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First published online 15 April 2009
doi: 10.1242/dev.031161
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Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, ON, M5S 3G5, Canada.
* Author for correspondence (e-mail: ashley.bruce{at}utoronto.ca)
Accepted 10 March 2009
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Goosecoid, Chordin, Noggin, Follistatin-like, Axis formation, DV patterning, Zebrafish, Organizer
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) and later in fish
(Ho, 1992;
Oppenheimer, 1934;
Saúde et al., 2000;
Shih and Fraser, 1995), chick
(Waddington, 1932) and mouse
(Beddington, 1994),
transplantation of dorsal organizer tissue to ventral regions of a host embryo
leads to secondary axis formation. Whereas the transplanted organizer
contributes predominantly to axial mesoderm, it also has the remarkable
capacity to non cell-autonomously re-specify host cells from their original
fates and recruit them to compose the remaining tissues of the secondary
axis.
In zebrafish, the organizer/shield elicits a secondary axis when
transplanted ventrally (Saúde et
al., 2000; Shih and Fraser,
1996). By convention the shield marks dorsal but, because dorsal
and anterior development are linked, dorsal and anterior tissues arise from
the shield side of the embryo, whereas posterior and ventral structures arise
from non-shield regions (Schier and
Talbot, 2005). The shield contributes to axial mesoderm and makes
smaller contributions to paraxial mesoderm, ventral neural tissue, and skin
(Saúde et al., 2000;
Shih and Fraser, 1995;
Shih and Fraser, 1996).
In Xenopus and zebrafish, maternal Wnt signaling results in
nuclear accumulation of β-catenin on the presumptive dorsal side of the
embryo, where it activates expression of zygotic organizer genes. Nodal
signaling is also involved in initial organizer formation
(Erter et al., 1998;
Feldman et al., 1998), whereas
later in development both Nodal and Wnt are inhibitory to organizer function.
Historically, the organizer was thought to express inducers of dorsal cell
fates; however, it is now well established that a main function of the
organizer is to repress factors secreted from ventral regions of the embryo
(Niehrs, 2004).
Secreted ventralizing factors of the BMP, Wnt and Nodal families function
in gradients in the early embryo and organizer-derived molecules attenuate the
activity of these factors (Niehrs,
2004). Ventrally expressed Wnt8 restricts the size of the
zebrafish organizer in the late blastula/early gastrula by regulating the
expression of the transcriptional repressors Vox and Vent, whereas BMP
signaling is required to maintain expression of these genes during late
gastrulation (Ramel et al.,
2005; Ramel and Lekven,
2004). Thus, Wnt and BMP act together to limit the organizer and
to promote ventral development.
A distinctive feature of the organizer is its ability to influence distant cell fates as well as to generate axial tissues. Surprisingly, investigation of the interrelationship between these short- and long-range organizer activities has not been a major research focus. It is known that many genes implicated in organizer activity have highly overlapping functions, making it difficult to determine the precise roles that individual genes play. Here we investigate basic questions about organizer activity, including: its short- and long-range signaling functions, the extent to which they are linked and the mechanisms underlying the redundancy of organizer gene activity.
We focused on the first organizer gene discovered, goosecoid
(gsc), which encodes a homeobox transcription factor
(Blumberg et al., 1991).
gsc is expressed in all vertebrate organizers examined, suggesting a
fundamental role in organizer function
(Blum et al., 1992;
Blumberg et al., 1991;
Izpisua-Belmonte et al., 1993;
Schulte-Merker et al., 1994;
Stachel et al., 1993).
gsc has been most studied in Xenopus, where gain-of-function
experiments demonstrated that Gsc induces secondary axes, similar to an
organizer transplant (Cho et al.,
1991; Niehrs et al.,
1993; Sander et al.,
2007). However, secondary axes were often incomplete, lacking head
and notochord. Loss-of-function experiments using dominant-negative and
antisense gsc constructs led to either head or head and notochord
reductions (Ferreiro et al.,
1998; Steinbeisser et al.,
1995; Yao and Kessler,
2001). Results of gsc morpholino oligonucleotide
injections demonstrated that Gsc is required for head but not notochord
formation (Sander et al.,
2007). In addition, Sander et al. suggested that a major function
of Gsc is to block the expression of the ventralizing transcription factors
Vent1/2. It has also been shown that ectopic Gsc represses expression of the
ventralizing factors XWnt-8 and BMP4
(Christian and Moon, 1993;
Fainsod et al., 1994;
Steinbeisser et al.,
1995).
A gene that is activated by ectopic Gsc, presumably indirectly, is the
organizer gene chd, which encodes an extracellular inhibitor of BMPs
(Piccolo et al., 1996;
Sasai et al., 1994).
Xenopus Chd is an essential downstream effector of Gsc function
(Sander et al., 2007). The
Xenopus work suggests that Gsc plays an important role in organizer
function. In sharp contrast, targeted gsc knockout in mouse had no
effect on early DV patterning
(Rivera-Perez et al., 1995;
Yamada et al., 1995). However,
in heterotypic transplantation experiments the neural-inducing properties of
the mutant organizer were impaired (Zhu et
al., 1999). Technical issues might explain some of the differences
in the severity of these effects, whereas real differences between species
could stem from the known redundancy in DV patterning mechanisms, which might
vary between different organisms. In either case, there are many unanswered
questions that warrant further investigation.
In zebrafish, gsc transcripts are present maternally, with zygotic
expression beginning at the midblastula transition in the region of the future
organizer (Schulte-Merker et al.,
1994; Stachel et al.,
1993). Typically, embryos with mutations that disrupt the
organizer have little or no gsc expression and reduced dorsal
structures (Feldman et al.,
1998; Gritsman et al.,
1999; Kelly et al.,
2000; Koos and Ho,
1999; Nojima et al.,
2004; Sampath et al.,
1998; Schier et al.,
1997; Yamanaka et al.,
1998), whereas gsc expression is expanded in embryos
dorsalized by lithium chloride treatment
(Stachel et al., 1993). Thus
gsc transcript levels correlate with organizer activity. In
loss-of-function studies, injection of gsc morpholinos cause head
truncations in a small fraction of embryos without affecting the notochord,
whereas head defects occur at higher frequency when morpholinos against
gsc and foxa3 were combined
(Seiliez et al., 2006). As in
Xenopus, ectopic zebrafish Gsc represses wnt8
(Seiliez et al., 2006).
To investigate the molecular basis of organizer activity, we examined Gsc function in zebrafish. Ventral injection of low doses of gsc mRNA produced partial secondary axes that reflect a short-range activity of the organizer, whereas higher doses led to complete secondary axes, mimicking the long-range signaling activity of the organizer. We propose that short-range signaling is accompanied by BMP repression and long-range signaling by BMP and Wnt repression. Surprisingly, Chd is not essential for Gsc function in zebrafish, unlike in Xenopus. In fact, Gsc exhibited organizer activity in the absence of three secreted BMP antagonists, suggesting that Gsc functions in a parallel pathway to BMP inhibitors.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Animals were treated in accordance with the policies of the
local animal care committee.
