/. Embryol. exp. Morph. 75, 21-30 (1983)
Printed in Great Britain © The Company of Biologists Limited 1983
21
Nature of the hypoblastic influence on the chick
embryo epiblast
By E. MITRANI 1 , Y. SHIMONI 1 AND H. EYAL-GILADI 1
From the Embryology Section, Department of Zoology, The Hebrew
University of Jerusalem
SUMMARY
Stage XIII chick blastoderms deprived of the marginal zone, the area opaca and the posterior half of the hypoblast, when incubated further developed axes whose orientation in 50 %
of the cases was according to the original blastoderm's orientation, whilst in 50 % of the cases
they developed at 90° from the posterior side. Those results illustrate the quantitative differences in inductivity between the anterior and the posterior hypoblastic halves. Normally the
posterior region has the highest effect but other regions can also bring about the development
of an embryonic axis if allowed to act upon the epiblast for a sufficiently long period of time.
The possible ways in which a chick hypoblast influences the epiblast to develop an embryo are
examined in the light of recent findings and of new experiments described below.
INTRODUCTION
Since the early experiments by Waddington (1937) the importance of the
hypoblast on determining the future of embryonic development of the epiblast
has become apparent. Waddington noted that a short primitive streak (PS) of a
chick blastoderm would tend to deflect towards the anterior end of the hypoblast
when the latter was placed at right angles to the epiblast. Later Azar & EyalGiladi (1979) in a carefully controlled experiment showed clearly that a denuded
stage XIII (Eyal-Giladi & Kochav, 1976) epiblast also deprived of the marginal
zone when allowed to generate a second layer, or defective hypoblast, cannot
generate embryonic structures. The need for the presence of a normal hypoblast,
formed from two different cellular sources, for the epiblast to develop into an
embryo, was clearly demonstrated. Furthermore, Azar & Eyal-Giladi (1981)
showed that if the hypoblast of a stage XIII chick blastoderm is rotated by 90 °
the PS direction will develop according to the orientation of the hypoblast.
Even though it was thus generally agreed that the hypoblast does perform an
inductive action on the epiblast we raised the question whether the hypoblast's
function is not just one of dictating the future polarity of the embryo or whether
it also performs a specific inductive function. We found that if the polarity of the
hypoblast is destroyed the orientation of the primitive streak can be determined
1
Authors' address: Department of Zoology, The Hebrew University of Jerusalem, 91904
Jerusalem, Israel.
22
E. MITRANI, Y. SHIMONI AND H. EYAL-GILADI
by the epiblast (Mitrani & Eyal-Giladi, 1981). The finding that there exists a
stored polarity in the epiblast confirmed the notion that the need for an hypoblast
in order for the epiblast to develop into an embryo is not solely related to polarity
but that also some inductive property is required from it. Although the nature
of the induction process performed by a normal hypoblast is not yet known we
argued then that the hypoblast's function is not just one of a physical layer that
provides that sort of cover to an otherwise denuded epiblast and/or that provides
non-specific anchorage or guidance for the movement of the future mesodermal
cells (Mitrani & Eyal-Giladi, 1981). The possibility still remained that as a result
of a specific interaction between the epiblast and the hypoblast, a particular
extracellular network is produced between the epiblast and the hypoblast and
that this network would be necessary for axial development to occur. This idea
became attractive since we found that fibronectin, a cell-cell, cell-substrate
mediator glycoprotein, was found between the hypoblast and the epiblast during
hypoblast formation and not during earlier developmental stages. Fibronectin
was found between the two layers indicating that a network does indeed exist
between the epiblast and the hypoblast (Mitrani & Farberov, 1982).
The present work is an attempt to examine further the nature of the necessity
for the interaction between the epiblast and the hypoblast. Even though there
has been some sort of concensus in that there must be some sort of gradient
system within the hypoblast (see Azar & Eyal-Giladi, 1981) evidence is so far
inconclusive as to whether only the posterior part of the hypoblast possesses the
primitive-streak-inducing capacity or whether this property is spread throughout
the entire hypoblast. The experiments reported below test the inductive
capacities of the anterior versus the posterior half of the hypoblast. It is examined
whether the anterior half presumably of low hypoblastic inductivity when acting
upon a normal epiblast for a sufficiently long period of time, after the elimination
of the posterior half, can bring about the formation of an ectopic embryonic axis.
