1 Department of Molecular Oncology, Graduate School of Medicine, Osaka
University, Suita, Osaka 565-0871, Japan
2 Laboratory for Vertebrate Axis Formation, Center for Developmental Biology,
RIKEN, Kobe, Hyogo 650-0047, Japan
3 Department of Biological Sciences, Graduate School of Science, The University
of Tokyo, Tokyo 113-0033, Japan
4 Department of Frontier Biosciences, Graduate School of Frontier Biosciences,
Osaka University, Suita, Osaka 565-0871, Japan
* Author for correspondence (e-mail: hibi{at}cdb.riken.go.jp)
Accepted 20 March 2003
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SUMMARY |
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Key words: Zebrafish, Ogon, Sizzled, Bmp, Chordin, Dorsoventral patterning, Feedback inhibitor
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INTRODUCTION |
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Roles for Bmp signaling in DV axis formation are also supported by analyses
of zebrafish mutants that display abnormalities in DV patterning. The
dorsalized mutants swirl (swr) and snailhouse
(snh) have defective bmp2b and bmp7 genes,
respectively (Dick et al.,
2000; Kishimoto et al.,
1997
; Schmid et al.,
2000
). somitabun, captain hook and piggy tail
all have defects in the zebrafish gene for Smad5 (madh5
Zebrafish Information Network) (Hild et
al., 1999
; Kramer et al.,
2002
), which functions as a signal transducer for Bmp signaling.
lost-a-fin encodes the gene for the type I Bmp receptor Alk8
(Bauer et al., 2001
;
Mintzer et al., 2001
), and
mini fin encodes Tolloid (Connors
et al., 1999
). All of these data indicate that Bmp2 and Bmp7 and
their signaling play essential roles in the formation of ventral tissues. In
contrast to the dorsalized mutants, there are only two mutants, namely
dino and ogon, that have been reported to display clearly
ventralized phenotypes without other abnormalities in the early specification
of the dorsal organizer (such as those seen in the bozozok mutants)
(Hammerschmidt et al., 1996a
;
Solnica-Krezel et al., 1996
).
These ventralized mutant embryos display an expansion of ventral tissues, such
as the ventral tail fin, posterior somatic mesoderm, blood and pronephron, and
a reduction, to various degrees, of the anterior somites and the
neuroectoderm. The dino (din) locus encodes the zebrafish
ortholog of Chordin (Chordino; Chd Zebrafish Information Network),
whereas the molecular identity of the ogon locus has not yet been
elucidated.
Complementation and mapping analyses revealed that
ogonm60, mercedestm305 and short
tailb180 are allelic, and thus commonly referred to as
ogon (ogo)
(Miller-Bertoglio et al.,
1999). The ogom60 and
ogob180 mutations are deficiencies in the distal part
(close to the telomere) of linkage group 25 (LG25), indicating that the
ogo locus is localized to the deleted region. The
N-ethyl-N-nitrosourea (ENU)-induced allele ogotm305
displays viable phenotypes, which are less severe than those of the
ogom60 and ogob180 mutants, suggesting
that ogotm305 is a hypomorphic allele or that
ogom60 and ogob180 harbor the loss of
additional gene(s) in the deletion. The ogom60 mutant
embryo displays neural degeneration in addition to the ventralized phenotypes.
The ventralized phenotypes of ogo are similar to those of
chordino mutants, except that the reduction of the anterior
neuroectoderm is less severe in the ogo than in the din
mutants. It has also been reported that a maternally derived ogo gene
contributes to dorsoventral patterning
(Miller-Bertoglio et al.,
1999
). The ventralized phenotypes of ogo are fully
suppressed by the overexpression of the Bmp antagonists Chordin and Noggin, or
the expression of a dominant-negative type II Bmp receptor
(Miller-Bertoglio et al.,
1999
). Epistatic analyses revealed that swr/bmp2b and
snh/bmp7 are epistatic to ogo in DV patterning
(Miller-Bertoglio et al.,
1999
; Wagner and Mullins,
2002
). In ventral tail fin formation, lost-a-fin/alk8, is
epistatic to ogo (Wagner and
Mullins, 2002
). All of these data consistently indicate that
ogo encodes a dorsalizing factor that inhibits Bmp signaling either
directly or indirectly. In contrast to chordino, ogo does not show an
epistatic relationship with mini fin/tolloid
(Wagner and Mullins, 2002
),
suggesting that ogo functions differently from chordin. It
has been reported that elimination of both the zygotic ogo and
chordin genes additively ventralizes the embryo, implying distinct
requirements for these genes in DV axis formation
(Hammerschmidt et al., 1996a
;
Miller-Bertoglio et al.,
1999
). The molecular identification of ogo is required to
elucidate the precise relationship between ogo and chordin,
and the molecular mechanisms by which ogo regulates the formation of
the DV axis.
