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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.
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