Constructs and morpholinos
gsc and follistatin-like 1b (fstl1b) ORFs were
cloned from cDNA into pCS2+
(Rupp et al., 1994). mRNAs
were transcribed using the SP6 mMESSAGE mMACHINE Kit (Ambion, Austin, TX,
USA). mRNAs from chordin-pCS2+ and
noggin1-pCS2+ (nog1-pCS2+) were
prepared as described (Dal-Pra et al.,
2006; Miller-Bertoglio et al.,
1997).
Morpholino (MO) sequences (5' to 3'):
); );
gsc constructs
The gsc homeobox and 3' sequence (codons 126 to 241) were
cloned into pVP16-N and pENG-N (Kessler,
1997) to generate VP16-gsc (VP-gscHD) and
engrailed-gsc (eng-gscHD), respectively. A Xenopus
construct consisting of the Xenopus gsc ORF with two minimal VP16
domains at the C-terminus was a gift from J. Smith
(Latinkic and Smith, 1999). To
create a zebrafish version (gsc-VP2), Xenopus gsc was
removed and replaced with the zebrafish gsc ORF.
Microinjections
Initial gsc microinjections were done double blind. Approximately
4 pl of mRNA was injected into a single cell of 8-cell stage embryos as
described (Bruce et al., 2003).
gsc mRNA (12, 24 or 48 pg) and chd mRNA (200, lower
concentrations had little effect, or 664 pg) were injected. gfp mRNA
doses ranged from 130 to 340 pg. gsc constructs were injected at the
following doses: 660 pg VP-gscHD, 24 pg eng-gscHD and
200-520 pg gsc-VP2. For subthreshold experiments gsc was
injected at 4 pg and gsc-VP2 at 100 pg.
Approximately 100 pl of gsc, chd, nog1, fstl1b, and control MOs
were injected into the yolk at the 1- to 2-cell stage. gsc-MO (330
pg) was injected and nonspecific phenotypes, which were not rescued by
coinjection of gsc mRNA, included mild head necrosis and general
developmental delay. The presence of the MO target site was confirmed by PCR
and sequencing. chd-MO injected at 22 pg produced no phenotype and
chd-MO injected at 100 pg ventralized embryos. nog1- and
fstl1b-MOs (3 ng) were injected, producing results as described
(Dal-Pra et al., 2006).
Dal-Pra et al. observed a range of phenotypes falling into three phenotypic
classes, whereas we observed phenotypes that fell predominantly into one
class. This is likely to be due to the fact that we injected into the
chd mutants, whereas Dal-Pra et al. primarily employed
chd-MO.
Morpholino rescue experiments
Xenopus chd mRNA (Sasai et
al., 1994) was used to rescue the effects of the chd-MO,
as described (Nasevicius and Ekker,
2000). Other mRNAs were generated from constructs that either did
not contain the MO binding site (gsc, fstl1b) or contained four
silent mutations to prevent MO binding (nog1).
Genotyping
Genotyping of embryos from chdtt250 heterozygous
parents was performed as described by Oelgeschläger
(Oelgeschläger et al.,
2003) and ZIRC.
Time-lapse
Shield stage embryos were mounted in 3% methylcellulose. Volocity
(Improvision, Lexington, MA, USA) was used to acquire DIC and fluorescent
images at 5-minute intervals.
In situ hybridization
In situ hybridizations were performed as described
(Jowett and Lettice, 1994)
using riboprobes to bmp2b and bmp4
(Martinez-Barbera et al.,
1997), chd
(Miller-Bertoglio et al.,
1997), dkk (Hashimoto
et al., 2000), dlx2a
(Akimenko et al., 1994),
flh (Talbot et al.,
1995), shha (Krauss
et al., 1993) and wnt8
(Kelly et al., 1995).
Immunohistochemistry
Embryos were fixed, blocked and incubated in primary antibody as described
(Bruce et al., 2001). The
peroxidase anti-peroxidase method was used. Anti-Ntl antibody was diluted
1:5000 (Schulte-Merker et al.,
1992), goat anti-rabbit secondary antibodies and rabbit peroxidase
anti-peroxidase tertiary antibodies were diluted 1:100 and 1:500, respectively
(Jackson ImmunoResearch, West Grove, PA, USA).
Cell counts
Embryos between bud and 2-somite stages, stained with anti-Ntl antibody,
were flat-mounted and photographed on a Zeiss AxioImager Z1 compound
microscope using an Orca-ER camera (Hamamatsu, Bridgewater, NJ, USA). Stained
nuclei visible in each focal plane, excluding the tailbud, were traced onto
transparencies and counted.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Helde et al.,
1994; Kimmel and Law,
1985; Kimmel and Warga,
1987), clone location was documented relative to the shield and
embryos were sorted into dorsal, dorsolateral, lateral, ventral and
ventrolateral classes at 6 hours post-fertilization (hpf)
(Fig. 1L). At 1 day
post-fertilization (dpf), sorted embryos were scored for GFP-expressing cells
in shield-derived structures: hatching gland, notochord, floor plate of the
spinal cord and hypochord (Saúde et
al., 2000; Shih and Fraser,
1995; Shih and Fraser,
1996). In striking contrast to gfp control clones, every
gsc clone labeled one or more shield derivative, regardless of its
initial location (see Table S1 in the supplementary material). This occurred
in two ways: gsc-expressing cells contributed either to enlarged
dorsal axial structures or to axial structures in a secondary axis. Thus,
gsc-overexpressing cells populated tissues normally derived from the
shield.
Analysis of ventral clones allowed us to examine Gsc function in isolation
from other organizer factors. gsc-injected embryos with
ventral/ventrolateral clones gave rise to secondary axes at high frequency,
whereas dorsal/dorsolateral gsc clones and gfp control
clones did not (see Table S2 in the supplementary material). As expected, in
controls with ventral clones (Fig.
1A), GFP-positive cells were located posteriorly at the 1-somite
stage and were absent from axial mesoderm
(Fig. 1B) and, at 1 dpf,
GFP-positive cells were located mainly in the myotomes of the trunk and tail
(Fig. 1C). By contrast,
gsc-injected embryos with ventral clones
(Fig. 1D) often had two axes on
opposite sides of an elongated yolk cell at bud stage, one of which was GFP
labeled (Fig. 1E). Elongated
yolk morphology is also seen with dorsalized embryos
(Mullins et al., 1996).
GFP-positive cells populated axial tissues of the secondary axis, including an
apparent prechordal plate (Fig.
1E,F) and a secondary notochord
(Fig. 1F). Secondary axes did
not always contain notochord at bud stage (see Table S3 in the supplementary
material) although they did by 1 dpf.
