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First published online May 16, 2007
doi: 10.1242/10.1242/dev.000901

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Cerberus is a feedback inhibitor of Nodal asymmetric signaling in the chick embryo

¹ Instituto Gulbenkian de Ciência, 2781-901 Oeiras, Portugal.
² Institute for Biotechnology and Bioengineering, Centro de Biomedicina Molecular e Estrutural, Universidade do Algarve, 8005-139 Faro, Portugal.

View larger version (73K): [in this window] [in a new window]   Fig. 1. Regulation of chick Cer and Nodal expression by Nodal and Shh signaling pathways. (A-H) Beads soaked in phosphate-buffered saline (PBS, control; A,C,E,G), Nodal protein (B,D,F) or Shh protein (H) were implanted on the right side at Hamburger and Hamilton stage 7 (HH7; A-F) or HH4 (G,H). Chick embryos were fixed after 1 hour (A,B), 2 hours (C,D), 4 hours (E,F) or 5 hours (G,H) and processed for single-label [chick Cer (cCer); A,B] or double-label (cCer and Nodal; C-H) whole-mount in situ hybridization (cCer, purple; Nodal, orange). Nodal protein induced the right-side expression of cCer transcripts in less than 1 hour (B; arrow), and Nodal transcripts in approximately 2 hours (D; arrows). After 4 hours, both cCer and Nodal expression levels were higher on the right than on the left side (F; arrows). On the other hand, Shh protein took approximately 5 hours to induce the transcription of both cCer and Nodal (H; arrow). (I-L) cCer transcripts detected by whole-mount in situ hybridization. (I',J',K',L') Merge of bright-field and RFP red fluorescence images. (I-J') Effect of the Nodal antagonist Xenopus CerS (XCerS) on cCer expression. (J,J') Chick embryos were electroporated with pCAGGS-XCerS on the left side of the node at HH4 (i.e. in the cells that express Nodal). pCAGGS-RFP was electroporated alone (control; I,I') or with pCAGGS-XCerS (J,J'), and used to label the populations of electroporated cells. In contrast to the control electroporation (I; arrowhead), the inhibition of Nodal by XCerS suppressed the left-sided expression of cCer (J; arrowhead). (K-L') Effect of the Nodal antagonist XCerS on Shh-induced cCer expression. HH4 chick embryos were electroporated with pCAGGS-RFP alone (control; K,K') or co-electroporated with pCAGGS-RFP and pCAGGS-XCerS (L,L'), and grafted on the right side with beads soaked in Shh protein. Ectopic induction of cCer expression by Shh (K; arrow) was suppressed by the Nodal inhibitor XCerS (L; arrow). All embryos are viewed from the ventral side. cCer, chick Cer; RFP, red fluorescent protein; XCerS, Xenopus CerS.

