) gave a faint precipitin line in the reaction with anti-adult-Hb, implying that antigenic determinants of adult minor Hb are different from those of adult major Hb. It was shown that two Hb components produced in the definitive erythrocytes of adult chickens are identical with the Hb components synthesized in the definitive erythrocytes found in the embryonic circulation (Bruns and Ingram, 1973a; Brown and Ingram, 1974; Moss and Hamilton, 1974). It was also demonstrated that embryonic definitive erythrocytes contain a minor Hb component, H, or fetal type Hb (Godet, Schiirch and Nigon, 1970; Shimizu, 1972a), in addition to two adult Hbs (Moss and Hamilton, 1974). One common polypeptide chain was detectable between adult major and fetal Hbs, while not between adult minor and fetal Hbs (Shimizu, 1972a; Moss and Hamilton, 1974). Furthermore, it was shown that embryonic minor and fetal Hbs share one polypeptide chain, but embryonic major and fetal Hbs do not share any chains (Shimizu, 1972a). Maniatis and Ingram (19711 found that tadpole erythrocytes were stained faintly with fluorescent antibodies against tadpole Hb, but frog erythrocytes did not stain with fluorescent antibodies against tadpole or frog Hbs, because of insufficient exposure of the Hb in the erythrocyte to the antibodies. However, they found that the positive fluorescent staining was obtained after pretreatment of the fixed smears with Tween-80. Similarly, primitive or definitive erythroid cells of late chicken embryos and adults were stained with fluorescent antibodies only after pretreatment of the fixed smears with Tween80, while primitive erythroid cells of early chicken embryos stained well with or without the pretreatment. All of the erythroid cells from the circulating blood of early embryos fluoresced green with anti-embryonic-Hb-FITC, as shown in Fig. 4A, while none of the cells fluoresced green with anti-adult-Hb-FITC,
VOLUME 48,
1976
as shown in Fig. 4B. Sometimes the nuclear portion of the erythroid cells from chicken embryos fluoresced white rather than green. This nuclear fluorescence was particularly frequent when immature cells were stained with antibodies. The interpretation of the biological meaning of such nuclear fluorescence so far remains for further study. All of the circulating erythrocytes from late embryos fluoresced green with antiadult-Hb-FITC, as shown in Fig. BB, while any of the cells did not fluoresce green with anti-embryonic-Hb-FITC, as shown in Fig. 8A. Cells stained with anti-adultHb-FITC from embryos, presumably definitive erythroid cells derived from yolk-sac, fluoresced not so obviously as cells stained from late embryos or adult chickens, presumably definitive cells derived from bone marrow. It is difficult to say that all of the definitive erythroid cells of yolk-sac origin stained with anti-adult-Hb-FITC as shown in Figs. 6B and 7B, probably because of the low sensitivity of the method used here with anti-adult-Hb. Table 1 gives the relative content of the erythrocytes stained with anti-embryonicHb-FITC or with anti-adult-Hb-FITC as the percentage of the total cells in the smears as compared with the percentage of primitive and definitive erythroid cells in the circulation (Bruns and Ingram, 1973a,b), and that of embryonic and adult type Hbs (Shimizu, 1972a) at a given age. The main finding of this report using specific fluorescent antibodies against embryonic and adult-type Hbs is that embryonic major Hbs and adult minor Hb are not present inside the same erythrocyte during ontogenesis. The result presented here strongly suggests that primitive erythroid cells, derived from yolk sac, synthesize exclusively embryonic type Hb, while definitive erythroid cells, derived from yolk sac or bone marrow, synthesize only adulttype Hb. The author is indebted to Professor T. S. Okada of Kyoto University for his critical reading of the man-
KOJI
Identification
SHIMIZU
of Hemoglobin
TABLE CHANGES
Days of incubation
4
IN CELL
Cells stained with anti-embryonic-HbFITC
POPULATIONS
Cells stained with antiadult-HbFITC
100
6 9
70 35 20
15 17
15
Adult a Each b After ’ After
75 100 100 100
0 0
number is presented Shimizu (1972a). Bruns and Ingram
as the percentage
Adult Type Hb*
0 0
100 61
39
25
75
3
97
0
97
0
100
ANTIBODIES~
Primitive erythroid cells’
Definitive erythroid cells’
0 o-5 18 26 70 75 87
100 95-100
82 74 30 25 13 0 0
100 100
0
100
of the total.
