Analysis of competence and of Brachyury autoinduction by use of hormone-inducible Xbra.
Analysis of gene function in Xenopus development frequently involves over-expression experiments, in which RNA encoding the protein of interest is microinjected into the early embryo. By taking advantage of the fate map of Xenopus, it is possible to direct expression of the protein to particular regions of the embryo, but it has not been possible to exert control over the timing of expression; the protein is translated immediately after injection. To overcome this problem in our analysis of the role of Brachyury in Xenopus development, we have, like Kolm and Sive (1995; Dev. Biol. 171, 267-272), explored the use of hormone-inducible constructs. Animal pole regions derived from embryos expressing a fusion protein (Xbra-GR) in which the Xbra open reading frame is fused to the ligand-binding domain of the human glucocorticoid receptor develop as atypical epidermis, presumably because Xbra is sequestered by the heat-shock apparatus of the cell. Addition of dexamethasone, which binds to the glucocorticoid receptor and releases Xbra, causes formation of mesoderm. We have used this approach to investigate the competence of animal pole explants to respond to Xbra-GR, and have found that competence persists until late gastrula stages, even though by this time animal caps have lost the ability to respond to mesoderm-inducing factors such as activin and FGF. In a second series of experiments, we demonstrate that Xbra is capable of inducing its own expression, but that this auto-induction requires intercellular signals and FGF signalling. Finally, we suggest that the use of inducible constructs may assist in the search for target genes of Brachyury.
Tada
M
M
Division of Developmental Biology, NIMR, Mill Hill, London, UK.
O'Reilly
M A
MA
Smith
J C
JC
D028341:Activins; D000818:Animals; D001757:Blastomeres; D046508:Culture Techniques; D003513:Cycloheximide / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000502:physiology; D003907:Dexamethasone / Q000494:pharmacology; D004475:Ectoderm; D005326:Fetal Proteins / Q000235:genetics* / Q000502:physiology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005775:Gastrula; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000502:physiology; D005938:Glucocorticoids / Q000494:pharmacology; D006801:Humans; D007265:Inhibins / Q000494:pharmacology; D008648:Mesoderm / Q000502:physiology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012313:RNA; D017468:Receptors, Fibroblast Growth Factor / Q000235:genetics / Q000502:physiology; D011965:Receptors, Glucocorticoid / Q000235:genetics*; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction; D020825:T-Box Domain Proteins; D014982:Xenopus laevis / Q000196:embryology / Q000235:genetics*; D000098908:Brachyury Protein
D028341:Activins; D003513:Cycloheximide; D004268:DNA-Binding Proteins; D003907:Dexamethasone; D005326:Fetal Proteins; D016222:Fibroblast Growth Factor 2; D005938:Glucocorticoids; D007265:Inhibins; D011500:Protein Synthesis Inhibitors; D012313:RNA; D017468:Receptors, Fibroblast Growth Factor; D011965:Receptors, Glucocorticoid; D011993:Recombinant Fusion Proteins; D020825:T-Box Domain Proteins; D000098908:Brachyury Protein