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/ . Embryo!, exp. Morph. Vol. 68, pp. 115-126, 1982
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1 J5
Hypoblast induction of multiple area
vasculosae, and stabilization of the area opaca
vasculosa in young chick blastoderm
By NIKOLAS ZAGRIS 1
From the Tissue Culture Laboratory, Department of Biological Sciences,
University of Patras
SUMMARY
Multiple vascular areas are formed in unincubated and prestreak chick blastoderms
under the influence of multiple transplanted hypoblasts. Though the transplanted hypoblasts merge into one continuous layer, they do not participate collectively in forming one
streak and embryonic axis with one area vasculosa at its postero-lateral end, but each
hypoblast seems to form an embryonic centre or fraction of a centre. It may be that the
merged hypoblasts do not lose their individuality or that they have induced their prospective
embryonic centre before merging. This is an indication that the hypoblast is able to initiate
certain events to which the epiblast responds. Composite blastoderms with stage-2 host
epiblasts and with two or more transplanted hypoblasts form the embryonic axis at its
prospective plane and two area vasculosae, the area opaca vasculosa (a.o.v.) as in the
control blastoderm, and an induced area vasculosa 180° anteriorly. However, composite
blastoderms with stage-3 epiblasts and with two or more transplanted hypoblasts behave
as the control blastoderms forming the embryonic axis and the a.o.v. at their prospective
sites. This indicates that the typical a.o.v. in the chick blastoderm is stabilized to blood
island formation at stage 3. The stage dependence which involves progressive restriction of
the areas in which blood islands will develop, suggests the existence of a centre which
creates organization by integrating short-lived fields. It seems that there are no particular
cell groups of the unincubated blastoderm determined to form erythrocytes but that the
organizing capacities of the area are necessary to induce the first early commitments of
prospective erythroblasts along this course.
INTRODUCTION
Blood islands are condensations of splanchnopleuric mesoderm which, in
close association with the underlying endoderm, form blood cells (Miura &
Wilt, 1969, 1970). Premesoderm. cells invaginate from the epiblast through
the primitive streak (PS) to establish the mesodermal layer (Rudnick, 1955;
Rosenquist, 1966). However, it is known that in the absence of a normal PS
and of the resulting axial mesoderm there is always present some mesenchyme
probably formed by a process of diffuse polyinvagination from the epiblast
1
Author's address: Tissue Culture Laboratory, Department of Biological Sciences,
University of Patras, Patras, Greece.
116
N. ZAGRIS
(Eyal-Giladi & Wolk, 1970; Azar & Eyal-Giladi, 1979; Zagris & Eyal-Giladi,
1982). By about 18 h of incubation, mesodermal cells from the area pellucida
in a horseshoe-shaped region posterior and posterolateral to the PS invade
the proximal portion of the area opaca adjacent to the area pellucida, and by
24 h of incubation they aggregate into cell clusters which are known as the
blood islands (Settle, 1954). This zone into which mesoderm has grown is
called the area opaca vasculosa (a.o.v.), because it is from this region that the
blood cells and yolk-sac blood vessels arise (reviewed by Wilt, 1967, and
Bellairs, 1971). Work of several investigators has contributed significantly to
our knowledge of the endodermal layer formation in the avian embryo (Vakaet,
1962, 1970; Modak, 1965, 1966; Nicolet, 1970; Rosenquist, 1972; Fontaine &
Le Douarin, 1977; Sanders, Bellairs & Portch, 1978). Though there is little
doubt that blood islands are mesodermal in origin, the presence of the endoderm
is necessary for the attainment of full haemoglobin (Hb)-forming capacity,
possibly because it transmits essential materials from yolk to mesoderm and/or
perhaps because it forms the endothelium of the blood islands (Wilt, 1965,
1967).
Zagris (1979, 1980) has shown that unincubated blastoderms in which the
PS and embryonic axis are inhibited mechanically are capable of forming
primitive and definitive erythrocytes and embryonic and adult Hbs. This is
evidence that the interacting components for erythroid cell formation need not
invaginate through a PS and do not require the continued presence of the
embryonic axis.
