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The initial synthesis of haemoglobin in
de-embryonated chick blastoderms.
I. Metabolism of the blastodisc cultured in vitro
by ANNA HELL 1
From the Laboratoire de Biophysique et Radiobiologie, Universite Libre de Bruxelles
WITH TWO PLATES
E N O R M O U S progress has been made in the last few years towards the
elucidation
of the mechanism of protein synthesis, and great interest is centred on the steps
leading to cellular differentiation and specific protein synthesis. We know that
genetic information is passed on from one generation of cells to the next by
deoxyribonucleic acid (DNA), and that this material directs all protein synthesis
by the intermediary of the different types of ribonucleic acid (RNA).
A simple in vitro system described by O'Brien (1959) seemed to offer an excellent
tool for the study of the differentiation of the blood islands, and the initial
formation of a well-known protein, haemoglobin (Hb), in chick embryonic
tissues. After de-embryonation, chick blastoderms, from the stage of primitive
streak onwards, can be cultured in vitro on a saline agar medium supplemented
with glucose. Cell migration is stopped, and growth very considerably reduced
in both the ecto- and theendoderm, while the mesodermal blood islands continue
to multiply and differentiate, the primitive erythroblasts synthesize Hb and
ultimately form primitive erythrocytes in a poorly defined tubular epithelium.
O'Brien (1959) described experiments in which the RNA precursor analogue,
azaguanine, inhibited the synthesis of Hb in this system only when added to the
culture medium before the blastodisc had reached a certain stage of morphological
differentiation. Added later, it seemed almost without effect on Hb synthesis.
He suggested that at this critical stage, the azaguanine interfered with the
formation of an RNA specifically associated with the subsequent production
ofHb.
We therefore thought it would be useful to study in more detail the morphological differentiation of the system, and to make quantitative estimations of
1
Author's address: Laboratoire de Biophysique et Radiobiologie, Faculte des Sciences,
1850, Chaussee de Wavre, Brussels 16, Belgium.
610
ANNA HELL
protein and nucleic acid production. We would then be able to follow up the
work of O'Brien, to see to what extent the effect of azaguanine was specific to
the differentiation of the Hb-synthesizing system. We hoped to extend the work
to other inhibitors, and thus try to define time-relationships between the successive
steps leading to the initial synthesis of Hb.
MATERIAL AND METHODS
Media
A modified Tyrode solution, which gave optimal growth and Hb production,
was prepared from the following solutions:
Solution 1: 8-0 g. NaCl, 0-2 g. KC1, 0-2 g. CaCl2, 0-1 g. MgCl2 in 125 ml.
distilled water.
2: 0-05 g. NaH 2 PO 4 .H 2 O 1
3: 1 • 0 g. NaHCO 3
I each in 125 ml. distilled water.
4: 6-25 g. glucose
J
5: 0-9 g. agar (Difco) in 30 ml. distilled water.
The solutions were sterilized in an autoclave and the bicarbonate subsequently
regenerated with CO 2 .
The operating medium, glucose saline solution, was prepared with 10 vol. of
each of solutions 1 to 4 added to 60 vol. sterile water. The agar medium similarly
contained 10 vol. of each of solutions 1 to 4, with 25 vol. solution 5 and 35 vol.
sterile water. The pH of the medium remained in the optimal range for chick
tissues (about pH 7 • 6 to 8 • 0) without a CO2-enriched atmosphere.
3
H-thymidine from the Radiochemical Centre, Amersham, was used in the
studies of DNA synthesis: the sample used had a specific activity of 12-1 c/mM,
and was added to the agar medium to give an activity of 2 /xc/ml. For some experiments, an incubation of 1 hr. on labelled medium was followed by washing three
times in sterile glucose saline solution containing 0-04 mg. 'cold' thymidine,
and incubation continued for 23 hr. on the agar medium containing this concentration of unlabelled thymidine.
Operations
Freshly laid chicken eggs of the Sex Sal Link breed of chickens kept by Mr
Latteur of Braine l'Alleud, were kept until required at 12° C , and used within
2 days. They were incubated at 37-38° C. up to about head process stage, which
took 22 to 32 hr. depending on the batch and the season. The blastodiscs were
explanted according to the method of New (1955) on to glass rings, and incubated
on 'fluid' albumin up to the desired stage. The rings were flooded with glucose
saline solution, the blastodisc detached from the vitelline membrane, the embryo
excised with fine scalpels, and the ring of vascular area was transferred, ventral
side uppermost, to a watchglass containing not less than 1 ml. agar medium.
