1 Department of Chemistry and Bioscience, Faculty of Science, Kagoshima
University, Kagoshima 890-0065, Japan
2 Department of Bioengineering, Yatsushiro National College of Technology, 2627
Hirayama Shin-Machi, Yatsushiro, 866-8501, Japan
3 Department of Molecular Biology, Kawasaki Medical School, Kurashiki, Okayama
701-0192, Japan
Author for correspondence (e-mail:
garu{at}sci.kagoshima-u.ac.jp).
Accepted 19 November 2003
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SUMMARY |
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Key words: Xenopus, Cytoplasmic determinants, VegT, Xwnt8, Organizer
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Introduction |
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However, dorsal determinants are not sufficient for the formation of the
Spemann organizer. When Xenopus eggs are UV-irradiated, the resulting
embryos lack entire dorsal structures
(Malacinski et al., 1977;
Scharf and Gerhart, 1980
;
Scharf and Gerhart, 1983
).
This is not because the dorsal determinants are destroyed, because the vegetal
pole cytoplasm of the UV-irradiated eggs still possesses transplantable dorsal
determinants (Fujisue et al.,
1993
; Holowacz and Elinson,
1993
). UV-irradiation is thought to interfere with the cortical
movement (Vincent et al.,
1986
; Gerhart et al.,
1989
), which is necessary for the transportation of the dorsal
determinants from the vegetal pole to the marginal region. Therefore, we have
proposed that in Xenopus eggs the organizer forms as a result of
mixing of two vegetally located determinants, the dorsal determinants in a
narrow region of the vegetal pole and the marginal determinants in the upper
part of the vegetal half (i.e. marginal zone)
(Sakai, 1996
;
Nagano et al., 2000
).
The marginal region is a special domain where gastrulation and
mesoderm-specific gene expression occurs. A preliminary experiment has shown
that `NG embryos' [NG is for non-gastrulating; re-named here as
permanent-blastula-type embryos (PBEs)] did not express the dorsal marker gene
chordin when treated with LiCl, although LiCl-treated GNEs formed
hyperdorsalized embryos (Sakai,
1996). This is probably because PBEs do not have competent
marginal cytoplasm to respond to LiCl treatment. Furthermore,
cortex-transplantation experiments have revealed that dorsal determinants are
active only when the cortex is transplanted into the sub-equatorial (marginal)
region but not into the animal or vegetal region
(Kageura, 1997
). These results
support the notion that putative marginal determinants are responsible for
gastrulation, mesoderm formation and dorsal axial development. However, for a
certain determinant, `the only really satisfactory proof is to transfer
cytoplasm from one place to another by microinjection and show that cells
inheriting the ectopic cytoplasm become structures normally formed by the egg
region from which the cytoplasm came'
(Slack, 1991
). Manes et al.
(Casal and Manes, 1999
;
Manes and Campos Casal, 2002
)
isolated the four animal cells from an eight-cell Bufo embryo, which
in this species only makes epidermis. Cytoplasmic transfers generated
mesoderm, with notochord and somite arising only with a cytoplasmic
combination of the ventroequatorial and the vegetal pole area. Further,
Shinagawa and Kobayashi (Shinagawa and
Kobayashi, 2000
) transferred marginal cytoplasm into the animal
pole region of Xenopus embryos and found a blastopore-like structure
in the injected region. We tried to show more solid evidence of marginal
determinant(s) by mRNA injection and cytoplasmic transfer experiments.
As recipients for cytoplasmic transfer, we used PBEs, which were made by
ablating more than 60% of the vegetal egg surface from an early one-cell stage
embryo (Fujii et al., 2002).
PBEs do not gastrulate, or express dorsal, neural and endo-mesodermal genes.
They express the epidermal marker EpK, and develop into simple
epidermal tissues (Fujii et al.,
2002
). Therefore, PBEs most probably lack two types of
determinants: the dorsal determinants in the vegetal pole and the marginal
determinants. Using PBEs as starting materials, we first tried to restore
gastrulation, mesoderm formation and finally the entire axial development.
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Materials and methods |
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Microinjection
Micropipettes for cytoplasm transfer was manually drawn from hard glass
capillary tubes (Drummond), cut to a flat, blunt end (tip external diameter:
40 µm). For mRNA injection, thinner micropipettes (5-7 µm) were used.