Dantschakoff (1907), Sabin (1917), and Murray (1932) have given classical
morphological accounts of the development of the blood islands. Haemoglobin
in these islands is first detectable at the 6- to 7-somite stage (Wilt, 1967), and
regulatory events occurring prior to the appearance of Hb have been described
by several investigations (reviewed by Wilt, 1967, and Bruns & Ingram, 1973).
Murray (1932) mapped the haemopoietic regions in chick blastoderms with
mature PS, and Rudnick (1938) reported the appearance of erythroblasts in
cultures taken from various stages of development of the PS. Settle (1954)
mapped the areas of the chick blastoderm capable of forming blood islands
in vitro from pre-streak to blastoderms of 4-6 somites and proposed that
certain areas of the chick blastoderm became committed to erythropoiesis
before the 6-7 h of incubation. Despite the extensive literature on the location
of the a.o.v. and erythroid cell development in the chick embryo (Fraser, 1963;
Wilt, 1967; Schalekamp, Schalekamp, Van Goor & Slingerland, 1972; Bruns
& Ingram, 1973; Brown & Ingram, 1974; Wainwright & Wainwright, 1974;
Cirotto, Scotto Di Telia & Geraci, 1975; Tobin, Selvig & Lasky, 1978; Zagris
& Melton, 1978; Chapman & Tobin, 1979; Martin, Beaupain & DieterlenLievre, 1980; Zagris, 1980), there is little information about the stabilization
of the a.o.v. The present work was undertaken to determine the time this
area, and not any other in the blastoderm, is committed and stabilized to blood
Stabilization of area opaca vasculosa in chick blastoderm
117
island formation. Various aspects of the hypoblast behaviour, such as the
organized influence it exerts on the epiblast, which emerge in this study are
discussed in concert with the a.o.v. formation.
MATERIALS AND METHODS
Culture
Freshly laid fertilized eggs (stage X - r o m a n numeral indicates stage of
development according to Eyal-Giladi & Kochav, 1976) of the White Leghorn
breed were used. Unincubated blastoderms, and blastoderms incubated up to
the definitive streak stage (stage 4 - arabic numeral indicates stage of development according to Hamburger & Hamilton, 1951) were removed from the
egg, washed free of the vitelline membrane and any adhering yolk, and carded
with a wide-mouthed pipette in a drop of Ringer solution on to a vitelline
membrane raft. The blastoderm was flattened, epiblast side against the surface
of the vitelline membrane which was stretched over a glass ring as described
by New (1955).
Hypoblasts from stage XIII blastoderms were loosened from the epiblast
and eventually peeled off from it with the use of sharpened fine dissecting
needles. Composite blastoderms were constructed by placing a series of two,
three or four hypoblasts on to host unincubated blastoderms (stage X)> or
on to denuded epiblasts from older blastoderms (up to stage 4). Hypoblasts
were manoeuvred so that they were stretched in a polar, triangular or quadrangular pattern.
The prospective anteroposterior axis was determined by means of the cell
population density which characterizes most unincubated blastoderms (Spratt
& Haas, 1960), and the plane of the axis was marked by a line of non-toxic,
non-diffusible carbon or carmine powder with a fine needle directly on the
hypoblast. The plane of the axis of the host epiblast was similarly marked. In
addition, control external marks were made close to the stretched blastoderm
on the vitelline membrane raft to serve as double checks of the blastoderm
orientation.
Blastoderms were cultured in plain Dulbecco's modified Eagle medium
(MEM), 1-5 ml/blastoderm in a small Petri dish (internal diameter 5 cm)
resting on a moist cotton ring inside a larger support Petri dish (9 cm). The
cultures were incubated at 38 °C as described elsewhere (Zagris, 1979). Blastoderms, culture media, and glassware were handled with sterile precautions.
