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Hemoglobin Types in Chick Embryos with Different Adult Hemoglobin Genotypes
464
D. B. KING
1964. Endocrine control of the adrenals in chickens. Endocrinology, 75: 192-200. Snedecor, G. W., 1956. Statistical Methods. 5th ed., The Iowa State College Press.
Staehelin, M., P. Barthe and P. A. Desaulles, 196S. On the mechanism of the adrenal gland response to adrenocorticotrophic hormone in hypophysectomized rats. Acta Endocrinol. 50: 55-64.
Hemoglobin Types in Chick Embryos with Different Adult Hemoglobin Genotypes C. R. DENMARK AND K. W. WASHBURN Department of Poultry Science, University of Georgia, Athens, Georgia 30601 (Received for publication August 15, 1968)
T HAS been demonstrated that genetic variation exists in hemoglobin types of the hatched chick. The hemoglobin types have been designated as Type I (homozygous normal), Type II (homozygous abnormal) and Type III (heterozygote) by Washburn (1968a). A "major band" is present in all birds in combination with either or both of two "minor", more acidic bands. The bands differ in their electrophoretic properties; the abnormal "minor" band migrates in an electrical field at a faster rate than that of the normal "minor" hemoglobin component. The differences in electrophoretic properties of the minor hemoglobin components are due to allelic, codominant genes (Washburn, 1968a). The adult hemoglobin type can vary, depending on the genotype of the bird. This variation could explain some of the disagreement found in the literature pertaining to the normal hemoglobin types of chick embryos, when the adult type hemoglobin (Hb) appears, and which components should be considered as embryonic. D'Amelio and Salvo (1961), using electrophoretic and immunoelectrophoretic analyses, found two distinct embryonic Hb components present at 68 hours of emUniversity of Georgia College of Agriculture Experiment Stations Journal Series Paper number 323, College Station, Athens.
bryonic development. By 88 hours of incubation, they found three additional hemoglobin fractions which they stated are identical to those present in hemoglobin from adult chickens. By the eleventh day of incubation, they reported that only traces of the embryonic hemoglobin were detectable in the embryo and that the adult type hemoglobins were the dominant respiratory blood proteins. Manwell et al. (1963) found that three and four day-old embryos had a distinctly different hemoglobin from that of late embryos or adults. Prior to the fifth day of incubation, only the two embryonic hemoglobin components were visible; but by the seventh day approximately one-half of the hemoglobin was of the adult type. In agreement with D'Amelio and Salvo (1961) they observed only traces of embryonic hemoglobin remaining after eleven days of incubation. The only apparent difference between their studies and those of D'Amelio and Salvo (1961), was whether there were three rather than two hemoglobin components in the adult chicken. Manwell et al. (1963) could detect only two adult components, both of which were visible by the seventh day of embryogenesis. They also found that there was no difference in the electrophoretic behavior or distribution of the hemoglobins between the various breeds of birds tested.
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I
H E M O G L O B I N T Y P E S I N EMBRYOS
imum of five hemoglobin components which differ from one another primarily in the structure of one polypeptide subunit and in concentration present. T h e y also found t h a t the five-day embryo possesses at least three distinct hemoglobin types, and these embryonic hemoglobin types differ from the adult in the structure of both polypeptide subunit classes. T h e purpose of this study was to determine the embryonic hemoglobin types and the approximate time of switch-over from production of embryonic to adulttype hemoglobin in chick embryos with different adult hemoglobin. MATERIALS AND METHODS The three groups of embryos used in this study were obtained from matings of an Athens-Canadian randombred subpopulation which was homozygous for the codominant hemoglobin alleles, described b y Washburn (1968a). These alleles result in two different minor hemoglobin components. Heterozygous embryos were obtained b y crossing populations pure breeding for these alleles. All types have the same major component. Pedigreed eggs were collected three times daily and immediately stored at 10-15°C. for a time period not exceeding two weeks. Incubation of the eggs was carried out in a forced-air incubator for 4, 5, 6, 7, 9, 11, 13, 15,17,19 or 21 days. After incubation for the desired periods, the eggs were candled to assure embryonic development and blood samples obtained at the same time of day for the prescribed periods. The samples were obtained b y opening the shell in the vicinity of the air sac and carefully dissecting away the shell membrane, using fine point forceps to assure t h a t the embryo or extra-embryonic membranes were not injured. T h e hole was then made larger by continued breaking of the shell radially from the original
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Fraser (1964), using electrophoresis on cellulose acetate, reported two readily detectable hemoglobins in the five-day chick embryo. I n the seven-day embryo and in all older stages three components were found, two of which were the same as those from the five-day embryos. H e concluded t h a t all three hemoglobins m a y be present in the five-day embryo, b u t t h a t the concentration of one is so low t h a t it is not detectable. Fraser (1961) separated all embryonic and adult chicken hemoglobin samples into two components, using both electrophoresis and carboxy methyl-cellulose chromotography. T h e only developmental difference reported was t h a t the relative amounts of the two components varied; five-day and sevenday embryos had 84 percent of the more acidic hemoglobin component which he termed "embryonic hemoglobin" and 16 percent of the more "basic hemoglobin". These proportions changed gradually until both newly hatched chicks and adults had 28 percent of the "embryonic hemoglobin" and 72 percent of the " a d u l t hemoglobin". T h e results of Huisman and Van Veen (1964) demonstrated the presence of three hemoglobin components in the red blood cells of the embryo. T h e y reported t h a t two of the three components were present in the adult chicken and t h a t the third, a slower-moving fraction, disappeared gradually from the blood after hatching. Using agar gel, starch gel, and immunoelectrophoresis, Wilt (1962) reported two kinds of hemoglobin in the adult chicken which were also present in two and three day old embryos. A third immunologically distinct globin-like component persisted only for 48 hours of development. This component behaved electrophoretically in the same way as the major adult component although it was immunologically different. Hashimoto and Wilt (1966) have reported t h a t the adult chicken contains a min-
465
466
C.