In this study, we isolated the gene responsible for the ogo
mutants by a positional cloning strategy and found that the ogo locus
encodes a zebrafish homolog of Secreted Frizzled (Sizzled). The
sizzled (szl) gene was originally identified in
Xenopus based on its ability to dorsalize the Xenopus embryo
(Salic et al., 1997). Szl
displays sequence similarity with a Wnt receptor Frizzled and is reported to
function as an inhibitor of Xenopus Wnt8 (XWnt8)
(Salic et al., 1997
). However,
szl and a szl-related gene sizzled2 reportedly do
not inhibit the activity of XWnt8, suggesting they have a different mode of
action in DV axis formation (Bradley et
al., 2000
; Collavin and
Kirschner, 2003
). We found that Ogo/Szl functions to inhibit Bmp
signaling in a manner that does not involve the inhibition of Wnt8-mediated
signaling. Ogo/Szl requires Chordin protein for its dorsalizing activity. In
contrast to other dorsalizing factors, ogo/szl is expressed on the
ventral side and requires Bmp signaling. Our results suggest that Ogo/Szl
functions as a negative-feedback regulator of Bmp signaling and provide a
novel mechanism by which the DV axis is established during gastrulation.
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MATERIALS AND METHODS |
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Positional cloning of ogon
ogonrk1 heterozygous fish were mated with wild-type
India fish to generate F1 families. Homozygous ogonrk1
mutant embryos were raised from the F1 cross and selected by morphological
criteria (expanded ventral tissue). We used samples of their genomic DNA to
carry out segregation analyses. We first examined the SSLP markers z1378,
z8380, z23415 and z14408, which are located on the distal region of linkage
group (LG) 25. This region is reported to be deleted in
ogonm60 and ogonb180
(Miller-Bertoglio et al.,
1999). We found that the SSLP marker z8380 was close to the
ogon locus (three recombinations out of 2998 meiotic segregations).
Using the PCR primers for z8380 as a probe, we obtained BAC and PAC lines: BAC
185L03, BAC 31 and PAC 203B16. We isolated PAC 35K5, PAC 251M11 and BAC 173L18
using the end sequence of PAC 203B16, and we isolated PAC 259J12 using the end
sequence of PAC 35K5. We isolated the fragments from the AB and India genomes,
which correspond to the end of the PAC and BAC clones, and found polymorphic
markers, SSLPs and STSs (sequence-tagged sites, detected by restriction
fragment length polymorphisms and PCR). The precise information on the markers
is available on request. ogo/szl cDNA was isolated by hybridizing a
lambda Ziplox zebrafish gastrula cDNA library with the inserts of PAC 203B16,
PAC 35K3 and a cosmid generated from PAC 203B16. A genomic fragment and cDNA
fragment of ogon/sizzled were sequenced by performing shot-gun
sequencing and reading the PCR products.
RNA and morpholino oligonucleotide injection
Capped RNAs for Noggin1, Dickkopf1, Chordin, Tlc (a constitutively active
type I Bmp receptor), Xenopus Frzb-1, Xenopus Sizzled and
Xenopus Crescent were generated as described previously
(Furthauer et al., 1999;
Hashimoto et al., 2000
;
Houart et al., 2002
;
Miller-Bertoglio et al., 1997
;
Nikaido et al., 1999
;
Pera and De Robertis, 2000
;
Salic et al., 1997
). The
coding region of zebrafish sizzled/ogon cDNA in pZL1 (from the lambda
Ziplox clone) was excised and inserted into a modified pCS2+ (pCS2+SN). The
sizzledtm305 cDNA was isolated by PCR from the
ogontm305 homozygous mutant embryos and inserted into
pCS2+SN. The sizzled cDNA containing four mispaired nucleotides
without amino acid changes was created by PCR and inserted into pCS2+SN.
pCS2+SN sizzled and sizzledtm305 were digested
with AscI, and the capped RNA was transcribed with SP6 RNA
polymerase. The antisense morpholino oligonucleotides used in this study were
szl MO (5'-ACAGCAGCAGA-CTGAATAGAGACAT-3') and control MO
with four mispaired bases (5'-ACAGgAG-CAcACTGAtTAGAcACAT-3'). The
chordin MO has previously been published
(Nasevicius and Ekker,
2000
).