Attention is paid to minimize the effect of additional regeneration integrative
mechanisms which might otherwise render the interpretation of results more
difficult.
MATERIALS AND METHODS
Blastoderms were obtained from White Leghorn or Hybrid New Hampshire
x Leghorn eggs and were incubated to the full hypoblast stage (Stage XIII EyalGiladi & Kochav, 1976). The blastoderms were removed from the egg, carefully
examined and only those found to be at stage XIII (see below) were used. Within
those, exclusively blastoderms whose posterior side could be clearly determined
were used. The posterior side was marked with carbon. In the first group the area
opaca and the marginal zone were removed with a hair loop to avoid any
regeneration of an inductive hypoblast and then the posterior half of the
hypoblast was removed with tungsten needles so that the whole epiblast proper
Hypoblast influence on chick epiblast
23
with only the anterior half of the hypoblast was incubated further (Group I).
Four additional groups of experiments were performed. In Group II only the
posterior half of the hypoblast of a stage XIII blastoderm was removed, the
marginal zone and the area opaca were left intact. In all remaining groups, the
marginal zone and the area opaca were removed from stage XIII blastoderms.
In Group III the anterior (instead of the posterior) side of the hypoblast was
removed. In Group IV the posterior half of the hypoblast was removed and the
anterior half was rotated 180 ° so that the most anterior aspect of the hypoblast
became aligned with the posterior epiblastic aspect. In Group V the posterior
Table 1.
Type of Experiment
Group 1
No marginal zone
No area opaca
No posterior half
of hypoblast
Deviation from original axis
0°
90° left
90° right
45°
0° + 90°
90° + 90°
180°
Group II
With marginal zone
With area opaca
No posterior half
of hypoblast
90° right
(15)
(5)
(4)
(2)
(2)
(2)
(4)
(21)
o
82%
(2)
(3)
0
O
18%
Group III
No marginal zone
No area opaca
No anterior half
of hypoblast
91%
(11)
(1)
o
9%
Group IV
No marginal zone
No area opaca
Anterior half of
hypoblast rotated 180
0°
90° left
90° right
(13)
(3)
(4)
0
(12)
Q
60%
e
40%
77%
O
23%
Group V
No marginal zone
No area opaca
Posterior half of
hypoblast replaced by
anterior half
0°
90° left
90° right
(2)
(6)
Deviation of PS axis from epiblast axis. Number in parenthesis indicates number of cases.
* Posterior epiblastic side. ||||| = anterior hypoblastic half, | g = posterior hypoblastic
half, I!!!! = Marginal zone.
24
E. MITRANI, Y. SHIMONI AND H. EYAL-GILADI
half of the hypoblast was removed and replaced by an additional anterior
hypoblastic half so that the epiblast was covered by two anterior hypoblastic
halves (see diagrams on Table 1).
All operated blastoderms were incubated further, lower side up, on a vitelline
membrane stretched on a glass ring (New, 1955) and put on semisolid albumin
for up to 72 h at 38 °C. Blastoderms were carefully examined after the initial 3 and
6 h and then every 12 h for the development of axes. The orientation of each axis
was carefully recorded.
RESULTS
There was a high percentage of blastoderms that did not develop into embryos.
The results are summarized in Table 1.
In Group I about 50 % of the blastoderms that had axes (by axis it is understood anything from a clear PS to an embryo with a few somites) developed them
at 0 °, namely from the original posterior side (Fig. 1). The posterior side of the
remaining 50 % of the axes was found to be shifted by 90 ° from the posterior side
of the epiblast (Fig. 2). In some cases two axes were obtained simultaneously.
