AN INDELIBLE LINEAGE MARKER FOR XENOPUS USING A MUTATED GREEN FLUORESCENT PROTEIN

We describe the use of a DNA construct (named GFP.RN3) encoding green fluorescent protein as a lineage marker for Xenopus embryos. This offe rs the following advantages over other lineage markers so far used in Xenopus. When injected as synthetic mRNA, its protein emits intense fl uorescence in living embryos. It is non-toxic, and the fluorescence do es not bleach when viewed under 380 nm light, It is surprisingly stabl e, being strongly visible up to the feeding tadpole stage (5 days), an d in some tissues for several weeks after mRNA injection. We also desc ribe a construct that encodes a blue fluorescent protein. We exemplify the use of this GFP.RN3 construct for marking the lineage of individu al blastomeres at the 32- to 64-cell stage, and as a marker for single transplanted blastula cells. Both procedures have revealed that the d escendants of one embryonic cell can contribute single muscle cells to nearly all segmental myotomes rather than predominantly to any one my otome. An independent aim of our work has been to follow the fate of c ells in which an early regulatory gene has been temporarily overexpres sed, For this purpose, we co-injected GFP.RN3 mRNA and mRNA for the ea rly Xenopus gene Eomes, and found that a high concentration of Eomes r esults in ectopic muscle gene activation in only the injected cells. T his marker may therefore be of general value in providing long term id entification of those cells in which an early gene with ephemeral expr ession has been overexpressed.