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BACKGROUND: Bone morphogenetic proteins (BMPs) transmit signals via the intracellular protein Smad1, which is phosphorylated by ligand bound receptors, translocates to the nucleus, and functions to activate BMP target genes. Recently, a subclass of Smad proteins has been shown to inhibit, rather than transduce, BMP signalling, either by binding to the intracellular domain of BMP receptors, thereby preventing phosphorylation-mediated activation of Smad1, or by binding directly to Smad1, thereby inhibiting its ability to activate gene transcription.
RESULTS: We have identified a Xenopus Smad (Smad6) that is 52% identical to mammalian Smad6, an inhibitory Smad. The spatial pattern of expression of Smad6 changes dynamically during embryogenesis and is similar to that of BMP-4 at the tailbud stage. Overexpression of Smad6 in Xenopus embryos phenocopies the effect of blocking BMP-4 signalling, leading to dorsalization of mesoderm and neuralization of ectoderm. Xenopus Smad6 completely blocks the activity of exogenous BMP-4, and, unlike human Smad6, partially blocks the activity of activin, in a mesoderm induction assay. We also find that Smad6 protein accumulates at the membrane in some cells but is partially or completely restricted to nuclei of most overexpressing cells.
CONCLUSIONS: We have identified an inhibitory Xenopus Smad, Smad6, that functions as an intracellular antagonist of activin and BMP-4 signalling. Our finding that Smad6 protein is partially or completely restricted to nuclei of most overexpressing cells suggests that it may employ a novel or additional mechanism of action to antagonize TGF-beta family signalling other than that reported for other inhibitory Smads.
Figure 1. Comparison of deduced amino acid sequences of Xenopus and mouse Smad6. Amino acid identity between mouse Smad6 (mSmad6) and Xenopus Smad6 (XSmad6) is indicated by black boxes. MH1, linker and MH2 domains are denoted above the sequences.
Figure 2. The spatial pattern of expression of Smad6 in developing Xenopus embryos analysed by whole-mount in situ and Northern blot hybridizations. (A) Animal-pole view of a gastrula stage (stage 10) embryo showing uniform Smad6 expression. (B) 10 μg of RNA dissected from the dorsal (D) or ventral (V) halves, or from the animal (Ani), marginal zone (MZ) or vegetal (Vg) thirds of early (stage 10) gastrulae was subjected to Northern blot analysis to detect Smad6 transcripts. The position of 18S (2.1 kb) and 28S (4.2 kb) ribosomal RNAs is indicated to the left of the blot. (C) Anterior-dorsal view of a neurula stage (stage 15) embryo showing Smad6 expression in lateral regions of the anterior neural plate (arrowheads) and weak expression in the prospective cement gland region (arrow) which becomes much stronger by stages 17–18 (D; anterior-dorsal view) and is restricted to the ventral half of the cement gland by stage 30 (E; lateral view, black arrow). (F) Lateral view of a stage 36 embryo cleared in benzyl benzoate:benzyl alcohol (2:1). (G) Transverse sections of stage 36 embryos at the levels of the eye (left) and otic vesicle (right). (E–G) Staining of the dorsal neural tube (grey arrowheads); dorsal eye (grey arrows); lateral otic vesicle (white arrowheads); olfactory placodes (asterisks) and heart anlage (white arrow) is denoted.
Figure 3. Smad6 antagonizes BMP signalling. (A) Overexpression of Smad6 in ventral cells induces the formation of a partial secondary axis (upper right, arrow) that contains immunoreactive muscle (middle right, arrow) while overexpression in dorsal cells does not induce patterning defects (upper and middle left). Overexpression of Smad6 in ectodermal cell explants (animal caps) causes formation of cement gland (lower right, arrows) while control explants maintain an epidermal appearance (lower left). (B) Smad6-injected, but not control ectodermal explants express cement gland-(XAG) and neural specific-(N-CAM, OtxA) genes but do not express the dorsal mesodermal marker α-actin (α-Act) as analysed by RT-PCR. EF-1α is an internal control. The faint Otx signal in the control explant lane was not observed in other experiments and is probably artefact.
Figure 4. Smad6 inhibits mesoderm induced by BMP-4 or activin. (A) Smad6, Smad6-myc or Myc-epitope tag (myc) RNAs were injected alone, or together with BMP-4 RNA, near the animal pole of two-cell embryos and animal caps were explanted at the blastula stage. Co-expression of Smad6 or Smad6-myc blocked BMP-4-mediated induction of Xwnt-8 or Xbra expression as analysed by RT-PCR in the presence (+) and absence (–) of reverse transcriptase (RT) at control stage 12. EF-1α is an internal control in all RT-PCR experiments. (B) Smad6, Smad6-myc or Myc-epitope tag mRNA was injected into animal pole of two-cell embryos. Animal caps were isolated at blastula stage and cultured in the presence or absence of human activin A (20 ng/mL) until sibling embryos reached stage 12 at which time expression of Xwnt-8 and EF-1α was analysed by RT-PCR. Smad6 or Smad6-myc partially blocked activin-mediated induction of Xwnt-8.
Figure 5. Smad6 protein accumulates at the plasma membrane and in the nuclei of overexpressing cells. Embryos were injected at the two-cell stage with 200 pg of plasmid pCS2+Smad6-myc, fixed at stage 12 and immunostained with antimyc antibody (9E10) which was visualized using a fluoroscein-conjugated anti-mouse secondary antibody (green; B,E) followed by nuclear staining with propidium iodide (red; A,D). Overlap of nuclear and antimyc expression appears light green (C,F).
smad6 (SMAD family member 6) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10, animal view.
smad6 (SMAD family member 6) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, anterior view, dorsal up and left.
smad6 (SMAD family member 6) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 36, lateral view, anteriorleft, dorsal up.
