Howard Hughes Medical Institute and Department of Biological Chemistry, University of California, Los Angeles, CA 90095-1662, USA
* Author for correspondence (e-mail: ederobertis{at}mednet.ucla.edu)
Accepted 12 May 2005
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SUMMARY |
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Key words: BMP, Chordin, Sizzled, Morpholino, Spemann organizer, Brain induction, Dorsoal-ventral patterning
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Introduction |
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Three BMPs stand out as candidates for regulating DV patterning in
Xenopus. These are Bmp2 and Bmp4, which can replace the function of
the DPP morphogen in Drosophila
(Padgett et al., 1993), and
Bmp7, which when mutated in zebrafish results in dorsalized phenotypes
(Hammerschmidt and Mullins,
2002
). During gastrulation in Xenopus, Bmp4 is expressed
in a ventral domain diametrically opposed to the dorsal Spemann organizer. A
number of secreted proteins are co-expressed with Bmp4 in this region, which
has been designated the ventral gastrula center
(De Robertis and Kuroda, 2004
).
Extracellular proteins expressed in the ventral center include Twisted
Gastrulation (Tsg) (a co-factor of both Bmp4 and Chordin), the
metalloproteinase Xolloid-related (which cleaves Chordin), the Chordin-related
protein Crossveinless-2, the Bmp inhibitory pseudoreceptor Bambi, and the
secreted Frizzled-related protein Sizzled (reviewed by
De Robertis and Kuroda, 2004
).
Expression of ventral center genes coincides with high levels of BMP
signaling, which can be monitored using phospho-specific antibodies directed
against the carboxy-terminal region of the transcription factor Smad1, which
is phosphorylated by BMP receptors (Faure
et al., 2000
; Kurata et al.,
2001
).
At the opposite pole of the gastrula lies the Spemann organizer, a source
of secreted BMP antagonists and other factors that interact extracellularly
with ventral center gene products to generate a BMP morphogenetic gradient
(Harland and Gerhart, 1997;
De Robertis and Kuroda, 2004
).
Traditionally, two main biological activities of the Spemann Organizer have
been distinguished (Spemann,
1938
; Niehrs,
2004
). The head organizer located in the early blastopore lip
induces head and trunk structures after transplantation, whereas the
trunk-tail organizer located in the late dorsal lip induces tail structures.
Molecular investigations using Wnt pathway inhibitors or the multivalent
inhibitor Cerberus have suggested that head formation requires the double
inhibition of Wnt and BMP (Glinka et al.,
1997
), or the triple inhibition of Nodal-related, Wnt and BMP
signaling (Piccolo et al.,
1999
; Niehrs,
2004
). Inhibition of just the BMP pathway (by dn-BMPR, cm-BMPs or
individual BMP antagonists) in wild-type embryos results in the development of
partial secondary axes with trunk/tail structures that lack head and forebrain
tissues.
Recent work has shown that at earlier stages, during blastula, two distinct
dorsal centers are formed. On the dorsal animal and marginal zone, a blastula
Chordin- and Noggin-expressing (BCNE) center is formed in cells that will
later on give rise to the anterior CNS
(Kuroda et al., 2004). In more
vegetal dorsal cells, the Nieuwkoop center releases mesoderm-inducing
Xenopus Nodal-related (Xnrs) growth factors and the
antagonist Cerberus (Kuroda et al.,
2004
). Dorsal ß-Catenin accumulation is triggered by sperm
entry and can be blocked by irradiation with ultraviolet (UV) light during the
first cycle (De Robertis et al.,
2000
; Weaver and Kimelman,
2004
). Thus, the Xenopus DV pattern is currently thought
to arise from a series of cell-cell interaction events involving four
signaling centers, two at blastula and two at gastrula stage, that generate a
gradient of BMP signaling (reviewed by De
Robertis and Kuroda, 2004
). Inhibition of BMP signaling is
required for the initial neural induction in Xenopus
(Harland, 2000
;
Stern, 2005
). A second layer
of regulation is provided by further inhibition of Smad1 activity by MAPK
phosphorylation, which may mediate the neural-inducing effects of FGFs and
IGFs (Pera et al., 2003
;
Sater et al., 2003
;
Kuroda et al., 2005
).