In Fig. 3 a case is shown where both axes developed at 90° one from the left and
one from the right. Figures 4 and 5 demonstrate cases in which one axis
originated from the posterior side and another at 90° from it. In very few cases
(10%) embryos developed at 180°.
The experiments of Groups II and III were performed in order to determine
whether the results obtained in Group I were not due to experimental artifacts
which might have been caused by cutting or manipulating the embryos (see EyalGiladi & Spratt, 1965). When the anterior, instead of the posterior half of the
hypoblast was removed together with the marginal zone and the area opaca, only
8 % the axes developed along the cut edge i.e. at 90 ° (Group III). In blastoderms
of which the posterior half of the hypoblast was removed but the area opaca and
the marginal zone were left intact, (Group II), in 82 % of the cases the embryos
developed according to the posterior side of the epiblast.
In Group I 63 % of the 15 embryos that developed at 0 ° reached either the
Fig. 1. Blastoderm from Group I in which an embryonic axis developed according
to the posterior end of the epiblast. All blastoderms were marked with carbon (c)
prior to surgery on the posterior end of the epiblast.
Fig. 2. Blastoderm from Group I in which an embryonic axis developed at 90 ° to the
epiblastic axis, (c) carbon mark.
Fig. 3. Blastoderm from Group I in which two axes both perpendicular to the
epiblast axis developed, (c) carbon mark.
Fig. 4. Blastoderm from Group I in which two axes developed; one according to the
epiblastic axis, and one perpendicular to it, (c) carbon mark.
Fig. 5. Same blastoderm as illustrated in Fig. 4 at a later stage of development. Note
how both axes cross each other, (c) carbon mark.
Hypoblast influence on chick epiblast
25
2
•-,
..4
•
'
•
.
*
J
3
1mm
5
Figs 1-5
26
E. MITRANI, Y. SHIMONI AND H. EYAL-GILADI
head process stage or somite stages. Whilst only 50% of the 11 embryos that
developed at 90 ° in Group I reached the head process or somite stages. The other
50 % regressed.
Both in Groups II and III in about 90 % of the cases that developed axes, the
latter did not deviate from the posterior side of the epiblast, and in most of the
cases they developed into normal embryos with several pairs of somites.
Experiments of Groups IV and V test further the inductive capacity of the half
of the hypoblast. In Group IV 77 % of the blastoderms developed axes at 0°
whilst 23 % developed axes at 90 °. In Group V the additional anterior hypoblast
half did not alter to a great extent the results of Group IV so that 69 % of the
blastoderms developed axes at 0° whilst 31 % developed axes at 90°. In Group
IV 70 % and in Group V 80 % of the blastoderms developed into normal embryos with several pairs of somites.
DISCUSSION
The aim of the experiments presented above was to check the hypothesis that
in the primary hypoblast there is a gradient of inductivity for PS with the maximal
concentration at the median posterior area and a gradual drop in anterior as well
as in lateral directions. We therefore tried to create as far as possible a situation
according the above hypothesis where the epiblast could be submitted to different quantitative hypoblastic influences. Because of the impracticability of
such an experiment a compromise experiment was designed (Group I) with the
appropriate controls (Groups II and III). In Group I the posterior epiblastic half
was left under no hypoblastic influence from the time of the operation until the
time the regenerating hypoblast reached again the posterior side. Meanwhile,
the anterior epiblastic side was left under an uninterrupted influence of the
presumably 'weaker' anterior hypoblastic half. The maximal inductive capacity
of which, should have been according to our prediction at its most posterior part,
namely the cut surface. The results of Group I experiments suggest that there are
quantitative differences within the hypoblast so that the anterior area of lower
hypoblastic inductivity acting upon an epiblastic area for a longer period of time,
can achieve the same effect, mainly the bringing about of an embryonic axis, as
a posterior area of higher hypoblastic inductivity acting for a shorter period of
time. In some cases each area was capable of generating its own axis.