Synthesis of haemoglobin in chick blastoderms. I
611
The embryos and media were handled with sterile precautions, incubations being
carried out in Petri dishes containing moist cotton wool.
In our experiments, we used blastodiscs de-embryonated at 1, 4, 7 and 10
somites. Before 1 somite, potential haemopoietic tissue would be removed with
the posterior part of the primitive streak (Settle, 1954); by 10 somites, Hb is
visibly being formed, so that the Hb-forming system must be differentiated at
least in part of the vascular area.
After the 24 hr. incubation (in a few cases it was 36 hr.), the agar medium was
flooded with glucose saline solution to detach the disc. Blastodiscs to be mounted
whole were stained for Hb by the pseudoperoxidase reaction of Hb with hydrogen
peroxide and o-dianisidine, as described by O'Brien (1961): the reaction was
modified in that the H 2 O 2 was used at 10 vol. rather than at 100 vol. concentration, which caused less evolution of oxygen and thus allowed better preservation
of the tissues. The preparations were counterstained with Light Green F.S.,
dehydrated with dioxane, and, after clearing in toluene, were mounted in
Canada balsam.
Blastodiscs for sectioning were cut into half along the antero-posterior axis:
one half was fixed with the o-dianisdine staining mixture, with 10 vol. hydrogen
peroxide; the other half was fixed with acetic acid-alcohol, 5 to 10 per cent., for
15 min. and washed for £ hr. with 70 per cent alcohol. The two parts were
dehydrated together in dioxane, and mounted adjacent in paraffin for sectioning
at 10/z, or at 5/x for autoradiography. Sections were stained (i) with Unna
(methyl green-pyronine), (ii) with Feulgen and counterstained with light green,
or (iii) with light green only as counterstain to the o-dianisidine.
Sections for autoradiography after incorporation of 3H-thymidine were
treated with 1 per cent PCA for 20 min. at 4°C. to remove free 3H-thymidine,
followed by washing for 1 hr. in running water and 1 hr. in 'cold' thymidine
solution containing 0-04 mg./ml. The sections were dehydrated to absolute
alcohol before allowing the slides to dry. Uford liquid emulsion L4 was used,
as described by Ficq (1955) with an exposure of 1 day. Sections over which
counts were to be compared were mounted on the same slide, to ensure identical
treatment.
Estimations
Blastodiscs on which quantitative measurements were to be made were
transferred to the glucose saline solution. The surrounding tissue was cut away
from the vascular area, taking great care not to damage the terminal sinus, and
the ring of tissue was homogenized in a glass homogenizer with a drop of water.
Determination ofHb
Hb estimations were made on each disc individually, to give a measure of the
variation between individual samples at a given stage. The pseudoperoxidase
reaction of Hb with hydrogen peroxide and o-dianisidine, described by O'Brien
40
612
ANNA HELL
(1961), was adapted to quantitative estimations. In whole mounts of blastodiscs,
staining was seen to be limited to the red cells and not to be visible before Hb
could be expected—i.e. 1-2 hr. before the red pigment is visible to the naked eye.
In fact haematin alone will catalyse the reaction, but quantitative tests showed it
to be about ten times less effective (on a molecular basis) than Hb. The reaction
is very sensitive: the absorption of Hb at 415 mp is 0 • 5 for a solution of 90 /xg/ml.,
whereas extraction of the oxidized o-dianisidine in 3 ml. xylene gives a reading
of 0-5 for 3 • 5 fig Hb. The reading is equivalent to only 0-1 to 0• 5 /*g/mg. total
protein in tissues where there is known to be no Hb (as in the vascular and
430 m/x
500 m/x
20
/xg Hb
TEXT-FIG.
1. Standard curves for the estimation of haemoglobin by its pseudoperoxidase
reaction with hydrogen peroxide and o-dianisidine.
perivascular areas of 1 or 4 somite embryos). The following staining mixture
was prepared :
o-Dianisidine, 100 mg./70 ml. ethanol
Acetate buffer, 1 • 5 M, pH 4 • 7
Water
Hydrogen peroxide, 100 vol.
20 parts
5 parts
7 parts
1 part
The o-dianisidine, buffer and water were mixed and cooled in an ice-bath, the
peroxide being added immediately before use. The reaction was carried out in
the cold avoiding sunlight. With each experiment a standard curve was run,
with 0 to 20 /xg Hb in 0-2 ml. solution. A Sigma bovine Hb preparation, twice
crystallized, was used. It was dissolved, 1 mg./0-l ml. N/10 NaOH, diluting
with 0 • 8 ml. water, and finally neutralizing with 0 • 1 ml. N/10 HC1. This solution
remains stable 2 to 4 weeks, if kept at 4°C.
