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First published online September 28, 2005
doi: 10.1242/10.1242/dev.02043

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The novel Smad-interacting protein Smicl regulates Chordin expression in the Xenopus embryo

¹ Department of Developmental Biology (VIB-07), Flanders Interuniversity Institute for Biotechnology (VIB), and Laboratory of Molecular Biology (Celgen), University of Leuven, B-3000 Leuven, Belgium
² Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Zoology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK

View larger version (61K): [in a new window]   Fig. 1. Smicl is a Smad-interacting protein that is expressed maternally and then zygotically during Xenopus development. (A) Amino acid sequence of XtSmicl, a novel Smad interacting protein. Conserved cysteines and histidines in the zinc fingers are underlined. (B) Smicl interacts with Smad3 and Smad4. HA-tagged XtSmicl was expressed in HEK293T cells with plasmids encoding Myc-tagged Smad1, Smad2, Smad3, Smad4 or Smad5 in the presence or absence of the appropriate constitutively active type I receptor. Samples were immunoprecipitated (IP) using an anti-Myc antibody and the presence of HA-XtSmicl was analysed by western blotting (W). (C) Smicl interacts with endogenous Smad3. HA-tagged XtSmicl was expressed in HEK293T cells in the presence or absence of caALK4. Samples were immunoprecipitated with anti-HA coupled beads and the presence of endogenous Smad3 was analysed by western blotting. (D) Expression pattern of Xenopus tropicalis Smicl studied by whole-mount in situ hybridisation. Stages (st) are indicated. a, animal; v, vegetal; D, dorsal; V, ventral; A, anterior; P, posterior; mes, mesoderm; ect, ectoderm. Smicl is expressed maternally and transcripts are particularly abundant in the animal hemisphere of the fertilised egg. These results were confirmed by in situ hybridisation experiments carried out on bisected embryos to facilitate penetration of the probe in the vegetal hemisphere of the embryo. In situ hybridisation using a sense probe revealed no background staining (data not shown). (E) Quantitative RT-PCR confirms the presence of high levels of Smicl RNA in the fertilised egg of Xenopus tropicalis. Significant zygotic expression of Smicl begins at tadpole stage 32.

View larger version (64K): [in a new window]   Fig. 2. Analysis of Smicl function in Xenopus laevis and Xenopus tropicalis. (A) The antisense morpholino oligonucleotides used in this study are aligned with their Xenopus tropicalis (Xt) and Xenopus laevis (Xl) target sequences. (B) XtMO1 inhibits translation of RNA encoding HA-tagged XtSmicl in a dose-dependent fashion. This is not observed with coMO. XtMO1 and coMO were injected in Xenopus embryos at the one-cell stage at the indicated concentrations, followed by RNA encoding HA-tagged XtSmicl. Embryos were cultured to early gastrula stage 10 and subjected to western blotting using an anti-HA antibody and an anti-Gapdh antibody as a loading control. (C) Injection of the morpholino oligonucleotides described in A disrupts gastrulation and axis formation in both Xenopus laevis and Xenopus tropicalis. Dbl, dorsal blastopore lip; A, anterior; P, posterior; D, dorsal; V, ventral. (D) Injection of mRNA encoding mouse Smicl into embryos of Xenopus laevis can `rescue' the phenotype caused by XlMO. Overexpression of mouse Smicl alone causes a `spina bifida' phenotype. Quantitation of morpholino defects and rescues are indicated on the figure. (E) Injection of antisense morpholino oligonucleotide XtMO1 causes upregulation of Smicl mRNA. RNA was extracted at stage 10.5 from embryos injected with 5 ng XtMO1 or coMO and analysed by quantitative RT-PCR.

