EMBRYONIC HEMOGLOBIN SYNTHESIS
113
methods in order to localize Hb formation have been hampered by the
very high background incorporation of iron in all the blastoderm cells,
presumably due to cytochrome synthesis and nonspecific binding of
inorganic iron to yolk. Granick and Levere (1965) have elaborated an
elegant fluorescence-microscopic method for heme localization, and they
report significant heme localization in blood island cells is first seen at
the 6-somite stage. In general, the application of immunological techniques conforms to the expectations outlined above. D'Amelio and Salvo
prepared antisera to adult chicken Hb and used agar-gel diffusion
(1959c) and immunoelectrophoretic methods (1961). They did not concentrate on the early embryonic stages, and cross reactions with yolk
were encountered. Extracts of 36-hr-old embryos contained antigens
giving several lines on Ouchterlony analysis, but all reacting systems
were distinct from the adult antigens. Thereafter, antigens serologically
related to adult Hb appeared between 36 and 72 hr of incubation, and
the agar-gel diffusion reactions were similar to those of the adult by
136 hr of incubation. Using immunoelectrophoresis in a later study, they
examined 47-hr-old embryos and obtained reactions suggesting presence
of a true embryonic Hb. Wilt (1962) also used antisera against adult
Hb as a test reagent. The antisera were absorbed with yolk and only
gave reactions with chicken Hb, i.e., they were species specific. Globin
also reacted with the antiserum. Very faint reactions were obtained with
extracts of blastoderms younger than the 7-somite stage, and 2 lines
were seen on Ouchterlony analysis. One of these lines disappeared between 36 and 48 hr of incubation, while the other persisted. At 36 hr of
development (7- to 10-somite stages) reactions of identity were first
found with the predominant antigen-antibody system (cf. Fig. 3). At
this same stage, the first significant incorporation of Fe
59
and leucine-H
3
into antibody precipitable material obtained from extracts of prelabeled
blastoderms was obtained. Wilt (1962) proposed that blastoderms prior
to 7 somites may contain some globin (perhaps 2 serological types), but
that embryonic Hb synthesis begins about the 7-somite stage. Extracts
of hundreds of pooled 4-somite blastoderms produce only colorless precipitate when treated with these antisera, but extracts of a similar
number of 8-somite blastoderms definitely produce a red precipitate
when incubated with the antisera. A very wide distribution of the antigen
was found in 12- to 14-somite blastoderms, but the concentration of
reacting antigen was considerably higher in extracts from the area opaca.
The role of cell division in formation of the first red blood cells should
be explored thoroughly. The number of cell divisions that prospective
blood island cells undergo from the time of laying is not known. It has
been noted, however, that blastoderm cells may be somewhat "synchronized" during the first two days of incubation (Sabin, 1920). Hell
113
methods in order to localize Hb formation have been hampered by the
very high background incorporation of iron in all the blastoderm cells,
presumably due to cytochrome synthesis and nonspecific binding of
inorganic iron to yolk. Granick and Levere (1965) have elaborated an
elegant fluorescence-microscopic method for heme localization, and they
report significant heme localization in blood island cells is first seen at
the 6-somite stage. In general, the application of immunological techniques conforms to the expectations outlined above. D'Amelio and Salvo
prepared antisera to adult chicken Hb and used agar-gel diffusion
(1959c) and immunoelectrophoretic methods (1961). They did not concentrate on the early embryonic stages, and cross reactions with yolk
were encountered. Extracts of 36-hr-old embryos contained antigens
giving several lines on Ouchterlony analysis, but all reacting systems
were distinct from the adult antigens. Thereafter, antigens serologically
related to adult Hb appeared between 36 and 72 hr of incubation, and
the agar-gel diffusion reactions were similar to those of the adult by
136 hr of incubation. Using immunoelectrophoresis in a later study, they
examined 47-hr-old embryos and obtained reactions suggesting presence
of a true embryonic Hb. Wilt (1962) also used antisera against adult
Hb as a test reagent. The antisera were absorbed with yolk and only
gave reactions with chicken Hb, i.e., they were species specific. Globin
also reacted with the antiserum. Very faint reactions were obtained with
extracts of blastoderms younger than the 7-somite stage, and 2 lines
were seen on Ouchterlony analysis. One of these lines disappeared between 36 and 48 hr of incubation, while the other persisted. At 36 hr of
development (7- to 10-somite stages) reactions of identity were first
found with the predominant antigen-antibody system (cf. Fig. 3). At
this same stage, the first significant incorporation of Fe
59
and leucine-H
3
into antibody precipitable material obtained from extracts of prelabeled
blastoderms was obtained. Wilt (1962) proposed that blastoderms prior
to 7 somites may contain some globin (perhaps 2 serological types), but
that embryonic Hb synthesis begins about the 7-somite stage. Extracts
of hundreds of pooled 4-somite blastoderms produce only colorless precipitate when treated with these antisera, but extracts of a similar
number of 8-somite blastoderms definitely produce a red precipitate
when incubated with the antisera. A very wide distribution of the antigen
was found in 12- to 14-somite blastoderms, but the concentration of
reacting antigen was considerably higher in extracts from the area opaca.
The role of cell division in formation of the first red blood cells should
be explored thoroughly. The number of cell divisions that prospective
blood island cells undergo from the time of laying is not known. It has
been noted, however, that blastoderm cells may be somewhat "synchronized" during the first two days of incubation (Sabin, 1920). Hell