Download The development of animal cap cells in Xenopus: a measure of the

Development 101. 557-563 (1987)
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557
The development of animal cap cells in Xenopus: a measure of the start
of animal cap competence to form mesoderm
E. A. JONES and H. R. WOODLAND
MRC Animal Development Group, Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK
Summary
Grafting, together with tissue identification by monoclonal antibodies, has been used to study the allocation
of Xenopus animal cap cells to the ectodermal or
mesodermal lineages. Animal cap cells become responsive at stage 61 and lose responsiveness to
mesodermal induction at, or just after, stage 10i
(depending on the batch of embryos). The ability of
the vegetal yolky cells to induce mesoderm disappears
between stages 10i and 11. It is present at stage 6-6£
and may exist before this. The emergence of competence to respond at stage 6i, coupled with the fact
that the endoderm is already capable of induction at
this stage, suggests that mesodermal induction begins
at this point in the intact embryo.
Introduction
interactions can be demonstrated when animal cap
cells are grafted into endodermal regions of a whole
embryo (Jones & Woodland, 1987).
In this paper, we seek to determine the time when
animal cells gain or lose their responsiveness to the
mesodermal inductive signal, and the period during
which vegetal cells are capable of producing this
signal. The results presented in this paper confirm,
within a stage, those published by others describing
the end points of inductiveness and competence
(Nakamura, Takasaki & Ishihara, 1971; Dale et al.
1985; Gurdon, Fairman, Mohun & Brennan, 1985). It
was necessary to make very accurate determinations
of these somewhat controversial time points as part of
our strategy for detecting the onset of competence to
respond and induce. This has provided an indication
that mesodermal induction begins very early in the
whole embryo.
The early amphibian embryo can be considered to
consist of two different types of cell, the pigmented
animal cap cells which primarily give rise to the
epidermis and nervous system (Keller, 1975; Cooke
& Webber, 1985) and the vegetal pole cells which will
mainly form the gut. In isolation the animal cap
region forms only differentiated epidermis (Holtfreter & Hamburger, 1955; Asashima & Grunz, 1983;
Slack, 1984; Jones & Woodland, 1986). However,
fate-mapping studies using 16- and 32-cell embryos
show that the animal region also forms substantial
amounts of the mesoderm (Moody, 1987; Cooke &
Webber, 1985; Dale & Slack, 1987). It is thought that
this mesoderm is formed by the inducing action of
cells in presumptive vegetal cells on competent animal cells, the latter being reported to be able to
respond to this induction up to the start of gastrulation (Nieuwkoop, 1969, 1973; Dale, Smith & Slack,
1985). This interaction can also be demonstrated in
experimental tissue combinations. Thus, if a competent animal cap is placed in association with an
inductive vegetal plug, the animal portion is induced
to form mesoderm, which is often dorsal in nature
(notochord and somite). We call these Nieuwkoop
combinations, after one of their originators. Similar
Key words: Xenopus laevis, animal cap, mesoderm,
grafting, monoclonal antibody, cell lineage, induction.
Materials and methods
Embryos were cultured and explants made as described
by Jones & Woodland (1986). Nieuwkoop grafts were made
by isolating animal caps from blastulae and early gastrulae
and combining them with isolated vegetal pole explants
in MBS (88mM-NaCl; lmM-KCl; 24mM-NaHCO3;
15mM-Tris-HCl; 0-33mM-Ca(NO3)2; lmM-MgSO4; lmM-
558
E. A. Jones and H. R. Woodland
NaHCO3; 2mM-sodium phosphate pH7-4 and 0-lmMNa2EDTA; Gurdon, 1977). Grafts healed within half an
hour and were then allowed to develop until control
embryos were stage 25-30. Animal and vegetal combinations were made between X. laevis and X. borealis; X.
borealis cells were identified by the presence of intensely
fluorescent chromatin granules after quinacrine staining
(Thie'baud, 1983).
Fixation, embedding, sectioning and staining with antibodies were as described in Jones & Woodland (1986). The
monoclonal antibody 5A3.B4, raised against adult Xenopus
skeletal muscle, was used to identify induced muscle. This
antibody reacts with striated muscle from stage 20 onwards
and reacts with no other tissue type. Notochord was usually
identified on morphological grounds, but confirmed in a
few cases using a notochord marker (Smith & Watt, 1985).
In no case did cells with apparent notochord morphology
prove to be negative.