Staining and determination of haemoglobin
The control and experimental blastoderms were cuHured for at least 3 days
before their staining with benzidine - peroxide solution (Zagris, 1980) to show
presence of Hb for photography. At the end of the culture period, the MEM
was removed and the blastoderms were flooded with benzidine - peroxide
118
N. ZAGRIS
solution. Colour developed within the first few minutes after application of
the staining solution which was removed after about 5 min, and the blastoderms
were photographed.
For Hb determination, blastoderms, which were in culture usually for 5 days,
were lifted from the vitelline membrane carefully, and were homogenized in
l-9mlH 2 O. The homogenate was centrifuged for 10 min at 700rev./min in
a clinical centrifuge, and the supernatant was used for Hb estimation by the
O-dianisidine-H2O2 procedure (Hell, 1964).
The results and conclusions are based on the study of more than 20 blastoderms per group.
RESULTS
Two, three or four hypoblasts transplanted on to unincubated blastoderms
and on to denuded epiblasts of older blastoderms in a polar, triangular, and
quadrangular pattern merge to form a continuous layer about 8 h after their
transplantation. Four hypoblasts transplanted onto an unincubated blastoderm
in a quadrangular pattern have merged into a continuous layer as shown after
20 h in culture (Fig. 1 A). Multiple vascular areas which form synchronously
one at the site of each transplanted hypoblast are orientated each around a
prominent tissue aggregation which is usually without distinct morphology as
shown after 6 d in culture (Fig. 1B). These vascular areas which are localized
usually in a U-shaped configuration resemble the a.o.v. and are referred to as
Abbreviations: «, aborted axis; aov, area opaca vasculosa; ax, embryonic axis;
chl, composite hypoblastic layer; h, head region; iav, induced area vasculosa.
Fig. 1. Stage-X blastoderm with four hypoblasts placed in a quadrangular pattern
shows merging of the hypoblasts into a continuous hypoblastic layer after 20 h
(A) in MEM culture. Shown after 6 days (B) in culture, it displays i.a.v. (dark areas),
one at the site of each transplanted hypoblast, stained with benzidine-peroxide
solution. Tracings (B) mark individual i.a.v. Scale bar = 500 fim.
Fig. 2. Stage-X blastoderm with four hypoblasts placed randomly forms four i.a.v.
as shown after 6 days in culture. Tracings mark individual i.a.v. Scale bar = 500 /tm.
Fig. 3. Stage-2 blastoderm with two additional hypoblasts forms the embryonic
axis and the a.o.v. at their prospective sites, and one i.a.v. 180° anteriorly as
shown after 6 days in MEM culture. Scale bar = 500 ftm.
Fig. 4. Stage-3 blastoderm with two additional hypoblasts forms the embryonic
axis and a.o.v. at their prospective sites shown after 5-5 days in MEM culture.
Scale bar = 500 fim.
Fig. 5. Stage-3 blastoderm with two additional hypoblasts forms the a.o.v. at
its prospective site, and the embryonic axis turned 180° as shown after 6 days in
MEM culture. Scale bar = 500 fim.
Fig. 6. Stage-3 control blastoderm forms the embryonic axis and the a.o.v. at their
prospective sites as shown after 6 days in MEM culture. Scale bar = 500 /im.
Fig. 7. Stage-10 to -11 blastoderm developed in ovo, shows normal formation of
the embryonic axis and a.o.v. at their prospective sites. Blastoderm stained with
benzidine-peroxide solution. Scale bar = 1000 ftm.
Stabilization of area opaca vasculosa in chick blastoderm
1A
hiV
.
2
A7V
aov
6 .
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i
i
119
120
N. ZAGRIS
induced area vasculosae (i.a.v.). However, presence of the prominent tissue
aggregation is not necessary and blood formation occurs in its absence. When
the hypoblasts are placed randomly and in close proximity, the neighbouring
i.a.v. which form interconnect to an intricate, massive plexus but they do
not lose their U-shaped orientation around the prominent tissue aggregation.