Two distinct axes were often no longer apparent by 1 dpf, and would have
been missed if bud-stage embryos had not been examined. At 1 dpf, the severely
abnormal embryo contained a broad head with two eyes
(Fig. 1G) and a partially
GFP-labeled notochord (Fig.
1I), thus the two axes moved together between bud and 1 dpf. When
double axes were generated, they moved together in 36% (13/36) of cases,
whereas in 58% (21/36) of cases the two axes remained distinct, although they
shared a single posterior trunk and tail. Expression of the neural marker
dlx2a (Akimenko et al.,
1994) was often normal in secondary axes, indicating the presence
of telencephalic and diencephalic tissue
(Fig. 1J,K; see Table S4 in the
supplementary material). Therefore secondary axes were essentially complete,
containing notochord and anterior-most brain, indicating that gsc
overexpression mimicked an organizer transplant.
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). The distribution of dorsal/dorsolateral gsc
clones was similar to controls (Fig.
2E-H) and contrasted with results in Xenopus, in which
dorsal gsc-overexpressing cells were excluded from axial tissues
(Niehrs et al., 1993).
The single axes in gsc-injected embryos often had enlarged shield
derivatives, but thinner posterior trunks and tails, suggesting the presence
of excess dorsal/anterior tissue at the expense of ventral/posterior tissues.
For example, the notochord domain of an embryo with a dorsolateral
gsc clone was much larger than the control (see Figs S2B and S2F in
the supplementary material). To quantify this observation, notochord nuclei
were stained in late gastrula stage embryos using the No tail (Ntl) antibody
(Schulte-Merker et al., 1992).
There was a 40% increase in Ntl-positive nuclei in embryos with dorsal,
dorsolateral, or lateral gsc clones
(Fig. 2I). The notochord was
often shorter, but disproportionately wider, in gsc-injected embryos
(Fig. 2J), although by 1 dpf
embryos were of similar lengths as controls. Recruitment of more cells to the
notochord domain, compared with controls, suggested that increasing the
gsc dose dorsally enhanced organizer activity.
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We used a molecular readout to measure the distance over which Gsc exerts
its non cell-autonomous effect by examining the expression of the organizer
gene chd in embryos with ventral gsc clones. Double labeling
of membrane GFP (to mark the clone) and chd showed that ectopic
chd was induced at a distance of up to ten cell diameters away from
the gsc clone (Fig.
3D). Signaling over this large distance constitutes long-range
signaling, as defined by Chen and Schier for the zebrafish protein
Nodal-related 1 (Chen and Schier,
2001). Thus, Gsc exhibits long-range signaling activity by a
molecular criterion, which is probably responsible for the long-range
organizer activity observed morphologically. Another potential explanation is
that dorsal chd-expressing cells migrated towards the ventral
gsc clone. We eliminated this possibility by labeling the shield with
Rhodamine Dextran in embryos with ventral gsc clones and observing no
movement of Rhodamine positive cells towards ventral gsc clones in
live embryos (not shown).
We next asked whether gsc had dose dependent effects on axis formation by injecting half the standard dose (12 pg). Partial secondary axes were produced that lacked heads and notochords and consisted only of neural and somitic tissue that was nearly completely GFP labeled (Fig. 3I-K). Only a very few unlabeled cells were recruited to the partial axis (Fig. 3K, insets), suggesting that short-range signaling occurred. Examination of ectopic chd expression in ventral gsc clones revealed that chd was primarily confined to the clone itself and only extended at most one or two cell diameters away (Fig. 3E). Thus, lower gsc doses acted predominantly cell-autonomously and were unable to elicit long-range signaling.
gsc also had dose dependent effects on DV gene expression at shield stage. At doses of gsc sufficient to induce complete secondary axes, we observed a reduction in wnt8, bmp2b and bmp4 expression and ectopic expression of the organizer genes chd, floating head (flh) and dickkopf 1 (dkk1) (Fig. 3L-O; Table 1). In embryos injected with a lower dose of gsc, chd was induced at similar frequencies but dkk1 (a Wnt signaling inhibitor) was induced only about half as often, and the frequency of wnt8 inhibition was also moderately but consistently reduced (Table 1). These results suggest that partial secondary axes produced by low gsc doses might result from inhibition of BMP signaling but insufficient inhibition of Wnt signaling. In addition, we noticed that at the standard gsc dose, wnt8 was always repressed in a relatively small domain, whereas chd was consistently induced in a large domain, that extended outside the gsc clone. This spatial arrangement mimics what is normally seen in the organizer, suggesting that Gsc is able to induce this organizer pattern in a dose dependent fashion.
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). To
determine if Chd was required for Gsc function in zebrafish, Gsc activity was
examined in the absence of Chd by overexpressing gsc in
chdtt250 null mutant embryos
(Schulte-Merker et al., 1997).
chdtt250 mutants have expanded ventral tissues, most
notably blood, blood island and tail fin and have reduced anterior neural
tissue, whereas gsc expression is normal in most mutants
(Fisher et al., 1997;
Hammerschmidt et al., 1996;
Schulte-Merker et al., 1997).
gsc was injected into one cell at the 8-cell stage and embryos were
genotyped at the end of the experiment. At the standard gsc dose (24
pg), the majority of mutant embryos with ventral/ventrolateral clones
(Fig. 4A) contained partial
secondary axes with neural and somitic tissue but not notochord
(Fig. 4D), similar to the
overexpression of a low dose of gsc in wild-type embryos. However,
these axes occasionally had well developed heads, separate from the endogenous
head (Fig. 4B). Interestingly,
the endogenous head was often more fully developed than in uninjected
chd mutants (Fig. 4B),
suggesting that ventral gsc injection could partially rescue the
endogenous axis, indicative of a long-range effect.
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chd and gsc overexpression are not equivalent
Since Chd facilitates the ability of Gsc to induce secondary axes, we next
addressed whether it was sufficient to induce complete secondary axes by
overexpressing chd ventrally. Global chd overexpression in
zebrafish dorsalizes embryos
(Miller-Bertoglio et al.,
1997) however, localized overexpression was not examined. To test
the axis-forming ability of Chd in zebrafish, 200 pg of chd mRNA was
injected into one cell at the 8-cell stage. Ventral/ventrolateral chd
clones generated partial secondary axes that never contained head or notochord
and that merged with the endogenous axis posteriorly. Morphologically,
secondary axes contained neural tissue
(Fig. 5C,D), most had ectopic
myotomes (Fig. 5E,F), some had
beating cardiac tissue (not shown) and ectopic or enlarged otic vesicles
(Fig. 5G,H). Notably, these
partial axes resembled those induced by low gsc doses. Chd did not
induce ectopic Gsc (see Fig. S3 in the supplementary material). dlx2a
expression was not detected in the secondary neural tube and flh and
wnt8 expression were normal at shield stage (not shown).
