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.

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