Two millilitres of the fresh staining mixture were added to each sample and
Synthesis of haemoglobin in chick blastoderms. I
613
to the standard tubes. After exactly 15 min., the colour was extracted by shaking
with 3 ml. xylene, the mixture was centrifuged, and the upper xylol layer removed
with a Pasteur pipette. The optical density was measured at the maximal
absorption of 430 mfx, at 500 m^u, and sometimes at 520 m/x, where readings were
lower but still proportional. Readings were taken against xylene, and not against
the blank, which seemed to be more variable than the standard values. The
optical density increases steadily over more than 2 hr., so that readings must be
taken after a fixed time. One to 1£ hr. after the initial addition of the mixture
was chosen, and on the Jobin-Yvon electronic spectrophotometer, about fortyfive solutions can be read at three to four wavelengths within | hr. A set of
standard curves is shown (Text-fig. 1).
Determination of RNA, DNA and total protein
To the turbid solution remaining in each tube after the removal of xylene were
added exactly 5 ml. alcohol-ether, 3:1, to precipitate nucleic acids and proteins.
The system was buffered at pH 4 • 7 by the staining mixture for Hb, and further
acidification of the supernatant did not yield more precipitate. Three to five
precipitates were combined at this stage, for the extraction of nucleic acids; they
were washed with alcohol-ether, then for 10 min. with alcohol-ether at 50°C,
and lastly with pure ether.
RNA was extracted with 10 per cent. PCA for 18 hr. at 4 ° C , and estimated by
the orcinol method of Miller et al. (1950) slightly modified. To one volume of
test solution was added an equal volume of freshly prepared orcinol solution
(10 mg. orcinol per ml. concentrated HC1 containing 4 ju.g. ferric alum per ml.).
After heating for 30 min. in a boiling water bath, and cooling, an equal volume
of 94° C. alcohol was added to stabilize the colour, and readings were taken in
the spectrophotometer at 690 m/x. Yeast RNA from Schwartz Laboratories was
used for the standard.
DNA was subsequently extracted with 5 per cent. PCA for 20 min. at 70° C ,
and estimated with indol by the method of Keck (1956). Standard solutions were
prepared from salmon sperm DNA, A Grade, California Biochemicals.
Protein was estimated by hydrolysing the remaining precipitate in N NaOH,
and using aliquots for the determination with the Folin-phenol reagent as
described by Lowry et al. (1951). The standard solution was prepared from bovine
albumin fraction V from bovine plasma (Armour).
OBSERVATIONS
The differentiation of the vascular area during normal development in the
time from 1 to 10 somites was observed. The mesoderm forms vessels, and
by 7 somites one sees blood islands budding from them. At 10 somites, the
release of cells into the lumen begins, and some cells show light Hb staining.
Plate 1, Fig. B, illustrates the appearance of sections of tissue after the 24 hr.
614
ANNA HELL
incubation in vitro, a picture which is similar whether the explant was taken at
1 or at 10 somites. The cells are seen to be free in the lumen: they are characteristically round and mononucleate with one or two nucleoli. After a comparable
time in vivo, the circulation between embryo and vascular area would be well
established, since this occurs around the 16-somite stage.
lOOi
2h
4h
8h
12 h
r-i
24 h
=5 50-1
o
o\
-Tfh
Tin
0 5 1O152O>2O
n-H
10 20>20
10 20>20
Grains per nucleus -
10 20 >20
10 20 >20
2. Incorporation of 3H-thymidine into blood island cells. Incubations 2 to 24 hr.;
2 /xc/ml. 3H-thymidine, autoradiography with Ilford L4 emulsion, exposure 1 day.
TEXT-FIG.
In her survey of the origin of blood vessels and blood cells in the first 2 days of
development of the chick embryo, Sabin (1920) noted that there was some synchronization of cell division in the differentiating tissues. Emanuelsson (1962)
also states that though the average rate of cell division in embryos of 0 to 50 hr.
100,
50-
4
TEXT-FIG.