Micropipettes were held in a Narishige micromanipulator. Microinjections were
performed using air pressure from a tuberculin syringe connected to 1 mm
diameter polyethylene tubing. For withdrawal of the cytoplasm, the
micropipette tip was placed on the cell surface and then negative pressure was
increased to break the cell surface. The suction was carried out very slowly.
It took about 5 minutes to withdraw 50 nl of cytoplasm. Soon after the
suction, the cytoplasm was injected into the host PBEs.
For fluorescent microscopy, the cytoplasmic donor was soaked in 0.01% Neutral Red for 5 minutes to stain donor cytoplasm. This stain could be clearly seen under a fluorescent microscope optimized for rhodamine.
Histology and in situ hybridization
Embryos at various stages were fixed with Bouin d'Hollande (2-3 hours) and
embedded in paraffin wax after dehydration. Serial 6 µm sections were
stained for 10 minutes with 0.5% aniline blue-2% orange G and for 15 minutes
with 0.5% Aniline Blue.
Whole-mount in situ hybridization was carried out following the method of
Shain and Zuber (Shain and Zuber,
1996). Embryos were fixed in MEMFA for 90 minutes at 20°C,
washed, and stored in -20°C 100% methanol. Before addition of anti-DIG
antibody, embryos were treated overnight in 10% hydrogen peroxide to bleach
out the pigment. In situ hybridization on sections was performed using 6 µm
paraffin wax sections based on the method of Endo et al.
(Endo et al., 2002
). Embryos
were fixed in MEMFA, embedded in paraffin wax and sectioned at 6 µm.
Sections were mounted on silan-coated slides pretreated with Vectabound.
Before hybridization, the slides were soaked in 1% gelatin. Detailed protocols
are available on request.
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Results |
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Some MC-injected PBEs were fixed for in situ hybridization soon after the injection. VegT was seen in 13 out of 15 MC injected PBEs (Fig. 4; Table 2). In these cases, the VegT stain had a spherical shape, resembling the shape of the donor cytoplasm in the epifluorescent view and in the histological section (Fig. 3A,E,F). Negative controls that were injected with PBE-animal cytoplasm (AC) showed no positive stain for VegT (Fig. 4; Table 2).
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As we had expected, injection of Xwnt8 alone did not dorsalize PBEs, though GNEs formed an almost normal axis in response to Xwnt8 injection (Fig. 6B,D). Most of the Xwnt8 injected GNEs formed a cement gland, two eyes, melanocytes and a tail fin (Table 1), and thus showed an almost normal appearance (Fig. 6D). These embryos expressed the organizer marker gene chordin at the upper blastopore region (Fig. 6C, Table 3). By contrast, PBEs injected with Xwnt8 showed no morphological signs of dorsalization except for cement gland formation at a low frequency (Table 1). These embryos never showed chordin expression (Fig. 6C; Table 3).
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We therefore co-injected Xwnt8 and MC into a single cell of eight-cell stage PBEs. MC was first injected into the bottom of PBEs at the one-cell stage. At the eight-cell stage, the injected MC was seen as a white area encompassing several blastomeres in the bottom region. Xwnt8 mRNA was injected into the white/black border in one blastomere of MC-injected PBEs. The resulting embryos had regenerated axis formation and chordin expression (Fig. 7A,D). NCAM and Krox20 expression showed near-normal patterns in MC/Xwnt8 injected embryos (Fig. 7E,F). Negative control embryos receiving animal pole cytoplasm (AC) instead of MC and then injected with Xwnt8 did not form dorsal structures nor express NCAM or Krox20 (Fig. 7G).
|
Double injections of marginal cytoplasm and vegetal-pole cytoplasm into PBEs restored normal axial structures
In our final experiment, we tried to restore normal embryos by double
injections of marginal cytoplasm (MC) and vegetal pole cytoplasm (VPC). As
described above, injection of VPC into PBEs resulted in the formation of
hyperdorsalized embryos. This is probably because these embryos had circular
dorsalizing region, the situation similar to LiCl-treated embryos
(Kao and Elinson, 1988).
