Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
* Author for correspondence (e-mail: marc{at}hms.harvard.edu)
Accepted 22 November 2002
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SUMMARY |
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Key words: Sizzled, Wnt signaling, Ventral blood islands, sFRP, Embryonic patterning, Xenopus
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INTRODUCTION |
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Although the dominance of the dorsal signals is obvious (transplanting ventral cells to the dorsal side does not produce an ectopic ventral axis), it does not follow that all of the remaining tissue merely responds to levels of signals dictated by the organizer. It is possible that patterning in the ventral and lateral domains is only weakly dependent on the dorsal signals, although still subservient to them. The ventral region may rely on self-organizing processes to regulate behavior and fate of the cells at a distance from the organizer. When Wnt and BMP inhibitors diffusing from the dorsal side fall below a certain threshold, these ventral organizing activities would be activated and much of the pattern would be generated by them, rather than by the concentration of the inhibitors. Most phenomenological experiments, such as those of Spemann and Mangold do not distinguish between these possibilities.
There is much complexity to control on the ventral side, including the
allocation of cells to the lateral plate mesoderm, muscle, pronephros and
blood (Davidson and Zon, 2000;
Hemmati-Brivanlou and Thomsen,
1995
), and there is some evidence for processes of ventral
organization being more complex than simply reading out a gradient of BMP and
Wnt signals created by inhibitors secreted from the organizer
(Munoz-Sanjuan and Hemmati-Brivanlou,
2001
). For example, not all mesoderm is converted to blood in
UV-irradiated embryos. Analogously, in embryos ventralized by dorsal injection
of BMP4 or DNxTCF3, it is possible to distinguish a defined domain of globin
expression in the absence of the organizer
(Kumano and Smith, 2000
). The
BMP and Wnt pathways themselves interact and are capable of generating
considerable complexity (Hoppler and Moon,
1998
; Marom et al.,
1999
). Part of the patterns of ventrolateral mesoderm undoubtedly
originates from reciprocal modulation of these two signals
(Dale and Jones, 1999
;
De Robertis et al., 2001
). But
are different ventrolateral fates determined simply by the distance from the
dorsal organizer, or are there self-organizing activities that generate
semi-independent patterns? If so, what role do they play in regulating cell
differentiation and cell behavior on the ventral side?
Exactly 180° opposite the organizer at early gastrulation is a
restricted expression domain for the gene sizzled (szl),
encoding a secreted Frizzled-related protein similar to Wnt inhibitors
expressed in the dorsal mesendoderm (Salic
et al., 1997). Secreted Frizzled-related proteins (sFRP) contain a
conserved cysteine rich domain similar to the ligand-binding domain of the Wnt
receptors (Frizzleds), and can inhibit signaling by directly competing for
secreted Wnt proteins (Wang et al.,
1997
; Wodarz and Nusse,
1998
; Xu et al.,
1998
). Szl is the only known sFRP expressed ventrally
during and after gastrulation, eventually overlapping with the ventral blood
islands at tailbud stages (Bradley et al.,
2000
; Salic et al.,
1997
). In embryos where the balance of mesoderm is shifted from
ventral to dorsal by early exposure to Li+, expression of Szl is
abolished. Reciprocally, when embryos are ventralized by UV irradiation of the
egg cortex, Szl expression extends around the entire marginal zone
(Salic et al., 1997
).
Additional studies have shown that Szl RNA is induced by BMP4 in a
dose-dependent manner in animal caps, whereas it is downregulated in embryos
injected with a truncated BMP receptor
(Marom et al., 1999
).
Therefore, it is likely that Szl expression depends on BMP4 signaling, either
directly or indirectly. Based on its behavior and its structural features, we
had previously proposed that Szl generates a domain in the extreme
ventral side with high BMP and reduced Wnt signals
(Salic et al., 1997
).
In the present work, we have attempted to define the biological role of Sizzled by examining the effects of morpholino-mediated suppression of Szl translation, and by studying the effects of Szl on Wnt8 activity. The results suggest that Szl is not an inhibitor of Wnt8, and that compared with other sFRPs its activity may be unique. The data further show that Szl is required for proper development of ventral posterior mesoderm, and in particular of the ventral blood islands (VBI). Specifically, Szl knockdown expands ventral posterior mesoderm and the VBI, whereas its overexpression restricts the ventral mesoderm and the VBI. Therefore, Szl appears to function in a negative feedback loop regulating allocation of cells to the most ventral fate. These observations suggest the existence of some limited self-organizing properties of the extreme ventral mesoderm, and reveal unexpected complexity within the ventral marginal zone.
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MATERIALS AND METHODS |
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Design and characterization of morpholino oligos and a rescue Szl
cDNA
The morpholino oligo used to knockdown Szl expression, MOSZL
(5'-GAGGAGCAGGAAGACTCCGGTCATG-3'), was purchased form Gene-Tools
LLC. It spans nucleotides -1 to +24 with respect to the ATG of Xenopus
szl, and anneals to each paralog of the gene with a single mismatch. Two
different morpholino oligos were used as negative controls: MOC, which is the
standard control provided by Gene-Tools; and MOC2
(5'-GCAGACCTTGTTTAATGAACTCAAC-3'), which was designed within the
coding region of szl. MOC2 anneals perfectly to each szl
paralog but, being downstream of the ATG, has no effect on translation. As
morpholino oligos diffuse very rapidly in Xenopus blastomeres
(Nutt et al., 2001), we always
injected MOSZL in both blastomeres at the two-cell stage for even distribution
to the entire embryo. The construct pCS2-wSzl-MT used in rescue experiments
contains appropriate conservative mutations so that the resulting mRNA has
nine mismatches to MOSZL. Efficacy of MOSZL was tested in vivo according to
the following protocol: 10 ng of MOSZL were injected in each blastomere of
two-cell embryos; subsequently, 200 pg of an expression plasmid encoding
Myc-tagged versions of either Szl (pCS2-Szl-MT) or wSzl (pCS2-wSzl-MT) were
injected in the marginal zone of the same embryos at the four-cell stage.