(1973a,b).
uscript. This work was supported in part by Research Grant No. 74275 from the Ministry of Education, Japan. REFERENCES D. W., SCHROEDER, W. A., and BALOG, J. (1958). Observation on the chromatographic heterogeneity of normal adult and fetal human hemoglobin: A study of the effects of crystallization and chromatography on the heterogeneity and isoleutine content. J. Amer. Chem. Sot. 80, 1628-1634. BETKE, K., and KLEIHAUER, E. (1958). Fetaler und bleibender Blutfarbstoff in Erythrozyten und Erythroblasten von menschlichen Feten und Neugebornen. Blut 41, 241-246. BROWN, J. L., and INGRAM, V. M. (1974). Structural studies on chick embryonic hemoglobins. J. Biol. Chem. 249, 3960-3972. BRUNS, G. A. P., and INGRAM, V. M. (1973a). Erythropoiesis in the developing chick embryo. Deuelop. Biol. 30, 455-459. BRUNS, G. A. P., and INGRAM, V. M. (1973b). The erythroid cells and haemoglobins of the chick embryo. Philos. Trans. Roy. Sot. London, Ser. B 266, 225-305. D’AMELIO, V. (1966). The globins of adult and embryonic chick hemoglobin, Biochim. Biophys. Acta 127, 59-65. DAN, M., and HAGIWARA, A. (1967). Detection of two types of hemoglobin (HbA and HbF) in single erythrocytes by fluorescent antibody technique. Jap. J. Human Genet. 12, 55-61. DRABKIN, D. L. (19491. A simplified technique for a large scale crystallization of human oxyhemoglobin Isomorphous transformations of hemoglobin and myoglobin in the crystalline state. Arch. Biothem. Biophys. 21, 224-231. ALLEN,
FLUORESCENT
100
50 60
10 0
WITH
Embryonic Type Hbb
10 11
1
STAINED
0 0
100
325
Types
FANTONI, KIND,
A.,
DE LA CHAPELLE,
A.,
CHUI,
D.,
RIF-
R. A., and MARKS, P. A. (1969). Control mechanism of the conversion from synthesis of embryonic to adult hemoglobin. Ann. N. Y. Acad. Sci. 165, 194-204. GITLIN, D., SASAKI, T., and VUOPIO, P. (1968). Immunochemical quantitation of proteins in single cells. I. Carbonic anhydrase B&chain hemoglobin and u-chain haemoglobin in some normal and abnormal erythrocytes. Blood 32, 796-810. G~DET, J., SCH~~RCH, D., and NIGON, V. (1970). Caracmrisation et evolution des hemoglobines dans le tours du developpement postembryonnaire chez la Poule. J. Embryol. Exp. Morph. 23, 153-167. HOSOI, T. (1965). Studies of hemoglobin F within single erythrocyte by fluorescent antibody technique. Exp. Cell Res. 37, 680-683. JURD, R. D., and MACLEAN, N. (1970). An immunofluorescent study of the haemoglobins in metamorphosing Xenopus laevis. J. Embryol. Exp. Morph. 23, 299-309. KAWAMURA, A. (19691. Preparations of materials. In “Fluorescent antibody techniques and their applications” (Kawamura, A., ed.), pp. 11-64. Univ. of Tokyo Press, Tokyo, Japan. KEANE, R. W., ABBOTT, U. K., BROWN, J. L., and INGRAM, V. M. (1974). Ontogeny of hemoglobins: Evidence for hemoglobin M. Develop. Biol. 38, 229-236. KLEIHAUER, E. F., TANG, T. E., and BETKE, K. (1967). Die intrazellulare verteilung von embryonalem haemoglobin in roten blutzellen menschlicher embryonen. Acta Haemat. 38, 264-272. LEMETZ, L. (1964). The blood of chick embryo: Quantitative embryology at a cellular level. In “Advances in Morphogenesis” (Abercrombie, M., and Brachet, J., eds.), Vol. 3, pp. 197-245. Academic Press, New York/London.