Interestingly, secondary axes were typically well separated from the primary
axis, in contrast to those seen following gsc overexpression. In
addition, partial secondary axes were nearly completely GFP labeled,
suggesting that Chd did not have long-range inductive effects.
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gsc overexpression induces secondary axes in the absence of three BMP inhibitors
The ability of Gsc to induce secondary axes in chd mutant embryos
could be explained by the presence of other, compensatory, BMP inhibitors. The
BMP inhibitors Noggin1 (Nog1) and Follistatin-like 1b (Fstl1b) were reported
to be zygotically expressed and to function redundantly with Chd
(Dal-Pra et al., 2006;
Fürthauer et al., 1999).
As shown previously, coinjection of nog1- and fstl1b-MOs
into chd mutant embryos enhances the mutant phenotype (see Fig. S3 in
the supplementary material) (Dal-Pra et
al., 2006). mRNA rescue experiments demonstrated morpholino
specificity (see Fig. S4 in the supplementary material). When gsc was
injected ventrally into embryos injected with nog1- and
fstl1b-MOs, both partial (Fig.
5I,J) and complete (Fig.
5K,L) secondary axes were observed. Therefore, Gsc induced
complete secondary axes when all known zygotic BMP antagonists expressed in
the early embryo were reduced or eliminated. In addition, the head of the
endogenous axis was partially rescued in embryos with ventral gsc
clones, further demonstrating the ability of Gsc to trigger long-range effects
(not shown).
|
).
Crucially, ventral coinjection of chd, fstl1b and nog1 mRNAs
in wild-type embryos also induced only partial secondary axes
(Fig. 5M,N). These data
indicate that Gsc can function in the absence of all three secreted BMP
inhibitors.
Distinct mechanisms underlie long- and short-range signaling
We next addressed whether Gsc is normally involved in DV patterning. No
gsc mutant exists, and maternal gsc expression raises the
possibility that maternal protein is present. For these reasons, we initially
took a dominant-negative approach to block Gsc function. Gsc is a
transcriptional repressor in other systems
(Danilov et al., 1998;
Ferreiro et al., 1998;
Latinkic and Smith, 1999;
Mailhos et al., 1998;
Smith and Jaynes, 1996).
Therefore, we made two activator constructs designed to antagonize endogenous
Gsc function. VP-gscHD contains the gsc homeodomain (HD)
fused to the VP16 transcriptional activator
(Kessler, 1997) and is
predicted to bind to and activate transcription of Gsc target genes. The
second construct, gsc-VP2, contains the entire gsc coding
region plus two minimal VP16 domains and, in Xenopus, the equivalent
construct blocks Gsc function by binding to Gsc target genes without
activating their transcription (Latinkic
and Smith, 1999). Thus, gsc-VP2 should produce a
phenotype more akin to Gsc loss-of-function than VP-gscHD. We also
generated a repressor construct, consisting of the gsc HD fused to
the engrailed repressor domain (eng-gscHD)
(Kessler, 1997), that should
mimic endogenous Gsc function. Similar constructs were originally used in
Xenopus (Kessler,
1997; Latinkic and Smith,
1999) and our zebrafish versions performed as expected in
Xenopus embryos (see Fig. S5 in the supplementary material and not
shown).
Ventral injection of eng-gscHD (24 pg) into zebrafish often produced complete secondary axes with GFP-positive cells located in organizer derivatives (Fig. 6A,B), demonstrating that Gsc functions as a transcriptional repressor in zebrafish. As predicted, ventral injection of VP-gscHD had no effect, even at high doses (660 pg, not shown). To test whether VP-gscHD could inhibit Gsc function, we examined its ability to block secondary axis formation by Gsc and found, surprisingly, that it could not (not shown). However, VP-gscHD blocked eng-gscHD from eliciting complete secondary axes, leading to partial axes instead (Fig. 6H,I). This suggested that VP-gscHD could block the long-range organizer activity of eng-gscHD, producing an effect similar to injection of a low gsc dose.
Unexpectedly, ventral injection of gsc-VP2, at a moderate dose
(200 pg) that was predicted to block Gsc function, occasionally produced
partial secondary axes that were almost completely labeled by GFP
(Fig. 6C,D). High
concentrations (520 pg) also produced partial secondary axes. Thus, complete
axes could not be induced at every dose tested, again mimicking the effect of
low dose gsc. An analysis of chd expression in
gsc-VP2-injected embryos revealed that ectopic chd was
transiently induced, presumably accounting for the infrequent partial
secondary axes observed (Fig.
6E-G and see Table S5 in the supplementary material). Ectopic
expression of Xenopus gsc-VP2
(Latinkic and Smith, 1999) or
dominant-negative Xenopus myc-tagged gsc
(Ferreiro et al., 1998) also
occasionally produced partial secondary axes in zebrafish (not shown).
Significantly, we never observed partial secondary axes in Xenopus
using either the frog or zebrafish constructs.
These findings suggest that constructs designed to block Gsc function in Xenopus could partially mimic the short-range function of Gsc in zebrafish. To confirm this, gsc was injected at a subthreshold dose (4 pg). This gsc dose had no effect alone but dorsalized embryos (6/7, 86%) when coinjected with a reduced dose of gsc-VP2 (100 pg), suggesting a synergistic effect.
Gsc and endogenous dorsal specification
Since the dominant-negative constructs failed to fully block Gsc function,
we used gsc morpholinos instead
(Seiliez et al., 2006).
gsc-MO had mild ventralizing effects, as shown previously
(Seiliez et al., 2006).
However, morphologically, most embryos appeared normal
(Fig. 7E). To further
investigate the extent to which Gsc requires Chd and to explore potential
synergistic effects, we used a fixed concentration of gsc-MO in
combination with chdtt250 mutant embryos and two different
doses of chd-MO to generate embryos possessing no, low or
intermediate levels of Chd (Nasevicius and
Ekker, 2000). Morpholino control experiments confirmed that the
effects were specific (see Fig. S4 in the supplementary material).