View larger version (27K): [in this window] [in a new window]   Fig. 2. Identification of the chick Cer left-side enhancer. (A) Deletion analysis of chick Cer (cCer) cis-regulatory sequences. The genomic organization of cCer is depicted at the top. cCer 5' sequences (black boxes) were fused to the reporter EGFP gene (green boxes) to determine the activity of each DNA fragment. The FoxH1 elements (red; F1 and F2) and the SMAD element (orange; S) are depicted in the reporter constructs. The presence (+) or absence (-) of EGFP expression in the anterior mesendoderm and its derivatives (AM) and in the left-side mesoderm (LSM) from electroporated chick embryos is listed on the right. Each result is representative of at least 12 embryos. LSM expression was disrupted in embryos electroporated with Cer0.34 or shorter constructs. Cer0.12-EGFP expression was very weak and ubiquitous (low). (B) Nucleotide sequence of the 5'-flanking region of cCer. Binding sites for the transcription factors FoxH1 (F1 and F2; orange), SMAD (S; yellow), GATA (green) and Nkx-2.5 (light blue), and a putative TATA box (purple), are outlined. Two transcription initiation sites were identified by RLM-RACE at positions -26 and -29 upstream of the ATG (arrowheads). Point mutations were introduced into the F1, S and F2 sites, as indicated. The morpholino antisense oligo sequence (MO) and its control oligo with five mismatches (CoMO) are outlined in pink. (C) Site-directed mutagenesis analysis of FoxH1- and SMAD-binding elements. LSM expression was specifically abolished in embryos transfected with constructs carrying deletions or mutations (*) in the FoxH1 (F1del, F1mut, F2del and F2mut) or SMAD (Sdel and Smut) elements. (D) Enhancer analysis of potential regulatory sequences of cCer. Fragments of the cCer 5' region (PCR1-5) and sequences of the FoxH1 and SMAD elements (FSF, FF and FS) were subcloned into an enhancer-less vector carrying the human beta-globin minimal promoter (blue boxes) upstream of the EGFP coding sequence. LSM expression was detected in embryos electroporated with the PCR3, PCR5 and FSF constructs (which contained all of the F1, F2 and S elements), but not in those electroporated with the PCR1, PCR2, PCR4, FF and FS constructs (which lacked at least one of those sites). EGFP fluorescence was observed in the AM of embryos electroporated with each of the EGFP reporter constructs tested, with the exception of PCR4. FS-EGFP expression was not tested in the AM cells (nd). +/-, presence/absence of EGFP expression; AM, anterior mesendoderm and its derivatives; CoMo, control morpholino oligo sequence; EGFP, enhanced green fluorescence protein; F1/F2, FoxH1-binding sites; LSM, left-side mesoderm; MO, morpholino antisense oligo sequence; nd, not tested. S, SMAD-binding site.

View larger version (95K): [in this window] [in a new window]   Fig. 3. Expression analysis of Cer-EGFP reporter constructs. (A-H) Cer-EGFP reporter expression in electroporated chick embryos. Different embryos were co-transfected with one Cer-EGFP reporter construct (green fluorescence) and the pCAGGS-RFP construct (positive control; red fluorescence). (A,C) Cer0.4-EGFP; (B,D) F2mut; (E) PCR3; (F) PCR2; (G) FSF; (H) FF (see Fig. 2 for construct details). (A,B) EGFP fluorescence was observed in the anterior mesendoderm (AM) of embryos electroporated with Cer0.4 (A) and F2mut (B) reporter constructs. Embryos were electroporated at Hamburger and Hamilton stage 3 (HH3) and fixed at HH6. (C-H) Asymmetric EGFP expression was detected in the left-side mesoderm (LSM) of embryos electroporated with Cer0.4 (C), PCR3 (E) and FSF (G), but not in those electroporated with the F2mut (D), PCR2 (F) or FF (H) reporter constructs. Embryos were electroporated at HH4-5 and fixed at HH8-9. Dashed line separates the right and left sides of the embryos. (I,J) Cer-Luc reporter activity in Xenopus animal cap luciferase assays. Luciferase reporter plasmids containing the indicated wild-type or mutant fragments of chick Cer (cCer) regulatory sequences were injected into Xenopus embryos in the absence (orange) or presence (green) of Nodal mRNA. Data are relative to the highest luciferase activity values (Cer0.36+Nodal in I; PCR3+Nodal and FSF+Nodal in J). The activities of reporter constructs that either lack one of the FoxH1 elements (F1mut, F2mut, PCR1, PCR2 or FS) or lack the SMAD element (Smut and FF) were reduced. AM, anterior mesendoderm; L, left; LSM, left-side mesoderm; R, right.

View larger version (141K): [in this window] [in a new window]   Fig. 4. Regulation of the chick Cer left-side enhancer by Shh, FGF and Nodal signaling pathways. (A-F) Analysis of Cer-EGFP expression in the left-side mesoderm (LSM) of embryos electroporated at HH4-5 and fixed at HH8-9. (G,H) Analysis of Cer-EGFP expression in the anterior mesendoderm (AM) of embryos electroporated at HH3 and fixed at HH6. (A-D) Chick embryos were electroporated with Cer0.4-EGFP and grafted with beads (arrowheads) soaked in Shh protein (A), the FgfR1 inhibitor SU5402 (B), phosphate-buffered saline (PBS, control; C) or Nodal protein (D). EGFP expression was ectopically induced on the right side by Shh, SU5402 and Nodal (arrows). (E-H) Effect of the Nodal antagonist Xenopus CerS (XCerS) on chick Cer (cCer) left-side enhancer activity. Chick embryos were electroporated either with pCAGGS-RFP and PCR5-EGFP (control; E,G) or with these plus pCAGGS-XCerS (F,H). XCerS repressed the transcription of PCR5-EGFP in the LSM (E,F), whereas it had no effect on AM expression (G,H). AM, anterior mesendoderm; LSM, left-side mesoderm; XCerS, Xenopus CerS.