Transcript detection
Whole-mount in situ hybridization was performed using BM purple (Roche) as
the alkaline phosphatase substrate. The detection of six3.2, bmp2b,
chordin, goosecoid, fused somites/tbx24 and eve1 was as
described previously (Joly et al.,
1993; Kobayashi et al.,
1998
; Miller-Bertoglio et al.,
1997
; Nikaido et al.,
2002
; Oxtoby and Jowett,
1993
; Stachel et al.,
1993
). The SalI-BamHI fragment of
sizzled was subcloned into pZL1. The sizzled RNA probe was
generated by digestion of pZL1-5' sizzled and transcription with SP6 RNA
polymerase. Photographs were taken using an AxioPlan2 microscope and AxioCam
(Zeiss).
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RESULTS |
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DISCUSSION |
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The phenotypes of the ogom60 and
ogob180 mutant embryos are more severe than the phenotype
of the ogotm305 mutant embryos
(Miller-Bertoglio et al.,
1999). ogom60 and ogob180
are deficiencies in the chromosome, and ogotm305 is
suggested to be a hypomorphic allele. However, misexpression of large amounts
of the szl gene from ogotm305 did not dorsalize
the embryo (Fig. 3E),
suggesting that ogotm305 is a functionally null allele.
The loss of Szl protein following the injection of the szl MO led to
ventralized phenotypes similar to those of the ogom60 and
ogob180 mutants (Fig.
2), indicating that the loss of function of the single gene
szl is solely responsible for the ventralized phenotypes of
ogo. The embryonic lethality of ogom60 and
ogob180 is probably due to the deletion of additional
gene(s) in LG25.
A contribution from maternally derived ogo has been reported
(Miller-Bertoglio et al.,
1999). Consistent with this, we detected maternally deposited
ogo/szl transcripts by RT-PCR (data not shown), and embryos receiving
injections of large amounts of szl MO display phenotypes similar to
those of maternal-zygotic (MZ) ogo mutant embryos
(Miller-Bertoglio et al.,
1999
) (Fig. 2).
However, as discussed below, the dorsalizing activity of ogo/szl
requires the presence of chordin, which is expressed after the
mid-blastula transition (Miller-Bertoglio
et al., 1997
). Maternally provided Ogo/Szl should support the
function of the zygotic Ogo/Szl in dorsalization.
ogon/sizzled expression depends on Bmp signaling
Expression of ogo/szl was detected in the ventral blastoderm from
the late blastula through the gastrula stages
(Fig. 5). Ventral expression of
ogo/szl at the gastrula stages strongly depended on Bmp signaling
(Fig. 6). Zygotic Bmp
signal-dependent expression has been reported for bmp2b/swr and
bmp7/snh (Dick et al.,
2000; Kishimoto et al.,
1997
; Schmid et al.,
2000
). The ventral expression of bmp2b and bmp7
decreases after the mid-gastrula stage in the swr and snh
mutant embryos, suggesting that the ventral expression of these genes depends
only on Bmp signaling after the mid-gastrula stage. Similarly, the ventral
ogo/szl expression was not affected at the late blastula stage in the
swr and din mutant embryos (data not shown). These data
indicate that ventral ogo/szl expression after the mid-gastrula stage
depends on zygotic Bmp signaling. However, the ogo/szl expression was
restricted to the ventral side from the time of its initiation at the late
blastula stage. It has recently been shown that maternally provided
smad5 is involved in the early specification of ventral tissue at the
late blastula stage (Kramer et al.,
2002
). Ventral ogo/szl at the late blastula and early
gastrula stages might be regulated by the maternally derived Bmp signal.
Alternatively, the early ventral expression of ogo/szl might be
regulated by the interaction between the dorsal-specific homeobox gene
bozozok and ventrally expressed homeobox genes vox
(previously vega1), vent (previously vega2) and
ved, which play a role in early DV specification before the zygotic
Bmp signaling occurs (Imai et al.,
2001
; Kawahara et al.,
2000a
; Kawahara et al.,
2000b
; Shimizu et al.,
2002
).
Compared with the expression of bmp2b, bmp4 and bmp7,
ogo/szl expression was restricted to the more ventral blastoderm
(Fig. 5). Similarly, in
Xenopus embryos, the expression of szl and szl2 is
confined to the ventral-most part of the ventral blastoderm
(Bradley et al., 2000;
Salic et al., 1997
). The
injection of increasing amounts of bmp4 RNA and the injection of
chordin MOs in Xenopus embryos revealed that a high level of
Bmp signaling is required for the expression of szl
(Marom et al., 1999
;
Oelgeschlager et al., 2003
).