326
DEVELOPMENTAL
BIOL~Y
G. M., and INGRAM, V. M. (1971). Erythropoiesis during amphibian metamorphosis. III. Immunochemical detection of tadpole and frog hemoglobins (Rana catesbeianu) in single erythro-
MANIATIS,
cytes. J. Cell Biol. 49, 390-404. MANWELL, C., BAKER, C. M. A.,
and BETZ, T. W. (1966). Ontogeny of hemoglobin in the chicken. J. Embryol. Exp. Morph. 16, 65-79. Moss, B. A., and HAMILTON, E. A. (1974). Chicken definitive erythrocyte haemoglobins. Biochim. Biophys. Actu 371, 379-391. ROSENBERG, M. (19701. Electrophoretic analysis of hemoglobin and isozymes in individual vertebrate cells. Proc. Nut. Acad. Sci. USA 67. 32-36. SCHALEKAMP, M., SCHALEKAMP, M., VAN GOOR, D., and SLINGERLAND, R. (1972). Re-evaluation of the presence of multiple haemoglobins during the ontogenesis of the chicken. J. Embryol. Exp. Morph. 28, 681-713. SHIMIZU, K. (1972al. Ontogeny of chicken hemoglobin. I. Electrophoretic study of the heterogeneity
VOLUME
48, 1976
of hemoglobin
in development. Develop. Growth 14, 43-55. (1972b). Ontogeny of chicken hemoglobin. II. Chromatographic study of the heterogeneity of hemoglobin in development. Deuelop. Growth Differentiation 14, 281-295. SHIMIZU, K., and HAGIWARA, A. (1973). Ontogeny of chicken hemoglobin. III. Immunological study of the heterogeneity of hemoglobin in development. Develop. Growth Differentiation 15, 285-306. TOMODA, Y. (1964). Demonstration of fetal erythrocyte by immunofluorescent staining. Nature (LenDifferentiation SHIMIZU, K.
don) WILT,
202,910-911.
F. H. (19621. The ontogeny of chick embryo hemoglobin. Proc. Nut. Acud. Sci. USA 48, 1582-
1589. WILT, F.
H. (1967). The control of embryonic hemoglobin synthesis. In “Advances in Morphogenesis” (Abercrombie, M., and Bracket, J., eds.), Vol. 6, pp. 89-125. Academic Press, New York/London.
BIOLOGY
48,
317-326
(1976)
Identification of Hemoglobin Types Contained in Single Erythrocytes by Fluorescent Antibody Technique KOJI Department
of Morphology,
Institute
SHIMIZU
of Developmental 480-03, Accepted
Chicken
Research, Japan
September
Aichi
Prefecture
Colony,
Kasugai.
Aichi
17, 1975
It has been suggested that the switch in hemoglobin (Hb) types (from embryonic to adult) during chicken embryonic development is associated with the substitution of one erythroid cell line (“primitive”) for another (“definitive”). For the detection of two Hb types inside single erythroid cells, rabbit antibodies specific for embryonic and adult Hbs were prepared. Rabbit antibody specific for embryonic Hb cross-reacted only with embryonic major Hb components, while antibody specific for adult Hb did solely with adult minor Hb component. The antibodies were conjugated with fluorescein isothiocyanate. The conjugated antibodies were used for the fluorescent staining of blood smears of developing chicken embryos at different ages. Direct fluorescent antibody technique demonstrated that the major components of embryonic Hb and the minor component of adult Hb were not present within the same erythrocyte during chicken ontogenesis. It strongly suggested that embryonic-type Hb and adult-type Hb do not coexist within the same cell.
During the course of vertebrate development, successive changes occur in the erythroid cell populations, in the sites of erythropoiesis, and in the hemoglobin (Hb) types produced. It would be of basic importance to know whether two or more Hbs coexist within a single cell or whether ontogenie changes in Hbs are due to the appearance of a new population of erythroid cells with exclusively new Hb type. In the human, it was found that both fetal and adult Hbs are present within the same erythrocyte at the time of a switch in Hb types (Betke and Kleihauer, 1958; Tomoda, 1964; Hosoi, 1965; Kleihauer et al., 1967; Dan and Hagiwara, 1967; Gitlin et al., 1968). Similarly, in metamorphosing Xenopus laevis, it was demonstrated that erythrocytes in the circulation contain both tadpole and adult-type Hbs (Jurd and Maclean, 1970). On the other hand, in metamorphosing Rana cates beiana, it was shown that circulating erythrocytes of the tadpoles contain either tadpole or frog Hb but not both (Rosenberg, 1970; Maniatis and Ingram, 1971). In developing mice, Fantoni et al. (1969) suggested that the switch in the
types of Hb from embryonic to adult is associated with the substitution of one erythroid cell line for another. In chickens, many of the reports presented up to this time suggested that circulating erythroid cells of the embryos contain either embryonic or adult-type Hb, but not both (Wilt, 1967; Shimizu, 1972a,b; Shimizu and Hagiwara, 1973; Bruns and Ingram, 1973a,b; Keane et al., 1974). There are many conflicting reports concerning subunit compositions of chicken Hbs belonging to embryonic and adult types (Manwell et al., 1966; D’Amelio, 1966; Schalekamp et al., 1972; Shimizu, 1972a; Brown and Ingram, 1974; Moss and Hamilton, 1974). Most of them, however, demonstrated that two adult Hb components share one polypeptide chain, which embryonic Hb components do not possess, in common, and that only one or two subunits are common between two types of chicken Hb. It must be expected that antibodies specific to embryonic or adult-type Hb components are obtainable. Shimizu and Hagiwara (1973) showed that antiserum against Hb from 5-day chicken embryos, after being absorbed with Hb from 317
Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
318
DEVELOPMENTAL
BIOLOGY
adults, reacted exclusively with embryonic type Hb components on agar gel immunodiffusion tests. Now that antibodies specific to the major components of embryonic type Hb and to the minor component of adult-type Hb were obtained recently, the present work was planned to clear the question of a suggestive correlation between the populations of erythroid cells and the Hb molecules produced by fluorescent antibody technique.