In the present study, we developed BMP-specific antisense morpholino oligomers (MO) and used them to investigate the individual roles of Bmp2, Bmp4 and Bmp7 in early Xenopus development. In embryonic explants, inhibition of Bmp4 and Bmp7 signals caused neural differentiation in ectoderm, and differentiation of dorsal fates in mesoderm. Triple knockdown of Bmp2, Bmp4 and Bmp7 compromised trunk and tail development, giving rise to embryos with enlarged dorsal structures. However, these embryos retained a considerable degree of DV polarity. Unexpectedly, we found that knockdown of BMPs caused the formation of extensive head and brain structures in embryos in which dorsal development had been prevented by UV treatment. Similarly, striking radial brain structures were obtained in embryos ventralized by injection of ß-Catenin MO. Formation of the Spemann organizer at gastrula stage, or of Nieuwkoop or BCNE centers at blastula stage, was not restored by BMP MOs in dorsalized embryos. Thus, in the absence of the dorsal organizing centers, inhibition of BMP signaling is sufficient to cause extensive head and CNS induction in embryos that would otherwise develop without any neural tissue at all.
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Materials and methods |
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Morpholinos were resuspended in sterile water to a concentration of 1 mM,
which was then further diluted to give a working solution of 0.25 mM. When
co-injected with ß-Catenin MO
(Heasman et al., 2000) or
Chordin MO [a mixture of two MOs targeting both pseudoalleles of Chordin
(Oelgeschläger et al.,
2003
)], a mixture was prepared and embryos were injected four
times radially at the two- to four-cell stage with 4 nl (3 ng MO/injection).
For rescue experiments mouse Bmp4 mRNA was microinjected at 25 pg in
each blastomere at the four-cell stage.
Embryological methods
Microinjections and mRNA synthesis were performed as described
(Kuroda et al., 2004). RT-PCR
conditions and primers, as well as the protocol for whole-mount in situ
hybridization, are described at
http://www.hhmi.ucla.edu/derobertis/index.html,
except for the modification that after hybridization all embryos (pigmented or
albino) were bleached overnight (in 10% H2O2, 20%
H2O, 80% methanol) under intense neon light to enhance contrast.
Digital photographs were taken with a Leica DC500 digital camera. For each
embryo, at least three pictures were taken in successive focal planes and
merged with Photoshop® CS.
Protein injections and western blots
Injections of recombinant mouse Fgf8b protein (R&D Systems) was carried
out as described (Pera et al.,
2003). Western blot analysis of endogenous levels of phospho-Smad1
in Xenopus whole embryos (Faure et
al., 2000
) used an anti-phospho-hSmad1 antibody at a 1 in 500
dilution (Persson et al.,
1998
).
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Results |
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Embryos depleted of Bmp4 exhibited the most severe phenotypes; at early
tailbud stage, enlarged brain structures (marked by Six3 in forebrain
and eye, and Krox20 in hindbrain) were observed (compare
Fig. 1E and 1F). Bmp4 MO
embryos appear larger and longer than controls because of an expanded
archenteron cavity, presumably caused by increased convergence-extension
(Myers et al., 2002). This
dorsalized phenotype could be rescued by microinjection of mouse Bmp4
mRNA (Fig. 1G), which has a
different nucleotide sequence in the region targeted by Xenopus Bmp4
MO. At the swimming tadpole stage, a posteriorization of the proctodeum (anus)
and a severe loss of ventral fin tissues could be observed
(Fig. 1I,J). These tail
phenotypes are consistent with a decrease of BMP signaling in Xenopus
(see Fig. S1 in the supplementary material) and zebrafish
(Wagner and Mullins, 2002
).
The onset of the Bmp4 depletion phenotype could be traced back to alterations
in gene expression patterns at gastrula, the stage at which Bmp4
expression is maximal in Xenopus
(Dale et al., 1992
). As shown
in Fig. S2 (see supplementary material), knockdown of Bmp4 decreased
expression of Bmp4, eliminated the ventral center marker
Sizzled, and downregulated the BMP downstream target genes
Mix1 and Vent1. The neural plate, visualized through the
pan-neural Sox2 and the forebrain/midbrain Otx2 markers, was
expanded.
|
Dorsalization in double and triple BMP knockdowns
To further characterize the requirements for Bmp2, Bmp4 and Bmp7, double
and triple knockdowns were performed in all possible combinations and analyzed
by in situ hybridization (Fig.
