Tada M et al. (1997), Analysis of competence and of Brachyury autoind...

XB-ART-16442

Development 1997 Jun 01;12411:2225-34. doi: 10.1242/dev.124.11.2225.

Show Gene links Show Anatomy links

Analysis of competence and of Brachyury autoinduction by use of hormone-inducible Xbra.

Tada M , O'Reilly MA , Smith JC .

???displayArticle.abstract???
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.

???displayArticle.pubmedLink??? 9187148
???displayArticle.link??? Development

Species referenced: Xenopus laevis
Genes referenced: actc1 actl6a evx1 fgf2 gsc nr3c1 odc1 tbx2 tbxt
???displayArticle.antibodies??? Notochord Ab2 Somite Ab1

???attribute.lit??? ???displayArticles.show???

	Fig. 1. Construction of Xbra-GR. The hormone binding domain of the human glucocorticoid receptor (hGR) amino acids 512-777 was fused to the carboxy-terminus of Xbra (1-432) to generate Xbra-GR. This fusion replaces the stop codon of Xbra with two amino acids (DL) generated by the insertion of a BglII site (see Materials and methods). The DNA-binding and activation domains of Xbra are shown by shaded and hatched boxes, respectively (see Kispert and Herrmann, 1993; Conlon et al., 1996). The hormone-binding domain of hGR is shown by a solid box.
	Fig. 2. Xbra-GR activates transcription of a synthetic target gene. (A) Schematic representation of the 2T-CAT reporter plasmid. The plasmid contains two copies of the palindromic Brachyury binding site (Kispert and Herrmann, 1993) upstream of a thymidine kinase (tk) minimal promoter driving expression of chloramphenicol acetyl transferase (CAT) (Conlon et al., 1996). (B) Analysis of transcription activation by Xbra-GR in Xenopus embryos. Xbra-GR RNA (400 pg) and the 2T-CAT reporter plasmid (20 pg) were coinjected into both cells of Xenopus embryos at the 2- cell stage. Animal caps were dissected at stage 8, then treated with 10-6 M DEX and cultured to stage 10.5, when they were analysed for expression of CAT by RNAase protection. Addition of DEX causes activation of CAT transcription.
	Fig. 3. Morphological and histological analyses of animal caps derived from embryos injected with RNA encoding Xbra, Xbra-GR or GR. Embryos at the one-cell stage were left uninjected (A-D) or injected with 400 pg of RNA encoding Xbra (E-H), Xbra- GR (I-L) or GR (M-P). Animal caps were dissected at stage 8, and in some cases treated with 10-6 M DEX (C,D,G,H,K,L,O,P). After culture to stage 42, caps were photographed (A,C,E,G,I,K,M,O) and subjected to histological analysis (B,D,F,H,J,L,N,P). Addition of DEX has no effect on animal caps from uninjected embryos, or those injected with RNA encoding Xbra or GR, but caused caps derived from embryos injected with RNA encoding Xbra-GR to form compact masses of muscle (L).
	Fig. 4. Mesoderm formation by Xbra-GR. RNAase protection analysis of mesoderm formation in animal caps derived from uninjected embryos or embryos injected with 200 pg of RNA encoding Xbra, Xbra-GR or GR. Animal caps were dissected at stage 8, treated with 10-6 M DEX, and cultured to stage 12 for analysis of goosecoid, to stage 14 for analysis of Xhox3 or to stage 23 for analysis of cardiac actin gene expression. ODC, EF-1a and cytoskeletal actin serve as loading controls.
	Fig. 5. Increasing amounts of RNA encoding Xbra-GR induce different types of mesoderm. Histological (A-C) and immunohistochemical (D-F) analyses of animal caps derived from embryos injected with increasing amounts of RNA encoding Xbra-GR. Animal caps were dissected at stage 8, treated with 10-6 M DEX, and cultured to stage 42 when they were fixed and sectioned at 7 mm for histological analysis or at 10 mm for immunohistochemical analysis. For histological analysis (A-C), the sections were stained by the Feulgen technique and with Light Green and Orange G. For immunohistochemical analysis (D-F), the sections were stained with 12/101 monoclonal antibody for muscle. (A,D) Untreated caps treated with DEX form atypical epidermis. (B,E) DEX-treated animal caps derived from embryos injected with 25 pg of RNA encoding Xbra-GR form ventral mesodermal vesicles, which contain mesothelium and mesenchyme. (C,F) DEX-treated animal caps derived from embryos injected with 400 pg of RNA encoding Xbra-GR form clumps of muscle cells. This muscle tissue is typical of caps derived from embryos injected with 100 pg of RNA encoding Xbra-GR or more.
	Fig. 6. Effects of injected RNA encoding Xbra-GR in whole embryos. (A-D) One animal pole blastomere (tier A) of embryos at the 32-cell stage received injections of RNA encoding Xbra-GR (10 pg). Embryos were left untreated (A) or exposed to 10-6 M DEX at stage 10 (B-D). They were allowed to develop to stage 34. DEX-treated embryos typically possess tail-like protrusions (B,C). When protrusions are present in the head, embryos lack eyes (C) and when they occur near the tail, they contain fin-like structures (D). The tail-like protrusions are shown by arrows. Treatment with DEX at stage 10 was more effective than that at stage 8 or 12. (E-H) Embryos were injected in one blastomere of tier A (E,F) or tier C (G,H) at the 32-cell stage with 10 pg of RNA encoding Xbra-GR together with