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),
whereas wnt8 was normal in chd-MO injected embryos (not
shown). By contrast, there was an obvious difference in the expression of
wnt8 in double morpholino injected embryos versus control and single
morpholino injected embryos. In almost half of double morpholino injected
embryos, wnt8 was ectopically expressed dorsally in the shield and
this expression was more robust than in gsc morphants
(Fig. 7S). Thus, reducing
gsc and chd levels resulted in a more than additive effect
on wnt8 expression. This is consistent with our overexpression
experiments showing that a higher gsc concentration was necessary to
induce complete secondary axes in the absence of chd, suggesting that
they could function cooperatively. Taken together, these results suggest a
synergistic interaction between Gsc and Chd and that these two proteins
operate in parallel pathways.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Gsc and short-range signaling
The hallmark of the organizer is its ability, upon transplantation, to
pattern distant cells to form a secondary axis. Organizer function, therefore,
requires cell-cell signaling. Our studies suggest that Gsc can trigger both
short- and long-range signaling. Ventral injection of low doses of
gsc into wild-type embryos resulted in nearly completely GFP-labeled
partial secondary axes, containing neural and somitic tissue. Labeled cells
were located in tissues that never normally express gsc, such as
retina and muscle, suggesting that gsc does not interfere with the
differentiation of specific cell types nor does it appear to directly induce
them. The observed effect is mainly cell-autonomous but not entirely, as a few
unlabeled cells are incorporated into the partial secondary axes. We suggest
that the short-range effects result from Gsc acting in cells to trigger
autocrine, short-range signaling and that only a few unlabeled cells that
receive this local signal are incorporated into the partial axes. This is
consistent with our molecular analysis showing that induction of ectopic
chd extends only one or two cell diameters away from the gsc
clone.
|
). In addition, zebrafish BMP pathway mutants and embryos
globally overexpressing chd have expanded neural and somitic tissue
but not axial mesoderm (Miller-Bertoglio
et al., 1997; Mullins et al.,
1996; Tucker et al.,
2008). Xenopus Gsc can repress bmp4
transcription (Fainsod et al.,
1994; Steinbeisser et al.,
1995) and we showed that Gsc can repress bmp2b and
bmp4 transcription in zebrafish, although it is not known whether
this is direct. Ventral chd overexpression also induced partial
secondary axes, indicating that it is capable of short-range signaling.
However, Chd is not required for axis induction by Gsc, suggesting that
additional factors are involved.
What is the identity of the short-range signal? BMP signaling is maintained
by a positive autoregulatory feedback loop, thus any reduction in BMP levels
will be amplified as a result of disrupting this loop
(Little and Mullins, 2006).
There is also in vitro evidence that mouse, human, chick and zebrafish
bmp2b transcript stability is reduced in cells expressing no or low
levels of BMP (Fritz et al.,
2004). It therefore seems likely that incorporation of a few
unlabeled cells into the partial secondary axes occurs simply as a result of
the local absence or reduction of BMP. How does Gsc repress BMP? The zebrafish
transcriptional repressor Bozozok (Boz; Dharma - ZFIN), which acts upstream of
zygotic Gsc, can directly bind and repress the bmp2b promoter
(Leung et al., 2003). Both Boz
and Gsc are paired-type homeodomain proteins, thus raising the possibility
that Gsc binds the paired-type binding sites in the bmp2b promoter.
Alternatively, Gsc might function via an unknown factor
(Fig. 8, Factor X) to repress
BMP signaling.
Gsc and long-range signaling
Increasing the gsc dose ventrally mimics both the short- and
long-range activities of the organizer, resulting in complete secondary axes,
in which GFP-labeled cells are predominantly confined to organizer
derivatives. Large-scale recruitment of unlabeled cells indicates that
long-range signaling occurred. A demonstration of long-range signaling at the
molecular level is that ectopic chd was induced at a distance from
gsc clones; however, as discussed below, chd cannot mediate
the long-range signal. We and others have observed that complete secondary
axes move towards and often merge with the primary axis. By contrast, we found
that partial secondary axes did not merge, suggesting that long-range signals
are responsible for this phenomenon.
Analysis of DV marker expression demonstrated that high gsc doses
resulted in inhibition of both BMP and Wnt signaling. In Xenopus, low
levels of BMP and Wnt signaling are required dorsally for notochord formation
and BMP, and Wnt signaling must be repressed ventrally for ectopic notochord
induction (Yasuo and Lemaire,
2001). In zebrafish, overexpression of wnt8a or
inhibition of the Wnt antagonist dkk1 results in head truncations
(Seiliez et al., 2006),
whereas overexpression of dkk1 and deletion of wnt8 produces
expanded brain and axial mesoderm
(Hashimoto et al., 2000;
Lekven et al., 2001). Although
axial mesoderm is unaffected in BMP mutants, when both wnt8 and
bmp2b function are removed, axial mesoderm expands
(Ramel et al., 2005). Thus,
the ability of Gsc to induce complete secondary axes ventrally and increase
notochord cell number dorsally is probably the result of combined inhibition
of Wnt and BMP signaling. Gsc directly represses wnt8 transcription
in Xenopus and we propose that this is likely to be the case in
zebrafish as well (Fig. 8). Gsc
might also directly inhibit expression of the ventralizing factors
ved/vox/vent as it does in Xenopus
(Sander et al., 2007).
What is the molecular identity of the long-range signal(s)? Induction of
head and notochord was always accompanied by cell recruitment, indicative of
long-range signaling. However, it is notable that we could see evidence of
long-range signaling in the absence of notochord induction. Ventral injection
of gsc into chd mutants at the dose that gave rise to
partial secondary axes often resulted in more fully developed endogenous
heads. We also found that ventral overexpression of dkk1 led to
enlarged endogenous heads, suggesting that it could be the long-range signal,
although dkk1 did not elicit complete secondary axes when
overexpressed ventrally (our unpublished data). We also tested whether
coinjection of dkk1 and chd ventrally could elicit complete
secondary axes and found that it could not (our unpublished data). One
possibility is that a combination of known factors is required, for example,
Dkk1 might act in combination with other Wnt signaling inhibitors.
Alternatively, an unknown factor(s) might be involved
(Fig. 8, Factor Y). A recent
study showed that implanted cells from early zebrafish gastrula and pharyngula
cell lines could induce secondary axes by inducing organizer gene expression
in host tissue, but this was not mediated by known organizer inducers,
including Nodals, Bozozok, and FGFs, leading the authors to suggest that an
unknown secreted factor was responsible
(Hashiguchi et al., 2008).
Clearly, additional experiments are required to identify these factors and to
characterize their interactions.
Gsc does not require Chd
Xenopus Gsc function relies entirely on Chd
(Sander et al., 2007).
Furthermore, Xenopus Chd is required for complete secondary axis
induction by ventral organizer transplantation
(Oelgeschläger et al.,
2003). Thus, it appears that Xenopus Chd can mediate both
short- and long-range organizer activities. We present evidence suggesting
that Chd is not required for Gsc function in zebrafish. Gsc induced complete
secondary axes in chdtt250 null mutant embryos and
injection of gsc-MO further ventralized chdtt250
mutants, suggesting that Gsc has Chd-independent functions in DV patterning.
Our database searches have not provided evidence for a second chd
gene in zebrafish, nor is chd maternally expressed
(Miller-Bertoglio et al.,
1997). Moreover, our data suggest that in zebrafish, Gsc and Chd
operate in parallel rather than in a simple linear pathway. We also showed
that Gsc does not require two other zygotic BMP inhibitors, Nog1 and Fstl1b.