View larger version (39K): [in a new window]   Fig. 3. Inhibition of Smicl function causes down regulation of Chordin expression, but not of other mesodermal and endodermal markers. (A-E) Expression levels, normalised to that of ornithine decarboxylase, of Xbra (A), Sox17 (B), Xnr3 (C), Goosecoid (D) and Chordin (E) in uninjected embryos, embryos injected with morpholino oligonucleotide coMO (80 ng), or embryos injected with morpholino oligonucleotide XlMO (80 ng). RNA was extracted at the indicated stages, and RNA levels were analysed by quantitative RT-PCR. (F,G) Inhibition of Smicl function by means of morpholino oligonucleotide XtMO1 (5 ng; F) or XtMO2 (30 ng; G) also causes downregulation of Chordin expression in Xenopus tropicalis. Embryos were assayed at stages 9, 9.5, 10 and 10.5 (F), or at stage 10.5 (G). (H) Injection of RNA encoding mouse Smicl causes the upregulation of Chordin in embryos injected with morpholino oligonucleotide coMO and rescues the downregulation of Chordin caused by morpholino oligonucleotide XlMO. Embryos were injected with the indicated RNAs (1 ng) or morpholino oligonucleotides (50 ng). RNA was extracted at stage 10.5 and expression of Chordin and ornithine decarboxylase was assayed by quantitative RT-PCR. (I) In situ hybridisation of Xenopus laevis embryos at the early gastrula stage confirms that inhibition of Smicl function causes the downregulation of Chordin. The embryo on the left was injected with coMO (80 ng; n=15) and the one on the right with XlMO (80 ng; n=15). (J) Injection of Chordin mRNA can partially rescue the phenotype caused by XlMO. Numbers indicate how many embryos out of 50 display this phenotype.

View larger version (17K): [in a new window]   Fig. 4. Indirect induction of Chordin by Xnr1 and ß-catenin. Induction of Chordin by ß-catenin (A) and by Xnr1 (B) is inhibited by cycloheximide. RNA (100 pg) encoding Xnr1 or constitutively active ß-catenin was injected into Xenopus laevis embryos at the one-cell stage and animal caps were dissected at mid blastula stage 8 (before the mid blastula transition). The animal pole regions were allowed to develop for 3 hours in the presence or absence of cycloheximide and RNA was analysed by quantitative RT-PCR for expression of Chordin. Induction of Chordin by both Xnr1 and ß-catenin is inhibited by cycloheximide and is therefore indirect.

View larger version (12K): [in a new window]   Fig. 5. Siamois induces expression of Chordin in a direct manner that does not require Smicl. (A) RNA encoding Siamois (500 pg) was injected into embryos of Xenopus laevis at the one-cell stage and animal pole regions were dissected at mid blastula stage 8 (before the mid blastula transition). The animal pole regions were cultured for 3 hours in the presence or absence of cycloheximide, and expression of Chordin was assayed by quantitative RT-PCR. Induction of Chordin is not inhibited by cycloheximide. (B) Expression of Siamois is not inhibited by antisense morpholino oligonucleotides directed against Smicl. Morpholino oligonucleotide XlMO (80 ng) was injected into Xenopus laevis embryos at the one cell stage and expression of Siamois was analysed by quantitative RT-PCR at the indicated stages. (C) RNA encoding Siamois (500 pg) was injected into Xenopus embryos at the one-cell stage either alone or in the presence of the indicated morpholino oligonucleotides. Animal caps were dissected at mid blastula stage 8 and they were cultured to the equivalent of stage 10.5 before being analysed for Chordin expression by quantitative RT-PCR.