2). In addition, western blot analyses showed that phosphorylation
of endogenous Smad1 at gastrula stage was increasingly inhibited by combined
depletion of Bmp2, Bmp4 or Bmp7 activities
(Fig. 1D). This suggested that
BMP signaling in the Xenopus embryo results from the integration of
multiple BMP signals that act in concert to promote ventral development. In
agreement with this, more severe phenotypes were consistently observed in
double or triple depletions than in single MO injections. For example, doubly
depleted Bmp4/Bmp7 tadpoles were unable to develop tail structures
(Fig. 1L).
Combinations of Bmp2, Bmp4 and Bmp7 MOs were injected and their effects on the expression domains of four markers genes, Otx2, Krox20, MyoD and Sizzled, analyzed at tailbud (Fig. 2). Sizzled expression in the entire ventral region was eliminated by Bmp2 MO in combination with either Bmp4 or Bmp7 MO (Fig. 2E',F'). MyoD, a paraxial mesoderm marker, enveloped the tailbud ventrally, forming a ring in Bmp4 MO-injected embryos (Fig. 2C,C') or any of the double BMP-depleted embryos (Fig. 2E-G'), indicating an expansion of paraxial tissue into ventroposterior mesoderm. Krox20 was unaffected or modestly increased in any single or double depletion, but rhombomere 5 expression was radially expanded in triple Bmp2/Bmp4/Bmp7 MO-injected embryos (arrowheads in Fig. 2H,H''). Thus, triple Bmp2/Bmp4/Bmp7-depleted embryos were dorsoanteriorized and consisted mainly of tissues anterior to rhombomere 5. Surprisingly, the forebrain/midbrain marker Otx2 was only moderately enhanced in Bmp-depleted embryos at tailbud stage, even in triple-knockdown embryos (Fig. 2H).
|
|
We conclude from these results that the dorsalized phenotype observed in single Bmp2, Bmp4 or Bmp7 knockdowns is increased in double and triple MO injections. Although development of the tail is lost, even triple Bmp2/Bmp4/Bmp7 knockdowns retain a significant amount of DV and anteroposterior (AP) pattern.
BMPs are required for histotypic differentiation
We next analyzed cell differentiation in Bmp4/Bmp7-depleted ventral
marginal zone (VMZ) and animal cap (AC) explants
(Fig. 4A-H). RT-PCR analyses
showed that VMZs depleted of Bmp4 or Bmp7 acquired dorsal fates, expressing
muscle markers such as Myf5 and neural markers such as Ncam,
N-Tubulin and Otx2 (Fig.
4A, lanes 2 to 4). In double Bmp4/Bmp7 knockdowns, midline
mesodermal markers such as Sonic Hedgehog (Shh) and
Xnot were induced, indicating that the VMZs differentiated into
dorsal marginal zone (DMZ) tissues (Fig.
4A, lanes 5 and 6). Histological analyses revealed the formation
of a large amount of neural and cement gland tissue accompanied by greatly
increased cell numbers (Fig. 4, compare B
with C). The ectodermal AC explant system provides the gold
standard for neural induction studies in Xenopus. We found that
although Bmp4 or Bmp7 MOs were sufficient to cause some neural induction
alone, their combined knockdown was much more potent, activating the
expression of neural markers such as N-Tubulin
(Fig. 4D-G), Ncam and
Otx2 (Fig. 4H) in
ectoderm.
|
Bmp4 is epistatic to Chordin
In Xenopus, Chordin knockdown causes a ventralized phenotype that
may result from excess BMP signaling
(Oelgeschläger et al.,
2003). To investigate this, we compared Bmp4 MO- or Chordin
MO-injected embryos with double Bmp4/Chd MO-injected embryos
(Fig. 5A-L). Sizzled
expression, which provides an excellent readout for BMP signaling and ventral
center formation, was greatly expanded in Chordin morphants at gastrula
(Fig. 5G). Bmp4 MO- and double
Bmp4/Chd MO-injected embryos were virtually devoid of Sizzled
expression (Fig. 5D,J). At
tadpole stages, a similar result was obtained: double Bmp4/Chd MO-injected
embryos had the same dorsalized phenotype as Bmp4 MO alone
(Fig. 5, compare H with K). We
conclude that Xenopus Bmp4 is epistatic to Chordin. Xenopus
Chordin, as is the case in zebrafish
(Hammerschmidt and Mullins,
2002
), serves as a dedicated BMP antagonist.