Expressed proteins were detected at different developmental stages by
immunoblotting. Under these conditions, MOSZL efficiently blocked szl
translation up to 3 days after injection (stage 30/35), while expression from
the rescue construct pCS2-wSzl-MT was only minimally affected (data not
shown).
Benzidine and o-dianisidine staining for blood cells
Both methods stain erythrocytes by forming a precipitate upon oxidation by
the heme group of hemoglobin in the presence of hydrogen peroxide. Benzidine
staining was performed as described
(Hemmati-Brivanlou and Thomsen,
1995); reactions were monitored closely, and embryos were promptly
photographed before significant damage by corrosion. o-dianisidine staining
was performed as described previously
(Huber et al., 1998
).
Reactions were terminated by transferring the embryos in ethanol.
In situ hybridization
Whole-mount in situ hybridization was performed as previously described
(Harland, 1991;
Salic et al., 1997
) using
digoxigenin- or fluorescein-labeled antisense RNA probes. Details on the
constructs and probes used in the present work are available from the authors.
For double in situ hybridization, the two probes were detected successively;
the first probe was usually detected using 5-bromo-4-chloro-3-indolyl
phosphate 4-toluidine salt (BCIP) and, after inactivating the alkaline
phosphatase, the second probe was detected using Magenta Phos. Embryos were
imaged on a Zeiss StemI stereoscope equipped with a Sony 3-chip color CCD
camera controlled by OpenLab 3.0 software (Improvision).
Plasmids
The coding regions of Xenopus Frzb1, Crescent and Dkk1 were PCR
amplified from corresponding plasmids using high-fidelity Pfx Polymerase
(Invitrogen/Life Technology) and cloned in pCS2-MT so that 6xMyc epitope tags
were added at the C terminus. pCS2-DNxWnt8 has been described previously
(Hoppler et al., 1996), as
well as pCS2-DNxWnt11 (Tada and Smith,
2000
).
RT-PCR
Blood induction in animal caps was performed as described
(Huber et al., 1998;
Mead et al., 1998
). Animal
caps were dissected at stage 8-9, and incubated in 0.5xMMR until sibling
embryos reached stage 30-35. When indicated, 50 ng/ml of recombinant human
bFGF (Life Technology/Invitrogen) were added to the incubation medium. Total
RNA was extracted using the RNAeasy procedure with an additional DNAseI step
to remove contaminant genomic DNA (Qiagen). Radioactive semi-quantitative
RT-PCR was performed on random primed cDNA using previously described primers
for xGata1,
T3 globin (Kelley et
al., 1994
) and EF1
.
(Agius et al., 2000
). The
primers used to amplify endogenous szl in embryos injected with wSz1
were EndoSzl-U 5'-CATGTCCGGAGTCTTCCTGC-3' and EndoSzl-L
5'-GGATGAACGTGTCCAGGCAG-3'.
Immunoblotting and luciferase assays
For immunoblotting, embryos were crushed by pipetting in low salt buffer
(25 mM HEPES pH 7.6, 50 mM NaCl, 0.2% Tween-20, 2mM PMSF, protease
inhibitors). Lysates were cleared by centrifugation and 2x Laemmli
sample buffer was added to the supernatant. A volume corresponding to one
quarter of an embryo was separated by SDS-PAGE and transferred to
nitrocellulose membranes (Schleicher & Schuell). Myc-tagged proteins were
detected using the 9E10 monoclonal antibody (Santa Cruz). Actin was detected
using a polyclonal antibody (Sigma). For luciferase assays, embryos were
crushed by pipetting in Luciferase Lysis Buffer (20 mM Tris pH 7.5, 125 mM
NaCl, 1 mM MgCl2, 1% Triton-X100, 1 mM DTT, protease inhibitors) and lysates
were cleared by centrifugation. A volume of the supernatants corresponding to
one half embryo was added directly to Luciferase Assay Reagent (Promega) and
light emission was measured in a Turner Design luminometer.
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RESULTS |
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As shown in Fig. 1A, injection of increasing amounts of MOSZL produce increasingly severe morphological defects. The affected embryos develop normally through gastrulation, with the first defects being visible after the neurula stage, when they appear enlarged ventrally around the remnant of the blastopore. At tailbud stage, Szl knockdown embryos have a significant expansion of the ventral posterior tissue; in fact, high doses of MOSZL induce a dramatic enlargement of the caudal region, a shortened tailbud and often a bent axis. These embryos develop into tadpoles with an excess of posterior ventral tissue, very often with edema (not shown). Embryos injected with similar or greater amounts of two different control morpholino oligos (MOC and MOC2) are indistinguishable from uninjected siblings (Table 1). The morphological effects of szl knockdown are opposite to those of Szl injection, which results in a dramatic reduction of the ventral posterior tissue. This can be readily appreciated in Fig. 1A, where Szl overexpressing embryos are directly compared with Szl knockdown siblings. Anterior structures are moderately affected; Szl knockdowns have smaller heads, whereas Szl-injected embryos have larger heads. Yet, heads in both cases are fully formed and appear normal in all of their features. Normal, beating hearts develop both in Szl knockdown and overexpressing tadpoles.
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To understand better the basis of these phenotypes, we followed the development of sibling embryos injected with Szl RNA or with the antisense morpholino oligo by time lapse video microscopy. As shown in Fig. 1B, embryos are indistinguishable up to the neurula stage. Subsequently, when embryos lengthen along the anteroposterior axis, those with reduced levels of Szl protein retain most of the ventral tissue in the posterior region. At the same time, embryos overexpressing Szl accumulate ventral tissue in the anterior region, assuming a characteristic shape with large heads and reduced posterior ventral structures. Although superficially one could refer to these as dorsalized or ventralized phenotypes, they appear to involve a redistribution of cells from posterior ventral in embryos with low Szl levels, to anterior ventral in embryos with high Szl levels.