Although Gsc might require other BMP inhibitors for its function, we have
eliminated the known zygotic BMP inhibitors that are expressed at the
appropriate time and place.
Conclusions
Our findings suggest that Gsc has dose dependent effects on axis induction
and provide new insights into molecularly distinct short- and long-range
signaling activities of the organizer. In addition, we show that Gsc functions
in parallel to three secreted BMP inhibitors. Our work also suggests that Gsc
function in Xenopus and zebrafish is different. Thus, despite the
conservation of organizer genes among vertebrates, divergent functions have
evolved for key organizer genes in different species.
|
Footnotes |
|---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/136/10/1675/DC1
We thank Stephanie Lepage for Fig. 8 and Fig. 1L; Olivia Luu for the Xenopus work; Brian Ciruna, Marnie Halpern, Mary Mullins, Ian Scott and Jim Smith for reagents; and Brian Ciruna, Tony Harris, Robert Ho, Stephanie Lepage, Hiro Ninomiya, Ulli Tepass and Rudi Winklbauer for comments on the manuscript. A.E.E.B. thanks Rudi Winklbauer for many helpful discussions. ZIRC is supported by NIH-NCRR. A.E.E.B. was supported by NSERC and CFI.
|
REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Abdelilah, S., Solnica-Krezel, L., Stainier, D. Y. and Driever,
W. (1994). Implications for dorsoventral axis determination
from the zebrafish mutation janus. Nature
370,468
-471.[CrossRef][Medline]
Akimenko, M. A., Ekker, M., Wegner, J., Lin, W. and Westerfield,
M. (1994). Combinatorial expression of three zebrafish genes
related to distal-less: part of a homeobox gene code for the head.
J. Neurosci. 14,3475
-3486.[Abstract]
Beddington, R. S. P. (1994). Induction of a
second neural axis by the mouse node. Development
120,613
-620.[Abstract]
Blum, M., Gaunt, S. J., Cho, K. W., Steinbeisser, H., Blumberg,
B., Bittner, D. and De Robertis, E. M. (1992). Gastrulation
in the mouse: the role of the homeobox gene goosecoid.
Cell 69,1097
-1106.[CrossRef][Medline]
Blumberg, B., Wright, C. V. E., DeRobertis, E. M. and Cho, K. W.
Y. (1991). Organizer-specific homeobox genes in Xenopus
laevis embryos. Science
253,194
-196.
Bruce, A. E., Oates, A. C., Prince, V. E. and Ho, R. K.
(2001). Additional hox clusters in the zebrafish: divergent
expression patterns belie equivalent activities of duplicate hoxB5 genes.
Evol. Dev. 3,127
-144.[CrossRef][Medline]
Bruce, A. E., Howley, C., Zhou, Y., Vickers, S. L., Silver, L.
M., King, M. L. and Ho, R. K. (2003). The maternally
expressed zebrafish T-box gene eomesodermin regulates organizer formation.
Development 130,5503
-5517.
Chen, Y. and Schier, A. F. (2001). The
zebrafish Nodal signal Squint functions as a morphogen.
Nature 411,607
-610.[CrossRef][Medline]
Cho, K. W., Blumberg, B., Steinbeisser, H. and De Robertis, E.
M. (1991). Molecular nature of Spemann's organizer: the role
of the Xenopus homeobox gene goosecoid. Cell
67,1111
-1120.[CrossRef][Medline]
Christian, J. L. and Moon, R. T. (1993).
Interactions between Xwnt-8 and Spemann organizer signaling pathways generate
dorsoventral pattern in the embryonic mesoderm of Xenopus. Genes
Dev. 7,13
-28.
Dal-Pra, S., Fürthauer, M., Van-Celst, J., Thisse, B. and
Thisse, C. (2006). Noggin1 and Follistatin-like2 function
redundantly to Chordin to antagonize BMP activity. Dev.
Biol. 298,514
-526.[CrossRef][Medline]
Danilov, V., Blum, M., Schweickert, A., Campione, M. and
Steinbeisser, H. (1998). Negative autoregulation of the
organizer-specific homeobox gene goosecoid. J. Biol.
Chem. 273,627
-635.
Erter, C. E., Solnica-Krezel, L. and Wright, C. V.
(1998). Zebrafish nodal-related 2 encodes an early mesendodermal
inducer signaling from the extraembryonic yolk syncytial layer.
Dev. Biol. 204,361
-372.[CrossRef][Medline]
Fainsod, A., Steinbeisser, H. and De Robertis, E. M.
(1994). On the function of BMP-4 in patterning the marginal zone
of the Xenopus embryo. EMBO J.
13,5015
-5025.[Medline]
Feldman, B., Gates, M. A., Egan, E. S., Dougan, S. T.,
Rennebeck, G., Sirotkin, H. I., Schier, A. F. and Talbot, W. S.
(1998). Zebrafish organizer development and germ-layer formation
require nodal-related signals. Nature
395,181
-185.[CrossRef][Medline]
Ferreiro, B., Artinger, M., Cho, K. and Niehrs, C.
(1998). Antimorphic goosecoids.
Development 125,1347
-1359.[Abstract]
Fisher, S., Amacher, S. L. and Halpern, M. E.
(1997). Loss of cerebum function ventralizes the zebrafish
embryo. Development 124,1301
-1311.[Abstract]
Fritz, D. T., Liu, D., Xu, J., Jiang, S. and Rogers, M. B.
(2004). Conservation of Bmp2 post-transcriptional regulatory
mechanisms. J. Biol. Chem.
279,48950
-48958.
Fürthauer, M., Thisse, B. and Thisse, C.
(1999). Three different noggin genes antagonize the activity of
bone morphogenetic proteins in the zebrafish embryo. Dev.
Biol. 214,181
-196.[CrossRef][Medline]
Gritsman, K., Zhang, J., Cheng, S., Heckscher, E., Talbot, W. S.
and Schier, A. F. (1999). The EGF-CFC protein one-eyed
pinhead is essential for nodal signaling. Cell
97,121
-132.[CrossRef][Medline]
Hammerschmidt, M., Pelegri, F., Mullins, M. C., Kane, D. A., van
Eeden, F. J., Granato, M., Brand, M., Furutani-Seiki, M., Haffter, P.,
Heisenberg, C. P. et al. (1996). dino and mercedes, two genes
regulating dorsal development in the zebrafish embryo.
Development 123,95
-102.[Abstract]
Hashiguchi, M., Shinya, M., Tokumoto, M. and Sakai, N.
(2008). Nodal/Bozozok-independent induction of the dorsal
organizer by zebrafish cell lines. Dev. Biol.
321,387
-396.[CrossRef][Medline]
Hashimoto, H., Itoh, M., Yamanaka, Y., Yamashita, S., Shimizu,
T., Solnica-Krezel, L., Hibi, M. and Hirano, T. (2000).