|
|
|
Inhibition of Bmp4/Bmp7 does not induce expression of dorsal genes
Because ventralized embryos were so strongly affected by Bmp4/Bmp7 MOs, our
expectation was that expression of dorsal genes, which is lost in
ß-Catenin MO and UV embryos, would be hyper-rescued. Surprisingly, we
found that at the blastula stage neither Nieuwkoop center genes (such as
Xnr6, Fig. 8A-D) nor
BCNE center markers (such as Pintallavis/Hnf3ß and
Chordin, Fig. 8E-L)
were induced in embryos depleted for ß-Catenin and Bmp4/Bmp7. RT-PCR
analysis confirmed that, as was the case for the single ß-Catenin
knockdown, all dorsal genes tested (Chordin, Noggin, Xnr3, Siamois, Xtwn,
Pintallavis/Hnf3ß, and Cerberus) were downregulated in
ß-Catenin/Bmp4/Bmp7 MO co-injections at the blastula stage
(Fig. 8Q, lane 4). Even at
gastrula, Spemann organizer expression of Chordin
(Fig. 8M-P) or
Goosecoid (not shown) was not restored by Bmp4/Bmp7 MOs in embryos
depleted of ß-Catenin.
These findings show that the induction of radial CNS structures caused by depletion of Bmp2, Bmp4 and Bmp7 in ventralized embryos is independent of Nieuwkoop or BCNE center formation, and ultimately of Spemann organizer function. As the extent of neural induction caused by ß-Catenin/Bmp2/Bmp4/Bmp7 MOs is much greater than in wild-type embryos, the results suggest that the dorsal organizing centers, in addition to providing dorsalizing growth factor antagonists, may be the source of additional ventralizing signals that are lacking in UV-treated or ß-Catenin-depleted embryos, as explained below.
|
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Discussion |
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BMP loss-of-function in Xenopus
Individual knockdowns of Bmp2, Bmp4 and Bmp7 resulted in mild
dorsalizations, characterized by an increase of dorsoanterior structures and a
reduction of ventral and posterior markers. In particular, Bmp4 seems to play
a crucial role in Xenopus development, as it does in mouse
(Winnier et al., 1995;
Hogan, 1996
;
Zhao, 2003
), because its
inhibition produced the strongest dorsalized phenotypes. All three BMPs had to
be knocked down to obtain PGCs loss in Xenopus.
Ventral fin tissues were very sensitive to decreased BMP signaling levels.
Both Bmp4 and Bmp7 MO-injected embryos had varying degrees of ventral fin
truncations and a posteriorization of the anus
(Fig. 1I-K), indicating that
the posterior ventral-most region of the embryo requires maximal BMP
signaling. This is in accordance with the co-expression of Bmp4 and
Bmp7 on the ventral side of the closing blastopore
(Hawley et al., 1995). In
zebrafish, it is well established that the ventral fin requires high levels of
BMP signaling (Mullins et al.,
1996
; Wagner and Mullins,
2002
). Interestingly, Tucker and Slack
(Tucker and Slack, 2004
) have
recently found that ventral fin cells derive from the ventral blastopore in
Xenopus. In the mouse, defects in BMP signaling can cause siren
(mermaid-like) phenotypes, in which ventral-posterior mesoderm development is
impaired (Zakin et al.,
2005
).
Given that the transplantation of the dorsal organizer has potent effects
but ventral grafts do not, ventral development has been viewed as subordinate
to dorsal Spemann organizer signals. However, more recent studies indicate
that diametrically opposite to the Spemann organizer, a ventral center, which
expresses a number of secreted molecules, is formed at gastrula stage
(De Robertis and Kuroda, 2004).
The present loss-of-function study shows that Bmp4 is a key positive regulator
of the expression of ventral center genes, such as Sizzled and
Bmp4 itself. Furthermore, epistatic studies showed that the
ventralization caused by knockdown of Chordin can be compensated by loss of
Bmp4 function (Fig. 5). In
zebrafish, the strongest dorsalized phenotypes are caused by Bmp2b
(swirl) and Bmp7 (snailhouse) mutations
(Hammerschmidt and Mullins,
2002
). Double mutations in bmp2b and bmp7 in
zebrafish do not increase the phenotype
(Schmid et al., 2000
), whereas
in Xenopus double and triple knockouts show increasing degrees of
dorsalization (Fig. 2). This
could be caused by a difference in transcriptional regulation, as zebrafish
bmp2b or bmp7 single mutants display greatly decreased
bmp2b, bmp4 and bmp7 expression
(Schmid et al., 2000
). In
Xenopus, however, Bmp2 MO does no affect Bmp4/Bmp7
transcription (data not shown).
BMP inhibition and CNS formation
Does BMP inhibition play a role in neural induction in Xenopus?