Szl affects ventral extension during the neurula to tailbud
transition
Characteristic morphogenetic movements occur in the ventral mesoderm and
ectoderm during the transition from neurula to tailbud, when the shape of the
embryo changes from spherical to elongated. Evidence that these movements are
autonomous and not passive has come from studies of isolated ventral explants,
which lengthen as much as the ventral sides of intact embryos. Ventral
elongation is primarily due to morphogenetic rearrangements of mesodermal and
ectodermal cells (Larkin and Danilchik,
1999). We asked whether the morphological aberrations of
szl knockdown or overexpressing embryos derived from defects in
dorsal or ventral elongation. Embryos were injected with MOSZL or with Szl
RNA, and dorsal and ventral explants were dissected at neurula stage. As shown
in Fig. 2, ventral explants
from uninjected and Szl-injected embryos elongated to similar lengths, while
ventral explants from MOSZL-injected embryos did not lengthen at all. There
was a subtler anomaly in the elongation of ventral explants from
Szl-overexpressing embryos, where the bulk of ventral tissue remained
anterior. As there were no significant differences in lengthening of the
corresponding dorsal explants (not shown), these results suggest that the
morphological features of affected embryos arise primarily from altered
morphogenetic processes of the ventral tissue.
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Szl negatively regulates development of the ventral blood
islands
In Xenopus, the primitive site of embryonic hematopoiesis is in
the ventral blood islands (VBI), which are located along the ventral midline
of tailbud embryos. Blood is therefore a marker of the most ventral mesoderm,
independent of its pre-gastrula origins
(Davidson and Zon, 2000;
Kumano et al., 1999
;
Lane and Sheets, 2002
). As
shown in Fig. 3A, embryos
injected with increasing amounts of MOSZL developed abnormally large VBIs.
Correspondingly, embryos injected with increasing amounts of Szl RNA had
severely reduced or absent VBI (see also
Table 1). To examine specific
stages of the blood differentiation pathway, we analyzed expression of xSCL
and xGata2 (not shown) as markers of hematopoietic precursors, and xGata1 and
T3 globin as markers of the committed erythroid lineage
(Davidson and Zon, 2000
;
Mead et al., 2001
;
Mead et al., 1998
;
Zon et al., 1991
). Using in
situ hybridization, we found that all of these markers were clearly expanded
in embryos injected with the Szl morpholino, when compared with control
embryos (Fig. 3B). In some
instances, we analyzed the same markers in sibling embryos injected with Szl
RNA, and we found that levels of xSCL, xGata1 (not shown) and
T3 globin
were all significantly reduced (Fig.
3B, parts c,d). Notably, Szl restricted the VBI when injected at
levels that have only a mild effect on the overall morphology of the embryo.
Taken together these results indicate that Szl inhibits development of the
ventral blood islands; expansion of the VBI in Szl knockdown embryos indicates
that this inhibitory activity is actually required for proper formation of
this ventral derivative.
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Szl does not directly affect hematopoietic differentiation
To understand whether Szl might affect blood differentiation per se, we
took advantage of a well-established experimental system whereby embryonic
hematopoiesis is recapitulated in animal pole explants, and expression of
blood markers is monitored by RT-PCR
(Huber et al., 1998). Large
numbers of blood cells are formed in animal caps after treatment with basic
fibroblast growth factor (bFGF), which induces mesoderm, while injection of a
moderate amount of BMP4 specifies such mesoderm to ventral fate
(Huber et al., 1998
;
Mead et al., 2001
).
Alternatively, blood differentiation can be induced in animal caps by
injection of high levels of BMP4 RNA even in the absence of bFGF treatment
(Xu et al., 1999
). Finally, it
has been shown that bFGF treatment alone can induce some blood differentiation
in animal caps, albeit producing few erythrocytes
(Miyanaga et al., 1999
). As
shown in Fig. 4A, injection of
Szl RNA had no detectable effect on xGata1 and
T3 globin expression in
animal caps treated with any of the methods described above. Given that Szl
transcription is induced by BMP4 in animal caps
(Marom et al., 1999
), we also
injected MOSZL in these experiments. As shown in
Fig. 4A, preventing Szl
translation had no detectable effect on BMP4-induced erythropoiesis. However,
in agreement with the in situ experiments, Szl depletion or overexpression had
dramatic effects on the same blood markers in the context of the whole embryo
(Fig. 4B). Within the
limitations of the experimental system, these results suggest that Szl does
not directly interfere with the blood differentiation program.
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Szl protein levels regulate the expansion of ventral mesoderm at post
gastrula stages
The effects of Szl protein levels on the hematopoietic lineage prompted us
to look at its effects on a variety of genes expressed at gastrula stage.
First, we analyzed Brachyury (Xbra), a T-box transcription factor expressed in
the prospective mesoderm during gastrulation
(Smith et al., 1991). As shown
in Fig. 5A, both the ring of
Xbra expression and the rim of cells not expressing Xbra just above the
blastopore lip (leading edge mesoderm) appeared normal in morpholino injected
embryos. As a marker of dorsal mesoderm, and particularly of the Spemann
organizer, we analyzed the secreted BMP inhibitor Chordin
(Sasai et al., 1994
); at the
same time we analyzed Sox 17ß, a transcription factor involved in
specification of the endoderm (Hudson et
al., 1997
). As markers of ventral mesoderm, we analyzed the
transcription factors Vent2/Xom (Rastegar
et al., 1999
) and Xpo (Sato
and Sargent, 1991
). As shown in
Fig. 5A, there was no
difference in the expression of any of these markers in MOSZL injected or
uninjected gastrulae.
|
We also analyzed expression of xMyoD, a lateral mesoderm gene which was
shown to be downregulated upon Szl overexpression
(Salic et al., 1997), and we
analyzed szl itself, the most restricted marker for the most ventral
sector of the prospective mesoderm at gastrula stage. As shown in
Fig. 5A (parts i,j), Szl
expression was increased dramatically in MOSZL-injected gastrulae, while xMyoD
was not significantly affected. At later stages, Szl RNA was even more
dramatically expanded both laterally and anteriorly on the ventral side in
embryos treated with the Szl morpholino
(Fig. 5A, parts k,l), while
xMyoD was correspondingly reduced in its lateral extension at the posterior
end of the embryo. szl expression was also dramatically increased at
stage 20 (Fig. 5A, parts m,n)
in the knockdown experiments, similar to what observed for xSCL
(Fig. 3B, part a).