Each of these areas gives the impression of an embryonal organization centre
as is shown after 6 days of culture of a host unincubated blastoderm onto
which four hypoblasts were transplanted (Fig. 2). The i.a.v. arise synchronously
at the site of the transplanted hypoblasts of which anteroposterior orientation
on the host epiblast is not important, and blood islands can form at the new
posterior end of the transplanted hypoblast which is oriented towards the
edge of the composite blastoderm.
Host epiblasts from pre-streak blastoderms display the same behaviour as
the unincubated blastoderm in that they also support formation of multiple
i.a.v. depending on the number of the hypoblasts transplanted.
Composite blastoderms with host epiblasts from initial streak (stage-2)
blastoderms show formation of only two vascular areas one posteriorly as in
the control, the other 180° anteriorly. In these epiblasts the early streak is
already imprinted, and, in all cases, it disperses after transplantation of the
hypoblasts, and a new streak forms at the original axis plate. Transplantation
of two hypoblasts onto the anterior end of a stage-2 blastoderm opposite the
host streak and with the anterior end of the transplanted and host hypoblasts
almost touching one another results in formation of a definite, although morphologically abnormal, embryonic axis according to its original anteroposterior
orientation, and to the formation of two area vasculosas, the a.o.v. as in the
control blastoderm, and an i.a.v. 180° anteriorly (Fig. 3).
Host epiblasts from intermediate streak (stage-3) and older blastoderms
on to which two or more hypoblasts are transplanted form a definite embryonic
axis at the original axis plate, and only one vascular area, the a.o.v. at its
expected topographical location on the host epiblast. Such a host epiblast on
to which two hypoblasts were transplanted forms the a.o.v. at the expected
normal location as is shown after 5-5 days of culture (Fig. 4). A similar composite blastoderm shows a posteroanterior turning of its embryonic axis, the
head occupying the original tail region in the host blastoderm, thus giving
the impression of a 180° shift of the a.o.v. (Fig. 5). This reversal of the anteroposterior polarity, which occurs rarely, apparently involves extensive reorganization of the embryonic axis, but the fact that the a.o.v. was formed at its
prospective site provides strong evidence of the stability of the a.o.v. by the
intermediate streak stage.
A control blastoderm explanted at stage 3 is shown after 6 days in culture
(Fig. 6). This specimen was chosen because it shows the typical embryonic
axis and also size and location of the a.o.v. as displayed by most blastoderms
in the plain MEM culture. It is of interest to note that the a.o.v. of the control
Stabilization of area opaca vasculosa in chick blastoderm
121
Table 1. Formation of area vasculosae in epiblasts from chick blastoderms at
various stages of development on to which two, three, or four hypoblasts were
transplanted
Stage*
2-3
3-4
Composite
Hypoblastsf Area vasculosae blastodermst
(no. transplanted) (no. formed)
(no. examined)
2
3
4
2
3
4
2
3
4
2
3
4
2
3
4
2
3
4
2
2
2
1
1
1
25
25
10
25
25
15
25
25
10
25
25
10
* Stage, marks the developmental time blastoderms were explanted and cultured as
composite or control blastoderms in MEM for at least 3 days.
t Composite blastoderms, were constructed by placing two, three, or four hypoblasts on
to unincubated blastoderms or on to denuded epiblasts of the stages indicated.
blastoderms is less prominent than the total surface area occupied by the
area vasculosae of the composite blastoderms.
A stage 10-11 blastoderm which developed in ovo is presented to serve as
reference point for the location of the a.o.v. relative to the embryonic axis
(Fig. 7). The blastoderm was stained with benzidine-peroxide solution.
The number of area vasculosae formed in composite blastoderms at various
stages of development can be summarized as shown in Table 1. Each transplanted hypoblast induces formation of one area vasculosa, that is four i.a.v.
are formed under the influence of four hypoblasts, in blastoderms at stages X
and 1, two area vasculosas, the a.o.v. and one i.a.v., are formed in stage-2
blastoderms, while only the a.o.v. is formed in stage-3 blastoderms. The results
in each group were consistent. Some cultures were lost because of technical
error, such as puncturing or overstretching the vitelline membrane of the
raft which resulted in disfiguring of the blastoderm, but these were discarded.