Few embryos (10 %) in Group I developed at 180 °. These results are in agreement with the previous results from Group I and may provide additional information. When only the anterior hypoblastic half is present the dominant part
seems to be the most posterior aspect of it. The most anterior aspect (i.e. axes
that developed at 180 °) being able to generate an axis only on a few percent of
the cases. (Compare also results of Group I with Group IV.) A word of caution
however should be said when attempting to interpret these results. It is often
difficult to distinguish at stage XIII between the posterior and the anterior
Hypoblast influence on chick epiblast
27
hypoblastic side and an error in the marking would have meant that the anterior
rather than the posterior side of the hypoblast would have been removed in
which case these experiments would form part of Group III and the set of results
would be comparable to those blastoderms from Group III that developed at 0 °.
The present experiments essentially differ from Spratt & Haas's earlier works
(1960a,6,1961) mainly in that with the exception of Group II the marginal zone
was removed so that the regenerative integrative mechanisms for which the
marginal zone is largely responsible could be avoided. Azar & Eyal-Giladi
(1979) have recently shown that the marginal zone has the capacity of regenerating an inductive hypoblast. Spratt & Haas (1961) have discussed the importance of the marginal zone in determining the capacity of an operated blastoderm to regenerate a new embryonic field, and the importance of directed
movement of cells from what has been called the growth centre. This growth
centre was believed to be largely responsible for the creation of the new
embryonic field.
In Group I 50 % of the embryonic axes developed along the cut edge, that is
at 90 ° from the posterior side of the blastoderm. In contrast in Group III virtually
no embryonic axes developed along the same cut edge suggesting that whatever
directed cell movements may be triggered by the cut edge this is not sufficient
to determine the direction and formation of an embryonic axis.
It must thus be concluded that hypoblastic cells located in the anterior half
must have been responsible for the induction of the axes at 90° in Group I.
Group II, although not being a direct control for Group I was meant to check
to what extent the introduction of the marginal zone component would alter the
equilibrium obtained when only the anterior half of the hypoblast was present.
As can be seen, the introduction of the marginal zone causes an increase in the
percentage of embryos that developed from the posterior end (0 °). This is understandable on the basis of the known observation that within the marginal zone
itself the region of higher inductivity lies in the posterior end (Azar & EyalGiladi, 1979). It is interesting nevertheless to note that the regenerative mechanisms and the cell movements that must have been triggered from the lateral
marginal zone by the cut hypoblastic edge were not sufficient in most cases to
overcome the inductivity of the posterior side of the marginal zone.
Experiments in Groups IV and V were performed to investigate further the
inductive capacity of the anterior hypoblastic half and in particular of the most
anterior hypoblastic aspect. The aim being to examine whether this hypoblast
inductive capacity extends to the most anterior end or whether on the contrary,
the most anterior aspect may have a primitive streak-forming inhibiting effect.
The fact that the percentage of embryos that developed at 0° was higher in
Groups IV and V (particularly in Group IV) than in Group I indicates that the
most anterior aspect of the hypoblastic half is not a primitive-streak-inhibiting
centre but it has as well as the other hypoblastic areas, inducing capacity. It must
be pointed out however that this capacity is clearly the result of a sinergistic
28
E. MITRANI, Y. SHIMONI AND H. EYAL-GILADI
process between a previously acted upon posterior epiblastic area and the most
anterior hypoblastic aspect.
Results from Group V indicate a relative increase in the number of axes that
developed at 90°. This could be an indication of some sort of reinforcement of
the hypoblastic inductivity at the plane where both anterior halves meet. Yet
even in this case it seems that this is not enough to counteract the inductive effect
of the most anterior hypoblastic aspect on the posterior epiblastic side.
By comparing the results from Groups I, IV, and V (with the appropriate
controls Groups II and III) a general picture appears in which, as expected, the
most posterior hypoblastic aspect has the strongest primitive streak inducing
capacity. The middle hypoblastic region has also primitive streak inducing capacity and even the most anterior aspect of the hypoblast has the capacity of inducing
a primitive streak.
We believe the experiments above provide one way of examining the effect of
the duration of contact between the epiblast and the different hypoblastic areas
being considered, whilst minimizing the effect of integrative mechanisms. The
fact that in Group 150 % of the embryonic axes that developed at 90 ° regressed,
is in agreement with previous observations (Azar & Eyal-Giladi, 1981) and is
perhaps another indication of the lower inductive capacity of the anterior
hypoblastic half.