VOLUME
48, 1976
phate buffer (pH 7.1) and 0.01% sodium merthiolate. The results were obtained after incubation at 20°C for 6 days. The precipitate lines were stained with o-dianisidine for Hb. Zmmunofluorescent staining. Both antisera were made specific for the homologous antigens by absorbing antiserum to Hb from 5-day embryos with Hb from adults (anti-embryonic-Hb), and antiserum to Hb from adults with Hb from 5-day embryos (anti-adult-Hb). Absorption was carried out at 37°C for 2 hr, and subseMATERIALS AND METHODS quently at 4°C for 2 days. Absorbed antiAnimal source. Fresh fertilized eggs and sera were first fractionated in 50% ammoadult hens of White Leghorn chickens for nium sulfate saturation at 4”C, then globucommercial source were obtained from the lin fraction was obtained by three cycles of Yatsurughi Agricultural Cooperative Asso- precipitation with 33% saturated ammociation near Nagoya. Eggs were immedinium sulfate (Kawamura, 1969). Conjugaately incubated blunt end up to 38°C in an tion with fluorescein isothiocyanate electric cabinet incubator. (FIT0 was carried out according to the Hemoglobin and erythrocyte preparaprocedure given by Kawamura (1969). Contions. Early embryos were bled from the jugated antibodies were absorbed before heart and the blood vessels on the yolk sac. use with both mouse liver powder and defatted chicken yolk powder at 20°C for 1 hr. Late embryos were bled from the umbilical vessels, while adult chickens were bled Fluorescent antibodies were employed in from a jugular vein. Erythrocytes were pre- the direct technique on blood smears prepared by the procedure of Dan and Hagipared by conventional methods (Wilt, 1962). Hemoglobin was prepared by the wara (19671, but after fixation the slides method of Drabkin (19491, and cyanmetwere treated with 3% Tween-80 (Sigma) in hemoglobin was prepared in the cold from the saline according to the method of Mancrystallized oxyhemoglobin according to iatis and Ingram (1971). The preparations the procedure of Allen, Schroeder, and were examined by fluorescence microscopy Balog (1958). with a Nikon FL. Zmmunization. Male New Zealand RESULTS White rabbits were immunized subcutaneously with cyanmethemoglobins from 5 Properties of antibodies. Anti-embryday chicken embryos or adults by the proce- onic-Hb reacted with Hb from 5-day dure mentioned previously (Shimizu and chicken embryos by forming precipitate lines positive for o-dianisidine staining on Hagiwara, 1973). Complement inhibition of the antisera obtained was carried out at agar gel immunodiffusion test, but did not 56°C for 30 min. The maximum dilution of with Hb from adults (Fig. 1A). Anti-adultthe antisera to give positive reaction in the Hb produced a clear precipitin line in the inter-facial ring test against cyanmethemoreaction with Hb from adult chickens, globins from &day embryos or adults was while its reaction with Hb from 5-day embryos was extremely feeble (Fig. 1B). The 64. Agar gel immunodiffusion tests. Agar reaction of the antibodies obtained to Hbs gel immunodiffusion tests were carried out from lo- and 15-day embryos was variable in 1% agarose gel plate with 0.15 M phos- in each test. Sometimes no visible lines
Ko.n
SHIMIZU
Identification
appeared (Figs. 2A and 2B), while in other tests very weak reactions were discernible. The reasons why no clear precipitate lines appeared in the tests were obscure. Anti-embryonic-Hb reacted with the major components of embryonic Hb (E-I and E-II), isolated from Hb of &day embryos on starch gel electrophoresis, by forming three precipitate lines positive for o-dianisidine staining, while it did not with the minor component of embryonic Hb (E-III), isolated from Hb of &day embryos, and both components of adult Hb (A-I and A-II) isolated from adult chicken Hb (Fig. 3A). Anti-adult-Hb reacted with the minor adult Hb (A-I), by forming a very weak precipitate line on immunodiffusion test, but it did not with the major adult Hb (AII) and any components of embryonic Hb (Fig. 3B).