Based on this analysis, szl itself is an early marker of the extreme ventral mesoderm that is expanded when Szl protein levels are reduced by morpholino knockdown. If this is true, szl RNA should be in turn downregulated by Szl overexpression. To test this hypothesis, we used a modified version of Szl designed for rescue experiments (wSzl, see below). We injected wSzl RNA in the marginal zone of four-cell embryos, and monitored expression of endogenous szl at gastrula stage by RT-PCR using primers that do not recognize wSzl. As shown in Fig. 5B, injection of wSzl inhibited expression of endogenous szl in a dose-dependent manner. The same primer pair was also used to detect endogenous Szl in embryos injected with MOSZL and, as expected, translational knockdown of Szl protein increased expression of Szl mRNA. Both repression and accumulation of endogenous szl are more dramatic at stage 12 than at stage 10.5.
In situ hybridization analysis of two markers expressed in lateral mesoderm
after gastrulation further supported the notion that decreased Szl protein
levels result in expansion of the most ventral mesoderm. For example, when we
analyzed expression of Xlim-1 (Taira et
al., 1994) in MOSZL-injected embryos, we found the two
characteristic lateral stripes demarcating the future pronephros to be weaker
and closer to the dorsal midline in the posterior part of the embryo
(Fig. 5C, part a). Similarly,
when we analyzed Flk-1/VEGF-R2, a marker of vascular endothelial precursors
(Cleaver et al., 1997
), we
noticed that the vitelline veins that run parallel along the ventral side of
tailbud embryos are more dorsal in Szl knockdowns. This means that more
ventral mesoderm separates the vitelline veins, while less lateral mesoderm
divides the vitelline veins from the cardinal veins located at the base of the
somites (Fig. 5C, part
c,d).
Ventral injection of exogenous Szl fully rescues the phenotype of Szl
knockdown embryos
To prove that the observed phenotype is specifically due to knockdown of
Szl translation, we asked whether MOSZL-injected embryos could be rescued by
exogenous Szl expression. As MOSZL anneals to the coding region, we built a
rescue construct in which the corresponding nucleotides were mutated
preserving the amino acid sequence. This construct was dubbed wSzl, as
`wobble' bases of codons were changed (see Materials and Methods). In rescue
experiments, MOSZL was injected in both blastomeres at the two-cell stage, and
wSzl RNA was injected in the ventral marginal zone (VMZ) of the same embryos
when they reached the four-cell stage (Fig.
6). The same amount of wSzl RNA was also injected in the VMZ of
control embryos. Under these conditions, wSzl rescued both the morphological
abnormalities and expansion of the VBI in Szl knockdown embryos
(Fig. 6, part d). Notably,
injection of MOSZL reciprocally rescued the phenotype induced by wSzl
overexpression. In fact, control embryos injected with the same amount of wSzl
RNA as MOSZL-injected embryos displayed the expected Szl phenotype
(Fig. 6, part c;
Table 2).
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Increased Wnt expression does not recapitulate the effects of Szl
knockdown
Given that Szl has the structural features of a secreted Wnt inhibitor, we
asked whether overexpression of Wnts could produce an effect similar to
injection of the antisense morpholino or whether inhibition of Wnt signaling
could phenocopy Szl overexpression. We analyzed xWnt8 and xWnt11, because both
are transcribed in domains overlapping with szl at gastrulation.
Expression plasmids encoding either xWnt8 or xWnt11 were injected into the
future ventral marginal zone of four-cell stage embryos, and larval globin RNA
was detected by in situ hybridization at tailbud stage. As shown in
Fig. 7, the VBI were not
expanded upon zygotic overexpression of xWnt8 or xWnt11. Specifically, xWnt8
induced severe head truncations and dose-dependent reduction of the VBI, both
consistent with previously published observations
(Hoppler and Moon, 1998;
Kumano et al., 1999
).
Injection of xWnt11 also caused reduction of the VBI, along with
dose-dependent gastrulation defects (Fig.
7). At the same time, injection of RNAs encoding dominant negative
Wnt8 (Hoppler et al., 1996
) or
Wnt11 (Tada and Smith, 2000
)
failed to reproduce the effects of Szl overexpression. In fact, DNxWnt8
induced a dorsalized phenotype with smaller but clearly detectable VBIs, while
injection of DNxWnt11 induced characteristic morphogenetic defects
(Tada and Smith, 2000
), but no
suppression of the VBI.
|
Other sFRPs cannot phenocopy Szl activity: evidence that Szl function
does not involve inhibition of the canonical Wnt pathway
Given the complexity of the Wnt signaling pathway and the many effects that
Wnts have on cell differentiation and behavior, overexpression of xWnt8 and
xWnt11, or their dominant-negative versions, may induce pleiotropic effects
that are unrevealing. We therefore asked whether other sFRPs could
recapitulate the activity of Szl in the ventral marginal zone. We injected
Crescent, which is closely related to Szl (44% identity)
(Pera and De Robertis, 2000;
Shibata et al., 2000
) and
Frzb1, which is perhaps the best characterized sFRP
(Leyns et al., 1997
;
Wang et al., 1997
). We also
injected Dikkopf1 (xDkk1), which is not a sFRP but is a potent and specific
inhibitor of the canonical Wnt pathway
(Bafico et al., 2001
;
Glinka et al., 1998
). To
verify that proteins were expressed at comparable levels, we used
epitope-tagged versions that could be detected by immunoblotting.