Results from Group I also render unlikely the possibility that some sort of
extracellular network is the primary mediator of the interaction between the
epiblast and the hypoblast. More recent experiments have indicated that the
fibronectin network found between the epiblast and the hypoblast probably
forms part of a basement membrane. The specific basement membrane component Laminin has been found to codistribute with fibronectin on the lower side
of the epiblastic layer facing the hypoblast both in stage XIII and in earlyprimitive-streak stages. Furthermore this basement membrane has been found
to be specifically broken at the streak region (Mitrani, 1982). We believe this is
an interesting observation in the context of the present work. It may be that
hypoblastic cells, or epiblastic cells through the direct action of the hypoblast,
selectively break the basement membrane in the streak region. Areas of higher
hypoblast inductivity having a higher chance of bringing about a localized breakdown of the basement membrane.
Another aspect which requires mentioning when performing the experiments
presented here is that although the passage from stage XIII to the physical
appearance of the primitive streak must be a gradual one, morphologically there
is no way of determining whether a full hypoblast stage XIII blastoderm is just
within stage XIII or is an old stage XIII which is about to generate a visible
streak. We therefore suggest that once a blastoderm has been classified as stage
XIII, if used in an experiment, its stage must be further determined retrospectively. Blastoderms should be examined for up to 4 h after further incubation
period. If within this period a primitive streak becomes apparent this would
Hypoblast influence on chick epiblast
29
become a clear indication that the blastoderm was an 'old' stage XIII and in most
cases as in the experiments described above, discarded. Indeed we have found in
apparent contradiction to earlier findings, that an epiblast proper, deprived of the
marginal zone and of the area opaca can proceed to generate a streak even in the
absence of the hypoblast. In such cases a PS will develop only from 'old' stage
XIII epiblasts since the streak will only be generated either 3 to 4 h after the
removal of the hypoblast or it will not be generated at all. It is therefore important
in designing experiments meant to determine the action of the hypoblast on the
epiblast, to take this fact into consideration. We believe that a young stage XIII
epiblast deprived of the marginal zone, the area opaca and without a hypoblast
provides an excellent bioassay to study primitive streak induction. The epiblast
proper will be fully competent to respond to a hypoblastic stimulus by forming a
complete embryo but will not respond by forming an embryonic axis in the absence of the hypoblastic component. The factors discussed above have however
to be carefully taken into consideration in future experiments.
The complex set of events by which a hypoblast can change, within a few hours,
an epiblastic layer of seemingly totipotential cells into a manifested polarized
heterogeneous system are still far from being elucidated. It now becomes clear
however that not only the posterior side of the hypoblast can induce an embryonic
axis. There seems to be some kind of a gradient-like influence so that an anterior
hypoblastic half (including its most anterior aspect) if allowed to act upon the
epiblast for a sufficiently long period of time could induce the development of an
embryonic axis.
Whether this inductivity gradient is distributed in the form of substances
throughout the hypoblast or whether the gradient itself is constituted by different
cell populations within the hypoblast is not known. In the developing chick limb,
a small group of cells - the polarizing region - acts to specify the pattern of
structures which develop. The quantity of these cells being responsible to a certain extent for the amount of duplicated digits that may be experimentally formed
(Tickle, 1981). The primary hypoblast itself is known to be formed by at least two
different cell populations - one, presumably non inductive - is obtained from
polyinvaginating cells coming directly from the epiblast, and the other, which is
inductive, develops from the posterior marginal zone and progresses anteriorly
(Eyal-Giladi & Kochav, 1976; Kochav etal. 1980; Azar & Eyal-Giladi, 1979). It
is thus tempting to suggest that the inductivity gradient within the hypoblast
might be directly established by the way the hypoblast is formed so that the
relative concentration of marginal zone-derived cells in any particular hypoblast
region might be what determines the inductivity of that area.
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