R.
D E N M A R K AND K.
opening. Once the hole was sufficiently large to expose the extra-embryonic vessels, a 75 mm. heparinized capillar}' tube, which had been pulled on heating to a fine point, was used to prick the largest of exposed vessels and to collect the escaping blood. T h e samples of blood collected in the heparinized capillary tubes were washed with physiological saline in the
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early stages of the experiment; however, this was omitted in later stages because the results obtained were the same as with nonwashed samples. Each time blood from several different embryos of each group was pooled until the thirteenth day of incubation. Normally, fourteen four-day embryos per group were needed to provide sufficient
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WASHBURN
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DAYS OF INCUBATION 7
11
I'IO. 1. Hemoglobin bands observed in three different hemoglobin genotypes of various ages of the embryo. Type I. Homozygous for allele producing normal minor band. Type H. Homozygous for allele producing abnormal minor band. Type III. Heterozygous for both alleles. Ei=embryonic band t ; M = major adult band; E> = embryonic band 2; T = t r a c e band; nii = normal minor band; ms=abnormal minor band. t = point of application. — = cathode. -)- = anode.
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I
W.
467
HEMOGLOBIN T Y P E S IN EMBRYOS
TYPE
I
5 DAY
TYPE
I
7 DAY
u
TYPE
U
7 DAY
5 DAY
in
5 DAY
TYPE rrx 7 DAY
TYPE TH 11 DAY
FIG. 2. Analytrol tracings of hemoglobin bands observed in Figure 1.
blood for a concentrated hemoglobin sample with a decreasing number needed as the age of the embryos increased. The filled capillar}' tubes were centrifuged for three minutes at 11,500 R P M to separate the erythrocytes from the plasma. A modification of the semi-micro method of preparing hemoglobin samples, as well as the method for electrophoretic analysis, was then used (Washburn, 1968b). RESULTS AND DISCUSSION
Description of hemoglobin bands: The number of different hemoglobin bands observed in the developing chick embryo varied from a minimum of three to a
maximum of six, depending on the adult hemoglobin genotype and the developmental stage of the embryo. The variation in the presence or absence of the bands could be divided into three time periods, within which the number of bands were the same for a particular genotype, but differed between genotypes. These periods were 3-5 days, 6-7 days, and 11-21 days of incubation and are illustrated in Figure 1 and Figure 2 by hemograms and analytrol tracings obtained at 5, 7 and 11 days of incubation. For the three-five day period, the minimum number of hemoglobin bands for all three genotypes were observed. Those em-
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TYPE
TYPE
468
C. R. DENMARK AND K. W.
addition to those bands previously observed. By the seventh day of incubation, the percentage concentration of the mi and m2 components in the respective genotypes was greatly increased so that it was distinctly visible in all samples. A gradual change in the electrophoretic mobility of the M-major band and/or the E2-embryonic band which had been observed from the third through the ninth day of incubation caused these hemoglobin fractions to become indistinguishable after the ninth day of incubation. The electrophoretic mobility of the Ttrace band which had been observed in both the homozygous abnormal and the heterozygote at 3 days gradually changed so that by the eleventh day of incubation it was indistinguishable from that of the mi-minor band in heterozygous embryos. In homozygous abnormal embryos the
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DAYS OF INCUBATION FIG.
3, Percentage concentration of embryonic hemoglobin component (Ei) at various ages of the embryo.
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bryos of adult hemoglobin Type I (normal homozygote) showed three distinct hemoglobin components: embryonic band 1 (Ei), the adult major band (M), and a previously unreported band, embryonic band 2 (E 2 ). Those embryos with adult hemoglobin Type II (abnormal homozygote) were observed to have the Ei-embryonic band, M-major band and a band which had previously been designated as a "Trace" adult band (T) by Washburn (1968b). The E2-embryonic band was absent in all embryos of this genotype. All the bands observed in both the homozygous normal and homozygous abnormal were observed in embryos with adult h e moglobin Type III (heterozygote). Starting at six days of incubation, small amounts of the minor adult hemoglobin components (mi and m2) could be determined in the respective genotypes, in
WASHBURN
469
H E M O G L O B I N T Y P E S I N EMBRYOS TABLE 1.-
Source Genotype (G) Period (P) PXG Error Total
-A nalysis of variance of concentration in various hemoglobin bands from three (liferent hemoglobin genotypes over a 21 day incubation period
M.S.
M M.S.
.46 1,465.95** 10.70** 2.68
168.59** 409.71** 43.48** 5.63
d.f. 2 10 20 231 263
E2 M.S.
T M.S.
370.53** 965.70** 66.51** 1.59
920.30** 164.61** 9.92** 2.91
d.f. 1 10 10 154 175
d.f. 2 10 20 231 263
Minor 1 M.S. 125.14** 4,168.19** 17.65** 3.00
*P<.05 **P<.01 1 Minor bands were combined in heterozygote.
TABLE 2.—Percent concentration of the various
Type I Type II Type I I I
E!