Measurements of the total amount of Hb were made on blastoderms which
were explanted at the stages X, 1, 2-3 and 3-4. Control, and composite blastoderms constructed by transplantation of two, and three hypoblasts on denuded
epiblasts were cultured in MEM for 5 days. There was about a 2-fold, and a
5- to 6-fold increase of Hb content in composite blastoderms with the two
and three transplanted hypoblasts, respectively, in the stage-X and stage-1
groups as compared to their control. Composite blastoderms of the stage-2 to
122
N. ZAGRIS
Table 2. Measurement of Hb in control and composite blastoderms
(Values expressed in /tg per blastoderm ± standard deviation.
The results are the average of three experiments.)
Stage*
Control
blastoderms
X
1
2-3
3-4
018 ±002
0-22 ±003
0-90 ±013
0-91 ±003
,
Composite blastodermsf
*
»
Two hypoblasts Three hypoblasts
0-27 ±001
0-55 ±.006
0-82 ±009
115 ±009
0-6 ±003
1-3 ±002
0-88 ±008
110±004
* Stage, marks the developmental time blastoderms were explanted and cultured as
composite or control blastoderms in MEM for 5 days.
t Composite blastoderms, as defined in Table 1.
-3 and stage-3 to -4 groups contain about the same amount of Hb as their
control blastoderms. More Hb is present in control blastoderms explanted at
older stages, compared to those at younger stages, at the end of the same
culture period (Table 2).
Blastoderms cultured in MEM form the embryonic axis with a distinctive
anteroposterior orientation at its prospective site. The embryonic axis shows
a pronounced brain region and neural tube, a lateral mesoderm which rarely
becomes segmented, and, in many cases, cardiac tissue which pulsates rhythmically (62 pulses/min). However, the U-shaped a.o.v. forms at its prospective
site characteristic of normal in ovo development, and normal growth of the
blastoderm is not affected. The MEM supports substantial Hb formation as
compared to culture in plain albumin in which, although there is blood island
formation, Hb presence is rarely detected under the stereoscope even after
staining with benzidine (Zagris & Eyal-Giladi, 1981). In addition to its enhancing
Hb formation, the MEM provides favourable conditions for prolonged blastoderm survival for at least 10 days, in contrast to culture in plain albumin in
which it shows the typical distinct signs of degeneration 2-3 days after the
beginning of culture. Enhancement of Hb formation and prolonged embryonic
survival in culture are distinct advantages which make the MEM a useful culture
medium for the study of erythropoiesis in the early chick blastoderm.
In all the experiments described above, the first blood islands appear as
numerous cell thickenings in the almost transparent blastoderm at the beginning
of the second day in culture. The cell thickenings become slightly tinged with
yellow at the end of the second day in culture, and a few hours later appear as
dense bright red clusters due to the presence of Hb apparent even on casual
observation. Blood islands become more intensely red the following days in
culture (Zagris, 1979). The presence of Hb was confirmed by a sharply positive
benzidine stain, and was not found, after staining, in any place in which it had
not been detected in the unstained state.
Stabilization of area opaca vasculosa in chick blastoderm
123
DISCUSSION
The unincubated blastoderm, a relatively unstructured tissue with no apparent
plane of symmetry, can form multiple embryonal centres or fractions of
centres as evidenced by cellular aggregations each associated with a vascular
area under the influence of multiple transplanted hypoblasts (Figs. 2, 3). It is
well known that the endoderm plays an important role in organizing the
mesoderm into blood islands (Miura & Wilt, 1970). In this connexion, it should
be noticed that the hypoblast affects both the PS and the a.o.v. Thus, an
induced streak is accompanied by an accumulation of blood islands opposite
its posterior end. In our results, vascular rings are orientated around tissue
aggregations which we interpret as aborted attempts to form embryonic axes.