of Hemoglobin
Types
319
ZmmunofZuorescent staining of erythrocytes. Figure 4 shows smears of erythroid cells in the circulation from 5day embryos, which possess almost embryonic type Hb, when it is said that erythroblasts of the second, or definitive, cell line appear in their circulation (Lemez, 1964). In Fig. 4A, all cells were stained with FITC-conjugated anti-embryonic-Hb (anti-embryonicHb-FITC), while in Fig. 4B no cells stained with FITC-conjugated anti-adult-Hb (antiadult-Hb-FITC). More than 70% of the circulating erythroid cells from 6-day embryos, which have definitive cells as well as primitive cells, were stained with antiembryonic-Hb-FITC, while less than 15% of the cells stained with anti-adult-HbFITC. Figure 5 shows smears of the circulating erythroid cells from 7-day embryos. More than 35% of the cells were stained
FIG. 1. Specificity of absorbed antibodies examined by agar gel immunodiffusion tests. A, Hb prepared from adult chickens; 5, Hb prepared from 5-day chicken embryos. Each outer well contains 2-4 cog of proteins. (A) Anti-embryonic-Hb in the center well. (B) Anti-adult-Hb in the center well. Each center well contains 2-4 mg of proteins.
FIG. 2. The time of disappearance or appearance of embryonic type Hb or adult-type Hb demonstrated by immunodiffusion tests on agar gel plate with specific antibodies. 5, Hb from 5day embryos; 10, Hb from IOday embryos. 15, Hb from 15-day embryos; A, Hb from adult chickens; S, adult chicken whole serum. L, mouse liver powder. Each outer well contains 2-4 cog of proteins. (A) Anti-embryonic-Hb in the center well. rB) Anti-adult-Hb in the center well. Each center well contains 2-4 mg of proteins.
320
DEVELOPMENTAL
0Eli
BIOLOGY
VOLUME
48. 1976
\ 0 II
@>II
FIG. 3. Specificity of each absorbed antibody examined further with isolated Hb components by immunodiffusion tests. A-I, adult minor Hb; A-II, adult major Hb; E-I, embryonic major Hb; E-II, embryonic major Hb; E-III, embryonic minor Hb. Each Hb component was isolated from starch gel after electrophoresis at pH 6.8 for 16 hr. Each outer well contains 2 pg of Hb. (A) Anti-embryonic-Hb in the center well. (B) Anti-adultHb in the center well. Each center well contains 2-4 mg of proteins. Anti-embryonic-Hb reacted exclusively with embryonic major Hb components. The reactions of anti-embryonic-Hb with Hb components E-I and E-II were those of identity. Anti-adult-Hb reacted solely with adult minor Hb component by forming a very weak precipitate line.
FIG. 4. Direct fluorescent staining chicken embryos with almost embryonic with anti-adult-Hb-FITC. x 900.
of circulating erythroid type Hb. (A) Staining
cells, almost primitive with anti-embryonic-Hb-FITC.
cells,
from 5-day (B) Staining
KOJI
SHIMIZU
Identification
of
Hemoglobin
Types
321
with anti-embryonic-Hb-FITC, while less FITC (Fig. 6B). Figure 7 represents smears of erythroid cells from 11-day emthan 50% of the cells stained with antiadult-Hb-FITC. In the circulation of g-day bryos. Less than 10% of erythroid cells embryos, less than 20% of erythroid cells were stained with anti-embryonic-HbFITC,while more than 75% of erythroid were stained with anti-embryonic-HbFITC (Fig. 6A), while more than 60% of cells stained with anti-adult-Hb-FITC. In erythroid cells stained with anti-adult-Hbthe circulating erythrocytes of 15-day em-
FIG. 5. Direct fluorescent staining embryonic type Hb and 75% adult-type anti-adult-Hb-FITC. x 900.
FIG. 6. Direct definitive erythroid (B) Staining with
of circulating erythroid Hb. (A) Staining with
fluorescent staining of circulating erythroid cells prevails over primitive erythroid cells. anti-adult-Hb-FITC. x 900.
cells from ‘I-day chicken anti-embryonic-Hb-FITC.
cells from (A) Staining
embryos with 25q (B’I Staining with
g-day embryos, at which with anti-embryonic-Hb-FITC.
time
322
DEVELOPMENTAL
BIOLOGY
bryos, when erythropoietic function begins in the bone marrow, cells stained with anti-embryonic-Hb-FITC were scarcely detected. Figure 8 shows smears of erythrocytes of 17-day embryos, that is, definitive cells, which contain solely adult type Hb.