M
14.09 13.94 14.02
58.68 61.45 60.12
bryonic band over the entire experimental period was 14.09 for the T y p e I, 13.94 for the Type I I , and 14.02 for the T y p e I I I (Table 2). Major band (M): T h e percentage concentration of the major adult band (M) for the three hemoglobin genotypes from embryos of various ages is shown in Figure 4. There was a sharp decline in the concentration of the (M) major band for all three hemoglobin types at the sixth and seventh day of incubation; thereafter, the percent concentration gradually increased for the remainder of the experimental period. T h e average percent concentration for the genotypes were 58.68 for T y p e I, 61.45 for T y p e I I , and 60.12 for T y p e I I I (Table 2). The differences between types as well as differences between ages and age-genotype interaction were all found t o be highly significant (Table 1). These results are in agreement with those of Washburn (1968b), who found the average percent concentration of the (M) major band of T y p e I I to be greater than t h a t of T y p e I and I I I for a 365 day period, with the heterozygote (Type I I I ) being intermediate. in bands averaged over the experimental period mi
7.05 4.15
ni2
mi+m2
17.88 7.29
19.55
20.19 6.73 2.16
12.26
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(T) trace band was present at all stages since the mutation in the minor band changed its electrophoretic properties and thus did not mask it. Differences in concentration of hemoglobin components: Embryonic band 1 (Ei): T h e Ei-embryonic band was present in an easily detectable quantity at 3 days of incubation and at all other ages of the chick embryo of all three hemoglobin types. As shown in Figure 3, the percent concentration of the embryonic band was greatest at the sixth day of incubation with the concentration decreasing rapidly thereafter for all three hemoglobin genotypes. There were no differences in concentration between the genotypes for the first thirteen days of incubation. Thereafter, the concentration in the homozygous normal (Type I) was consistently higher t h a n t h a t of T y p e I I and I I I , with the heterozygote (Type I I I ) being intermediate. T h e differences resulted in a highly significant age by genotype component of variance with no significant differences between the three types over the experimental period (Table 1). The average percent concentration for the Ei-em-
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DAYS OF INCUBATION FIG. 4. Percentage concentration of the major hemoglobin component (M) at various ages of the embryo. Embryonic band 2 (£2): T h e E 2 -embryonic band was found in only T y p e I and I I I genotypes. This band was present at 3 days of incubation in both types. There were marked differences in the percentage composition of the E2 component u p through the seventh day of incubation in T y p e I and I I I embryos (Figure 5). Since the (E 2 ) embryonic band was not separable from t h a t of the (M) major band after 7 days, the percent concentration was taken to be zero in both types throughout the remainder of the experimental period. T h e lack of separation of the (E 2 ) embryonic, band from the (M) major band after 7 days was apparently due to changes in the electrophoretic migration of the (E 2 ) embryonic band a n d / o r the (M) major band observed from the third through the seventh day of inclubation in T y p e I and I I I hemograms. T h e average percent concentration of the E 2 -embryonic band for the two genotypes was 7.05 for T y p e I and 4.15 for
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FIG. 5. Percentage concentration of embryonic hemoglobin component (E2) at various ages of the embryo.
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O Q
471
HEMOGLOBIN T Y P E S IN EMBRYOS
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DAYS OF INCUBATION FIG. 6. Percentage concentration of the trace band (T) at various ages of the embryo.
T y p e I I I (Table 2). These differences were found to be highly significant (Table 1). When recalculated on the basis of no (T)band, the average percent concentration of the (E 2 ) embryonic band of Type I I I embryos was 4.38. Therefore, the higher relative concentration of the E2-embryonic band in T y p e I embryos was apparently not caused by the additional band (Ttrace band) in T y p e I I I embryos. Trace band (T): T h e (T) trace band was found only in birds with T y p e I I and I I I genotypes. As shown in Table 2 and Figure 6, the percentage concentration for the T-trace band in T y p e I I hemograms (6.73%) was much higher t h a n t h a t of T y p e I I I (2.16%). This resulted in highly significant differences between types (Table 1). There were also highly significant differences between ages and an agegenotype interaction (Table 1). As shown
in Figure 6, the percent concentration decreased sharply in both types. By the eleventh day of incubation, the (T) trace band had become indistinguishable from t h a t of the (mi) minor band in T y p e I I I hemograms; therefore, the percent concentration in this type was taken to be zero throughout the remainder of the experimental period. The percentage concentration of the (T) trace band in T y p e I I embryos progressively decreased after the fifth day of incubation, and by hatching comprised approximately 2.6 percent of the total hemoglobin. This value compares favorably with the 1.7 percent in the hatched chick and the adult as reported b y Washburn (1968b). Minor components {m\ and nia): T h e percentage composition of the minor components was analyzed in two ways. First, the total amount of the minor components
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u. O
472
C. R. DENMARK AND K.
W.
WASHBURN
' 4 5 6 7
17 19 21 DAYS OF I N C U B A T I O N
i-
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FIG. 7a. Percentage concentration of the minor hemoglobin component (mi) at various ages of the embryo.
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was compared. This included comparisons between the (mi) minor component of Type I, (m2) minor component of Type II, and the (mi+m 2 ) minor components of Type III. Secondly, the concentration of individual bands were compared. This included comparisons of the (mi) minor band between Type I and the (m2) minor band of Type II as well as comparisons of the (mi) and (m2) bands within the heterozygote. When the total concentrations of the minor bands are compared (Figure 7 a and 7b), it is evident that the concentration of the minor band (mi) normally found in the embryo is considerably higher than that of the abnormal (m2) minor band
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FIG. 7b. Percentage concentration of the minor hemoglobin component (m2) at various ages of the embryo.
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throughout most of the experiment. These differences persist after hatching (Washburn, 1968b). The differences in concentration of these components were not great until after the ninth day of incubation. When the average concentration of the (mi) minor band of the normal homozygote is compared with the total concentration (average) of the two minor components in the heterozygote, the concentration of the (mx) minor band of the normal hemoglobin type is greater than the combined concentration of the (mi+m 2 ) minor bands in the heterozygote. The concentration of the (m2) minor band is significantly lower than that of the (mx) minor band in comparison between homozygous types and comparisons of these bands within the heterozygote. These results support the con-
HEMOGLOBIN T Y P E S IN EMBRYOS
I t would appear from these results t h a t the embryonic hemoglobin in the domestic fowl consist of the (Ei) embryonic band in all chickens and the (E 2 ) or the (T) band (depending on the genotype), while the (mi) and (m 2 ) minor bands represent the adult hemoglobin components. T h e TABLE 3.—Comparison oj various hemoglobin bands in chick embryos with different adult hemoglobin genotypes
Type I Type 11 Type 111
Ei
M
E2
+ + +
+ + +
+
_
+
+ +
+ =band present. — =band absent.
