100
M. Hild et al.
in the L3 loop region of the MH2 domain of Smad5 protein. This
threonine residue is conserved in all currently known receptor-regulated
Smad protein. Mutational in vitro analyses carried out for mouse Smad2
have shown that this threonine residue is involved in the binding of
receptor-regulated Smad proteins to the cytoplasmic domain of the
Bmp/TGF~ receptors and to Smad4, while the formation of homotrimers is not affected. This might explain the dominant negative,
antimorphic activity of the sbn Smad5 mutant, assuming that mutant
protein traps wild-type Smad5 in inactive trimers which cannot bind to
the Bmp/TGF~ receptors or to Smad4.
When mutant smad5 mRNA is injected into wild-type embryos, the
sbn mutant phenotype (C4 dorsalization) is phenocopied, while injection of high amounts of wild-type smad5 mRNA into sbn mutant embryos leads to a rescue of the mutant phenotype to wild-type condition.
In addition to mRNA, phenocopy and rescue experiments were also
carried out with plasmid DNAs that drive smad5 expression under the
control of two promoters with different temporal activation profiles; the
Xenopus EFla promoter, which is activated right after midblastula
transition when zygotic gene expression starts (Kane and Kimmel
1993), and the cytoskeletal actin (CSKA) promoter, which is strongly
activated during gastrulation, but very weakly expressed at earlier
stages (Hammerschmidt, et al. 1998). Injection of wild-type smad5
DNA into sbn embryos only led to a rescue, and injection of mutant
smad5 DNA into wild-type embryos to a phenocopy of the sbn phenotype, when gene expression was under the control of the EF 1 a promoter;no effect was observed upon injection of the corresponding
CSKA constructs. These data suggest that smad5 acts after midblastula
transition and before gastrulation.
The antimorphic nature of the sbn smad5 mutation was also confirmed in Xenopus animal cap explants. Wild-type smad5, when overexpressed in animal caps, induces the expression of ventral mesodermal
markers like Xhox3. Upon injection of sbn smad5, however, no induction of Xhox3 transcription is observed, indicating that sbn Smad5 has
retained no, or very weak, ventralizing activity. Coexpression of wildtype and mutant smad5 revealed that the ventralizing effect of wild-type
smad5 RNA was inhibited approximately 80% by equal amounts of
co-injected sbn tc24 smad5 RNA, and 90% by twofold amounts.
M. Hild et al.
in the L3 loop region of the MH2 domain of Smad5 protein. This
threonine residue is conserved in all currently known receptor-regulated
Smad protein. Mutational in vitro analyses carried out for mouse Smad2
have shown that this threonine residue is involved in the binding of
receptor-regulated Smad proteins to the cytoplasmic domain of the
Bmp/TGF~ receptors and to Smad4, while the formation of homotrimers is not affected. This might explain the dominant negative,
antimorphic activity of the sbn Smad5 mutant, assuming that mutant
protein traps wild-type Smad5 in inactive trimers which cannot bind to
the Bmp/TGF~ receptors or to Smad4.
When mutant smad5 mRNA is injected into wild-type embryos, the
sbn mutant phenotype (C4 dorsalization) is phenocopied, while injection of high amounts of wild-type smad5 mRNA into sbn mutant embryos leads to a rescue of the mutant phenotype to wild-type condition.
In addition to mRNA, phenocopy and rescue experiments were also
carried out with plasmid DNAs that drive smad5 expression under the
control of two promoters with different temporal activation profiles; the
Xenopus EFla promoter, which is activated right after midblastula
transition when zygotic gene expression starts (Kane and Kimmel
1993), and the cytoskeletal actin (CSKA) promoter, which is strongly
activated during gastrulation, but very weakly expressed at earlier
stages (Hammerschmidt, et al. 1998). Injection of wild-type smad5
DNA into sbn embryos only led to a rescue, and injection of mutant
smad5 DNA into wild-type embryos to a phenocopy of the sbn phenotype, when gene expression was under the control of the EF 1 a promoter;no effect was observed upon injection of the corresponding
CSKA constructs. These data suggest that smad5 acts after midblastula
transition and before gastrulation.
The antimorphic nature of the sbn smad5 mutation was also confirmed in Xenopus animal cap explants. Wild-type smad5, when overexpressed in animal caps, induces the expression of ventral mesodermal
markers like Xhox3. Upon injection of sbn smad5, however, no induction of Xhox3 transcription is observed, indicating that sbn Smad5 has
retained no, or very weak, ventralizing activity. Coexpression of wildtype and mutant smad5 revealed that the ventralizing effect of wild-type
smad5 RNA was inhibited approximately 80% by equal amounts of
co-injected sbn tc24 smad5 RNA, and 90% by twofold amounts.