Fig. 4. Aboral distribution of LvTbx2/3 protein (red) in prism-stage embryos, demonstrated by co-staining with 5a7 (EctoV; green). The EctoV antigen is expressed from late gastrula stages onward, solely in the oral ectoderm and foregut. Prism-stage embryos viewed aborally (A) and in a vegetal cross-section (B). Complementary expression is observed, indicating that LvTbx2/3 is distributed asymmetrically about the oral/aboral axis, and localized in aboral territories of the endoderm, ectoderm and mesoderm. In many prism and pluteus-stage embryos, a patch of cilia on the oral surface crossreacts with Cy3 secondary antibodies nonspecifically, as observed in the LvTbx2/3 (red) images. (C) Whole-mount, immunofluorescent analysis of fixed embryos probed with preimmune serum. No nuclear staining is observed at any stage (early prism stage shown). (D) Whole-mount, immunofluorescent analysis of fixed embryos probed with with polyclonal LvTbx2/3 serum that had been preincubated with recombinant fusion protein. No nuclear staining is observed at any stage examined (early pluteus stage shown).

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