—
T
mi
m2
+
_
+
+ +
—
(M) major band is present at a relatively high concentration at 3 days of embryonic development. However, its concentration increases and is maintained at a high level throughout the adult life. The percentage concentration of the (Ei) embryonic band began to decrease in all three genotypes after the sixth day of incubation, and b y hatching comprised only approximately 6 percent of the total hemoglobin. T h e percentage concentration of the (T) trace band progressively decreased after the fifth day of incubation in T y p e I I and Type I I I embryos respectively. Previous investigation, using only T y p e I embryos have concluded that an embryonic band (presumably E 2 ) is replaced by the (M) adult band. However, our results demonstrate t h a t the (M) adult band exists from 3 days of incubation throughout the development of the embryo. The observed change in the electrophoretic mobility of the (M) adult band a n d / o r the (E 2 ) embryonic band was the apparent cause for the disappearance of the (E 2 ) embryonic band. This is supported by comparison of the normal (Type I) and m u t a n t type (Type I I ) . Both the (M) adult band and the (E 2 ) embryonic band exist in the normal type, whereas only the (M) adult band is present in the m u t a n t type. SUMMARY
A study was conducted to determine the hemoglobin types in chick embryos with different adult hemoglobin genotypes and the approximate time of switch-over from the production of embryonic to the production of the adult hemoglobin. Using cellulose acetate electrophoresis, a minim u m of three to a maximum of six different hemoglobin bands were observed in the developing embryo, depending on the adult hemoglobin genotype and the developmental stage of the embryo. For all
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elusions of Washburn (1968b), who showed t h a t the m u t a n t gene resulting in the production of the abnormal (m2) minor hemoglobin component does not function as efficiently in producing hemoglobin as does the normal allele. I t was evident from this study t h a t there is a definite association of the (E 2 ) embryonic band with the normal minor component (mi) and the (T) trace band with the abnormal minor component (m 2 ). Since the heterozygote contains both minor components (mi and m2) both the (E 2 ) embryonic band and (T) trace band were also found to be present (Table 3). The (mi) and (m2) minor components first appeared in their respective genotypes in the hemograms of 6 day old embryos and progressively increased in concentration to approximately the thirteenth day of incubation. Therefore, the approximate time of switch-over from the production of embryonic hemoglobin to the production of adult hemoglobin was observed to be between the fifth and sixth day of incubation in all genotypes, which is in agreement with the results of Manwell et al. (1963), Fraser (1964), Hashimoto and Wilt (1966).
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C. R. DENMARK AND K.
After 9 days of incubation, the (E2) embryonic band became indistinguishable from the (M) major band in Type I and III embryos. The (T) trace band became indistinguishable from the (mi) minor com-
WASHBURN
ponent in heterozygous embryos (Type III). This study indicated a definite association of the (E2) embryonic band with the normal minor component (mi) and the (T) trace band with the abnormal minor component (m2). The heterozygote was found to have both the (E2) embryonic band and (T) trace band, since it contains both minor components (mi and m2). REFERENCES D'Amelio, V., and A. M. Salvo, 1961. Further studies on the embryonic chick hemoglobin. An electrophoretic and immunoelectrophoretic analysis. Acta. Embryol. Morph. Exper. 4: 250-259. Fraser, R. C , 1961. Hemoglobin formation in the chick embryo. Exp. Cell. Res. 25: 418^27. Fraser, R. C , 1964. Electrophoretic characteristics and cell content of the hemoglobins of developing chick embryos. J. Exp. Zool. 156: 185-196. Hashimoto, K., and F. H. Wilt, 1966. The heterogeneity of chicken hemoglobin. Proc. Nat. Acad. Sci. 56: 1477-1483. Huisman, T. H. J., and J. M. Schillhorn Van Veen, 1964. Studies on animal hemoglobins. III. The possible role of intercellular inorganic phosphate on the oxygen equilibrium of the hemoglobin in the developing chicken. Biochimica et Biophysica acta. 88: 367-374. Manwell, C , C. M. A. Baker, J. B . Roslansky, and M. Foght, 1963. Molecular genetics of avian proteins. II. Control genes and structural genes for embryonic and adult hemoglobins. Proc. Nat. Acad. Sci. 49: 496-503. Washburn, K. W., 1968a. Inheritance of an abnormal hemoglobin in a random-bred population of domestic fowl. Poultry Sci. 47: 561-564. Washburn, K. W., 1968b. Effects of age of bird and hemoglobin type on the concentration of adult hemoglobin components of the domestic fowl. Poultry Sci. 47: 1083-1089. Wilt, F. H., 1962. The ontogeny of chick embryo hemoglobin. Proc. Nat. Acad. Sci. 48: 1582-1590.
NEWS AND NOTES {Continued from page 453) to hold the 1974 World's Poultry Congress in the United States. The proposed host city is Chicago, Illinois. The next meeting of the U.S.A. Branch is sched-
uled to be held during the annual meeting of the Poultry Science Association next August in Fort Collins, Colorado, at the Colorado State University.
(Continued on page 523)
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three genotypes, the minimum number of bands was observed from 3-5 days of incubation; the maximum number from 6-7 days. From 11-21 days of incubation, the number and type of bands found were the same as for the hatched chick. From 3-5 days of incubation, Type I embryos (normal homozygote) had three distinct components: (Ei) embryonic band, (M) major band and (E2) embryonic band. Type II embryos (abnormal homozygote) had the (Ei) embryonic band, (M) major band, and a (T) trace band, but no (E2) embryonic band. All the bands observed in Type I and II embryos were seen in the heterozygote (Type III). At six days of incubation, the minor adult hemoglobin components (mi and m2) first appeared in their respective genotypes and rapidly increased in concentration to approximately the thirteenth day of incubation. After the sixth day, the concentration of the (Ei) embryonic band, (E2) embryonic band and (T) trace band progressively decreased throughout the remainder of the experimental period. Therefore, the approximate time of switch-over from the production of embryonic hemoglobin to the production of the adult hemoglobin was observed to be between the fifth and sixth day of incubation in all genotypes.
W.