Therefore, we tried to create a posteriorizing domain outside of the VPC
injected dorsalizing region. To this end, PBEs at the one-cell stage were
first injected with 50 nl MC into the bottom, and at eight-cell stage injected
with 18 nl VPC into the white/black border in one blastomere.
Most of the resulting embryos formed near-normal body axes, with a cement gland, two eyes, melanocytes, muscle tissue and a tail fin (Fig. 7H; `PBE+MC+VPC' in Table 1). Interestingly, the overall shape of these embryos was more normal when compared with mRNA-injected embryos (compare Fig. 7H with 7A,B). Naturally, these embryos expressed chordin (Fig. 7D, Table 3).
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Discussion |
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Previous studies have shown that when maternal VegT mRNA is depleted by
antisense oligonucleotides, the vegetal masses of the resulting embryos do not
produce mesoderm-inducing signals. Mesoderm formation in these embryos
occurred ectopically in the vegetal region rather than the equatorial region
(Zhang et al., 1998;
Kofron et al., 1999
). However,
these VegT-depleted embryos include
10% maternal VegT,
probably causing zygotic expression of VegT and Xbra and
gastrulation in the vegetal pole region. Furthermore, these embryos form some
axial structures. In contrast to VegT-depleted embryos, PBEs never
express zygotic VegT or Xbra nor form dorsal axial
structures.
The Xenopus cell fate is characterized by variable cell lineages.
Lineage tracing experiments show that Xenopus blastomeres develop
variably (Dale and Slack, 1987;
Moody, 1987
;
Masho, 1990
) so that it is
impossible to forecast the specific fate of a certain cell. This is quite
different from the invariant development of ascidian embryos in which the fate
of a cell is identical in all individual embryos
(Nishida and Satoh, 1985
;
Nishida, 1987
). The variable
fate of early Xenopus embryos makes it difficult to assess the
results of experimental manipulations. The fate of PBEs is always atypical
epidermis although they can make a full spectrum of embryonic structures.
Using PBEs as starting materials, we can assess the effects of experimental
manipulations by comparing the development of the resulting embryos with the
epidermal fate of PBEs.
VegT mRNA is a marginal determinant
When MC was injected into PBEs we found, by in situ hybridization, the
conspicuous presence of VegT mRNA. As VegT mRNA was entirely absent in PBEs,
the observed VegT in the MC-injected PBEs must have come from the
injected MC. To our knowledge, this is the first visualization that an
injection of any cytoplasm transfers a specific mRNA into chordate host cells.
The MC-injected PBEs gastrulated and expressed zygotic VegT and
Xbra at high frequencies. Furthermore, we found that injection of
synthetic VegT mRNA also resulted in gastrulation and Xbra
expression. These results show that the maternal VegT is a marginal
determinant, although VegT localization is not restricted to the
marginal zone.
It should be noted that the dose of injected VegT (12 pg) was very
low compared with previous studies. Zhang and King
(Zhang and King, 1996)
injected 0.7 or 2.5 ng of VegT mRNA into the ventral marginal zone and found
secondary axis formation. Kurth and Hausen
(Kurth and Hausen, 2000
)
injected 300 pg into animal pole, which resulted in weak gastrulation. In
comparison with the animal pole of normal embryos, PBEs are very sensitive to
VegT injection. We injected 12 pg VegT into the animal pole
of one-cell stage embryos and found no morphological signs of gastrulation
(data not shown), though the same dose induced gastrulation in all 16 PBEs
examined (Table 1). We did not
observe dorsal axis formation or chordin expression in PBEs injected
with 12 pg of VegT. When an excess amount of VegT (60-250
pg) was injected, the resulting embryo elongated and formed axial structures
(data not shown). We conclude that 12 pg of VegT is sufficient for
PBEs to develop into GNE-like embryos. Similarly, Darras et al.
(Darras et al., 1997
) found
that injection of vegetal pole cytoplasm into animal pole of Xenopus
embryos led to expression of both siamois and Xnr3 but not
Xbra in animal caps. Injection of vegetal pole cytoplasm into PBEs
resulted in a hyperdorsalized phenotype, indicating the activation of both
dorsalizing and mesodermalizing activity. The different results obtained by
our study and Darras et al. (Darras et al.,
1997
) may be due to a difference in sensitivity between PBEs and
animal caps.