It is of interest to note that, though the transplanted hypoblasts merge to
form a continuous thick layer, they do not participate collectively in forming
one streak and embryo body with one area vasculosa at the posterior end, It
may be that the merged hypoblasts do not lose their individuality, or, more
likely, that they have induced their prospective embryonal centre before their
merging. It seems that the unincubated blastoderm is a developmental mosaic
field out of which one or more embryonic fields may arise. The properties of
mosaic fields are discussed in a theoretical paper by Chandebois (1976). Another
point of interest which emerges from our results is that the hypoblast does
not seem to have an anteroposterior orientation but this is determined by the
epiblast. Thus, blood islands always form at the hypoblast posterior oriented
towards the periphery of the epiblast.
Prestreak blastoderms exhibit similar behaviour as that discussed with the
unincubated blastoderm and are not capable of complete embryonic integration.
However, embryonic segregation, especially that concerned with embryonic
axis formation, occurs in stage-2 and older blastoderms which display remarkable
regulative features. Blastoderm with stage-2 host epiblasts and with two or
more transplanted hypoblasts form one well centered embryonic axis and two
blood island areas one posteriorly as in the control, the other 180° anteriorly.
More Hb is present in composite as compared to control blastoderms at
stage X and stage 1, the amount of Hb increasing as the number of transplanted hypoblasts increases. This, in concert with the observation that the
vascular area(s) of the composite look more prominent morphologically than
that of the control blastoderms, may reveal that there is not a fixed pool of
prospective blood cells which is shared. It would seem either that there js a
non-fixed pool from which prospective blood cells are recruited or that each
transplanted hypoblast induces blood cell formation in situ.
Waddington (1933) has shown that the hypoblast plays an important tole
in directing tissue movements which build the streak in the epiblast but it was
not clear whether the hypoblast starts these movements. In our results, the
formation of multiple embryonal centres or fractions of centres at the site
124
N. ZAGRIS
of the transplanted hypoblast on unincubated blastoderm or pre-streak epiblast
shows that the hypoblast is able to initiate certain events to which the epiblast
responds. The experiment in which the embryonic axis formed at an 180° angle
(Fig. 7), in addition to providing strong evidence demonstrating the stability
of the definitive a.o.v. by stage 3, also indicates that the hypoblast can induce
a new set of movements to form a PS. This new set of movements seems to
coalesce with the original set and may be powerful enough to annul the original
thus reversing the posteroanterior orientation of the axis.
The fact that an already imprinted streak on the host epiblast disappears
in all cases demonstrates that its cells are not stabilized, and may show that
to accomplish integration, it is important to re-establish a simple low-layer
morphological pattern. According to Spratt & Haas (1960), complete integration
depends upon the dominance of one embryo-initiating centre over any others
which may be present in a composite system. Then, it is likely that movements
might merge into one another to give rise to a single PS in the prospective
axial plane of the blastoderm.
The stage dependence involving a progressive restriction of the areas in
which blood islands will develop, suggests the existence of a centre which
creates organization by integrating short-lived fields. It seems that there are
no particular cell groups of the unincubated blastoderm determined to form
erythrocytes, but that the organizing capacities of the area are necessary to
induce the first early commitments of prospective erythroblasts along this
course. It is at the stage-3 blastoderm that the distinctive horseshoe-shaped
a.o.v. surrounding the posterior and posterolateral parts of the area pellucida
is stabilized.
Some of the results of this work were presented at the XlVth International Embryological
Conference, 11-17 Sept. 1980, Patras, Greece.
REFERENCES
AZAR, Y. & EYAL-GILADI, H. (1979). Marginal zone cells- the primitive streak-inducing
component of the primary hypoblast in the chick. /. Embryol. exp. Morph. 52, 79-88.
BELLAIRS, R. (1971). Developmental Processes in Higher Vertebrates. London: Logos.