Thus, the ventral expression of sizzled is regulated by a mechanism
that is conserved between zebrafish and Xenopus, and a high level of
Bmp signaling activity, which exists in the ventralmost part of the
blastoderm, is required for the expression of sizzled on the ventral
side. Promoter analyses of ogo/szl will clarify this issue.
Bmp antagonist versus Wnt antagonist
Ogo/Szl has sequence similarity with the Wnt receptor Frizzled, suggesting
a role for Ogo/Szl in Wnt inhibition. However, the overexpression of Ogo/Szl
did not inhibit the Wnt8-dependent ectopic expression of bozozok
(Fig. 4). Misexpression of the
Wnt8 inhibitor Dkk1 and the sFrp Crescent anteriorized the neuroectoderm, but
did not dorsalize the embryo efficiently, whereas misexpression of Ogo/Szl
dorsalized the embryo but did not anteriorize the neuroectoderm, unlike the
Bmp inhibitor Noggin 1 (Fig.
4). These data suggest that Ogo/Szl functions as a Bmp inhibitor
rather than as a Wnt inhibitor.
We found that overexpression of Crescent and Dkk1 suppressed the
ventralized phenotypes of szl MO-injected embryos
(Fig. 4). However, Crescent and
Dkk1 also elicited expansion of the dorsal organizer
(Fig. 4)
(Hashimoto et al., 2000),
which produces the Bmp inhibitors Chordin and Noggin 1. Thus, Crescent and
Dkk1 might substitute for the function of Ogo/Szl indirectly by expanding the
expression domain of the organizer-derived Bmp inhibitors. It remains possible
that Crescent and Dkk1 (less likely) might have a dorsalizing activity other
than expansion of the dorsal organizer, just as Ogo/Szl does. However, Ogo/Szl
did not inhibit the Wnt8 activity; therefore, the dorsalizing activity of
Ogo/Szl should not depend on Wnt8 inhibition.
In zebrafish, the loss of wnt8 or of tcf3/headless, which
functions to inhibit Wnt8 signaling, strikingly affects the AP patterning in
the neuroectoderm in addition to causing abnormalities in the DV patterning
(Erter et al., 2001;
Kim et al., 2000
;
Lekven et al., 2001
). The
phenotypes of the ogo mutant and the ogo/szl-overexpressing
embryos were different from those of embryos with high (e.g. headless
mutant embryos) and low (wnt8 morphant embryos) Wnt8 activities,
respectively, further supporting the idea that Ogon does not function to
inhibit Wnt8 signaling. In addition to wnt8, there are several Wnt
genes reported to be expressed at the blastula and gastrula stages in
zebrafish. Among them, wnt5/pipetail and wnt11/silberblick,
which activate a non-canonical Wnt signal, are known to be involved in
convergent-extension movements during gastrulation
(Heisenberg et al., 2000
;
Rauch et al., 1997
), and thus
it is unlikely that the dorsalizing activity of Ogo/Szl is due to the
inhibition of Wnt5 and Wnt11. Consistent with this, misexpression of
ogo/szl did not affect the wnt5- or wnt11-mediated
inhibition of convergent extension (data not shown). Similarly, it was
reported that Xenopus szl2 does not inhibit the activities of
Xenopus Wnt3a, Wnt5a and Wnt8
(Bradley et al., 2000
). All of
these data indicate that Ogon/Szl and Xenopus Szls promote
dorsalization through interactions with factors other than the Wnts.
How does Ogon/Sizzled inhibit Bmp signaling?
Ogo/Sizzled requires the Chordin protein to dorsalize embryos
(Fig. 7), indicating that
Ogo/Szl displays a mode of action that is completely different from that of
other Bmp antagonists. This finding is consistent with a previous report that
misexpression of Xenopus sizzled cannot rescue UV-treated ventralized
embryos, which should not express chordin
(Salic et al., 1997).
How does Ogo/Szl inhibit Bmp signaling in a Chordin-dependent manner? Our
data and the data previously published imply the mode of function of Ogo/Szl:
(1) din and ogo mutant embryos have similar ventralized
phenotypes (Hammerschmidt et al.,
1996b; Miller-Bertoglio et
al., 1999
; Solnica-Krezel et
al., 1996
); (2) the ventralized phenotypes of ogo can be
suppressed by the expression of Chordin, Noggin and a dominant-negative Bmp
receptor (Miller-Bertoglio et al.,
1999
), but the din phenotypes cannot be suppressed by
misexpression of ogo/szl (Fig.
