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First published online March 30, 2004
doi: 10.1242/10.1242/dev.01070

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The Cerberus/Dan-family protein Charon is a negative regulator of Nodal signaling during left-right patterning in zebrafish

Hisashi Hashimoto^1,, Michael Rebagliati², Nadira Ahmad², Osamu Muraoka³, Tadahide Kurokawa¹, Masahiko Hibi^3,* and Tohru Suzuki^1,*

¹ National Research Institute of Aquaculture, Nansei, Mie 516-0193, Japan
² Department of Anatomy and Cell Biology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242, USA
³ Laboratory for Vertebrate Axis Formation, Center for Developmental Biology, RIKEN, Kobe 650-0047, Japan

View larger version (75K): [in a new window]   Fig. 5. Charon inhibits Nodal signaling. (A-F) Overexpression of Charon inhibited the effects of Nodal overexpression. The injection of 10 pg of cyclops (cyc) or squint (sqt) RNA, or 100 pg of southpaw (spaw) RNA elicited expansion and/or ectopic expression of goosecoid (gsc) at 6 hpf (A,C,E). Co-injection of 25 pg charon RNA inhibited the effects of cyc, sqt and spaw overexpression (B,D,F). (A-F) Animal pole views, with dorsal to the right. (G) Interaction between Charon and Southpaw. COS7 cells were transfected with expression vectors for Myc-tagged Charon (Charon-Myc), Myc-tagged PRDC (PRDC-Myc), or HA-tagged Spaw (HA-Spaw). The supernatants containing Charon-Myc, PRDC-Myc and HA-Spaw were mixed as indicated, and immunoprecipitated with anti HA- or anti-Myc epitope antibodies. The immunoprecipitates were immunoblotted with anti-HA or Myc antibodies. Arrowheads indicate the position of Charon-Myc (two bands, correspond to approximately 35 and 40 kD). A black asterisk indicates light chains of the antibodies, and white asterisks indicate the position of PRDC-Myc. (H-M) southpaw (spaw) expression (H,I) and charon expression (K,L) at the 12-somite stage (15 hpf). Cross-sections of 12-somite stage embryos showing charon (J) and spaw (M) expression. (N-Q) Two-color staining of charon (purplish) and spaw (red) at the 12 somite (N,O) and 18-somite (18 hpf; P,Q) stages. (H,K,N,P) Lateral views. (I,L,O,Q) Dorsal views of the tailbud.

View larger version (66K): [in a new window]   Fig. 1. Comparison of the amino acid sequence of fish Charon with the sequences of other Cerberus/Dan family members. (A) Alignment was done with the ClustalW program. Identities in the amino acid sequence of zebrafish (z) Charon to the sequences Fugu Charon, flounder (fl) Charon, Xenopus (X) Cerberus, chick (c) Caronte, and human Cerberus-related (hCerberus) are 41%, 41%, 18%, 25%, 20%, respectively. Fugu Charon and flounder Charon are the most similar to each other (63%). (B) A radial phylogenic tree of the Cerberus/Dan family proteins. The tree was calculated according to the Expansion of the ClustalW program by DDBJ (DDBJ; http://www.ddbj.nig.ac.jp) using the amino acid sequences of the cysteine-knot domain of the proteins. Zebrafish PRDC and zebrafish Gremlin are less similar (16% and 18%, respectively) to chick Caronte than to zebrafish Charon. c, chick; X, Xenopus; z, zebrafish.

View larger version (116K): [in a new window]   Fig. 2. charon expression in zebrafish. In situ hybridization analysis revealed that charon expression was initiated around 12 hpf at the 6-somite stage in the tailbud (A,B). charon expression became more obvious at 14 hpf (10-somite stage) (C,D). The charon transcripts were observed in the posterior half of the flanking domain of Kupffer's vesicle in a horseshoe shape (D, refer to E,F). The expression continued through 15 hpf to 18 hpf in the same tissue (16 hpf, 14-somite stage, G,H). The expression pattern of charon mRNA in Fugu (I,J) and flounder (K,L, refer to M) was very similar to that in zebrafish. The charon transcripts were not detected in any other domain throughout embryogenesis. There was no obvious L/R bias in the strength of charon expression in the majority of embryos. (A,C,E,G,I,K,M) Lateral views. (B,D,F,H,J,L) Vegetal pole views of the tailbud region. (E,F,M) Control non-stained embryos. Arrowheads indicate the position of Kupffer's vesicle.

View larger version (144K): [in a new window]   Fig. 3. Regulation of charon expression. Expression of charon in the wild-type (A), mutant (B-H) and antivin/lefty1 RNA-injected (atv inj.) embryos (I) at the 10-somite stage (13 hpf). Vegetal pole views of the tailbud region. In bozozok (boz) embryos, charon expression was reduced (C) or absent (B). In no tail (ntl) embryos, no charon expression was detected (D). In squint (sqt) embryos, charon expression was reduced (F) or absent (E). In cyclops (cyc) embryos, charon expression was not affected (G). In one-eyed pinhead (oep) embryos, the strength of charon expression was not affected, but the expression domain became smaller, in proportion to the size of Kupffer's vesicle (G). In embryos injected with 25 pg of antivin/lefty1 RNA, which displayed phenotypes similar to cyc:sqt double mutant and maternal-zygotic oep mutant embryos at 24 hpf (data not shown), no charon expression was detected (I). Variability of charon expression in boz and sqt embryos was consistent with the variable expressivity of these boz and sqt alleles. Arrowheads indicate weak expression of charon.