The early ß-Catenin signal induces the BMP antagonists Chordin and Noggin
in the BCNE center, and their transcripts are required for neural tissue
formation in the absence of mesoderm
(Kuroda et al., 2004). In
addition, the early ß-Catenin signal represses Bmp4
transcription on the dorsal side of the embryo
(Baker et al., 1999
). We show
that double Bmp4/Bmp7 depletion induced ectopic neurons in the posterior of
the embryo, in the region in which paraxial mesoderm surrounds the tailbud.
However, triple Bmp2, Bmp4 and Bmp7 knockdowns, although substantially
dorsalized, did not have a significant increase in forebrain/midbrain neural
tissues when compared with those of wild-type embryos at tailbud. By examining
just these results, one would conclude that BMP levels are not involved in
neural induction in Xenopus, as indeed has been proposed in other
species (Stern, 2005
). Even in
Xenopus, the inhibitor Smad6 failed to induce neural tissue when
injected into ventral ectoderm (Linker and
Stern, 2004
; Delaune et al.,
2005
). However, other experiments with an inducible construct of
the inhibitor Smad7, indicate that ectopic neural tissue can be obtained by
inhibiting BMP at pre-gastrula stages, but not at later stages of development
(Wawersik et al., 2005
). It
has also been found that the use of multiple dominant-negative BMPRs greatly
enhances secondary axis formation in the whole embryo
(Yamamoto et al., 2001
).
Importantly, it has been shown that triple inhibition of Chordin, Noggin and
Follistatin causes a catastrophic loss of dorsal structures, including the CNS
(Khokha et al., 2005
).
Neural induction can also be caused by Receptor Tyrosine Kinase (RTK)
ligands, such as FGFs and IGFs, which can increase BMP signaling inhibition
through inhibitory phosphorylation, via Ras/MAPK, of the linker region of
Smad1 (Massague, 2003;
Pera et al., 2003
). This
inhibitory phosphorylation is particularly importantly in dissociated animal
caps, which undergo a sustained activation of endogenous Ras/MAPK signals
(Kuroda et al., 2005
). FGF
signals may also induce neural tissue in Smad-independent ways
(Delaune et al., 2005
).
Although inhibition of Bmp4/Bmp7 by itself does not induce much neural tissue
in whole embryos, it greatly synergizes with Fgf8 protein microinjection,
causing patches of neuronal differentiation throughout the ectoderm
(Fig. 3). The loss-of-function
results provided here for animal cap and VMZ explants confirm that decreasing
endogenous BMP ligands promotes neural differentiation
(Munoz-Sanjuan and Brivanlou,
2002
).
Importantly, our results indicate that, in the intact embryo, regulatory mechanisms that limit the effects of depleting Bmp2/Bmp4/Bmp7 on neural differentiation must exist. When dorsal development is inhibited by ventralization by UV treatment or by ß-Catenin MOs, a massive induction of brain structures is triggered by reduced BMP levels (Fig. 7B,C). In this experimental situation, the effects of BMP inhibition become obvious, so that an embryo that completely lacked a CNS would now develop radial neural structures that cover half the ectoderm. These results support a key role for BMP inhibition in CNS induction in the intact Xenopus embryo.
Brain induction in the absence of Spemann organizer
The most striking result presented here is that extensive CNS structures
are generated by the knockdown of BMP signals in embryos ventralized by UV
irradiation or ß-Catenin depletion. In amphibians, head and trunk
induction are thought to be two separate processes, stemming from head and
trunk-tail organizing centers at early and late gastrula, respectively
(Spemann, 1938;
Niehrs, 2004
). It has been
proposed that double inhibition of Wnt and BMP
(Glinka et al., 1997
), or
triple inhibition of the Wnt, BMP and Nodal pathways
(Piccolo et al., 1999
), is
required to form heads, whereas trunk formation only requires BMP antagonism.
While these models are concerned with the role played by the organizer once it
has formed, we now find that the Spemann organizer is dispensable for brain
induction in embryos lacking both Spemann organizer and BMP signals (Figs
6,
7 and
8).
The diagrams shown in Fig.
8R-V attempt to explain these findings in terms of the cell-cell
signaling thought to take place at blastula stage (before the actual formation
of the Spemann organizer). We propose that, in Xenopus, brain
formation results from the regulated antagonism between BMP signals and the
two dorsal blastula signaling centers (the Nieuwkoop center and the BCNE
center) that form under the influence of nuclear ß-Catenin on the dorsal
side (Fig. 8R)
(De Robertis and Kuroda, 2004).