8 12
hrs incubation —
24
3. Percentage cells with more than 5 grains per nucleus after incubations of 2 to
24 hr.: data from Text-fig. 2.
of development is of the order of 10 to 15 hr., there are periods of intensified
multiplication around 5 hr., 18 to 20 hr., and 30 to 35 hr. In our sections, we
noted a mitotic index of about 7 at zero time, 7 and 10 somites, and in all explants
at 24 hr. incubation this had fallen to 4 or 5. Some cells in mitosis were seen to
have stained for Hb, which indicates that the beginning of Hb synthesis need
not exclude cell division. This is interesting in relation to Thorell's observations
(1947) of the continued Hb synthesis and cell division in bone marrow cells.
Closer examination of the cell division cycle and of the behaviour of the de-
J. Embryol. exp. Morph.
Vol. 12, Part 4
il
ect
B
ect
PLATE 1
Sections of the vascular area of blastodiscs.
FIG. A. At 10 somites.
Frc B. After 24 hr. incubation in vitro from 1 somite.
Sections stained with Unna. end =endoderm; ect = ectoderm; b.v. = blood vessel;
b.i. = blood island; p.e. = primitive erythrocyte.
A. HELL
{Facing page 614)
./. Embryol. exp. Morph.
Vol. 12, Part 4
end
ect
t
; : *
•:.t
*r
.ect
«
,;rS
$#
p.e/
* \m.
end
B
PLATE 2
Incubation of blastodiscs on medium containing 3H-thymidine 10 ^c/ml.
A. For 1 hr.
FIG. B. For 1 hr. followed by 23 hr. on 'cold' thymidine.
Autoradiography with Ilford L4 emulsion, exposure 1 day; stained after development with
Unna. Abbreviations as Plate 1.
FIG.
A. HELL
(Facing page 615)
Synthesis of haemoglobin in chick blastoderms. I
615
3
embryonated blastodisc in vitro was carried out by incorporation of H-thymidine
and autoradiography of sections of the tissue. In the first experiment, a series
of blastodiscs was taken at 4 somites, and incubated for 2 to 24 hr. on agar
containing 2 ^c/ml. 3H-thymidine. Counts of autoradiographs were taken
over 80 to 100 cells in each case.
The results of the counts are given in Text-fig. 2, and the timing of labelling
of blood island cells is summarized in Text-fig. 3. About 60 per cent, of the
100^
Blood island cells
Ectoderm
—t
Endoderm
50.
r-TI—n
-i-n-rfi-rL
0 5 10
20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 60
>
Grains per nucleus
(i) lh incubation: zero time, 10 somites
100
Ectoderm
Blood island cells
Endoderm
50
o
0
10 20 30
40
an
0 10 20
0 10 20 30 31- 5 1 Grains per nucleus
>
50_ 100
(ii) 1 h. +23h in 'cold' thymidine: zero time, 10 somites
TEXT-FIG. 4. Incorporation of 3 H-thymidine into the vascular area of the blastodisc: ^
dine at 2 /xc/ml.; autoradiography with Ilford L4 emulsion, exposure 1 day.
cells will be synthesizing DNA at any given time, and the cell division cycle,
taken as the time between DNA synthetic periods, or the time for all nuclei to
become labelled, is about 11 hr.
In the second experiment, explants were taken at 10 somites, and incubated
(i) on 3H-thymidine for 1 hr. and fixed, or(ii)for 1 hr. on 3H-thymidine followed
by 23 hr. on unlabelled thymidine before fixing. Autoradiographs were prepared
similarly. At a first glance, it is striking that grains over the nuclei of the blood
island cells are very much reduced over the period of incubation on 'cold'
thymidine, while the nuclei of the ecto- and particularly of the endoderm remain
heavily labelled. The effect is illustrated in Plate 2, which are photographs of
616
ANNA HELL
preparations of explants incubated in 10 /u-c/ml. 3H-thymidine. Text-fig. 4 shows
grain counts over the three cell types. Again 80 to 100 cells were counted in each
group. For the blood island cells, the number of nuclei with 0 to 5 grains has
increased from 50 to 75 per cent., and the maximum count after the 23 hr. incubation on unlabelled medium is about half to one-third of the maximum at the end
of the initial labelling period.
If the model of Watson & Crick (1953) was applicable to whole cells, and each
chromatid was composed of a single strand of DNA, then we should arrive at the
following picture. In the first generation after the labelling period, the same
proportion of cells should be labelled, but with half the density, each of the
chromatids of a labelled pair having complemented with new unlabelled material.