View larger version (93K): [in a new window]   Fig. 4. Overexpression of zebrafish charon leads to a lack of mesendoderm. Zebrafish charon RNA (25 or 100 pg) was injected into one-to-four cell stage embryos. The RNA injection led to variable levels of defects in the formation of mesendoderm, which is observed in one-eyed pinhead (oep) mutant and antivin/lefty1-injected embryos. Non-injected control embryos at 10 hpf [at the time of yolk plug closure (YPC), which is equivalent to bud stage (A) and at the pharyngula stage (24 hpf, B,C,D)]. The phenotypes of the injected embryos were classified into three categories (Class I-III) with increasing severity. Class I embryos at YPC (E) and at the pharyngula stage (F-I). Class II embryos at YPC (J) and at the pharyngula stage (K,L,M). Class III embryos at YPC (N) and at the pharyngula stage (O,P,Q). (A,B,E,F,J,K,N,O) Lateral views. (C,G,L,P) Ventral views of the head. (D,H,M,Q) Dorsal views of the head. (I) Lateral view of the trunk. Arrowheads indicate the anterior border of the dorsal axial mesendoderm and notochord. The numbers of each class of the embryos are shown in Table 1. (R-Y) Misexpression of charon caused a defect in the axial mesendoderm formation. The embryos receiving 25 pg of charon RNA lacked no tail (ntl) expression in the dorsal midline and dorsal forerunner cells at YPC (S,U) and goosecoid (gsc) expression at the 90% epiboly stage (W). In the charon RNA-injected embryos, the expression of six3.2 (a marker for forebrain, arrowhead) was slightly expanded and the expression domain of pax2.1 (a marker for the mid-hindbrain boundary, arrow) was slightly shifted posteriorly at YPC (Y). (R,T,W,X) Uninjected control embryos. (R,S) Dorsal views. (T-W) Lateral views. (X,Y) Animal pole views, with ventral to the top.

View larger version (74K): [in a new window]   Fig. 6. Knockdown of Charon leads to a defect in heart laterality. (A) A pharyngula-stage (24 hpf) embryo that received 25 pg of charon RNA. (B) An embryo that received both charon RNA (25 pg) and charon-MO (0.8 ng). (C-K) The charon morphant embryos displayed variable defects in heart positioning. The numbers of embryos showing each phenotype are shown in Table 2. nkx2.5 expression at 26 hpf (C-E), and cardiac myosin light chain (cmlc2) expression at 26 hpf (F-H) and 52 hpf (I-K). At 26 hpf, the heart jogged to the left (C,F), to the right (E,H), or did not jog (D,G) in charon morphant embryos. Likewise, heart looping was disrupted in charon morphant embryos at 52 hpf (D-loop, I; no-loop, J; L-loop, K). (L) The charon morphant embryos showed no gross morphological abnormalities at 40 hpf. (A,B,L) Lateral views. (C-E, I-K) Ventral views. (F-H) Dorsal views.

View larger version (104K): [in a new window]   Fig. 7. Charon is required for the early processes of L/R patterning. (A-I) Embryos were injected with charon-MO and examined for atv/lefty1 (20 hpf, A-C), spaw (18 hpf, E-F), and pitx2 (20 hpf, G-I) expression. In addition to the normal (left-sided) expression of the gene (A,D,G), bilateral (B,E,H) or reversed (C,F,I) expression of the left-side-specific markers was also observed. The numbers of embryos showing each phenotype are given in Table 3. Embryos that showed strong right-side stain and weaker left-side stain were recorded as bilateral (dorsal views). Arrowheads indicate the presence of the expression. (J-T) Midline barriers are not affected in charon morphant embryos. no tail (ntl) expression in notochord of wild-type (J,K) and charon morphant embryos (L,M) at 13 hpf. sonic hedgehog (shh) expression in notochord at 13 hpf (N,O) and in floor plate at 24 hpf (R,S) in wild-type (N,R) and charon morphant embryos (O,S). antivin/lefty1 expression in wild-type (P) and charon morphant embryos (Q) at the 6-9-somite stage. Saggital sections of ntl expression at 24 hpf in wild-type (T) and charon morphant embryos (U). (J,L,R,S) Lateral views. (K,M,N,O) Dorsal views. (P,Q) Dorso-lateral views.

View larger version (94K): [in a new window]   Fig. 8. Southpaw is epistatic to Charon. Control (uninjected) embryos (A,D,G), and embryos injected with charon-MO (0.8 ng, B,E,H), or co-injected with charon-MO (0.8 ng) and southpaw-MO (8 ng) (spaw-MO+charon-MO, C,F,I), were examined for pitx2 (22-24-somite stage, A-C), cyclops (cyc) (18-21-somite stage, D-F), and antivin/lefty1 (18-21-somite stage, G-I) expression. (A-C) `Face-on' views, optical sections. Similar effects were seen on pitx2 expression in the lateral plate. Arrowheads indicate pitx2 expression in dorsal diencephalons. (D-F) Dorsal views, with anterior to the top. Arrowheads indicate cyc expression in dorsal diencephalon and lateral plate mesoderm. (G-I) Dorsal views, with anterior to the left. Arrowheads indicate antivin/lefty1 expression in dorsal diencephalon and lateral plate mesoderm. Typical data are shown in this Figure and the numbers showing laterality defects are given in Table 3.

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