View larger version (81K): [in this window] [in a new window]   Fig. 5. Chick Cer regulatory regions are able to drive EGFP expression in the left lateral plate mesoderm of mouse embryos. (A,B) E8.5 Cer2.5-EGFP transgenic mouse embryos in ventral (A) and left-side (B) views. (Aa) Transverse section of embryo in A (line). (Bb) Longitudinal section of embryo in B (line). Cell nuclei are labeled with DAPI (blue). Green fluorescence was asymmetrically detected in the left lateral plate mesoderm (llpm; A,B), both in the splanchnopleure and in the somatopleure (Aa,Bb). Fluorescent cells were also found in the foregut (f) and heart (h; B,Aa). (C-D') Expression patterns of EGFP (purple) and Nodal (orange; C,C') or Lefty1,2 (orange; D,D') in E8.25 transgenic mouse embryos detected by double whole-mount in situ hybridization. (C,D) Anterior views. (C',D') Left-side views. The expression domain of EGFP overlapped with Nodal and Lefty2 in the left lateral plate mesoderm. A, anterior; f, foregut; h, heart; L, left; llpm, left lateral plate mesoderm; P, posterior; R, right.

View larger version (98K): [in this window] [in a new window]   Fig. 6. Regulation of Nodal expression by chick Cer. (A-C') Effect of chick Cer (cCer) overexpression. Chick embryos were electroporated with pCAGGS-cCerCDS (coding sequence) on the left (A-B') or right (C,C') side at Hamburger and Hamilton stage 4 (HH4) and fixed at HH8. pCAGGS-RFP was electroporated alone (control; A,A') or with pCAGGS-cCerCDS (B-C'). (A-C) Nodal transcripts detected by whole-mount in situ hybridization. (A',B',C') Merge of bright-field and RFP fluorescence images. cCer overexpression on the left side suppressed Nodal expression (A) in the left lateral plate mesoderm (B; arrow in A), whereas cCer misexpression on the right side had no effect (C; arrow). Nodal transcripts were always detected in the node (A-C; arrowheads). (D-F') Effect of cCer knockdown. HH4-6 chick embryos were electroporated on the left side with fluorescein-tagged morpholinos (MO) and fixed at HH8-11. (D-F) Nodal transcripts detected by whole-mount in situ hybridization. (D',E',F') Merge of bright-field and fluorescein green fluorescence images. (E,F) Nodal expression was ectopically induced by cCer MO in the right lateral plate mesoderm (black arrows), whereas it was normal in the left side (white arrows). Electroporation of a control morpholino (CoMo) did not perturb Nodal left-side expression (D; white arrow). cCer, chick Cer; cCerCDS, chick Cer coding sequence.

View larger version (69K): [in this window] [in a new window]   Fig. 7. Proposed model of the regulation and function of chick Cer in the left-side mesoderm. At Hamburger and Hamilton stage 5-6 (HH5-6), the early expression of Nodal in the node is activated on the left side by Notch and Shh signaling pathways, and is repressed on the right side by Fgf8. At HH7, the Nodal protein released by the node directly activates chick Cer (cCer) and Nodal expression in the adjacent left paraxial and lateral plate mesoderm (P+LPM). Nodal signal is transduced into the phosphorylation of SMAD2 and/or SMAD3, which then bind SMAD4, translocate into the nucleus and synergize with the FoxH1 transcription factor in the activation of cCer transcription. At HH8, Nodal protein produced by the P+LPM cells upregulates cCer and Nodal expression throughout the left lateral plate mesoderm (broken arrows). cCer protein is then required to downregulate the Nodal signal in the left lateral plate mesoderm and prevent it from crossing to the right side of the chick embryo. L, left; R, right.