Results
The technique we have used to achieve mesodermal
induction is to combine animal cap cells with vegetal
pole plugs; individually neither forms mesoderm
when incubated to an appropriate stage but, in
combination, the animal cap is induced to form
mesoderm. This technique was pioneered by Nieuwkoop (1969, 1973) and has recently been used extensively by Gurdon and his colleagues (1985) and Dale
el al. (1985), among others. In all grafted combinations, mesodermal structures were identified with
specific monoclonal antibodies and the animal cap
origin of the induced cells confirmed using Xenopus
borealis/Xenopus laevis graft combinations. Fig. 1
shows muscle staining and notochord structure in a
typical inducing Nieuwkoop combination. In all
cases, except where noted in the tables, blocks of
muscle and patches of notochord were of a similar,
and substantial, size and were not isolated cells,
indicating that these combinations either responded,
or did not respond, in an all-or-nothing fashion.
We have used these grafts to attempt to answer two
very simple questions: when is ectoderm first competent to respond to the inductive stimulus from
vegetal cells, and when can this stimulus first be
detected? One way in which one can ask questions
about the timing of these events is by grafting animal
and vegetal regions of early and late embryonic ages
together. If one of the tissues in these heterochronous
combinations is close to the end of its period of giving
or receiving the inductive signal, then the ability of
the other tissues to respond or induce can be
measured by the presence of induced mesoderm in
the grafted combination. If the combination fails to
generate mesoderm because of the early state of
origin of one of the tissues, the time when tissues
become responsive or inductive can be assigned to a
particular stage of development (see below). Importantly, two assumptions are not made in this approach,
whereas they are central to the detection of mesodermal induction by explanting the prospective mesodermal tissue. First, it is not necessary to assume that
pure tissues are isolated. Second, it is not necessary to
assume that the early steps in mesoderm formation
are irreversible. In order to carry out these experiments, the end of both the competent and inductive
periods must be established with high accuracy. We
felt that it was important to do this since there are
minor disagreements in the literature that would be of
significance in our kind of experiment. Having confirmed the end points of both competence and inductiveness, we have used animal tissue very near the
end of its period of competence in the assay concerned and combined it with progressively earlier
inductive tissue. If inductiveness is absent at early
stages, mesoderm should not be seen, because the
animal cells will have lost competence by the time it
appears. This assumes that the endoderm cells cannot
reset the timing of the animal cell programme. This
has been tested, with respect to a cardiac actin
mRNA appearance by Gurdon et al. (1985), who
found that this time was intrinsic to the animal cells.
We have also described it with respect to the appearance of the epidermal marker recognized by 2F7.C7
and observed no changes in timing (unpublished
data). The timing of expression of the antigens
recognized by 2F7.C7 and B4 are identical in X. laevis
and X. borealis embryos. Furthermore, the grafted
combinations have been carried out using both
species for the source of each tissue in the grafts.
These combinations give exactly comparable results
irrespective of the source of inducing or responding
tissue. For simplicity results have been pooled in
Tables 1-3.
The end of animal cap competence
We defined the time at which animal cap cells lost
their ability to form mesoderm by combining progressively later animal caps with fully inductive vegetal plugs (Table 1). Stage-10 animal cap was fully
induced by stage-7 to -9 endoderm whereas stage-11
ectoderm was not. Stage-10i ectoderm gave somewhat variable results. In some experiments, it was
totally refractory to mesoderm induction and in
others gave fairly low percentages of induction. This
indicates that the end of animal cap competence lies
at some time very close to stage 10i, variability in
results perhaps being due to the morphological criteria used for staging these embryos, or perhaps from
a genuine variation in the disappearance of competence.
Development of animal cap cells in Xenopus
The start of endodermal inductiveness
Since competence disappears close to stage 10£, we
used stage-10 animal cap, which should have a
maximum of 45 min responsiveness, to test when
inductiveness appeared in vegetal pole cells (Table 2).
559
Endoderm from the earliest stage tested, stage 5, was
clearly inductive in stage-10 combinations, resulting
in the formation of both notochord and muscle from
the grafted animal cap. This means that the endoderm must be inductive within 45 min of stage 5, that
Fig. 1. Nieuwkoop grafts sectioned to show
the origin, extent and variety of mesoderm
formed. (A) Graft of stage-9 X. borealis
animal cap on to the vegetal pole from
stage-9 X. laevis, showing muscle, identified
by B4 staining (arrow), in cells derived from
the grafted ectoderm. Inset shows the extent
of the induced mesoderm in the whole graft.