7); (3) overexpression of ogo/szl did not inhibit Wnt8
activity (Fig. 4); (4)
overexpression of ogo/szl induced a similar phenotype to that of
noggin1 but not that of dkk1 or crescent
(Fig. 4); (5) low levels of
chordin could act synergistically with ogo/szl in
dorsalization (Fig. 7); (6) a
mutation in ogo did not enhance the ventralized phenotypes of
din embryos (Fig. 8);
and (7) loss of tolloid/mini fin can suppress the ogon tail
phenotype (Wagner and Mullins,
2002
). All of these results indicate that Ogo/Szl can augment the
activity of Chordin, by inhibiting an inhibitor of Chordin, by directly making
Chordin more active, or by modulating the Bmp signal so that it becomes more
susceptible to the Chordin-mediated inhibition. The dorsalizing activity of
the Chordin protein is regulated by different mechanisms: the chordin protein
level is regulated through processing by Tolloid-related metalloproteinases,
and Chordin interacts physically and functionally with Bmp and Twisted
Gastrulation (Tsg) to modulate Bmp activity
(De Robertis et al., 2000
).
Tolloid-related proteins and Tsg might be involved in the function of Ogo/Szl.
Alternatively, Ogo/Szl may function in parallel with Chordin. Both Ogo/Szl and
Chordin are required for the formation of posterior dorsal tissues, and the
loss of either Ogo/Szl or Chordin might lead to ventralization. In this
scenario, the lowering of the Bmp signal by Chordin might work cooperatively
with Ogo/Szl to dorsalize the embryo.
A mutation in the cysteine-rich domain (CRD) of the Ogo/Szl in
ogotm305 implies an essential role for the CRD in the
activity of Ogo/Szl. As the CRD of Frizzled is known to interact with Wingless
and Wnts (Bhanot et al., 1996),
it is still possible that Ogo/Szl functions by inhibiting unidentified Wnt(s).
The identification of proteins that interact with Ogo/Szl will shed light on
the mechanisms by which Ogo/Szl inhibits Bmp signaling and regulates the
specification of the DV axis.
Ogon/Sizzled functions as a negative-feedback regulator of Bmp
signaling
Many feedback inhibitors play roles in the early patterning of vertebrate
embryogenesis (Freeman, 2000).
The Antivin/Lefties (Lefy1 and Lefy2) function as feedback inhibitors for the
Nodal-related molecules in zebrafish, Xenopus and mice
(Bisgrove et al., 1999
;
Cheng et al., 2000
;
Meno et al., 1999
;
Thisse and Thisse, 1999
).
Sprouty4 and Sef function in FGF signaling
(Furthauer et al., 2002
;
Furthauer et al., 2001
;
Tsang et al., 2002
). An
inhibitory Smad, Smad7 and Bambi/Nma function as feedback inhibitors of Bmp
signaling (Grotewold et al.,
2001
; Nakayama et al.,
1998
; Onichtchouk et al.,
1999
; Souchelnytskyi et al.,
1998
). ogo/szl is regulated positively by Bmp signaling
and in turn Ogo/Szl inhibits Bmp signaling, indicating that Ogon/Szl functions
as a negative-feedback regulator of Bmp signaling. In contrast to the other
feedback inhibitors described above, Ogo/Szl function requires the Chordin
protein. ogo/szl is expressed on the ventral side of the embryo in a
Bmp-signal-dependent manner, whereas chordin is expressed on the
dorsal side and is negatively regulated by Bmp signaling
(Hammerschmidt et al., 1996b
).
Thus, ogo/szl and chordin are regulated in completely
opposite manners, but cooperate in inhibiting the Bmp signal. The Ogo/Szl and
Chordin proteins that are diffused from the ventral and dorsal sides might be
colocalized at a specific position along the DV axis and function there
cooperatively to inhibit the Bmp signal. In support of this idea, Chordin and
Szl2 appear to diffuse a long distance from their source
(Bradley et al., 2000
;
Jones and Smith, 1998
).
Alternatively, molecules that function downstream of the Ogo/Szl-mediated
signaling might interact with Chordin or unknown regulator(s) of Chordin to
inhibit Bmp signaling. In any case, the functional interaction between Ogo/Szl
and Chordin provides a precise positional cue to cells along the DV axis
during gastrulation.
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ACKNOWLEDGMENTS |
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