In experiments analogous to those described in Fig. 6, none of the tested Wnt inhibitors could rescue the szl knockdown. Morphologically, the injected embryos often displayed a complex phenotype, with defects induced by the sFRPs superimposed on those induced by MOSZL (not shown). More easily measurable, expansion of the VBI was not rescued by injection of any of these genes (Table 3).
|
Next, we asked whether these Wnt inhibitors could inhibit formation of the
VBI. Increasing amounts of wSzl-MT, Frzb-MT, xCrescent-MT and xDkk1-MT were
injected in the marginal zone of four-cell stage embryos, and expression of
blood markers was analyzed by RT-PCR at stage 30/35. Some embryos were
collected at gastrula stage to verify the levels of injected proteins by
immunoblotting (Fig. 8B). As
shown in Fig. 8A, only Szl
repressed blood markers under these conditions. Embryos not used for RNA
extraction were fixed and stained for T3 globin by in situ
hybridization to evaluate morphological phenotypes; again, only Szl-injected
embryos showed efficient loss of the VBI
(Fig. 8C). Interestingly,
embryos dorsalized by Frzb appeared morphologically similar to embryos
dorsalized by Szl; however, analysis of erythroid markers uncovered a critical
difference in their ability to affect development of the VBI.
|
To verify that Myc-tagged Wnt inhibitors were biologically active, we used
a Luciferase reporter controlled by a TCF/ß-catenin-responsive promoter
(Korinek et al., 1997). We
coinjected the reporter construct with xWnt8 and different amounts of wSzl-MT,
Frzb-MT, Crescent-MT or Dkk1-MT into the animal pole of two-cell stage
embryos, and assayed luciferase activity at early gastrula (stage 10+ to
10.5). RNA dilutions were calibrated to express comparable levels of proteins.
Under these conditions, Szl had no detectable effect on the canonical Wnt
pathway and did not inhibit luciferase activation even when injected at an RNA
concentration 100-fold greater than xWnt8 (400 pg,
Fig. 8D). By contrast, xDkk1
and the other sFRPs efficiently suppressed luciferase activation when injected
at much lower doses (4 pg and 40 pg respectively). The minimal effective
concentration of Szl RNA that completely inhibited formation of the VBI in
whole embryos was 80 pg. Therefore, levels of Szl that were much higher than
needed to completely suppress the blood islands had no effect on the canonical
Wnt pathway.
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DISCUSSION |
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We found that Szl plays a crucial and surprisingly specific role in
coordinating development of specific ventral posterior structures, in
particular of the ventral blood islands (VBI). Given its similarity to
secreted Wnt inhibitors expressed in the Spemann organizer, we had previously
proposed that Szl protects the extreme ventral marginal zone from Wnt signals
inducing lateral mesoderm, so that this region could assume a ventral fate.
Antagonism toward xWnt8 satisfactorily explained the paradoxical evidence that
Szl overexpression actually dorsalizes the embryo
(Salic et al., 1997). Based on
this model, we had predicted that inhibiting Szl expression would reduce
ventral-most derivatives. Instead, we observed that Szl knockdown expanded
ventral-posterior tissue and the VBI. Such a complete disparity between
prediction and experiment clearly demonstrates the crucial importance of
combining loss-of-function with overexpression approaches to understand gene
function.
The morphological effects of Szl
Time lapse recordings and dissection of ventral explants indicate that Szl
levels affect lengthening of the ventral side of the embryo between neurula
and tailbud stage. It is possible that Szl directly or indirectly regulates
cell behavior, but it is also possible that these defects arise from altered
competence of the ventral cells to undergo extension in response to other
signals. In the intact embryo, lengthening reflects both the capacity of the
tissue to elongate and the number of cells in the elongating population. There
was ample evidence that Szl affects the recruitment of cells into a specific
population, as we observed a larger region of ventral mesoderm expressing Szl
RNA and early blood markers when Szl translation was inhibited. However, it is
possible that cell behavior and cell recruitment reinforce each other, with
cell movements required to place cells in a spatial context where they can be
properly exposed to inductive signals. Such a linkage between morphogenesis
and cell differentiation is observed in the dorsal mesoderm
(Sive et al., 1989;
Winklbauer and Keller, 1996
).
It is not easy to distinguish regulation of cell behavior from changes in cell
identity, as the two are tightly correlated; but if we accept Szl itself as a
marker of ventralmost mesoderm, the fact that its expression is affected long
before the ventral cell movements during the neurula to tailbud transition
would support a patterning phenomenon.
Ventral mesoderm patterning or differentiation of the VBI?
Careful lineage tracing studies have shown that the marginal zone at
gastrula stage is divided in two domains along the animal-vegetal axis: the
animal marginal zone gives rise mostly to muscle, while the vegetal marginal
zone (leading edge mesoderm, or LEM) gives rise to blood
(Keller, 1991;
Kumano et al., 1999
;
Lane and Sheets, 2002
). The
two domains are distinguished by differential expression of Xbra, which is
excluded from the LEM. Patterning of the marginal zone along the
animal-vegetal axis derives from interaction of nodal and FGF signals;
inhibition of FGF signaling eliminates Xbra expression and expands
hematopoietic mesoderm (Kumano and Smith,
2000
; Kumano and Smith,
2002
). Using in situ hybridization, we observed normal Xbra
expression at gastrulation in embryos injected with the Szl morpholino,
suggesting that early FGF signaling and the primary animal-vegetal
regionalization of the marginal zone are unaffected. Yet, at later stages we
observed that the domains of ventral markers such as szl and xSCL are
expanded, while lateral markers such as xMyoD and Xlim-1 are shifted dorsally
in the posterior region of the embryo. These data are consistent with Szl
playing a role in patterning the LEM during and after gastrulation.
The VBI are affected by variations in Szl protein levels that induce only
mild morphological defects. Although this might suggest that the two
phenotypes (formation of the VBI and allocation of ventral-posterior tissue)
are not correlated, it seems unlikely that Szl plays a direct role in
differentiation of embryonic blood. In fact, the earliest hematopoietic
markers are expanded in embryos treated with the Szl morpholino and, most
importantly, Szl knockdown or overexpression had no effect on BMP4-induced
erythropoiesis in animal caps (Fig.
4). Although animal cap experiments would not detect activity
upstream of BMP4, the current understanding of embryonic hematopoiesis defines
BMP4 as the earliest signal in the blood differentiation pathway
(Davidson and Zon, 2000).