Xwnt8 acts in synergy with VegT in a cell autonomous manner to form dorsal axial structures
Previously, we have proposed that the organizer forms by the mixing of
vegetal pole determinants and marginal determinants. However, the present
study shows that the marginal determinant is most probably VegT mRNA scattered
all over the vegetal half of the egg. Therefore, vegetal pole cytoplasm (VPC)
must have both dorsal and marginal determinants, and injection of VPC should
result in dorsal axis formation in recipient PBEs. In fact, VPC-injected PBEs
showed VegT translocation and hyperdorsalized phenotypes. We injected
Xwnt8 mRNA in place of natural dorsal determinant. Xwnt8 is a dorsalizing
factor that acts upstream of ß-catenin. ß-catenin protein
translocates in nuclei of the dorsal side of cleavage stage Xenopus
embryos (Schneider et al.,
1996), and causes dorsal axial development
(Brannon et al., 1997
). When
ß-catenin is depleted early in the cleavage stage, resulting embryos do
not develop dorsal axial structures
(Heasman et al., 1994
).
Injection of Xwnt8 does not rescue the ß-catenin-deleted
non-axial phenotype (Heasman et al.,
1994
).
The present results show clearly that injection of Xwnt8 alone
does not cause the formation of dorsal structures in recipient PBEs, while the
same dose (3 pg into a single cell of and embryo at the eight-cell stage) into
GNEs resulted in well-organized axial development with chordin
expression in the appropriate position. As described above, VegT
alone also did not dorsalize PBEs. Double injections of VegT and
Xwnt8 into PBEs resulted in chordin expression and
development of a near-normal body axis. Although VegT has been
proposed to play roles in the early wnt dorsalizing pathway
(Zhang et al., 1998;
Houston et al., 2002
) the
present deletion-injection experiments provide the first direct proof that
VegT is required for the wnt dorsalizing process. Baker et al.
(Baker et al., 1999
) reported
that animal ectodermal tissue is neuralized by a single injection of
Xwnt8; however, the animal tissue in their experiments may contain
VegT mRNA. In relation to this, some Xwnt8-injected PBEs formed a
cement gland, a most anterior dorsal marker. In these cases, host PBEs might
contain a low level of VegT, which might act in synergy with injected
Xwnt8 to form a cement gland. Even if this is true, the results
presented here still showed that Xwnt8 injected PBEs did not express
chordin or NCAM. For strong expression of these genes, a
substantial amount of VegT is necessary. In any case, details of the
molecular characteristics of Xwnt8 injected PBEs are not clear at
present. For example, gene expression upstream of chordin (e.g. Xnr
genes and siamois) and nuclear transportation of ß-catenin were
not examined in the present study. Although Xwnt8 is thought to be a secreting
factor, the present study strongly suggests that Xwnt8 acts in a
cell-autonomous manner in synergy with VegT, as the injection of VegT
and Xwnt8 into separate cells did not cause dorsal axial development.
Furthermore, given that Xwnt8 acts upstream of ß-catenin and thus acts
before MBT, VegT also should act before MBT. This is in accordance with the
fact that the ß-catenin-Tcf-dependent transcription of the
Xenopus nodal genes Xnr5 and Xnr6 occurs as early
as the 256-cell stage (Yang et al.,
2002
).
In summary, VegT plays dual roles in early Xenopus development. First, VegT acts as an upstream factor of endo-mesodermal genes such as Xsox17, Xwnt8 and Xbra. Second, VegT acts as a co-factor of dorsal determinant(s) to activate the wnt-dorsalizing cascade.
It has been proposed that cortical rotation drives dorsal determinant(s) to
a marginal sector where translocation of ß-catenin protein in the dorsal
marginal zone nucleus results in siamois expression
(Brannon et al., 1997).
Possible dorsal determinant molecules are Xdsh and GBP
(Yost et al., 1998
); however,
there has been no evidence for their localization at the vegetal pole. After
the translocation of ß-catenin, the earliest gene expression has been
shown to occur at 256-cell stage (Yang et
al., 2002
). For this early expression of Xnr5 and
Xnr6, the two determinants presented in this paper most probably play
crucial roles.