View larger version (11K): [in a new window]   Fig. 6. Induction of Chordin by Smad3 is indirect and requires Smicl. (A) RNA (500 pg) encoding Smad2 or Smad3 was injected into embryos of Xenopus laevis at the one-cell stage and animal pole regions were dissected at mid blastula stage 8 (before the mid blastula transition). The animal pole regions were cultured for 3 hours in the presence or absence of cycloheximide and expression of Chordin was assayed by quantitative RT-PCR. Smad3 is a more efficient inducer of Chordin than is Smad2, and the action of both is indirect. In the same experiment, Xwnt8 and eFGF proved to be induced to higher levels by Smad2 than by Smad3, confirming that the two Smad family members have differential effects in the Xenopus embryo. Additional experiments showed that eFGF and Goosecoid are direct targets of Smad2 and Smad3, respectively (data not shown). (B) Inhibition of Smicl function prevents induction of Chordin by Smad3 and by Xnr1. Embryos of Xenopus laevis were injected with morpholino oligonucleotides coMO or XlMO (80 ng) either alone or in the presence of RNA encoding Smad3 (500 pg) or Xnr1 (100 pg). Animal caps were dissected at mid blastula stage 8 and they were cultured to the equivalent of stage 10.5 before being analysed for Chordin expression by quantitative RT-PCR. Both Smad3 and Xnr1 induced expression of Chordin, and this was inhibited by a morpholino oligonucleotide directed against Smicl. (C) A model based on the data presented so far: the induction of Chordin by Xnr1 and Smad3 is indirect and requires Smicl and the synthesis of another factor X.

View larger version (42K): [in a new window]   Fig. 7. Smad3 induces expression of Xlim1 in a direct manner; induction of Chordin by Xlim1-3m requires Smicl. (A) Smad3 but not Smad2 is a direct inducer of Xlim1. The experimental regime was identical to that described in Fig. 6A, except that expression of Xlim1 and not Chordin was analysed by quantitative RT-PCR. (B) Xlim1/3m is a direct inducer of Chordin. RNA encoding Xlim1/3m (400 pg) was injected into embryos of Xenopus laevis at the one-cell stage. Animal caps were dissected from such embryos and cultured in control medium or medium containing cycloheximide, as described in Fig. 6A. Induction of Chordin is not inhibited by cycloheximide. (C) Smicl is required for Xlim1/3-mediated induction of Chordin. RNA encoding Xlim1/3m (400 pg) was injected into embryos at the one-cell stage in the presence of 80 ng morpholino oligonucleotide coMO or XlMO. Animal pole regions were dissected from such embryos at mid blastula stage 8 and cultured until control embryos reached stage 11. (D) Smicl is not required for expression of Xlim1 in Xenopus laevis. Morpholino oligonucleotides XlMO or coMO (80 ng) were injected into Xenopus laevis embryos at the one-cell stage and RNA was extracted at early gastrula stage 10.5. Expression of Xlim1 is not inhibited by XlMO. (E) Smicl and Xlim1/3 can interact. The indicated combinations of expression constructs encoding HA tagged XtSmicl, Flag tagged Xlim1/3m, Myc tagged Smad3 and caALK4 were co-transfected into HEK 293T cells. Extracts were subjected to immunoprecipitation using an anti-Flag antibody and the presence of HA XtSmicl was analysed by western blotting by using an anti-HA antibody. Smad3 can bind Xlim1/3 in a Smic1-dependent fashion as shown by western analysis of Xlim1/3 immunoprecipitates using an anti-Myc antibody. (F) Binding of Flag-tagged Xlim and HA-tagged Smicl to Chordin promoter sequences containing Xlim-binding sites. Extracts of HEK293T cells transfected with Xlim and/or Smicl expression constructs were incubated with streptavidin-agarose beads and biotinylated double-stranded oligonucleotides. Upper panel: precipitated complexes were subjected to western blotting using anti-Flag and anti-HA antibodies. Xlim1, but not Smicl, interacts with this region of the Chordin promoter, but Smicl can form part of a ternary complex with the Chordin promoter region and Xlim1. Xlim does not bind to Chordin promoter oligonucleotides with mutations in the Xlim-binding sites (see Materials and methods). Lower panel: western blotting of whole extracts shows similar expression levels of Flag-tagged Xlim and HA-tagged Smicl.

View larger version (12K): [in a new window]   Fig. 8. A model describing the activation of Chordin. Briefly, signalling by Xenopus Nodal-related proteins such as Xnr1 activates Smad2 and Smad3. Smad3 induces expression of Xlim1 directly and Xlim1 and Smicl, perhaps in a ternary complex with Smad3 (indicated by `?'), then induce expression of Chordin.

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