Therefore, we think that the VBI respond to small variations in Szl levels
because they are the most ventral structure of the post-gastrula embryo.
What is the biochemical activity of Szl?
In an attempt to identify the molecular basis of Szl activity, we started
with the reasonable speculation that it affects some extracellular component
of the Wnt signaling pathway. Two Wnts are detected in ventral mesoderm at
gastrulation: xWnt8, which is expressed in a broad ventral sector overlapping
the domain of Szl expression but more extended in the dorsolateral direction;
and xWnt11, which is expressed in a ring around the blastopore, overlapping
with Szl only in the ventral sector. There are contradictory data on the
activity of Szl as an inhibitor of Wnt8: Salic et al. showed that Szl could
inhibit induction of Siamois by xWnt8 in animal caps
(Salic et al., 1997), whereas
Bradley et al. reported that it could not
(Bradley et al., 2000
).
Additionally, Szl does not inhibit axis duplication induced by ventral
injection of xWnt8 RNA, but zygotic overexpression of xWnt8 could rescue some
aspects of the dorsalization induced by Szl
(Salic et al., 1997
). One
possible explanation is that experiments using high RNA doses might have
exposed nonspecific activities. In the experiments reported here, we injected
only the reasonably small amounts of szl RNA (160 pg at most)
necessary to maximally support the morphological and differentiation effects
on the ventral side. When we compared the activity of szl and other
secreted Wnt inhibitors in a Luciferase assay in vivo
(Fig. 8C), we found that Szl
could not block activation of the canonical pathway by xWnt8. Accordingly,
injection of dominant negative Wnt8 or other Wnt8 inhibitors could not mimic
szl overexpression. Less direct evidence also suggests that
szl does not inhibit xWnt11. Specifically, overexpression of xWnt11
did not reproduce the effects of Szl knockdown, and injection of dominant
negative Wnt11 failed to reproduce the effects of Szl overexpression
(Fig. 7). In addition, Szl
could not inhibit axis duplication induced by co-injection of xWnt11 with
Frizzled-5 (L. C., unpublished) (He et
al., 1997
). It remains possible that Szl inhibits a novel,
unidentified Wnt molecule expressed in ventral mesoderm. Indeed, the existence
of an unknown ventralizing Wnt has been hypothesized
(Itoh and Sokol, 1999
). But we
also need to consider the possibility that Szl does not act as a Wnt
antagonist at all.
One confounding question concerning the rather limited number of signaling
pathways active in early development is the specificity of effects in
different target tissues. The situation is particularly acute for the Wnt
signaling pathway. In fact, few Wnts are expressed during the early stages of
embryonic development, yet they convey a wide spectrum of inductive signals
affecting differentiation, proliferation, morphogenesis and cell polarity
(Pandur et al., 2002;
Wodarz and Nusse, 1998
). Part
of this versatility might be accounted for by the selective activation of
different intracellular pathways (Kuhl,
2002
; Pandur et al.,
2002
), and an intriguing possibility is that secreted
Frizzled-related proteins, including Szl, may function as modulators rather
than simple antagonists of Wnt signaling. Specific intracellular pathways
could then be activated by specific combinations of Wnts, Frizzleds and sFRPs.
Although highly speculative, this hypothesis finds some support in the
evidence that different sFRPs induce different effects when overexpressed
(Bradley et al., 2000
;
Pera and De Robertis, 2000
).
Accordingly, none of the sFRPs we tested could mimic Szl in modulating the
fate of ventralmost mesoderm. It is worth noting that some inhibitors of the
canonical Wnt pathway (Frzb, Dkk1 and DNxWnt8, but not Crescent) produced
phenotypes morphologically similar to those induced by szl, but
failed to inhibit formation of the VBI. The simplest explanation is that these
proteins have different affinities for different Wnts; unfortunately,
biochemical characterization of these interactions is greatly impaired by the
difficulty in obtaining soluble, biologically active Wnts
(Cadigan and Nusse, 1997
;
Wodarz and Nusse, 1998
).
Szl as a negative feedback regulator of ventral mesoderm; unexpected
complexity of ventral signaling
The findings described in this paper suggest that szl acts in a
negative feedback circuit that limits recruitment of cells to extreme ventral
fate. Mechanistically, it is possible that Szl inhibits a graded ventralizing
signal, setting a threshold so that ventralmost mesoderm forms only in a
restricted region where the signal is strong enough to overcome such
inhibition. Alternatively, Szl may act at a longer range than the signal that
induces it, setting up a `remote inhibition' system similar to the Spitz-Argos
feedback loop in Drosophila eye development
(Freeman, 2000). Szl could
diffuse from its source, preventing cells at a distance from the ventralizing
signal from differentiating into ventralmost mesoderm, while cells expressing
Szl would be unaffected because they are already committed. Another
possibility is that Szl provides a completely independent specification signal
that, by interacting at different levels with ventralizing pathways,
eventually results in inhibition of extreme ventral fate. In any case, the
molecular nature of the ventralizing signal(s) antagonized by Szl remains to
be established.
In conclusion, knockdown experiments have uncovered an unexpected and complex function of Szl, raising important biochemical questions of specificity and action of this and other secreted Frizzled-related proteins. Our data suggest the existence of a self-regulatory circuit controlling the allocation of tissue in the ventral mesoderm, with direct consequences for both blood formation and morphogenesis. The ventral organizing activity involving Szl clearly differs from the Spemann organizer in the locality of its effects. However, it is likely that local organization with feedback control will be manifest in many regions of the embryo, raising interesting questions about how specificity is achieved with a limited number of signaling components.
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ACKNOWLEDGMENTS |
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REFERENCES |
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---|
Agius, E., Oelgeschlager, M., Wessely, O., Kemp, C. and de
Robertis, E. M. (2000). Endodermal Nodal-related signals and
mesoderm induction in Xenopus. Development
127,1173
-1183.
