120
FRED H. WILT
(cf. Ebert and Wilt, 1960). Malpoix found that addition of RNA from
chicken or rabbit reticulocytes to cultures of presumptive area opaca
vasculosa stimulated Hb synthesis during a 24-hr period by 4-39%.
Large amounts of RNA were used, the results were somewhat variable,
and the specificity of the response was not investigated in detail; it is
difficult at present to relate this finding to possible controls for normal
Hb synthesis.
The relation of heme and globin synthesis might be a useful avenue
for gaining an understanding of the initiation of hemoglobin synthesis
(Wilt, 1962), and the results using inhibitors have reinforced the probability that this would be fruitful. Studies mentioned earlier have shown
that heme could stimulate the rate of globin formation and ensuing
hemoglobin production, in vitro. Levere and Granick (1965) explanted
deembryonated chick blastoderms on media containing δ-aminolevulinic
acid (DAL) or succinate and glycine. DAL is the first distinctive precursor in the biosynthesis of heme; it is formed by condensation of
succinate and glycine by the enzyme DAL synthetase (Granick and
Levere, 1964). They examined the amount of hemoglobin which accumulated in 24-48 hr of culture, and they found a striking elevation of the
amount of hemoglobin formed in blastoderms exposed to DAL, as measured by spectrophotometric methods. The stimulation was transient, however, and the degree of response varied over a wide range in individual
blastoderms, being completely absent in some instances. The same effect
of DAL was independently discovered by Wilt (1966) using the o-diansidine stain for Hb (O'Brien, 1960) and HelFs colorimetric method (Hell,
1964a) to measure hemoglobin levels. Furthermore, Wilt found that when
whole blastoderms were cultured on DAL, a diffuse rose color became
faintly visible in the area pellucida of the blastoderm, although red blood
cells are not present in abnormal places. This effect deserves further exploration. Wilt (1966) has also briefly reported that when young blastoderms (head process-head fold) are explained on DAL containing media,
benzidine positive cells can be detected in the area opaca vasculosa by the
3- to 4-somite stage, several hours in advance of the normal appearance
of the hemoglobin. It must be pointed out that in none of these preliminary observations is there definitive proof that DAL accelerates or
precociously initiates production of globin by direct chemical methods,
and this kind of evidence is essential. The potential use of this type of
tool to analyze possible control mechanisms is obvious. Of course, the
same approaches can and should be employed with other possible
candidates: iron, ferritin, apoferritin, erythropoietin, etc.
V. Conclusions
Although many areas of research which impinge on the differentiation
of erythropoietic functions have not been discussed, it is hoped the selec-
FRED H. WILT
(cf. Ebert and Wilt, 1960). Malpoix found that addition of RNA from
chicken or rabbit reticulocytes to cultures of presumptive area opaca
vasculosa stimulated Hb synthesis during a 24-hr period by 4-39%.
Large amounts of RNA were used, the results were somewhat variable,
and the specificity of the response was not investigated in detail; it is
difficult at present to relate this finding to possible controls for normal
Hb synthesis.
The relation of heme and globin synthesis might be a useful avenue
for gaining an understanding of the initiation of hemoglobin synthesis
(Wilt, 1962), and the results using inhibitors have reinforced the probability that this would be fruitful. Studies mentioned earlier have shown
that heme could stimulate the rate of globin formation and ensuing
hemoglobin production, in vitro. Levere and Granick (1965) explanted
deembryonated chick blastoderms on media containing δ-aminolevulinic
acid (DAL) or succinate and glycine. DAL is the first distinctive precursor in the biosynthesis of heme; it is formed by condensation of
succinate and glycine by the enzyme DAL synthetase (Granick and
Levere, 1964). They examined the amount of hemoglobin which accumulated in 24-48 hr of culture, and they found a striking elevation of the
amount of hemoglobin formed in blastoderms exposed to DAL, as measured by spectrophotometric methods. The stimulation was transient, however, and the degree of response varied over a wide range in individual
blastoderms, being completely absent in some instances. The same effect
of DAL was independently discovered by Wilt (1966) using the o-diansidine stain for Hb (O'Brien, 1960) and HelFs colorimetric method (Hell,
1964a) to measure hemoglobin levels. Furthermore, Wilt found that when
whole blastoderms were cultured on DAL, a diffuse rose color became
faintly visible in the area pellucida of the blastoderm, although red blood
cells are not present in abnormal places. This effect deserves further exploration. Wilt (1966) has also briefly reported that when young blastoderms (head process-head fold) are explained on DAL containing media,
benzidine positive cells can be detected in the area opaca vasculosa by the
3- to 4-somite stage, several hours in advance of the normal appearance
of the hemoglobin. It must be pointed out that in none of these preliminary observations is there definitive proof that DAL accelerates or
precociously initiates production of globin by direct chemical methods,
and this kind of evidence is essential. The potential use of this type of
tool to analyze possible control mechanisms is obvious. Of course, the
same approaches can and should be employed with other possible
candidates: iron, ferritin, apoferritin, erythropoietin, etc.
V. Conclusions
Although many areas of research which impinge on the differentiation
of erythropoietic functions have not been discussed, it is hoped the selec-