Absence of gastrulation, mesoderm formation and dorsal gene expression in the vegetal pole
As described above, we propose that the mixing of vegetal half cytoplasm
(VegT mRNA) and the vegetal pole determinant(s) (unknown molecules) plays a
main role in organizer formation. VegT mRNA in the marginal zone is most
likely responsible and the cause of this region-specific gastrulation, as well
as Xbra expression, dorsal axis formation in response to dorsal
cortex transplantation (not to mention dorsalization by LiCl treatment) and
Xwnt8 overexpression. The absence of maternal VegT in the
animal pole is the likely cause of the lack of gastrulation. In addition, lack
of the competence to cortex transplantation
(Kageura, 1997) and the lack
of competence to LiCl treatment and Xwnt8 injection (present study)
are probably caused by the absence of maternal VegT. If the mixing of
the two determinants is necessary and sufficient, the vegetal pole of a
UV-irradiated embryo with both determinants should form the Spemann organizer.
In support of this idea, the present study shows that vegetal pole cytoplasm
with (most probably) both determinants, leads to dorsalized PBEs. Darras et
al. (Darras et al., 1997
) found
that siamois and Xnr3 expression occurs at the UV-irradiated
vegetal pole cells, indicating the activation of ß-catenin pathway;
however, the vegetal pole cells do not have any axis-forming activity at the
gastrula stage. Even when siamois is injected into the vegetal pole,
the injected cells do not have axis forming activity. In addition, the vegetal
pole region do not respond to the transplantation of dorsalizing cortex
(Kageura, 1997
). These results
show that the early dorsalizing process leading to siamois expression
can be active in the vegetal pole; however, the later dorsalizing process
leading to chordin expression is not active in the vegetal pole.
Furthermore, Xbra is not expressed in the vegetal pole region in
normal development. The absence of a dorsalizing pathway leading to
chordin expression and the absence of Xbra expression in the
vegetal pole requires some explanation other than being due to the absence of
maternal VegT. Injection of MC into PBEs resulted in the formation of
a MC sphere around which Xbra was expressed. VegT is
apparently a causal factor for Xbra expression; however,
Xbra was not expressed in the VegT-containing region.
We propose that the organizer forms in the margin of VegT containing cytoplasm, where both VegT and dorsal determinant(s) are present. When we injected vegetal pole cytoplasm, the recipient PBE has a margin of VegT cytoplasm where dorsal determinant(s) are also most likely present. It should be noted that the organizer should form in a cell-autonomous manner, as described above. By contrast, the UV-vegetal region does not develop an organizer as this region does not have the VegT margin. However, we do not know why this dorsalizing process occurs only in the VegT margin.
Proposed model
Our model for the formation of Spemann organizer is presented in
Fig. 8, which shows a unique
intersection (yellow) of the marginal region (broken rectangle) and the dorsal
determinant (red) (which comes from the vegetal pole region). Previously, we
postulated a specific `marginal determinant' as being present in the marginal
zone (Sakai, 1996). In the
present model, the marginal region is postulated to be a `margin' of
VegT containing cytoplasm, which is present all over the vegetal half
of one-cell stage egg (green). The present model is not special because this
model is based on a tradition of the determinant hypothesis
(Elinson and Kao, 1989
): For
the formation of the Spemann organizer, the importance of the spread of the
determinant(s) on the dorsal side has been suggested. Stewart and Gerhart
(Stewart and Gerhart, 1990
)
suggest that inductions to establish the organizer occur after the original
cytoplasmic inheritance. There is experimental support for the activity of the
Nieuwkoop center on the vegetal dorsal side; however, Kodjabachian and Lemaire
(Kodjabachian and Lemaire,
1998
) argue that this is dispensable in normal axial development.
Rather, they emphasized a dorsal determinant in the vegetal pole. Furthermore,
Sokol and Melton (Sokol and Melton,
1991
) indicate that the dorsal ectoderm is different from the
ventral ectoderm in the competence to activin.
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The present deletion/injection experiments provide direct proof for the marginal determinant, which is necessary for the formation of both endo-mesoderm and dorsal axial structures. Rigorous genetic proof for mechanism(s) of the synergy of dorsal and marginal determinants is lacking, but hopefully the present model will provide a basis for further studies. The present PBE system should serve as a good experimental system for investigating the molecular mechanisms of the synergistic process.
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ACKNOWLEDGMENTS |
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Footnotes |
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