A new triple staining method for double in situ hybridization in combination with cell lineage tracing in whole-mount Xenopus embryos.

To analyze cell to cell interaction effects on cell differentiation, we developed a new triple staining method for double in situ hybridization with cell lineage tracing in whole-mount Xenopus embryos. The method provides high color contrast views, and also enabled us to examine inside the embryos. Wild-type embryos whose blastomere(s) had been injected with a cell lineage tracer were cultured, fixed, hemisectioned when necessary, and first served for the double in situ hybridization, with two sequential chromogenic reactions. They were postfixed, and the labeled cells were retraced immunohistochemically. Finally, the pigment of the embryos was bleached to obtain a clear view. We applied this method to a blastomere transplantation experiment to examine whether the spatial gene expression patterns along the anteroposterior axis can be induced by cell to cell interactions. The presumptive organizer of a 32-cell embryo was replaced by the labeled presumptive epidermis of another synchronous embryo. The resultant triple-stained late gastrula showed quite similar anteroposterior expression patterns of gsc and Xbra to those of a normal embryo in the axial mesoderm derived from the transplanted presumptive epidermis, indicating that cell to cell interactions had induced these patterns.