BROWN, J. L. & INGRAM, V. M. (1974). Structural studies on chick embryonic hemoglobins.
J. biol. Chem. 249, 3960-3972.
BRUNS, G. A. P. & INGRAM, V. M. (1973). The erythroid cells and haemoglobins of the
chick embryo. Proc. Trans. R. Soc. B 265, 225-305.
CHANDEBOIS, R. (1976). Cell sociology: a way of reconsidering the current concepts of
morphogenesis. Acta biotheor. Leiden 25, 71-102.
CHAPMAN, B. S. & TOBIN, A. J. (1979). Distribution of developmentally regulated hemoglobins in embryonic erythroid populations. Devi Biol. 69, 375-387.
CIROTTO, C , SCOTTO, Di TELLA, A. & GERACI, G. (1975). The hemoglobins of the developing
chicken embryos. Fractionation and globin composition of the individual component of
total erythrocytes and of a single erythrocyte type. Cell Differ. 4, 87-99.
DANTSCHAKOFF, W. (1907). Ober der erste Auftreten der Blutelemente im Huhnerembryo.
Folia haemat. 4, 159-166.
Stabilization of area opaca vasculosa in chick blastoderm
125
H. & KOCHAV, S. (1976). From cleavage to primitive streak formation: a
complementaiy normal table and a new look at the first stages of development of the
chick. I. General morphology. Devi Biol. 49, 321-337.
EYAL-GILADI, H. & WOLK, M. (1970). The inducing capacities of the primary hypoblast as
revealed by transfilter induction studies. Wilhelm Roux1 Arch. EntwMech. Org. 165,
226-241.
FONTAINE, J. & LE DOUARIN, N. M. (1977). Analysis of endoderm formation in the avian
blastoderm by the use of quail-chick chimaeras. /. Embryol. exp. Morph. 41, 209-222.
FRASER, R. C. (1963). Electrophoretic characteristics and cell content of the haemoglobins
of developing chick embryos. /. exp. Zool. 156, 185-195.
HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in the development
of the chick embryo. J. Morph. 88, 49-92.
HELL, A. (1964). The initial synthesis of haemoglobin in de-embryonated chick blastoderms.
I. Metabolism of the blastodisc cultured in vitro. J. Embryol. exp. Morph. 12, 609-619.
MARTIN, C , BEAUPAIN, D. & DIETERLEN-LIEVRE, F. (1980). A study of the development of the hemopoietic system using quail-chick chimeras obtained by blastoderm
recombination. Devi Biol. 75, 303-314.
MIURA, Y. & WILT, F. H. (1969). Tissue interaction and the formation of the first erythroblasts of the chick embryo. Devi Biol. 19, 201-211.
MIURA, T. & WILT, F. H. (1970). The formations of blood islands in dissociated-reaggregated
chick embryo yolk sac cells. Expl Cell Res. 59, 217-226.
MODAK, S. P. (1965). Sur l'origine de l'hypoblaste chez les oiseaux. Experientia 21, 273.
MODAK, S. P. (1966). Analyse experimentale de l'origine de l'endoblaste embryonnaire
chez les oiseaux. Rev. Suisse Zool. 73, 877-908.
MURRAY, P. D. F. (1932). The development in vitro of the blood of the early chick embryo.
Proc. R. Soc. Lond. B 111, 497-521.
NEW, D. A. T. (1955). A new technique for the cultivation of the chick embryo in vitro.
J. Embryol. exp. Morph. 3, 326-331.
NICOLET, G. (1970). Analyse autoradiographique de la localization des differentes ebauches
presomptives dans la ligne primitive de l'embryon de Poulet. / . Embryol. exp. Morph.
23, 79-108.
ROSENQUIST, G. C. (1966). A radioautographic study of labeled grafts in the chick blastoderm. Carnegie Inst. Contrib. Embryol. 38, 111-121.