D. B. KING
1964. Endocrine control of the adrenals in chickens. Endocrinology, 75: 192-200. Snedecor, G. W., 1956. Statistical Methods. 5th ed., The Iowa State College Press.
Staehelin, M., P. Barthe and P. A. Desaulles, 196S. On the mechanism of the adrenal gland response to adrenocorticotrophic hormone in hypophysectomized rats. Acta Endocrinol. 50: 55-64.
Hemoglobin Types in Chick Embryos with Different Adult Hemoglobin Genotypes C. R. DENMARK AND K. W. WASHBURN Department of Poultry Science, University of Georgia, Athens, Georgia 30601 (Received for publication August 15, 1968)
T HAS been demonstrated that genetic variation exists in hemoglobin types of the hatched chick. The hemoglobin types have been designated as Type I (homozygous normal), Type II (homozygous abnormal) and Type III (heterozygote) by Washburn (1968a). A "major band" is present in all birds in combination with either or both of two "minor", more acidic bands. The bands differ in their electrophoretic properties; the abnormal "minor" band migrates in an electrical field at a faster rate than that of the normal "minor" hemoglobin component. The differences in electrophoretic properties of the minor hemoglobin components are due to allelic, codominant genes (Washburn, 1968a). The adult hemoglobin type can vary, depending on the genotype of the bird. This variation could explain some of the disagreement found in the literature pertaining to the normal hemoglobin types of chick embryos, when the adult type hemoglobin (Hb) appears, and which components should be considered as embryonic. D'Amelio and Salvo (1961), using electrophoretic and immunoelectrophoretic analyses, found two distinct embryonic Hb components present at 68 hours of emUniversity of Georgia College of Agriculture Experiment Stations Journal Series Paper number 323, College Station, Athens.
bryonic development. By 88 hours of incubation, they found three additional hemoglobin fractions which they stated are identical to those present in hemoglobin from adult chickens. By the eleventh day of incubation, they reported that only traces of the embryonic hemoglobin were detectable in the embryo and that the adult type hemoglobins were the dominant respiratory blood proteins. Manwell et al. (1963) found that three and four day-old embryos had a distinctly different hemoglobin from that of late embryos or adults. Prior to the fifth day of incubation, only the two embryonic hemoglobin components were visible; but by the seventh day approximately one-half of the hemoglobin was of the adult type. In agreement with D'Amelio and Salvo (1961) they observed only traces of embryonic hemoglobin remaining after eleven days of incubation. The only apparent difference between their studies and those of D'Amelio and Salvo (1961), was whether there were three rather than two hemoglobin components in the adult chicken. Manwell et al. (1963) could detect only two adult components, both of which were visible by the seventh day of embryogenesis. They also found that there was no difference in the electrophoretic behavior or distribution of the hemoglobins between the various breeds of birds tested.
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I
H E M O G L O B I N T Y P E S I N EMBRYOS
imum of five hemoglobin components which differ from one another primarily in the structure of one polypeptide subunit and in concentration present. T h e y also found t h a t the five-day embryo possesses at least three distinct hemoglobin types, and these embryonic hemoglobin types differ from the adult in the structure of both polypeptide subunit classes. T h e purpose of this study was to determine the embryonic hemoglobin types and the approximate time of switch-over from production of embryonic to adulttype hemoglobin in chick embryos with different adult hemoglobin. MATERIALS AND METHODS The three groups of embryos used in this study were obtained from matings of an Athens-Canadian randombred subpopulation which was homozygous for the codominant hemoglobin alleles, described b y Washburn (1968a). These alleles result in two different minor hemoglobin components. Heterozygous embryos were obtained b y crossing populations pure breeding for these alleles. All types have the same major component. Pedigreed eggs were collected three times daily and immediately stored at 10-15°C. for a time period not exceeding two weeks. Incubation of the eggs was carried out in a forced-air incubator for 4, 5, 6, 7, 9, 11, 13, 15,17,19 or 21 days. After incubation for the desired periods, the eggs were candled to assure embryonic development and blood samples obtained at the same time of day for the prescribed periods. The samples were obtained b y opening the shell in the vicinity of the air sac and carefully dissecting away the shell membrane, using fine point forceps to assure t h a t the embryo or extra-embryonic membranes were not injured. T h e hole was then made larger by continued breaking of the shell radially from the original
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Fraser (1964), using electrophoresis on cellulose acetate, reported two readily detectable hemoglobins in the five-day chick embryo. I n the seven-day embryo and in all older stages three components were found, two of which were the same as those from the five-day embryos. H e concluded t h a t all three hemoglobins m a y be present in the five-day embryo, b u t t h a t the concentration of one is so low t h a t it is not detectable. Fraser (1961) separated all embryonic and adult chicken hemoglobin samples into two components, using both electrophoresis and carboxy methyl-cellulose chromotography. T h e only developmental difference reported was t h a t the relative amounts of the two components varied; five-day and sevenday embryos had 84 percent of the more acidic hemoglobin component which he termed "embryonic hemoglobin" and 16 percent of the more "basic hemoglobin". These proportions changed gradually until both newly hatched chicks and adults had 28 percent of the "embryonic hemoglobin" and 72 percent of the " a d u l t hemoglobin". T h e results of Huisman and Van Veen (1964) demonstrated the presence of three hemoglobin components in the red blood cells of the embryo. T h e y reported t h a t two of the three components were present in the adult chicken and t h a t the third, a slower-moving fraction, disappeared gradually from the blood after hatching. Using agar gel, starch gel, and immunoelectrophoresis, Wilt (1962) reported two kinds of hemoglobin in the adult chicken which were also present in two and three day old embryos. A third immunologically distinct globin-like component persisted only for 48 hours of development. This component behaved electrophoretically in the same way as the major adult component although it was immunologically different. Hashimoto and Wilt (1966) have reported t h a t the adult chicken contains a min-
465
466
C.
R.
