Inductive interactions play a major role in early development, and one of the earliest such interactions in amphibian development, and perhaps the in development of all vertebrates, is mesoderm induction (1-5). Mesoderm induction occurs at blastula stages, when a signal from the vegetal hemisphere of the embryo acts on overlying equatorial cells, causing them to form mesoderm rather than ectoderm. This interaction was first discovered in experiments in which prospective ectodermal tissue of the embryo (the so-called "animal cap") is juxtaposed with future endoderm from the vegetal hemisphere (Fig. 1). When cultured alone, the animal caps form epidermis; when cultured adjacent to vegetal pole blastomeres, they form mesoderm.

In the last decade, great advances have been made in coming to understand the signals involved in mesoderm induction as well as in identifying the intra-cellular signal transduction pathways used by these factors. Progress has also been made in identifying target genes of mesoderm-inducing factors, and even in studying how the transcription of these genes is regulated. This chapter describes three assays for mesoderm induction. The first is the original assay described above, in which animal pole and vegetal pole tissue is juxtaposed. In the second, the inducing factor is supplied to the target tissue not from vegetal pole cells, but as a soluble protein, and in the third, the inducing agent is supplied to the responding cells by microinjecting RNA encoding the protein in question into the developing embryo.

This last method has proven particularly useful for studying proteins that cannot yet be obtained in purified, soluble form (6,7) and for studying the activities of intracellular proteins, such as components of the MAP kinase path-

From: Methods in Molecular Biology, Vol. 97: Molecular Embryology: Methods and Protocols Edited by: P. T. Sharpe and I. Mason © Humana Press Inc., Totowa, NJ

Fig. 1. Mesoderm induction. Cells from the animal hemisphere of the embryo are juxtaposed with cells from the vegetal hemisphere.

Vegetal 1

Fig. 1. Mesoderm induction. Cells from the animal hemisphere of the embryo are juxtaposed with cells from the vegetal hemisphere.

way (8-10). The technique can also be adapted to identify novel mesoderm-inducing agents by "expression-cloning" (11). Finally, mesoderm induction can also be used as a convenient read-out for ligand-receptor interactions, which have nothing to do with the in vivo process. For example coexpression of GDNF and the receptor tyrosine kinase Ret in Xenopus animal caps causes the formation of mesoderm, whereas expression of either protein alone has no effect, thus suggesting that GDNF signals through Ret (12).

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