doi: 10.1242/10.1242/dev.00506
Ogon/Secreted Frizzled functions as a negative feedback regulator of Bmp signaling
Taijiro Yabe1,
Takashi Shimizu1,2,
Osamu Muraoka2,
Young-Ki Bae1,2,
Tsutomu Hirata1,
Hideaki Nojima1,
Atsushi Kawakami3,
Toshio Hirano1,4 and
Masahiko Hibi1,2,*
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
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Fig. 1. The ogon locus encodes a zebrafish homolog of secreted
frizzled. (A) The positional cloning of the ogon gene. The
numbers on the linkage map and the ends of the BAC and PAC clones indicate the
number of recombinations in 2998 meiosis. (B) Amino acid sequence of zebrafish
Ogon/Sizzled and alignment with Xenopus Sizzled. The cysteine-rich
domain (CRD) is underlined. The position of Asp92 is indicated (red dot). (C)
A phylogenic tree of secreted Frizzled-related proteins (FRPs). z, zebrafish;
x, Xenopus; c, chick; m, mouse. (D) Mutations in the
ogon/sizzled genome and the Sizzled proteins of the
ogonrk1 and ogontm305 alleles. CRD is
indicated by yellow boxes.
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Fig. 2. sizzled is responsible for ogon. (A) Control 24 hpf
embryo. (B) Embryos that received an injection of 5 ng of an antisense
morpholino oligonucleotide for sizzled (szl MO) phenocopied
the ogon mutant embryos (n=38/38). (D) Injection of 5 ng of
control MO (szl 4mis MO, with four mispaired bases) did not have any
significant effect (n=45/45). (C) Co-injection of 5 ng of
szl MO and 10 ng of modified szl RNA, which contains four
mispaired bases without codon changes in the MO recognition site, rescued the
ogon phenotype (n=11/21). Rescue of maternal-zygotic (MZ)
ogonrk1 by injection of wild-type szl RNA (also
see Table 1). MZ
ogonrk1 embryos (E) that received an injection of 10 pg of
szl RNA displayed a dorsalized phenotype, normal phenotype (G) and
weakly ventralized phenotype (F).
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Fig. 3. Ogon/Sizzled is a dorsalizing factor. (A) Control 30 hpf embryo. (B)
Injection of 100 pg of ogo/szl RNA dorsalized wild-type embryos.
(C-E) chordin expression at the 80% epiboly stage. Lateral views with
dorsal towards the right. Control (C). Injection of 100 pg of ogo/szl
RNA (D) but not ogo/szltm305 RNA (E) elicited the ventral
expansion of chordin (din) expression. (F,G)
bmp2b/swirl (swr) expression at 80% epiboly stage. Lateral
views with dorsal towards the right. Control (F). (G) Injection of 100 pg of
ogo/szl RNA decreased the expression of bmp2b/swirl. (H-K)
goosecoid (gsc) expression at the shield (H,I; animal pole
views with dorsal towards the right) and 80% epiboly stages (J,K,
dorsoanterior views). (H,J) Control. Injection of 100 pg of ogo/szl
RNA did not affect gsc expression in the embryonic shield (I) or
prechordal plate (K).
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Fig. 4. Ogon/Sizzled functions differently from the Wnt inhibitor Dkk1 and other
Frps. (A) ogo/szl induced ventral expansion, but not anteriorization,
of the neuroectoderm. Embryos that received injections of ogo/szl
(100 pg), noggin1 (nog, 50 pg), dickkopf1
(dkk1, 50 pg) or Xcrescent (xcre, 600 pg) were
fixed and stained with the forebrain-specific marker six3.2 and the
mid-hindbrain boundary marker pax2.1/no isthmus. Lateral views with
dorsal to the right. (B) Ogo/Szl does not inhibit Wnt8. One nanogram of
ogo/szl RNA or 600 pg of Xenopus crescent RNA was
co-injected with 40 pg of wnt8.1 RNA. Wnt8.1-dependent ectopic
expression of bozozok (boz)/dharma (indicated by arrows) was
inhibited by Xcrescent (n=53/58) but not by ogo/szl
(n=9/67). Endogenous boz/dharma expression is indicated by
arrowheads. (C-J) Effects of Xcre and dkk1 overexpression on
szl morphant embryos. Embryos that received injections of
szl MO (5 ng) or szl MO (5 ng) together with Xcre
RNA (25 pg) or dkk1 RNA (12.5 pg) were fixed at shield (C-F) and 80%
epiboly stages (G-J), and stained with gsc or ogo/szl,
respectively. Xcre and dkk1 suppressed expansion of
ogo/szl expression, which was observed in szl morphant
embryos, but concomitantly elicited expansion of gsc at shield
stage.