D E N M A R K AND K.
opening. Once the hole was sufficiently large to expose the extra-embryonic vessels, a 75 mm. heparinized capillar}' tube, which had been pulled on heating to a fine point, was used to prick the largest of exposed vessels and to collect the escaping blood. T h e samples of blood collected in the heparinized capillary tubes were washed with physiological saline in the
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early stages of the experiment; however, this was omitted in later stages because the results obtained were the same as with nonwashed samples. Each time blood from several different embryos of each group was pooled until the thirteenth day of incubation. Normally, fourteen four-day embryos per group were needed to provide sufficient
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TYPE I
WASHBURN
M
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DAYS OF INCUBATION 7
11
I'IO. 1. Hemoglobin bands observed in three different hemoglobin genotypes of various ages of the embryo. Type I. Homozygous for allele producing normal minor band. Type H. Homozygous for allele producing abnormal minor band. Type III. Heterozygous for both alleles. Ei=embryonic band t ; M = major adult band; E> = embryonic band 2; T = t r a c e band; nii = normal minor band; ms=abnormal minor band. t = point of application. — = cathode. -)- = anode.
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I
W.
467
HEMOGLOBIN T Y P E S IN EMBRYOS
TYPE
I
5 DAY
TYPE
I
7 DAY
u
TYPE
U
7 DAY
5 DAY
in
5 DAY
TYPE rrx 7 DAY
TYPE TH 11 DAY
FIG. 2. Analytrol tracings of hemoglobin bands observed in Figure 1.
blood for a concentrated hemoglobin sample with a decreasing number needed as the age of the embryos increased. The filled capillar}' tubes were centrifuged for three minutes at 11,500 R P M to separate the erythrocytes from the plasma. A modification of the semi-micro method of preparing hemoglobin samples, as well as the method for electrophoretic analysis, was then used (Washburn, 1968b). RESULTS AND DISCUSSION
Description of hemoglobin bands: The number of different hemoglobin bands observed in the developing chick embryo varied from a minimum of three to a
maximum of six, depending on the adult hemoglobin genotype and the developmental stage of the embryo. The variation in the presence or absence of the bands could be divided into three time periods, within which the number of bands were the same for a particular genotype, but differed between genotypes. These periods were 3-5 days, 6-7 days, and 11-21 days of incubation and are illustrated in Figure 1 and Figure 2 by hemograms and analytrol tracings obtained at 5, 7 and 11 days of incubation. For the three-five day period, the minimum number of hemoglobin bands for all three genotypes were observed. Those em-
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TYPE
TYPE
468
C. R. DENMARK AND K. W.
addition to those bands previously observed. By the seventh day of incubation, the percentage concentration of the mi and m2 components in the respective genotypes was greatly increased so that it was distinctly visible in all samples. A gradual change in the electrophoretic mobility of the M-major band and/or the E2-embryonic band which had been observed from the third through the ninth day of incubation caused these hemoglobin fractions to become indistinguishable after the ninth day of incubation. The electrophoretic mobility of the Ttrace band which had been observed in both the homozygous abnormal and the heterozygote at 3 days gradually changed so that by the eleventh day of incubation it was indistinguishable from that of the mi-minor band in heterozygous embryos. In homozygous abnormal embryos the
29
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DAYS OF INCUBATION FIG.
3, Percentage concentration of embryonic hemoglobin component (Ei) at various ages of the embryo.
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bryos of adult hemoglobin Type I (normal homozygote) showed three distinct hemoglobin components: embryonic band 1 (Ei), the adult major band (M), and a previously unreported band, embryonic band 2 (E 2 ). Those embryos with adult hemoglobin Type II (abnormal homozygote) were observed to have the Ei-embryonic band, M-major band and a band which had previously been designated as a "Trace" adult band (T) by Washburn (1968b). The E2-embryonic band was absent in all embryos of this genotype. All the bands observed in both the homozygous normal and homozygous abnormal were observed in embryos with adult h e moglobin Type III (heterozygote). Starting at six days of incubation, small amounts of the minor adult hemoglobin components (mi and m2) could be determined in the respective genotypes, in
WASHBURN
469
H E M O G L O B I N T Y P E S I N EMBRYOS TABLE 1.-
Source Genotype (G) Period (P) PXG Error Total
-A nalysis of variance of concentration in various hemoglobin bands from three (liferent hemoglobin genotypes over a 21 day incubation period
M.S.
M M.S.
.46 1,465.95** 10.70** 2.68
168.59** 409.71** 43.48** 5.63
d.f. 2 10 20 231 263
E2 M.S.
T M.S.
370.53** 965.70** 66.51** 1.59
920.30** 164.61** 9.92** 2.91
d.f. 1 10 10 154 175
d.f. 2 10 20 231 263
Minor 1 M.S. 125.14** 4,168.19** 17.65** 3.00
*P<.05 **P<.01 1 Minor bands were combined in heterozygote.
TABLE 2.—Percent concentration of the various
Type I Type II Type I I I
E!