ROSENQUIST, G. C. (1972). Endoderm movements in the chick embryo between the $arly
short streak and head process stages. / . exp. Zool. 180, 95-104.
RUDNICK, D. (1938). Differentiation in culture of pieces of the early chick blastoderms. I.
The definitive streak and head process stages. Anat. Rec. 70, 351-369.
RUDNICK, D. (1955). Teleosts and birds. In Analysis in Development (ed. B. H. Wi)lier,
P. A. Weiss & V. Hamburger). Philadelphia, Pennsylvania: Saunders.
SABIN, F. R. (1917). Preliminary note on the differentiation of angioblasts and the method
by which they produce blood-vessels, blood-plasma and red blood-cells as seen in the
living chick. Anat. Rec. 13, 199-204.
SANDERS, E. J., BELLAIRS, R. & PORTCH, P. A. (1978). /// vivo and in vitro studies on the
hypoblast and definitive endoblast of avian embryos. /. Embryol. exp. Morph. 46, 187205.
EYAL-GILADI,
SCHALEKAMP, M., SCHALEKAMP, M., VAN GOOR, D. & SLINGERLAND, R. (1972). Re-evalu$tion
of the presence of multiple haemoglobins during ontogenesis of the chicken: electrophoretic
and chromatographic characterization, polypeptide composition and immunochettiical
properties. / . Embryol. exp. Morph. 28, 681-713.
SETTLE, G. W. (1954). Localization of the erythrocyte-forming areas in the early chick
blastoderm cultivated in vitro. Contr. Embryol. 241, 223-237.
SPRATT, N. T., JR. & HAAS, H. (1960). Integrative mechanisms in development of the early
chick blastoderm. I. Regulative potentiality of separated parts. /. exp. Zool. 145, 97-^138.
TOBIN, A. J., SELVIG, S. E. & LASKY, L. (1978). RNA synthesis in avian erythroid cells.
Devi Biol. 67, 11-22.
5
EMB 68
126
N. ZAGRIS
L. (1962). Some new data concerning the formation of the definitive endoblast in
the chick embryo. /. Embryol. exp. Morph. 10, 38-57.
VAKAET, L. (1970). Cinephotomicrographic investigations of gastrulation in the chick blastoderm. Archs Biol. {Liege) 81, 387-426.
WADDINGTON, C. H. (1933). Induction by the primitive streak and its derivatives in the
chick. /. exp. Biol. 10, 38-46.
WAINWRIGHT, S. D. & WAINWRIGHT, L. K. (1974). Isolation of two erythrocyte cell
populations from the early chick blastodisc and the further resolution of one into two
essential subpopulations. Expl Cell Res. 88, 143-152.
WILT, F. H. (1965). Regulation of the initiation of chick embryo haemoglobin synthesis.
J. molec. Biol. 12, 331-341.
WILT, F. H. (1967). The control of embryonic hemoglobin synthesis. In Advances in Morphogenesis (ed. M. Abercrombie & J. Brachet), pp. 89-125. New York, London: Academic
Press.
ZAGRIS, N. (1979). Differentiation capacity of unincubated chick blastoderm in culture.
/. Embryol. exp. Morph. 50, 47-55.
ZAGRIS, N. (1980). Erythroid cell differentiation in unincubated chick blastoderm in culture.
J. Embryol. exp. Morph. 58, 209-216.
ZAGRIS, N. & EYAL-GILADI, H. (1981). Thymidine: Inhibitor of differentiation in the young
chick blastoderm in culture. Experientia 37, 892-894.
ZAGRIS, N. & EYAL-GILADI, H. (1982). 5-bromodeoxyuridine inhibition of the epiblast
competence for primitive streak formation in the young chick blastoderm. Devi Biol.,
in press.
ZAGRIS, N. & MELTON, C. G. (1978). Hemoglobins in single chick erythrocytes as determined
by a differential elution procedure. Z. Naturf. 33 c, 330-336.
VAKAET,
{Received 6 July 1981, revised 9 October 1981)