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48, 1976
In Fig. 8A none of the cells were stained with anti-embryonic-Hb-FITC, while in Fig. 8B all of the cells stained with antiadult-Hb-FITC. Similar results were obtained with erythrocytes in the circulation of a 6-month old hen (Fig. 9).
FE. 7. Direct fluorescent staining of circulating erythrocytes from cells and 87% definitive cells. (A) Staining with anti-embryonic-Hb-FITC. FITC. x 900.
FIG. 8. Direct fluorescent ryos with almost adult-type :b-FITC. x 900.
staining of circulating Hb. (A) Staining with
almost ei rythrocytes, an ti-embryonic-Hb-FITC.
U-day embryos (B) Staining
with with
definitive cells, (B) Staining
13% primitive anti-adult-Hb-
from with
17-day emanti-adult-
KOJI
SHIMIZU
FIG. 9. Direct fluorescent staining ens with exclusively adult-type Hb. adult-Hb-FITC.
Identification
of circulating (A) Staining
of Hemoglobin
erythrocytes, definitive with anti-embryonic-Hb-FITC.
DISCUSSION
Previously, Shimizu and Hagiwara (1973) were successful in obtaining rabbit antiserum specific to chicken embryonic type Hb, while not in obtaining antiserum specific to adult-type Hb. In the present work, antibody specific to the minor component of adult-type Hb (anti-adult-Hb), as well as that to the major components of embryonic type Hb (anti-embryonic-Hb), was successfully obtained. It was, however, very difficult to obtain completely absorbed antibodies with sufficiently high titer. Fig. 2A shows that Hbs from lo- and l&day embryos did not react with antiembryonic-Hb, although immunologically embryonic type Hb was clearly detectable in lo-day chicken embryos with absorbed antiserum on agar gel immunodiffusion tests (Shimizu and Hagiwara, 1973). Fig. 2B also represents that Hbs from lo- and 15-day embryos did not react with antiadult-Hb, though adult-type Hb components are sufficiently detectable in those embryos by electrophoresis or chromatography. The reasons why Hbs from lo- and 15-day embryos did not react clearly with
Types
erythrocytes, from (B) Staining
323
adult chickwith anti-
absorbed antibodies were not proved in the present work. The agar gel immunodiffusion test given in Fig. 3A shows that embryonic major Hb components (E-I and E-11) were apparently distinguished from embryonic minor (EIII) and both adult Hb components (A-I and A-II), implying that embryonic major Hb components possess unique globin chain (or chains), which other Hb components do not have. The reasons why antiembryonic-Hb produced three precipitin lines in the reaction with embryonic major Hb components were not clear. Further studies were expected to make clear the chemical properties of embryonic type Hb components. In the previous work, Shimizu and Hagiwara (1973) failed to obtain the antiserum that enables us to distinguish adult major Hb from adult minor Hb. Brown and Ingram (1974) reported, however, that antigenie determinants of Hb D (adult minor Hb or A-I) and Hb A (adult major Hb or AII) are similar in the reaction with antiHb-D serum, but anti-Hb-A serum contains antibodies for such antigenic determi-
324
DEVELOPMENTALBIOLOGY
nants on Hb A that are not present in Hb D. Figure 3B shows that only adult minor Hb (A-1) gave a faint precipitin line in the reaction with anti-adult-Hb, implying that antigenic determinants of adult minor Hb are different from those of adult major Hb. It was shown that two Hb components produced in the definitive erythrocytes of adult chickens are identical with the Hb components synthesized in the definitive erythrocytes found in the embryonic circulation (Bruns and Ingram, 1973a; Brown and Ingram, 1974; Moss and Hamilton, 1974). It was also demonstrated that embryonic definitive erythrocytes contain a minor Hb component, H, or fetal type Hb (Godet, Schiirch and Nigon, 1970; Shimizu, 1972a), in addition to two adult Hbs (Moss and Hamilton, 1974). One common polypeptide chain was detectable between adult major and fetal Hbs, while not between adult minor and fetal Hbs (Shimizu, 1972a; Moss and Hamilton, 1974). Furthermore, it was shown that embryonic minor and fetal Hbs share one polypeptide chain, but embryonic major and fetal Hbs do not share any chains (Shimizu, 1972a). Maniatis and Ingram (19711 found that tadpole