Blocking early ß-Catenin signals by UV irradiation or ß-Catenin
depletion (Fig. 8S) prevents
the formation of all dorsal centers (the Nieuwkoop center, BCNE center and,
ultimately, the Spemann organizer) and the expression of BMP antagonists. This
leads to the unopposed activity of ventral BMPs, causing the development of
ventralized embryos (also called belly-pieces) lacking all dorsal tissues,
including CNS (Fig. 8S).
Knockdown of BMP activity (Fig.
8T) in wild-type embryos by MOs principally affects development of
the posterior ventral-most structures of the embryo, which arise from ventral
mesoderm, giving rise to tail-less tadpoles.
Remarkably, simultaneous knockdown of both dorsal and ventral signals by
co-injection of ß-Catenin and Bmp4/Bmp7 or Bmp2/Bmp4/Bmp7 MOs results in
radially symmetric embryos containing massive CNS structures
(Fig. 8U). These
hyperneuralized embryos retained AP pattern and in future it will be
interesting to determine whether this is caused by posteriorizing inputs such
as FGF, Wnt or retinoic acid. Radial head structures have also been reported
in zebrafish triple mutants deficient for bozozok, chordino and
bmp2b (Gonzalez et al.,
2000). In Xenopus, radially dorsalized embryos can be
obtained by LiCl treatment, but through a different molecular mechanism.
LiCl-treated embryos have radial expression of BMP antagonists such as
Chordin (Oelgeschläger et
al., 2003
), which inhibit ventralizing BMP signals and result in
expanded dorsal structures (Fig.
8V). We note, however, that the radial CNS structures formed by
LiCl treatment are much smaller than in those seen in
ß-Catenin/Bmp4/Bmp7-depleted embryos.
Lineage-tracing studies have shown that brain cell progenitors in
Xenopus can be traced back to cells of the BCNE center
(Kuroda et al., 2004). These
cells transiently secrete Chordin and Noggin at blastula, forming an early
domain devoid of BMP signaling, which, if cultured in isolation, can
self-differentiate into anterior CNS tissue
(Kuroda et al., 2004
). In
ß-Catenin/Bmp2/Bmp4/Bmp7 morphants, lower BMP levels are attained
throughout the embryo leading to hyperneuralization. Foley et al.
(Foley et al., 2000
) have
proposed that in the chick, as gastrulation proceeds, an important function of
the organizer, located in the anterior primitive streak in the chick embryo,
would be to posteriorize forebrain progenitors located in the epiblast. These
chick forebrain precursors (which are equivalent to the Xenopus BCNE
cells) transiently express Chordin and at all stages migrate ahead of
the organizer (Streit et al.,
1998
; Foley et al.,
2000
), and, consequently, are protected from its caudalizing
signals. The posteriorizing signal produced by the primitive streak of the
chick would correspond to the trunk/tail organizer in Xenopus. The
results presented here are consistent with the proposal that neural induction
precedes Spemann organizer formation. It appears that a principal role of the
Spemann organizer of the gastrula is the maintenance and caudalization of
neural tissue, rather than its initial induction. However, our results also
differ from the current view of chick neural induction
(Stern, 2004
), as we find a
crucial role for the inhibition of BMP activity in the initial induction of
neural tissue in Xenopus.
Yet, one surprising finding is difficult to accommodate in this model: Bmp2/Bmp4/Bmp7-depleted embryos were dorsalized but still retained much of their DV polarity, whereas embryos without a Spemann organizer (ß-Catenin depleted or UV-treated), which normally develop without a trace of a CNS, developed massive CNS structures in Bmp2/Bmp4/Bmp7 morphants. This indicates that the Spemann organizer is capable of partially compensating for the phenotypic effects associated with Bmp2/Bmp4/Bmp7 depletion, and that somehow it can mediate ventral development when Bmp2/Bmp4/Bmp7 signals are eliminated. If the organizer were responsible only for posteriorizing neural tissue, then its absence should mainly affect the AP pattern of the pre-existing CNS, but not trigger the massive formation of radial brain structures observed experimentally. We think the solution to this conundrum lies in the possibility that the Spemann organizer serves as a source of ventralizing signals, in addition to dorsalizing growth factor antagonists. Our ongoing research centers on the molecular identification of this ventralizing activity of the Spemann organizer.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/15/3381/DC1
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