(B) Graft of stage-7 X. borealis animal cap
on to stage-9 X. laevis vegetal pole, showing
induced muscle, identified by B4 staining
(arrow) and notochord. (C) Shows the
quinacrine staining of the same section as B,
confirming the animal cap origin of the
mesodermal tissues. The X. borealis animal
cap cells show typical punctate fluorescence
(arrow), an, animal cap cells; veg, vegetal
pole cells; nt, notochord: Bar, 35 /my.
560
E. A. Jones and H. R. Woodland
Table 1. Heterochronous Nieuwkoop grafts:
assessment of the competence of animal caps to form
mesoderm
Table 3. Heterochronous Nieuwkoop grafts: time at
which competence to respond to mesodermal induction
appears in the animal cap
Animal
pole stage
Vegetal
pole stage
Number
% induction
of mesoderm*
Animal
cap stage
Vegetal
pole stage
Number
% induction
of mesoderm
10
10
10-5
10-5
11
7
8-9
5-6
7-8
7-8
10
6
16
15
17
40
100
6-25
20
0
4
5
6-8
10-5
10-5
10-5
6
12
18
0
8*
56
4
5
6-7
10
10
10
12
11
9
8
72
77
5
6-7
9
•9
8
7
* In all instances in this and other tables, mesoderm was
substantially dorsal in nature. It included substantial blocks of
muscle, identified by B4 except in the instance inentioned in
Table 3.
Table 2. Heterochronous Nieuwkoop grafts: time of
disappearance of the ability to induce mesoderm
Animal
cap stage
Vegetal
pole stage
Number
% induction
of mesoderm
9-10
10
10
6-8
8-10
8-10
5
6-7
8-10
10-5
11
12
17
16
8
13
11
8
82
50
100
53
0
0
is by stage 6i, or 64 cells, and it is possible that the
endoderm is inductive from much earlier stages. We
do know, however, that the oocyte is not capable of
inducing mesoderm when animal cells are grafted on
to its vegetal pole (data not shown). Of course,
induction might not be actually happening at these
early stages, because there might be no responsive
animal cap cells.
The end of endodermal inductiveness
The time at which the endoderm in vegetal pole plugs
loses its capacity to induce mesoderm was tested in a
similar way (Table 2). Vegetal pole cells from stages
9-1CH induced mesoderm when in combination with
stage-8 to -10 animal cap, whereas vegetal pole cells
from stage 11 or greater never induced similar ectodermal cells.
The start of ectodermal competence
In combinations with endoderm at the end of its
inductive period, stage 10i, it is clear that stage-4 and
-5 animal caps are not competent to respond to the
inductive stimulus. In contrast, animal cells dissected
from stage 6 or later formed abundant dorsal mesoderm in such combinations (Table 3). The failure of
these early animal caps to form mesoderm was not a
consequence of total inability of these grafts to
62-5
85
*A very small patch of induced muscle was seen in one case.
respond, because when they were placed in combination with earlier endoderm, stage 9 or 10, they
formed mesoderm. Stage-4 ectoderm failed to respond in all except one case in combination with
stage-10 endoderm, but stage-5 ectoderm responded
in the majority of these combinations. In these
combinations the endoderm has respectively a maximum of 2| or lih of inductiveness remaining (see
Table 2), during which time the animal cap will have
aged beyond stage 6. However, stage-10i endoderm
can have only a maximum of 45 min inductiveness
remaining, during which time stage-4 animal caps
could advance to stage 6 and stage-5 animal cells to
stage 6i. This indicates that competence to respond to
mesodermal induction appears between stage 6 and
6i. Notochord was seen in many of the grafted
combinations, particularly when the animal cells were
taken from embryos earlier than stage 7.
These experiments suggest that vegetal pole cells
are already generating mesodermal inductive signals
close to the earliest point that they can be separated
from the animal cap cells, and that animal cells are
competent to respond to this signal from comparatively early stages - stage 6-6^. However, our results
suggest that animal cells cannot respond at stage 5 to
6, and hence that mesodermal induction cannot be
occurring at this point.
Discussion
In this paper, we are concerned with the times when
the animal cap is competent to respond to an inductive signal from the vegetal pole cells, and the times
when this signal is given. This is essential background
for understanding how the future mesoderm of the
embryo appears in its appropriate position and
amount.