erythrocytes were stained faintly with fluorescent antibodies against tadpole Hb, but frog erythrocytes did not stain with fluorescent antibodies against tadpole or frog Hbs, because of insufficient exposure of the Hb in the erythrocyte to the antibodies. However, they found that the positive fluorescent staining was obtained after pretreatment of the fixed smears with Tween-80. Similarly, primitive or definitive erythroid cells of late chicken embryos and adults were stained with fluorescent antibodies only after pretreatment of the fixed smears with Tween80, while primitive erythroid cells of early chicken embryos stained well with or without the pretreatment. All of the erythroid cells from the circulating blood of early embryos fluoresced green with anti-embryonic-Hb-FITC, as shown in Fig. 4A, while none of the cells fluoresced green with anti-adult-Hb-FITC,
VOLUME 48,
1976
as shown in Fig. 4B. Sometimes the nuclear portion of the erythroid cells from chicken embryos fluoresced white rather than green. This nuclear fluorescence was particularly frequent when immature cells were stained with antibodies. The interpretation of the biological meaning of such nuclear fluorescence so far remains for further study. All of the circulating erythrocytes from late embryos fluoresced green with antiadult-Hb-FITC, as shown in Fig. BB, while any of the cells did not fluoresce green with anti-embryonic-Hb-FITC, as shown in Fig. 8A. Cells stained with anti-adultHb-FITC from embryos, presumably definitive erythroid cells derived from yolk-sac, fluoresced not so obviously as cells stained from late embryos or adult chickens, presumably definitive cells derived from bone marrow. It is difficult to say that all of the definitive erythroid cells of yolk-sac origin stained with anti-adult-Hb-FITC as shown in Figs. 6B and 7B, probably because of the low sensitivity of the method used here with anti-adult-Hb. Table 1 gives the relative content of the erythrocytes stained with anti-embryonicHb-FITC or with anti-adult-Hb-FITC as the percentage of the total cells in the smears as compared with the percentage of primitive and definitive erythroid cells in the circulation (Bruns and Ingram, 1973a,b), and that of embryonic and adult type Hbs (Shimizu, 1972a) at a given age. The main finding of this report using specific fluorescent antibodies against embryonic and adult-type Hbs is that embryonic major Hbs and adult minor Hb are not present inside the same erythrocyte during ontogenesis. The result presented here strongly suggests that primitive erythroid cells, derived from yolk sac, synthesize exclusively embryonic type Hb, while definitive erythroid cells, derived from yolk sac or bone marrow, synthesize only adulttype Hb. The author is indebted to Professor T. S. Okada of Kyoto University for his critical reading of the man-
KOJI
Identification
SHIMIZU
of Hemoglobin
TABLE CHANGES
Days of incubation
4
IN CELL
Cells stained with anti-embryonic-HbFITC
POPULATIONS
Cells stained with antiadult-HbFITC
100
6 9
70 35 20
15 17
15
Adult a Each b After ’ After
75 100 100 100
0 0
number is presented Shimizu (1972a). Bruns and Ingram
as the percentage
Adult Type Hb*
0 0
100 61
39
25
75
3
97
0
97
0
100
ANTIBODIES~
Primitive erythroid cells’
Definitive erythroid cells’
0 o-5 18 26 70 75 87
100 95-100
82 74 30 25 13 0 0
100 100
0
100
of the total.
(1973a,b).
uscript. This work was supported in part by Research Grant No. 74275 from the Ministry of Education, Japan. REFERENCES D. W., SCHROEDER, W. A., and BALOG, J. (1958). Observation on the chromatographic heterogeneity of normal adult and fetal human hemoglobin: A study of the effects of crystallization and chromatography on the heterogeneity and isoleutine content. J. Amer. Chem. Sot. 80, 1628-1634. BETKE, K., and KLEIHAUER, E. (1958). Fetaler und bleibender Blutfarbstoff in Erythrozyten und Erythroblasten von menschlichen Feten und Neugebornen. Blut 41, 241-246. BROWN, J. L., and INGRAM, V. M. (1974). Structural studies on chick embryonic hemoglobins. J. Biol. Chem. 249, 3960-3972. BRUNS, G. A. P., and INGRAM, V. M. (1973a). Erythropoiesis in the developing chick embryo. Deuelop. Biol. 30, 455-459. BRUNS, G. A. P., and INGRAM, V. M. (1973b). The erythroid cells and haemoglobins of the chick embryo. Philos. Trans. Roy. Sot. London, Ser. B 266, 225-305. D’AMELIO, V. (1966). The globins of adult and embryonic chick hemoglobin, Biochim. Biophys. Acta 127, 59-65. DAN, M., and HAGIWARA, A. (1967). Detection of two types of hemoglobin (HbA and HbF) in single erythrocytes by fluorescent antibody technique. Jap. J. Human Genet. 12, 55-61. DRABKIN, D. L. (19491. A simplified technique for a large scale crystallization of human oxyhemoglobin Isomorphous transformations of hemoglobin and myoglobin in the crystalline state. Arch. Biothem. Biophys. 21, 224-231. ALLEN,