Bafico, A., Liu, G., Yaniv, A., Gazit, A. and Aaronson, S. A. (2001). Novel mechanism of Wnt signalling inhibition mediated by Dickkopf-1 interaction with LRP6/Arrow. Nat. Cell Biol. 3,683 -686.[CrossRef][Medline]
Bradley, L., Sun, B., Collins-Racie, L., La Vallie, E., McCoy, J. and Sive, H. (2000). Different activities of the frizzled-related proteins frzb2 and sizzled2 during Xenopus anteroposterior patterning. Dev. Biol. 227,118 -132.[CrossRef][Medline]
Cadigan, K. M. and Nusse, R. (1997). Wnt
signaling: a common theme in animal development. Genes
Dev. 11,3286
-3305.
Cleaver, O., Tonissen, K. F., Saha, M. S. and Krieg, P. A. (1997). Neovascularization of the Xenopus embryo. Dev. Dyn. 210,66 -77.[CrossRef][Medline]
Dale, L. and Jones, C. M. (1999). BMP signalling in early Xenopus development. BioEssays 21,751 -760.[CrossRef][Medline]
Davidson, A. J. and Zon, L. I. (2000). Turning mesoderm into blood: the formation of hematopoietic stem cells during embryogenesis. Curr. Top. Dev. Biol. 50, 45-60.[Medline]
De Robertis, E. M., Wessely, O., Oelgeschlager, M., Brizuela, B., Pera, E., Larrain, J., Abreu, J. and Bachiller, D. (2001). Molecular mechanisms of cell-cell signaling by the Spemann-Mangold organizer. Int. J. Dev. Biol. 45,189 -197.[Medline]
Freeman, M. (2000). Feedback control of intercellular signalling in development. Nature 408,313 -319.[CrossRef][Medline]
Glinka, A., Wu, W., Delius, H., Monaghan, A. P., Blumenstock, C. and Niehrs, C. (1998). Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction. Nature 391,357 -362.[CrossRef][Medline]
Graf, J. D. and Kobel, H. R. (1991). Genetics of Xenopus laevis. Methods Cell Biol 36, 19-34.[Medline]
Harland, R. and Gerhart, J. (1997). Formation and function of Spemann's organizer. Annu. Rev. Cell Dev. Biol. 13,611 -667.[CrossRef][Medline]
Harland, R. M. (1991). In situ hybridization: an improved whole-mount method for Xenopus embryos. Methods Cell Biol. 36,685 -695.[Medline]
He, X., Saint-Jeannet, J. P., Wang, Y., Nathans, J., Dawid, I.
and Varmus, H. (1997). A member of the Frizzled protein
family mediating axis induction by Wnt-5A. Science
275,1652
-1654.
Heasman, J. (2002). Morpholino oligos: making sense of antisense? Dev. Biol. 243,209 -214.[CrossRef][Medline]
Hemmati-Brivanlou, A. and Thomsen, G. H. (1995). Ventral mesodermal patterning in Xenopus embryos: expression patterns and activities of BMP-2 and BMP-4. Dev. Genet. 17,78 -89.[Medline]
Hoppler, S. and Moon, R. T. (1998). BMP-2/-4 and Wnt-8 cooperatively pattern the Xenopus mesoderm. Mech. Dev. 71,119 -129.[CrossRef][Medline]
Hoppler, S., Brown, J. D. and Moon, R. T. (1996). Expression of a dominant-negative Wnt blocks induction of MyoD in Xenopus embryos. Genes Dev. 10,2805 -2817.[Abstract]
Huber, T. L., Zhou, Y., Mead, P. E. and Zon, L. I.
(1998). Cooperative effects of growth factors involved in the
induction of hematopoietic mesoderm. Blood
92,4128
-4137.
Hudson, C., Clements, D., Friday, R. V., Stott, D. and Woodland, H. R. (1997). Xsox17alpha and beta mediate endoderm formation in Xenopus. Cell 91,397 -405.[Medline]
Itoh, K. and Sokol, S. Y. (1999). Axis
determination by inhibition of Wnt signaling in Xenopus. Genes
Dev. 13,2328
-2336.
Keller, R. (1991). Early embryonic development of Xenopus laevis. Methods Cell Biol. 36, 61-113.[Medline]
Kelley, C., Yee, K., Harland, R. and Zon, L. I. (1994). Ventral expression of GATA-1 and GATA-2 in the Xenopus embryo defines induction of hematopoietic mesoderm. Dev. Biol. 165,193 -205.[CrossRef][Medline]
Korinek, V., Barker, N., Morin, P. J., van Wichen, D., de Weger,
R., Kinzler, K. W., Vogelstein, B. and Clevers, H. (1997).
Constitutive transcriptional activation by a beta-catenin-Tcf complex in
APC-/- colon carcinoma. Science
275,1784
-1787.
Kuhl, M. (2002). Non-canonical Wnt signaling in Xenopus: regulation of axis formation and gastrulation. Semin. Cell Dev. Biol. 13,243 -249.[CrossRef][Medline]
Kumano, G., Belluzzi, L. and Smith, W. C.
(1999). Spatial and temporal properties of ventral blood island
induction in Xenopus laevis. Development
126,5327
-5337.
Kumano, G. and Smith, W. C. (2000). FGF signaling restricts the primary blood islands to ventral mesoderm. Dev. Biol. 228,304 -314.[CrossRef][Medline]
Kumano, G. and Smith, W. C. (2002). The nodal target gene Xmenf is a component of an FGF-independent pathway of ventral mesoderm induction in Xenopus. Mech. Dev. 118, 45.[CrossRef][Medline]
Lane, M. C. and Sheets, M. D. (2002). Primitive and definitive blood share a common origin in Xenopus: a comparison of lineage techniques used to construct fate maps. Dev. Biol. 248, 52-67.[CrossRef][Medline]
Larkin, K. and Danilchik, M. V. (1999). Ventral cell rearrangements contribute to anterior-posterior axis lengthening between neurula and tailbud stages in Xenopus laevis. Dev. Biol. 216,550 -560.[CrossRef][Medline]
Leyns, L., Bouwmeester, T., Kim, S. H., Piccolo, S. and de Robertis, E. M. (1997). Frzb-1 is a secreted antagonist of Wnt signaling expressed in the Spemann organizer. Cell 88,747 -756.[Medline]
Marom, K., Fainsod, A. and Steinbeisser, H. (1999). Patterning of the mesoderm involves several threshold responses to BMP-4 and Xwnt-8. Mech. Dev. 87, 33-44.[CrossRef][Medline]
Mead, P. E., Deconinck, A. E., Huber, T. L., Orkin, S. H. and
Zon, L. I. (2001). Primitive erythropoiesis in the Xenopus
embryo: the synergistic role of LMO-2, SCL and GATA-binding proteins.