Zebrafish Dkk1 functions in forebrain specification and axial mesendoderm
formation. Dev. Biol.
217,138
-152.[CrossRef][Medline]
Helde, K. A., Wilson, E. T., Cretekos, C. J. and Grunwald, D.
J. (1994). Contribution of early cells to the fate map of the
zebrafish gastrula. Science
265,517
-520.
Ho, R. K. (1992). Axis formation in the embryo
of the zebrafish, Brachydanio rerio. Semin. Dev.
Biol. 3,53
-64.
Izpisua-Belmonte, J. C., De Robertis, E. M., Storey, K. G. and
Stern, C. D. (1993). The homeobox gene goosecoid and the
origin of organizer cells in the early chick blastoderm.
Cell 74,645
-659.[CrossRef][Medline]
Jowett, T. and Lettice, L. (1994). Whole-mount
in situ hybridizations on zebrafish embryos using a mixture of digoxigenin-
and fluorescein-labelled probes. Trends Genet.
10, 73-74.[CrossRef][Medline]
Kelly, C., Chin, A. J., Leatherman, J. L., Kozlowski, D. J. and
Weinberg, E. S. (2000). Maternally controlled
(beta)-catenin-mediated signaling is required for organizer formation in the
zebrafish. Development
127,3899
-3911.[Abstract]
Kelly, G. M., Greenstein, P., Erezyilmaz, D. F. and Moon, R.
T. (1995). Zebrafish wnt8 and wnt8b share a common activity
but are involved in distinct developmental pathways.
Development 121,1787
-1799.[Abstract]
Kessler, D. S. (1997). Siamois is required for
formation of Spemann's organizer. Proc. Natl. Acad. Sci.
USA 94,13017
-13022.
Kimmel, C. B. and Law, R. D. (1985). Cell
lineage of zebrafish blastomeres. III. Clonal analyses of the blastula and
gastrula stages. Dev. Biol.
108,94
-101.[CrossRef][Medline]
Kimmel, C. B. and Warga, R. M. (1987).
Indeterminate cell lineage of the zebrafish embryo. Dev.
Biol. 124,269
-280.[CrossRef][Medline]
Kimmel, C. B., Warga, R. M. and Schilling, T. F.
(1990). Origin and organization of the zebrafish fate map.
Development 108,581
-594.
Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. and
Schilling, T. F. (1995). Stages of embryonic development of
the zebrafish. Dev. Dyn.
203,253
-310.[Medline]
Koos, D. S. and Ho, R. K. (1999). The
nieuwkoid/dharma homeobox gene is essential for bmp2b repression in the
zebrafish pregastrula. Dev. Biol.
215,190
-207.[CrossRef][Medline]
Krauss, S., Concordet, J. P. and Ingham, P. W.
(1993). A functionally conserved homolog of the Drosophila
segment polarity gene hh is expressed in tissues with polarizing activity in
zebrafish embryos. Cell
75,1431
-1444.[CrossRef][Medline]
Latinkic, B. V. and Smith, J. C. (1999).
Goosecoid and mix.1 repress Brachyury expression and are required for head
formation in Xenopus. Development
126,1769
-1779.[Abstract]
Lekven, A. C., Thorpe, C. J., Waxman, J. S. and Moon, R. T.
(2001). Zebrafish wnt8 encodes two wnt8 proteins on a bicistronic
transcript and is required for mesoderm and neurectoderm patterning.
Dev. Cell 1,103
-114.[CrossRef][Medline]
Leung, T., Bischof, J., Soll, I., Niessing, D., Zhang, D., Ma,
J., Jackle, H. and Driever, W. (2003). bozozok directly
represses bmp2b transcription and mediates the earliest dorsoventral asymmetry
of bmp2b expression in zebrafish. Development
130,3639
-3649.
Little, S. C. and Mullins, M. C. (2006).
Extracellular modulation of BMP activity in patterning the dorsoventral axis.
Birth Defects Res. C Embryo Today
78,224
-242.[CrossRef][Medline]
Mailhos, C., Andre, S., Mollereau, B., Goriely, A.,
Hemmati-Brivanlou, A. and Desplan, C. (1998). Drosophila
Goosecoid requires a conserved heptapeptide for repression of paired-class
homeoprotein activators. Development
125,937
-947.[Abstract]
Martinez-Barbera, J. P., Toresson, H., Da Rocha, S. and Krauss,
S. (1997). Cloning and expression of three members of the
zebrafish Bmp family: Bmp2a, Bmp2b and Bmp4. Gene
198, 53-59.[CrossRef][Medline]
Miller-Bertoglio, V. E., Fisher, S., Sanchez, A., Mullins, M. C.
and Halpern, M. E. (1997). Differential regulation of chordin
expression domains in mutant zebrafish. Dev. Biol.
192,537
-550.[CrossRef][Medline]
Mullins, M. C., Hammerschmidt, M., Kane, D. A., Odenthal, J.,
Brand, M., van Eeden, F. J., Furutani-Seiki, M., Granato, M., Haffter, P.,
Heisenberg, C. P. et al. (1996). Genes establishing
dorsoventral pattern formation in the zebrafish embryo: the ventral specifying
genes. Development 123,81
-93.[Abstract]
Nasevicius, A. and Ekker, S. C. (2000).
Effective targeted gene `knockdown' in zebrafish. Nat.
Genet. 26,216
-220.[CrossRef][Medline]
Niehrs, C. (2004). Regionally specific
induction by the Spemann-Mangold organizer. Nat. Rev.
Genet. 5,425
-434.[Medline]
Niehrs, C., Keller, R., Cho, K. W. and De Robertis, E. M.
(1993). The homeobox gene goosecoid controls cell migration in
Xenopus embryos. Cell
72,491
-503.[CrossRef][Medline]
Nojima, H., Shimizu, T., Kim, C. H., Yabe, T., Bae, Y. K.,
Muraoka, O., Hirata, T., Chitnis, A., Hirano, T. and Hibi, M.
(2004). Genetic evidence for involvement of maternally derived
Wnt canonical signaling in dorsal determination in zebrafish. Mech.
Dev. 121,371
-386.[CrossRef][Medline]
Oelgeschläger, M., Kuroda, H., Reversade, B. and De
Robertis, E. M. (2003). Chordin is required for the Spemann
organizer transplantation phenomenon in Xenopus embryos. Dev.
Cell 4,219
-230.[CrossRef][Medline]
Oppenheimer, J. M. (1934). Experiments on early
developing stages of Fundulus. Proc. Natl. Acad. Sci.
USA 20,536
-538.
Piccolo, S., Sasai, Y., Lu, B. and De Robertis, E. M.
(1996). Dorso-ventral patterning in Xenopus: inhibition
of ventral signals by direct binding of Chordin to BMP-4.
Cell 86,589
-598.[CrossRef][Medline]
Ramel, M. C. and Lekven, A. C. (2004).