Development of animal cap cells in Xenopus
The stage at which the animal cap loses its competence
to form muscle
As background to our study of the time at which
competence to form muscle appears, heterochronous
combinations of tissues were used to determine when
the animal cap loses its ability to form muscle, using
stage-7 to -9 tissue as an inducer. Dale et al. (1985)
found that the ability to form dorsal (though not
ventral) mesoderm was lost by stage 10, our results
are closer to Nakamura et al. (1971) and Gurdon etal.
(1985) and suggest that it is lost in a very short time
interval close to stage 10i. This is also consistent with
Asashima & Grunz's (1983) observation that a chick
embryo extract induced stage-10i animal cap to form
mesoderm. In agreement with these latter authors'
study using a heterologous inducer, we have found
that inner and outer animal cap layers can individually form mesoderm up till stage 10£, when naturally
induced with vegetal cells (data not shown).
Like Dale et al. (1985), we found that notochord
was formed more often when early animal caps were
used. However, this is not a fundamental change in
the responsiveness of the tissue, since in unpublished
experiments we find that animal cap as late as stage
10i can form notochord when grafted superficially
into the dorsal marginal zone of a stage-8 blastula.
This difference from Nieuwkoop grafts may derive
from the changing sensitivity of animal caps in development, coupled to the strength of the inductive
signal produced by parts of the endoderm which may
or may not be included in vegetal pole fragments.
In conclusion, we can confirm the results of some of
the previous workers, that the animal cap loses
mesodermal competence very close to stage 10£, with
some variability between batches of embryos.
Stage at which mesodermal inductiveness disappears in
the vegetal region
Using Nieuwkoop combinations of vegetal cells and
competent animal caps Nakamura et al. (1971) and
Gurdon et al. (1985) found a loss of inductiveness at
mid stage 9. The former identified ventral and dorsal
mesodermal tissues morphologically, whereas the
latter, like us, focused on muscle, using molecular
markers. On the other hand, Dale et al. (1985) with
Xenopus, Boterenbrood & Nieuwkoop (1973) with
axolotls, and Asashima (1975) with Triturus, obtained
mesoderm, including muscle, from stage-10 inducers.
However, all noted a decrease in dorsal induction.
Dale et al. (1985) felt that this might be because they
failed to dissect out the dorsal vegetal material. In our
experiments, we obtained strong muscle induction at
stage 10 and 104, using genetically marked animal cap
cells. It therefore seems that, judged by this assay,
mesodermal inductiveness disappears at late stage 10,
concomitantly with mesodermal competence in the
561
animal cap. Since the muscle was clearly striated, it
seems unlikely that our difference from the results of
Gurdon etal. (1985) relates to the fact that they used a
different marker, cardiac actin gene transcripts.
Stage at which mesodermal inductiveness and
competence arises
Once mesodermal competence and inductiveness
have arisen, induction should occur and mesoderm
should be determined. An obvious way to estimate
the onset of the two phenomena might therefore
appear to be to isolate the region fated to form
mesoderm from embryos of different stages and
establish when mesoderm first appears. This approach was adopted by Nakamura & Takasaki (1970),
who identified stage 6i as the stage at which much
mesoderm appeared. It could hardly be earlier, since
at stage 6 (32-cell) the cells that form most of the
mesoderm also form ectoderm (Cooke & Webber,
1985; Dale & Slack, 1987), so induction, as normally
conceived, could not have occurred. Nevertheless,
this experiment is certainly flawed, as pointed out by
Nieuwkoop (1973), since the region excised should
contain both future endoderm and ectoderm, and
therefore must be capable of self-induction after
isolation. Despite the severe criticisms of this work,
this has been the only study attempting to define the
actual start of mesodermal induction. [It has recently
been claimed that stage-6i cells are competent to
respond and induce because when grafted together at
this stage they later form muscle (Brennan, 1987). Of
course, this experiment does not bear on the point
because the cell interaction could have happened at
any subsequent time.]