Development 128,2301
-2308.
Mead, P. E., Kelley, C. M., Hahn, P. S., Piedad, O. and Zon, L.
I. (1998). SCL specifies hematopoietic mesoderm in Xenopus
embryos. Development
125,2611
-2620.
Miyanaga, Y., Shiurba, R. and Asashima, M. (1999). Blood cell induction in Xenopus animal cap explants: effects of fibroblast growth factor, bone morphogenetic proteins, and activin. Dev. Genes Evol. 209,69 -76.[CrossRef][Medline]
Munoz-Sanjuan, I. and Hemmati-Brivanlou, A. (2001). Early posterior/ventral fate specification in the vertebrate embryo. Dev. Biol. 237, 1-17.[CrossRef][Medline]
Nieuwkoop, P. D. and Faber, J. (1967).Normal Table of Xenopus laevis (Daudin) . Amsterdam: North-Holland Publishing Company.
Nutt, S. L., Bronchain, O. J., Hartley, K. O. and Amaya, E. (2001). Comparison of morpholino based translational inhibition during the development of Xenopus laevis and Xenopus tropicalis. Genesis 30,110 -113.[CrossRef][Medline]
Pandur, P., Maurus, D. and Kuhl, M. (2002). Increasingly complex: new players enter the Wnt signaling network. BioEssays 24,881 -884.[CrossRef][Medline]
Pera, E. M. and de Robertis, E. M. (2000). A direct screen for secreted proteins in Xenopus embryos identifies distinct activities for the Wnt antagonists Crescent and Frzb-1. Mech. Dev. 96,183 -195.[CrossRef][Medline]
Rastegar, S., Friedle, H., Frommer, G. and Knochel, W. (1999). Transcriptional regulation of Xvent homeobox genes. Mech. Dev. 81,139 -149.[CrossRef][Medline]
Salic, A. N., Kroll, K. L., Evans, L. M. and Kirschner, M.
W. (1997). Sizzled: a secreted Xwnt8 antagonist expressed in
the ventral marginal zone of Xenopus embryos.
Development 124,4739
-4748.
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.[Medline]
Sato, S. M. and Sargent, T. D. (1991). Localized and inducible expression of Xenopus-posterior (Xpo), a novel gene active in early frog embryos, encoding a protein with a `CCHC' finger domain. Development 112,747 -753.[Abstract]
Shibata, M., Ono, H., Hikasa, H., Shinga, J. and Taira, M. (2000). Xenopus crescent encoding a Frizzled-like domain is expressed in the Spemann organizer and pronephros. Mech. Dev. 96,243 -246.[CrossRef][Medline]
Sive, H. L., Grainger, R. M. and Harland, R. M. (2000). Early development of Xenopus laevis. A Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
Sive, H. L., Hattori, K. and Weintraub, H. (1989). Progressive determination during formation of the anteroposterior axis in Xenopus laevis. Cell 58,171 -180.[Medline]
Smith, J. C., Price, B. M., Green, J. B., Weigel, D. and Herrmann, B. G. (1991). Expression of a Xenopus homolog of Brachyury (T) is an immediate-early response to mesoderm induction. Cell 67,79 -87.[Medline]
Spemann, H. and Mangold, H. (1924). Uber induktion von embryonalanlagen durch implantation artfremder organisatoren. Roux's Arch. Entw. Mech. 100,599 -638.
Tada, M. and Smith, J. C. (2000). Xwnt11 is a
target of Xenopus Brachyury: regulation of gastrulation movements via
Dishevelled, but not through the canonical Wnt pathway.
Development 127,2227
-2238.
Taira, M., Otani, H., Jamrich, M. and Dawid, I. B.
(1994). Expression of the LIM class homeobox gene Xlim-1 in
pronephros and CNS cell lineages of Xenopus embryos is affected by retinoic
acid and exogastrulation. Development
120,1525
-1536.
Wang, S., Krinks, M., Lin, K., Luyten, F. P. and Moos, M., Jr (1997). Frzb, a secreted protein expressed in the Spemann organizer, binds and inhibits Wnt-8. Cell 88,757 -766.[Medline]
Winklbauer, R. and Keller, R. E. (1996). Fibronectin, mesoderm migration, and gastrulation in Xenopus. Dev. Biol. 177,413 -426.[CrossRef][Medline]
Wodarz, A. and Nusse, R. (1998). Mechanisms of Wnt signaling in development. Annu. Rev. Cell Dev. Biol. 14,59 -88.[CrossRef][Medline]
Wolpert, L. (1989). Positional information revisited. Development Suppl.3 -12.
Xu, Q., D'Amore, P. A. and Sokol, S. Y. (1998).
Functional and biochemical interactions of Wnts with FrzA, a secreted Wnt
antagonist. Development
125,4767
-4776.
Xu, R. H., Ault, K. T., Kim, J., Park, M. J., Hwang, Y. S., Peng, Y., Sredni, D. and Kung, H. (1999). Opposite effects of FGF and BMP-4 on embryonic blood formation: roles of PV.1 and GATA-2. Dev. Biol. 208,352 -361.[CrossRef][Medline]
Zon, L. I., Mather, C., Burgess, S., Bolce, M. E., Harland, R. M. and Orkin, S. H. (1991). Expression of GATA-binding proteins during embryonic development in Xenopus laevis. Proc. Natl. Acad. Sci. USA 88,10642 -10646.[Abstract]