M
14.09 13.94 14.02
58.68 61.45 60.12
bryonic band over the entire experimental period was 14.09 for the T y p e I, 13.94 for the Type I I , and 14.02 for the T y p e I I I (Table 2). Major band (M): T h e percentage concentration of the major adult band (M) for the three hemoglobin genotypes from embryos of various ages is shown in Figure 4. There was a sharp decline in the concentration of the (M) major band for all three hemoglobin types at the sixth and seventh day of incubation; thereafter, the percent concentration gradually increased for the remainder of the experimental period. T h e average percent concentration for the genotypes were 58.68 for T y p e I, 61.45 for T y p e I I , and 60.12 for T y p e I I I (Table 2). The differences between types as well as differences between ages and age-genotype interaction were all found t o be highly significant (Table 1). These results are in agreement with those of Washburn (1968b), who found the average percent concentration of the (M) major band of T y p e I I to be greater than t h a t of T y p e I and I I I for a 365 day period, with the heterozygote (Type I I I ) being intermediate. in bands averaged over the experimental period mi
7.05 4.15
ni2
mi+m2
17.88 7.29
19.55
20.19 6.73 2.16
12.26
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(T) trace band was present at all stages since the mutation in the minor band changed its electrophoretic properties and thus did not mask it. Differences in concentration of hemoglobin components: Embryonic band 1 (Ei): T h e Ei-embryonic band was present in an easily detectable quantity at 3 days of incubation and at all other ages of the chick embryo of all three hemoglobin types. As shown in Figure 3, the percent concentration of the embryonic band was greatest at the sixth day of incubation with the concentration decreasing rapidly thereafter for all three hemoglobin genotypes. There were no differences in concentration between the genotypes for the first thirteen days of incubation. Thereafter, the concentration in the homozygous normal (Type I) was consistently higher t h a n t h a t of T y p e I I and I I I , with the heterozygote (Type I I I ) being intermediate. T h e differences resulted in a highly significant age by genotype component of variance with no significant differences between the three types over the experimental period (Table 1). The average percent concentration for the Ei-em-
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WASHBURN
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DAYS OF INCUBATION FIG. 4. Percentage concentration of the major hemoglobin component (M) at various ages of the embryo. Embryonic band 2 (£2): T h e E 2 -embryonic band was found in only T y p e I and I I I genotypes. This band was present at 3 days of incubation in both types. There were marked differences in the percentage composition of the E2 component u p through the seventh day of incubation in T y p e I and I I I embryos (Figure 5). Since the (E 2 ) embryonic band was not separable from t h a t of the (M) major band after 7 days, the percent concentration was taken to be zero in both types throughout the remainder of the experimental period. T h e lack of separation of the (E 2 ) embryonic, band from the (M) major band after 7 days was apparently due to changes in the electrophoretic migration of the (E 2 ) embryonic band a n d / o r the (M) major band observed from the third through the seventh day of inclubation in T y p e I and I I I hemograms. T h e average percent concentration of the E 2 -embryonic band for the two genotypes was 7.05 for T y p e I and 4.15 for
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FIG. 5. Percentage concentration of embryonic hemoglobin component (E2) at various ages of the embryo.
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O Q
471
HEMOGLOBIN T Y P E S IN EMBRYOS
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DAYS OF INCUBATION FIG. 6. Percentage concentration of the trace band (T) at various ages of the embryo.
T y p e I I I (Table 2). These differences were found to be highly significant (Table 1). When recalculated on the basis of no (T)band, the average percent concentration of the (E 2 ) embryonic band of Type I I I embryos was 4.38. Therefore, the higher relative concentration of the E2-embryonic band in T y p e I embryos was apparently not caused by the additional band (Ttrace band) in T y p e I I I embryos. Trace band (T): T h e (T) trace band was found only in birds with T y p e I I and I I I genotypes. As shown in Table 2 and Figure 6, the percentage concentration for the T-trace band in T y p e I I hemograms (6.73%) was much higher t h a n t h a t of T y p e I I I (2.16%). This resulted in highly significant differences between types (Table 1). There were also highly significant differences between ages and an agegenotype interaction (Table 1). As shown
in Figure 6, the percent concentration decreased sharply in both types. By the eleventh day of incubation, the (T) trace band had become indistinguishable from t h a t of the (mi) minor band in T y p e I I I hemograms; therefore, the percent concentration in this type was taken to be zero throughout the remainder of the experimental period. The percentage concentration of the (T) trace band in T y p e I I embryos progressively decreased after the fifth day of incubation, and by hatching comprised approximately 2.6 percent of the total hemoglobin. This value compares favorably with the 1.7 percent in the hatched chick and the adult as reported b y Washburn (1968b). Minor components {m\ and nia): T h e percentage composition of the minor components was analyzed in two ways. First, the total amount of the minor components
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u. O
472
C. R. DENMARK AND K.
W.
WASHBURN
' 4 5 6 7
17 19 21 DAYS OF I N C U B A T I O N
i-
z UJ
FIG. 7a. Percentage concentration of the minor hemoglobin component (mi) at various ages of the embryo.
z
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o
was compared. This included comparisons between the (mi) minor component of Type I, (m2) minor component of Type II, and the (mi+m 2 ) minor components of Type III. Secondly, the concentration of individual bands were compared. This included comparisons of the (mi) minor band between Type I and the (m2) minor band of Type II as well as comparisons of the (mi) and (m2) bands within the heterozygote. When the total concentrations of the minor bands are compared (Figure 7 a and 7b), it is evident that the concentration of the minor band (mi) normally found in the embryo is considerably higher than that of the abnormal (m2) minor band
n I E a. O
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I / •I I 1 l I I I I I '4 5 6 7 9 1 1 13 15 17 DAYS OF INCUBATION
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FIG. 7b. Percentage concentration of the minor hemoglobin component (m2) at various ages of the embryo.
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throughout most of the experiment. These differences persist after hatching (Washburn, 1968b). The differences in concentration of these components were not great until after the ninth day of incubation. When the average concentration of the (mi) minor band of the normal homozygote is compared with the total concentration (average) of the two minor components in the heterozygote, the concentration of the (mx) minor band of the normal hemoglobin type is greater than the combined concentration of the (mi+m 2 ) minor bands in the heterozygote. The concentration of the (m2) minor band is significantly lower than that of the (mx) minor band in comparison between homozygous types and comparisons of these bands within the heterozygote. These results support the con-
HEMOGLOBIN T Y P E S IN EMBRYOS
I t would appear from these results t h a t the embryonic hemoglobin in the domestic fowl consist of the (Ei) embryonic band in all chickens and the (E 2 ) or the (T) band (depending on the genotype), while the (mi) and (m 2 ) minor bands represent the adult hemoglobin components. T h e TABLE 3.—Comparison oj various hemoglobin bands in chick embryos with different adult hemoglobin genotypes
Type I Type 11 Type 111
Ei
M
E2
+ + +
+ + +
+
_
+
+ +
+ =band present. — =band absent.