In the second generation, the number of labelled cells should decrease by 50 per
cent., since the labelled cells, being radioactive in only one chromatid, would
give rise to an equal number of labelled and of unlabelled cells. In fact, all species
of cells examined have been shown to undergo a certain degree of exchange
between chromatids (see Taylor, 1963). So, from the counts over the blood island
cells, one may conclude that they have passed through two complete cycles of
division in the 23-hr, period after labelling, in good agreement with the value
from the first experiment. In the ectoderm, a few cells may have divided, but in
the endoderm it is hard to judge what may have occurred. There is a definite
increase in the proportion of labelled cells, and grain counts were higher than
after the first hour. The cells are large and diffuse, and can probably store
nutrients which would not have been washed out in the rinses before the explant
was transferred to unlabelled medium.
Results of quantitative measurements
Measurements of Hb, total protein, RNA and DNA were made on the
vascular area of blastodiscs at zero time, i.e. at 1, 4, 7 and 10 somites, and after
24 hr. incubation on agar medium from each of these stages. After this period
there is an easily measurable amount of Hb synthesized by the explants taken at
1 somite, and it is very active in the older explants. There is, however, a considerable variation in individual results, since the number of somites and the
degree of development of the blood islands do not always run exactly parallel
(Sabin, 1920). Occasionally a blastodisc was seen to have either a poorly or a
particularly highly developed vascular area in relation to the number of somites
at the time of transfer, and was discarded at once.
An incubation period of 24 hr. was chosen as most convenient for quantitative
estimations, and would later allow the comparison of the effects of different
inhibitors on the system. The results were more reproducible than after 36 hr.;
earlier experiments had shown that there seemed to be no significant increase of
RNA or DNA between 24 and 36 hr., though total protein had increased about
40 per cent.; and there was an average increase of 6 to 10 jug Hb for the stages 1 to
7 somites.
Synthesis of haemoglobin in chick blastoderms. I
617
The measurements are given in Tables 1 (zero time values) and 2 (24 hr.
incubations).
TABLE 1
Measurements ofHb, totalprotein, RNA andDNA in the vascular area of the
blastodisc at zero time
Values in /ig per blastodisc ± standard deviations. Results are the average
of three experiments.
No somites
No embryos
Hb
Protein
RNA
DNA
1
47
0
87 ±5
5 •l±0-6
1 •0±0-2
4
36
0
108 ±20
6-7±0-5
l-3±O-3
7
38
0 •3±0-l
133±23
10 •1 ± 1-7
2 •0±0-3
10
36
0 •6±0-2
163 ±24
12 •4±3-4
2 •9±0-3
TABLE 2
Measurements ofHb, total protein, RNA and DNA in the vascular area after
24 hr. incubation from the stage indicated
Values in /x|I per blastodisc ± standard deviations. The results are the
average of eight experiments.
No somites
No embryos
Hb
Protein
RNA
DNA
1
32
3-7±0-5
179 ±42
13-3±2-9
1-9 + 0-8
4
36
6-6±2-8
244 ±65
19-2±5-3
3-2+1-6
7
26
ll-0±0-2
292 ±70
24-l±3-6
4-2±2-2
10
21
17-5±2-6
380 ±76
31-0±4-l
6 1 + 3-0
It is seen that protein and DNA approximately double, while RNA increases
two and a half times during the in vitro period. The figures, of course, include the
ecto- and endoderm which will, according to the observations with 3H-thymidine,
probably contribute much less to the increase than the differentiating mesoderm.
DISCUSSION
In the absence of the embryo, the vascular area is seen to continue metabolic
activity essentially for the development and differentiation of the mesoderm into
a primitive vascular system. The blood island cells divide and produce Hb, and
the latter has been seen not to exclude further cell division (compare Thorell,
1947). It is possible that nucleic acid synthesis stops after about 24 hr. due to
lack of nutrient on the very simple medium given. In fact, the tissue surrounding
the vascular area becomes notably depleted if the incubation is continued to 36 hr.,
becoming thin and transparent as if all reserves had been used by the growing
vascular area. For the comparison of the effects of inhibitors, it is advantageous
to have the simplest possible system, in order to minimize secondary effects; but
618
ANNA HELL
it would be interesting to see if the culture could be kept in active growth and Hb
synthesis over extended periods on a richer medium, and whether the tissue could
be cultured on a plasma clot on a cover-slip immersed in a rich medium. Preliminary experiments have allowed the differentiation of blood islands within
the original explants of tissue taken from primitive streak embryos, but the outgrowths were always of fibroblast nature. The cells can be dispersed by treatment
with trypsin in balanced salt solution, but attempts at culture in monolayers have
not produced Hb synthesizing cells.