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Fig. 5. ogon/sizzled expression. Expression of ogo/szl during
zebrafish development: sphere (A), 30% epiboly (EP; B), shield (C), 80%
epiboly (D), tailbud (TB; E), five-somite (5 ss; F) and mid-segmentation (17
hpf; G) stages. Lateral views with dorsal towards the right.
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Fig. 6. ogon/sizzled is regulated by Bmp signaling. ogo/szl
expression in wild-type embryos (A,E), swirltc300 (B,F)
and dinott250 mutant embryos (C,G), and embryos
that received injections of 200 pg of constitutively active Bmp receptor IA
RNA (D,H) at the shield (A-D) and 80% epiboly (E-H) stages. Animal pole views
with dorsal towards the right (A-D), and lateral views with dorsal towards the
right (E-H).
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Fig. 7. Requirement for Chordin in Ogon/Sizzled-mediated Bmp inhibition. (A)
Injection of ogo/szl RNA did not dorsalize the dino/dino
homozygous embryos. Embryos obtained by crossing heterozygous
dinott250 fish received injections of 200 pg of
ogo/szl RNA. The wild-type and heterozygous din embryos
(din/+) displayed dorsalized phenotypes, but the homozygous
din embryos (din/din) displayed ventralized phenotypes at 24
hpf (din/din was confirmed by genotyping), which are typical
phenotypes for the din/din embryos (right). (B) chordin
expression was not regulated by ogo/szl in the absence of Chordin
protein. Two hundred picograms of ogo/szl RNA or 50 pg of
noggin1 RNA was injected into embryos from the din/+ cross,
or embryos received an injection of 2 ng of chordin MO (din
morphant). The embryos were fixed at the 80% epiboly stage and stained with
the chordin probe. Lateral views, dorsal towards the right. The
din mutant and din morphant embryos displayed reduced or an
absence of lateral chordin expression. Overexpression of nog
but not ogo/szl in these embryos rescued and expanded the lateral
expression of chordin. (C) Ogo/Szl acts cooperatively with Chordin to
dorsalize the embryos. Embryos from the din/+ cross received
injections of 15 pg chordin RNA alone or 15 pg chordin RNA
and 200 pg ogo/szl RNA. The embryos were fixed at the 80% epiboly
stage and stained with a probe for fused somites
(fss)/tbx24, which marks the paraxial mesoderm, followed by
genotyping. fss expression in the chordin RNA-injected
embryos was slightly rescued or ectopically expanded, compared with that in
the uninjected din/din embryos, but was weaker than in the wild-type
or din/+ embryos. fss expression was strongly expanded in
the chordin and ogo/szl RNA-coinjected embryos.
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Fig. 8. ogon does not enhance the chordino phenotypes. (A-G)
Embryos obtained from crossing
ogork1/+;dintt250/+ parents (crosses
of five different parent pairs) were assigned to five groups by morphological
inspection at 24 hpf. (A-E) Lateral views with anterior towards the left and
(F,G) ventral views of tail region. The numbers of embryos in each
morphological category and their genotypes are shown in
Table 4. (B,F)
ogo-like embryo, displaying typical ogo phenotypes: caudal
cell accumulation (containing blood cells, arrowhead) and expansion of the
ventral tail fin (the ventral tail fin was expanded laterally), but no
reduction in anterior neuroectoderm. (C-E,G) din-like embryos,
displaying a reduction in the anterior neuroectoderm and caudal cell
accumulation (arrowhead). These embryos displayed variable phenotypes in the
tail fin (arrows): loss (din1, C), reduction (din2, D) or
expansion (din3, E,G) of the ventral tail fin. (H-M) Expression of
eve1 and krox20 in the eight-somite stage wild-type,
din, and ogo;din embryos. (H-J) Vegetal pole views with
dorsal towards the top and (K-M) anterior-dorsal views. din mutant
embryos show variable expansion of eve1 expression (I) and the
additional ogo mutation did not enhance this expansion (J). The
ogo mutation did not increase the reduction of krox20
expression, which marks rhombomeres 3 and 5, in the din mutant
embryos (L,M).
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© The Company of Biologists Ltd 2003