Repression of the vertebrate organizer by Wnt8 is mediated by Vent and Vox.
Development 131,3991
-4000.
Ramel, M. C., Buckles, G. R., Baker, K. D. and Lekven, A. C.
(2005). WNT8 and BMP2B co-regulate non-axial mesoderm patterning
during zebrafish gastrulation. Dev. Biol.
287,237
-248.[Medline]
Rivera-Perez, J. A., Mallo, M., Gendron-Maguire, M., Gridley, T.
and Behringer, R. R. (1995). Goosecoid is not an essential
component of the mouse gastrula organizer but is required for craniofacial and
rib development. Development
121,3005
-3012.[Abstract]
Rupp, R. A., Snider, L. and Weintraub, H.
(1994). Xenopus embryos regulate the nuclear localization of
XMyoD. Genes Dev. 8,1311
-1323.
Sampath, K., Rubinstein, A. L., Cheng, A. M., Liang, J. O.,
Fekany, K., Solnica-Krezel, L., Korzh, V., Halpern, M. E. and Wright, C.
V. (1998). Induction of the zebrafish ventral brain and
floorplate requires cyclops/nodal signalling. Nature
395,185
-189.[CrossRef][Medline]
Sander, V., Reversade, B. and De Robertis, E.
(2007). The opposing homeobox genes Goosecoid and
Vent1/2 self-regulate Xenopus patterning. EMBO
J. 26,2955
-2965.[CrossRef][Medline]
Sasai, Y., Lu, B., Steinbeisser, H., Geissert, D., Gont, L. K.
and De Robertis, E. M. (1994). Xenopus chordin: a novel
dorsalizing factor activated by organizer-specific homeobox genes.
Cell 79,779
-790.[CrossRef][Medline]
Saúde, L., Woolley, K., Martin, P., Driever, W. and
Stemple, D. L. (2000). Axis-inducing activities and cell
fates of the zebrafish organizer. Development
127,3407
-3417.[Abstract]
Schier, A. F. and Talbot, W. S. (2005).
Molecular genetics of axis formation in zebrafish. Annu. Rev.
Genet. 39,561
-613.[CrossRef][Medline]
Schier, A. F., Neuhauss, S. C., Helde, K. A., Talbot, W. S. and
Driever, W. (1997). The one-eyed pinhead gene functions in
mesoderm and endoderm formation in zebrafish and interacts with no tail.
Development 124,327
-342.[Abstract]
Schulte-Merker, S., Ho, R. K., Herrmann, B. G. and
Nusslein-Volhard, C. (1992). The protein product of the
zebrafish homologue of the mouse T gene is expressed in nuclei of the germ
ring and the notochord of the early embryo.
Development 116,1021
-1032.[Abstract]
Schulte-Merker, S., Hammerschmidt, M., Beuchle, D., Cho, K. W.,
De Robertis, E. M. and Nusslein-Volhard, C. (1994).
Expression of zebrafish goosecoid and no tail gene products in wild-type and
mutant no tail embryos. Development
120,843
-852.[Abstract]
Schulte-Merker, S., Lee, K. J., McMahon, A. P. and
Hammerschmidt, M. (1997). The zebrafish organizer requires
chordino. Nature 387,862
-863.[CrossRef][Medline]
Seiliez, I., Thisse, B. and Thisse, C. (2006).
FoxA3 and goosecoid promote anterior neural fate through inhibition of Wnt8a
activity before the onset of gastrulation. Dev. Biol.
290,152
-163.[CrossRef][Medline]
Shih, J. and Fraser, S. E. (1995). Distribution
of tissue progenitors within the shield region of the zebrafish gastrula.
Development 121,2755
-2765.[Abstract]
Shih, J. and Fraser, S. E. (1996).
Characterizing the zebrafish organizer: microsurgical analysis at the
early-shield stage. Development
122,1313
-1322.[Abstract]
Smith, S. T. and Jaynes, J. B. (1996). A
conserved region of engrailed, shared among all en-, gsc-, Nk1-, Nk2- and
msh-class homeoproteins, mediates active transcriptional repression in
vivo. Development 122,3141
-3150.[Abstract]
Spemann, H. and Mangold, H. (1924). Uber
induction von embryonanlagen durch implantation artfremder organis atoren.
Roux's Arch. Dev. Biol.
100,599
-638.
Stachel, S. E., Grunwald, D. J. and Myers, P. Z.
(1993). Lithium perturbation and goosecoid expression identify a
dorsal specification pathway in the pregastrula zebrafish.
Development 117,1261
-1274.[Abstract]
Steinbeisser, H., Fainsod, A., Niehrs, C., Sasai, Y. and De
Robertis, E. M. (1995). The role of gsc and BMP-4 in
dorsal-ventral patterning of the marginal zone in Xenopus: a loss-of-function
study using antisense RNA. EMBO J.
14,5230
-5243.[Medline]
Talbot, W. S., Trevarrow, B., Halpern, M. E., Melby, A. E.,
Farr, G., Postlethwait, J. H., Jowett, T., Kimmel, C. B. and Kimelman, D.
(1995). A homeobox gene essential for zebrafish notochord
development. Nature 378,150
-157.[CrossRef][Medline]
Tucker, J. A., Mintzer, K. A. and Mullins, M. C.
(2008). The BMP signaling gradient patterns dorsoventral tissues
in a temporally progressive manner along the anterioposterior axis.
Dev. Cell 14,108
-119.[CrossRef][Medline]
Waddington, C. H. (1932). Experiments on the
development of the chick and the duck embryo cultivated in vitro.Philos. Trans. R. Soc. London Ser. B
211,179
-230.
Yamada, G., Mansouri, A., Torres, M., Stuart, E. T., Blum, M.,
Schultz, M., De Robertis, E. M. and Gruss, P. (1995).
Targeted mutation of the murine goosecoid gene results in craniofacial defects
and neonatal death. Development
121,2917
-2922.[Abstract]
Yamanaka, Y., Mizuno, T., Sasai, Y., Kishi, M., Takeda, H., Kim,
C. H., Hibi, M. and Hirano, T. (1998). A novel homeobox gene,
dharma, can induce the organizer in a non-cell-autonomous manner.
Genes Dev. 12,2345
-2353.
Yao, J. and Kessler, D. S. (2001). Goosecoid
promotes head organizer activity by direct repression of Xwnt8 in Spemann's
organizer. Development
128,2975
-2987.
Yasuo, H. and Lemaire, P. (2001). Role of
Goosecoid, Xnot and Wnt antagonists in the maintenance of the notochord
genetic programme in Xenopus gastrulae. Development
128,3783
-3793.
Zhu, L., Belo, J. A., De Robertis, E. M. and Stern, C. D.
(1999). Goosecoid regulates the neural inducing strength of the
mouse node. Dev. Biol.
216,276
-281.[CrossRef][Medline]
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