The techniques described above have been applied to observations of the
effects of a number of metabolic inhibitors on the system (see following paper):
it was hoped thereby to come to a better understanding of the events leading
to the initial synthesis of Hb.
SUMMARY
1. The in vitro culture of the de-embryonated blastoderm of the chick embryo
is described.
2. Blastoderms were explanted at 1,4,7 and 10 somites, and the morphological
differentiation of the mesoderm to form blood islands was observed. The early
synthesis of haemoglobin was measured.
3. A method was described for the successive estimation of haemoglobin,
total protein, RNA and DNA in the vascular area of the culture.
4. The metabolism of DNA in the different cell types was studied by autoradiography after the incorporation of 3H-thymidine: an estimate was made of
the length of the division cycle of the blood island cells.
5. The possibility of the further culture of the blood island tissue was discussed.
RESUME
La synthese initiate de Vhemoglobine dans le blastoderme depoulet: I. metabolisme
de Vaire vasculaire cultivee in vitro
1. La culture in vitro des blastodermes de poulet, dont l'embryon meme a
ete enleve, est decrite.
2. Les blastodermes sont explantes aux stades de 1, 4, 7 et 10 somites, et la
differentiation morphologique du mesoderme pendant la formation des ilots
sanguins a ete observee, La synthese d'hemoglobine a ete mesuree.
3. Une methode est decrite, pour le dosage successif de l'hemoglobine, des
proteines totales, du RNA et du DNA dans l'aire vasculaire des blastodermes.
4. Le metabolisme du DNA dans les differents types de cellules a ete etudie
par autoradiographie apres l'incorporation de 3H-thymidine: on a estime la
duree de la division cellulaire dans les ilots sanguins.
5. La possibility de culture prolongee du tissu des ilots sanguins est discutee.
Synthesis of haemoglobin in chick blastoderms. I
619
ACKNOWLEDGEMENTS
I would like to express my warmest thanks to Professor M. Errera for his kind help and
encouragement throughout the work; also to Dr B. R. A. O'Brien for his practical advice;
to Dr F. Wilt, who sent me his manuscript prior to publication, and to Professors J. Brachet
and R. Thomas for their helpful comments on the manuscript.
This work was supported by the National Institutes of Health, U.S. Public Health Service,
Grant R.G. 6214; by Euratom, contract 016-61-10 ABIB, and by the 'Fonds de la Recherche
Scientifique Fondamentale Collective'.
REFERENCES
H. (1962). Growth and nucleic acid mobilisation in the early chick embryo.
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FICQ, A. (1955). Etude autoradiographique du metabolisme de l'oocyte d'Asterias rubens
au cours de la croissance. Arch. Biol., Paris et Liege, 66, 509-24.
KECK, K. (1956). An ultramicro technique for the determination of DNA. Arch. Biochem.
Biophys. 63,446-51.
LOWRY, O. H., ROSEBOROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurement with the Folin-phenol reagent. /. biol. Chetn. 193, 265-75.
MILLER, G. L., MILLER, E. E. & GOLDER, R. H. (1950). Studies on the orcinol method for
the estimation of pentoses. Fed. Proc. 9, 206.
NEW, D. A. T. (1955). A new technique for the cultivation of the chick embryo in vitro.
J. Embryol. exp. Morph. 3, 326-31.
O'BRIEN, B. R. A. (1959). 8-azaguanine inhibition of Haemoglobin synthesis in de-embryonated chick blastoderms. Nature, Lond. 184, 376-7.
O'BRIEN, B. R. A. (1961). Identification of haemoglobin by its catalase reaction with peroxide
and o-dianisidine. Stain Tech. 36, 57-61.
SABIN, F. (1920). Studies on the origin of blood vessels and red blood cells, as seen in the
living blastoderm of the chick during the second day of incubation. Carnegie Inst. Wash.
Publ. 272, Contribs. to Embryol. 9, 213-62.
SETTLE, G. W. (1954). Localisation of erythrocyte forming areas in the early chick blastoderm
cultivated in vitro. Carnegie Inst. Wash. Publ. 603, Contribs. to Embryol. 35, 223-37.
TAYLOR, J. H. (1963). In Molecular Genetics, Part I. New York: Academic Press.
THORELL, B. (1947). Studies on the Formation of Cellular Substances During Blood Cell
Production. London: Henry Kimpton.
WATSON, J. D. & CRICK, F. H. C. (1953). General implications of the structure of deoxyribonucleic acid. Nature, Lond. Ill, 964-7.
EMANUELSSON,
(Manuscript received 1st May 1964)