—
T
mi
m2
+
_
+
+ +
—
(M) major band is present at a relatively high concentration at 3 days of embryonic development. However, its concentration increases and is maintained at a high level throughout the adult life. The percentage concentration of the (Ei) embryonic band began to decrease in all three genotypes after the sixth day of incubation, and b y hatching comprised only approximately 6 percent of the total hemoglobin. T h e percentage concentration of the (T) trace band progressively decreased after the fifth day of incubation in T y p e I I and Type I I I embryos respectively. Previous investigation, using only T y p e I embryos have concluded that an embryonic band (presumably E 2 ) is replaced by the (M) adult band. However, our results demonstrate t h a t the (M) adult band exists from 3 days of incubation throughout the development of the embryo. The observed change in the electrophoretic mobility of the (M) adult band a n d / o r the (E 2 ) embryonic band was the apparent cause for the disappearance of the (E 2 ) embryonic band. This is supported by comparison of the normal (Type I) and m u t a n t type (Type I I ) . Both the (M) adult band and the (E 2 ) embryonic band exist in the normal type, whereas only the (M) adult band is present in the m u t a n t type. SUMMARY
A study was conducted to determine the hemoglobin types in chick embryos with different adult hemoglobin genotypes and the approximate time of switch-over from the production of embryonic to the production of the adult hemoglobin. Using cellulose acetate electrophoresis, a minim u m of three to a maximum of six different hemoglobin bands were observed in the developing embryo, depending on the adult hemoglobin genotype and the developmental stage of the embryo. For all
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elusions of Washburn (1968b), who showed t h a t the m u t a n t gene resulting in the production of the abnormal (m2) minor hemoglobin component does not function as efficiently in producing hemoglobin as does the normal allele. I t was evident from this study t h a t there is a definite association of the (E 2 ) embryonic band with the normal minor component (mi) and the (T) trace band with the abnormal minor component (m 2 ). Since the heterozygote contains both minor components (mi and m2) both the (E 2 ) embryonic band and (T) trace band were also found to be present (Table 3). The (mi) and (m2) minor components first appeared in their respective genotypes in the hemograms of 6 day old embryos and progressively increased in concentration to approximately the thirteenth day of incubation. Therefore, the approximate time of switch-over from the production of embryonic hemoglobin to the production of adult hemoglobin was observed to be between the fifth and sixth day of incubation in all genotypes, which is in agreement with the results of Manwell et al. (1963), Fraser (1964), Hashimoto and Wilt (1966).
473
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C. R. DENMARK AND K.
After 9 days of incubation, the (E2) embryonic band became indistinguishable from the (M) major band in Type I and III embryos. The (T) trace band became indistinguishable from the (mi) minor com-
WASHBURN
ponent in heterozygous embryos (Type III). This study indicated a definite association of the (E2) embryonic band with the normal minor component (mi) and the (T) trace band with the abnormal minor component (m2). The heterozygote was found to have both the (E2) embryonic band and (T) trace band, since it contains both minor components (mi and m2). REFERENCES D'Amelio, V., and A. M. Salvo, 1961. Further studies on the embryonic chick hemoglobin. An electrophoretic and immunoelectrophoretic analysis. Acta. Embryol. Morph. Exper. 4: 250-259. Fraser, R. C , 1961. Hemoglobin formation in the chick embryo. Exp. Cell. Res. 25: 418^27. Fraser, R. C , 1964. Electrophoretic characteristics and cell content of the hemoglobins of developing chick embryos. J. Exp. Zool. 156: 185-196. Hashimoto, K., and F. H. Wilt, 1966. The heterogeneity of chicken hemoglobin. Proc. Nat. Acad. Sci. 56: 1477-1483. Huisman, T. H. J., and J. M. Schillhorn Van Veen, 1964. Studies on animal hemoglobins. III. The possible role of intercellular inorganic phosphate on the oxygen equilibrium of the hemoglobin in the developing chicken. Biochimica et Biophysica acta. 88: 367-374. Manwell, C , C. M. A. Baker, J. B . Roslansky, and M. Foght, 1963. Molecular genetics of avian proteins. II. Control genes and structural genes for embryonic and adult hemoglobins. Proc. Nat. Acad. Sci. 49: 496-503. Washburn, K. W., 1968a. Inheritance of an abnormal hemoglobin in a random-bred population of domestic fowl. Poultry Sci. 47: 561-564. Washburn, K. W., 1968b. Effects of age of bird and hemoglobin type on the concentration of adult hemoglobin components of the domestic fowl. Poultry Sci. 47: 1083-1089. Wilt, F. H., 1962. The ontogeny of chick embryo hemoglobin. Proc. Nat. Acad. Sci. 48: 1582-1590.
NEWS AND NOTES {Continued from page 453) to hold the 1974 World's Poultry Congress in the United States. The proposed host city is Chicago, Illinois. The next meeting of the U.S.A. Branch is sched-
uled to be held during the annual meeting of the Poultry Science Association next August in Fort Collins, Colorado, at the Colorado State University.
(Continued on page 523)
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three genotypes, the minimum number of bands was observed from 3-5 days of incubation; the maximum number from 6-7 days. From 11-21 days of incubation, the number and type of bands found were the same as for the hatched chick. From 3-5 days of incubation, Type I embryos (normal homozygote) had three distinct components: (Ei) embryonic band, (M) major band and (E2) embryonic band. Type II embryos (abnormal homozygote) had the (Ei) embryonic band, (M) major band, and a (T) trace band, but no (E2) embryonic band. All the bands observed in Type I and II embryos were seen in the heterozygote (Type III). At six days of incubation, the minor adult hemoglobin components (mi and m2) first appeared in their respective genotypes and rapidly increased in concentration to approximately the thirteenth day of incubation. After the sixth day, the concentration of the (Ei) embryonic band, (E2) embryonic band and (T) trace band progressively decreased throughout the remainder of the experimental period. Therefore, the approximate time of switch-over from the production of embryonic hemoglobin to the production of the adult hemoglobin was observed to be between the fifth and sixth day of incubation in all genotypes.
W.