December 1, 2003;
The beta-catenin/VegT-regulated early zygotic gene Xnr5 is a direct target of SOX3 regulation.
In Xenopus laevis, beta-catenin-mediated dorsal axis formation can be suppressed by overexpression of the HMG-box transcription factor XSOX3
. Mutational analysis indicates that this effect is due not to the binding of XSOX3
to beta-catenin nor to its competition with beta-catenin-regulated TCF-type transcription factors for specific DNA binding sites, but rather to SOX3
binding to sites within the promoter of the early VegT
- and beta-catenin-regulated dorsal-mesoderm
-inducing gene Xnr5
. Although B1-type SOX proteins, such as XSOX3
, are commonly thought to act as transcriptional activators, XSOX3
acts as a transcriptional repressor of Xnr5
in both the intact embryo
and animal caps injected with VegT
RNA. Expression of a chimeric polypeptide composed of XSOX3
and a VP16 transcriptional activation domain or morpholino-induced decrease in endogenous XSOX3
polypeptide levels lead to an increase in Xnr5
expression, as does injection of an anti-XSOX3
antibody that inhibits XSOX3
DNA binding. These observations indicate that maternal XSOX3
acts in a novel manner to restrict Xnr5
expression to the vegetal hemisphere.
[+] show captions
Fig. 2. Immunocytochemical analysis of XSOX3. (A) In situ hybridization of fertilized eggs with an antisense probe for XSOX3 RNA reveals that XSOX3 mRNA is localized primarily to the fertilized egg's animal hemisphere (`an' and `vg' mark the animal and vegetal hemispheres, respectively, in all parts). (B) Whole-mount immunocytochemistry of a 64-cell embryo with the anti-XSOX3c antibody reveals a strong cytoplasmic reaction with the animal hemisphere; preincubation of the antibody with the peptide conjugate against which it was raised completely abolished staining (ab). (C) The nuclear nature of the staining becomes more pronounced as development proceeds but can be clearly seen in early stage embryos (∼128-cell stage). The protein is still primarily localized to the animal hemisphere but nuclei in vegetal blastomeres (arrows) clearly contain the immunoreactive polypeptide. Staining of early stage embryos with either anti-XTCF3n (D) or anti-XTCF3c (E) produced a pattern of staining similar to that seen for anti-XSOX3c. Nuclei are marked by arrows. (F) By mid-blastula stages, the XSOX3 polypeptide appears to be nuclear except in mitotic cells (arrows). Nuclear XSOX3 staining is seen throughout the embryo. (G) During gastrulation, anti-XSOX3 staining can be seen in the nuclei (arrows) of yolk plug (YP) cells. The blastopore (BP) is clearly visible and XSOX3 staining is seen throughout the surface ectoderm.