We have adopted a different approach to define
when mesodermal competence and inductiveness
start, using the heterochronous Nieuwkoop grafts
described earlier. To test when the inductive stimulus
is present we combined stage-10 with stage-5, and
later, vegetal cells. These grafts usually form muscle,
indicating that the inductive stimulus is present within
45min of stage 5; i.e. when the vegetal plug is stage
6-6i. Thus, we could not identify a negative time,
and inductiveness may exist from the start of development, though it is absent from the oocyte. Since
significant transcription is not seen until stage 8
(Bachrarova, Davidson, Allfrey & Mirsky, 1968;
Gurdon & Woodland, 1969; Newport & Kirschner,
1982), the production of the inducer most probably
depends on stored mRNA or protein.
The onset of animal cap competence to be induced
was judged by using stage-9 to -10i vegetal tissue to
induce progressively earlier animal caps. Stage-5 caps
produced muscle and notochord in combination with
stage-9 and -10 vegetal tissue, but not with stage 10i
562
E. A. Jones and H. R. Woodland
even though the latter can clearly induce later ectoderm. This suggests that stage-5 animal cap is not
competent to respond to mesodermal induction, but
becomes responsive at some later stage at least by
45min after stage 5, i.e. by the latest at stage 6i, as
indicated by the successful induction of stage-5 caps
and stage-10 vegetal combinations. Thus mesoderm
induction in the normal embryo is likely to begin at,
or just before, stage 64, this time being set solely by
the onset of competence. If mesodermal induction
starts at the 64-cell stage, it is relevant to ask if there
are cells of a purely mesodermal state at this stage.
Fate-mapping experiments at the 256-cell stage indicate that derivatives of equatorial cells enter all three
germ layers (Jacobsen, 1983). This result can be
reconciled with the appearance of mesodermal induction at 64 cells if mesodermal differentiation is
initially reversible (irreversibility is not assumed in
our experimental design). This is actually to be
expected since grafts conducted with late blastula
tissue indicate that mesodermal induction takes
li-2h, i.e. any changes that occur are reversible
within this period (Gurdon et al. 1985). Thus by
extrapolation to natural, earlier embryos, if induction
starts at stage 64, it would not be complete (i.e.
irreversible) for any cells until stage 8, which happens
to be the time of general genome activation.
Nakamura & Takasaki (1970) concluded that the
mesoderm becomes determined between stages 6 and
6i. This was based on the observation that isolated
stage-6i equatorial zones, but not those of stage 6,
showed subsequent mesodermal differentiation on
culturing. As pointed out above, the experiment is
flawed because it depends on the explant containing
only mesodermal progenitors. If this was so, and the
mesoderm was originally generated as a result of
induction, then this induction must have started
li-2£h earlier, at the 2- to 4-cell stage, when there
were not even animal and vegetal cells. Furthermore,
our results show that the vegetal pole cells separated
at the 16-cell stage are inductive, but that the animal
cells are not responsive at this stage. This lends
weight to Nieuwkoop's (1973) conclusion that Nakamura's experiment was faulty because the isolated
equatorial zone must have contained endodermal and
ectodermal progenitors and therefore could be
capable of continued induction after explantation.
Our conclusion differs from Nakamura's in that we
argue that mesodermal induction starts in the 64-cellstage embryo, but that 'committed' mesodermal cells
would not be expected before stage 8 (these would be
'committed' as defined by differentiation in isolated
fragments, as in Gurdon et al.'s (1985) separated
Nieuwkoop grafts, which were used to determine the
timing of mesodermal induction).
The biological significance of these observations
The early time of mesodermal induction might be
relevant to how much mesoderm is formed in this
early phase of mesoderm formation. A working
hypothesis is that in this early phase the mesoderm is
formed from an annulus of animal cells which make
contact with the yolky vegetal cells. It is proposed
that the mesoderm itself is noninductive and that the
large cells involved therefore act as a barrier to
propagation of the inductive stimulus.
The reason for the disappearance of mesodermal
competence and inductiveness in normal development seems clear. In the blastula, the animal cap cells
that neighbour vegetal cells all form mesoderm. The
rest is separated from it either by the future mesoderm itself, or by the blastocoel. After the tissue
rearrangements of gastrulation, prospective ectoderm comes close to endoderm, for example ventrally. If a mutant arose in which the former were
competent to be induced to form mesoderm, or the
latter was inductive, then more mesoderm would be
generated in inappropriate positions, probably with
lethal results. Thus the loss of pluripotency may be a
vitally important aspect of any particular differentiated phenotype, although there is as yet no reason
to believe that it is always permanent.
This work was funded by the Medical Research Council.
The authors acknowledge the clerical assistance of Mrs Len
Schofield and the technical assistance of P. Day.
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