FLUORESCENT
100
50 60
10 0
WITH
Embryonic Type Hbb
10 11
1
STAINED
0 0
100
325
Types
FANTONI, KIND,
A.,
DE LA CHAPELLE,
A.,
CHUI,
D.,
RIF-
R. A., and MARKS, P. A. (1969). Control mechanism of the conversion from synthesis of embryonic to adult hemoglobin. Ann. N. Y. Acad. Sci. 165, 194-204. GITLIN, D., SASAKI, T., and VUOPIO, P. (1968). Immunochemical quantitation of proteins in single cells. I. Carbonic anhydrase B&chain hemoglobin and u-chain haemoglobin in some normal and abnormal erythrocytes. Blood 32, 796-810. G~DET, J., SCH~~RCH, D., and NIGON, V. (1970). Caracmrisation et evolution des hemoglobines dans le tours du developpement postembryonnaire chez la Poule. J. Embryol. Exp. Morph. 23, 153-167. HOSOI, T. (1965). Studies of hemoglobin F within single erythrocyte by fluorescent antibody technique. Exp. Cell Res. 37, 680-683. JURD, R. D., and MACLEAN, N. (1970). An immunofluorescent study of the haemoglobins in metamorphosing Xenopus laevis. J. Embryol. Exp. Morph. 23, 299-309. KAWAMURA, A. (19691. Preparations of materials. In “Fluorescent antibody techniques and their applications” (Kawamura, A., ed.), pp. 11-64. Univ. of Tokyo Press, Tokyo, Japan. KEANE, R. W., ABBOTT, U. K., BROWN, J. L., and INGRAM, V. M. (1974). Ontogeny of hemoglobins: Evidence for hemoglobin M. Develop. Biol. 38, 229-236. KLEIHAUER, E. F., TANG, T. E., and BETKE, K. (1967). Die intrazellulare verteilung von embryonalem haemoglobin in roten blutzellen menschlicher embryonen. Acta Haemat. 38, 264-272. LEMETZ, L. (1964). The blood of chick embryo: Quantitative embryology at a cellular level. In “Advances in Morphogenesis” (Abercrombie, M., and Brachet, J., eds.), Vol. 3, pp. 197-245. Academic Press, New York/London.
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DEVELOPMENTAL
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G. M., and INGRAM, V. M. (1971). Erythropoiesis during amphibian metamorphosis. III. Immunochemical detection of tadpole and frog hemoglobins (Rana catesbeianu) in single erythro-
MANIATIS,
cytes. J. Cell Biol. 49, 390-404. MANWELL, C., BAKER, C. M. A.,
and BETZ, T. W. (1966). Ontogeny of hemoglobin in the chicken. J. Embryol. Exp. Morph. 16, 65-79. Moss, B. A., and HAMILTON, E. A. (1974). Chicken definitive erythrocyte haemoglobins. Biochim. Biophys. Actu 371, 379-391. ROSENBERG, M. (19701. Electrophoretic analysis of hemoglobin and isozymes in individual vertebrate cells. Proc. Nut. Acad. Sci. USA 67. 32-36. SCHALEKAMP, M., SCHALEKAMP, M., VAN GOOR, D., and SLINGERLAND, R. (1972). Re-evaluation of the presence of multiple haemoglobins during the ontogenesis of the chicken. J. Embryol. Exp. Morph. 28, 681-713. SHIMIZU, K. (1972al. Ontogeny of chicken hemoglobin. I. Electrophoretic study of the heterogeneity
VOLUME
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in development. Develop. Growth 14, 43-55. (1972b). Ontogeny of chicken hemoglobin. II. Chromatographic study of the heterogeneity of hemoglobin in development. Deuelop. Growth Differentiation 14, 281-295. SHIMIZU, K., and HAGIWARA, A. (1973). Ontogeny of chicken hemoglobin. III. Immunological study of the heterogeneity of hemoglobin in development. Develop. Growth Differentiation 15, 285-306. TOMODA, Y. (1964). Demonstration of fetal erythrocyte by immunofluorescent staining. Nature (LenDifferentiation SHIMIZU, K.
don) WILT,
202,910-911.
F. H. (19621. The ontogeny of chick embryo hemoglobin. Proc. Nut. Acud. Sci. USA 48, 1582-
1589. WILT, F.
H. (1967). The control of embryonic hemoglobin synthesis. In “Advances in Morphogenesis” (Abercrombie, M., and Bracket, J., eds.), Vol. 6, pp. 89-125. Academic Press, New York/London.