nucb-gal RNA. The embryos were treated with 10-6 M DEX at stage 10 (F,H) or were untreated (E,G), fixed at stage 34 and stained with X-gal. Embryos injected in tier A and treated with DEX possess tail-like protrusions (see Table 2). When Xbra-GR and nucb-gal RNAs were injected into a tier A blastomere in the absence of DEX, labelled cells were found in the surface ectoderm (E) or in neural tissues, but addition of DEX at stage 10 changed the fate of these cells causing them to form tail-like protrusions (98% of cases; n=67) (F). In contrast, following injection into a tier C blastomere, labelled cells were observed in the notochord or in the paraaxial cells (G) even after treatment with DEX (H). (I-L) Whole-mount immunostaining analysis of embryos injected with 10 pg of RNA encoding Xbra-GR into a tier A blastomere at the 32-cell stage. The embryos were left untreated (I,K) or treated with 10-6 M DEX at stage 10 (J,L), fixed at stage 34 and stained with MZ15 monoclonal antibody for notochord (I,J) or with 12/101 monoclonal antibody for muscle (K,L). Tail-like protrusions are negative for notochord (J), while they are positive for muscle (arrow in L).
	Fig. 7. Stability of Xbra-GR protein in Xenopus embryos. (A) Immunoprecipitation analysis of in vitro translated protein derived from Xbra-HA, Xbra-GR-HA and GR-HA, all of which include repeated nonapeptides from the influenza hemagglutinin protein (HA), using anti-HA monoclonal antibody 12CA5 (see Materials and methods). Immunoprecipitates were analysed by 12% acrylamide gel electrophoresis followed by fluorography. Positions of molecular mass markers (Â´10-3) are indicated. (B) Western blotting analysis of Xbra-GR protein levels in Xenopus embryos. Animal caps from embryos injected with 400 pg of RNA encoding Xbra-GR-HA were dissected at stage 8, treated with 10-6 M DEX where appropriate and collected at the indicated stages. Two cap equivalents of protein were analysed by western blotting using the anti-HA antibody after 12% acrylamide gel electrophoresis. Positions of molecular mass markers (Â´10-3) are indicated. The band corresponding to the protein of Xbra-GR-HA is indicated by an arrowhead. Note that Xbra-GR-HA protein levels are stable in the absence of DEX but fall rapidly after its addition.
	Fig. 8. Competence of animal caps to respond to mesoderm inducers. (A) Embryos at the one-cell stage were left uninjected or injected with 100 pg of RNA encoding Xbra-GR. Animal caps were dissected at stage 8, 9, 10 or 11, then treated with 100 ng/ml bFGF, 8 U/ml activin or 10-6 M DEX, and cultured to stage 23, when they were analysed for expression of actin genes by RNAase protection. Competence in response to bFGF and activin is gradually lost by the early gastrula stage, whereas the response to Xbra persists. (B) Competence of animal caps to respond to Xbra. Embryos at the one-cell stage were injected with 100 pg of RNA encoding Xbra-GR. Animal caps were dissected at stage 8, treated with 10-6 M DEX at the indicated stages, and cultured to stage 23, when they were analysed for expression of actin genes by RNAase protection. Competence of animal caps to respond to Xbra persists during until the late gastrula stage (stage 12).
	Fig. 9. Autoinduction of Xbra. (A) Autoinduction of Xbra in animal pole explants. Embryos were injected with 25 pg of RNA encoding Xbra-GR at the one-cell stage. Animal caps were dissected at stage 8, treated as appropriate with 10-6 M DEX and cultured to stage 10.5, when they were analysed for expression of Xbra by RNAase protection. ODC serves as a loading control. (B,C) Autoinduction of Xbra in whole embryos. Embryos were injected with 10 pg of RNA encoding Xbra-GR into a tier A blastomere at the 32-cell stage. The embryos were left untreated (B) or exposed to 10-6 M DEX at stage 10 (C) and allowed to develop to stage 11.5, when they were analysed by whole-mount in situ hybridisation. Ectopic expression of Xbra is seen in the prospective ectoderm.
	Fig. 10. Xbra autoinduction is indirect. (A) Time course of Xbra induction. Embryos were left uninjected or injected at the one-cell stage with 200 pg of RNA encoding Xbra-GR. Animal caps were dissected at stage 8, and treated as appropriate with 10-6 M DEX or with 50 ng/ml bFGF. They were harvested at the indicated times after treatment and analysed for expression of Xbra by RNAase protection. Expression of Xbra was elevated in response to bFGF within 2 hours, but the response to Xbra required 3 hours. (B) Xbra autoinduction requires intercellular signals. Embryos at the one-cell stage were injected with 25 pg of RNA encoding Xbra-GR or left uninjected. Animal caps were dissected at stage 8 and kept intact or dispersed by culture in medium lacking calcium and magnesium. The intact or dissociated caps were exposed to 10-6 M DEX or to 50 ng/ml bFGF and cultured for 3 hours to the equivalent of stage 10.5, when they were analysed for expression of Xbra by RNAase. ODC serves as a control for loading in samples in these experiments. Xbra autoinduction is not observed in dissociated cells, whereas induction by bFGF is unaffected.
	Fig. 11. Xbra function requires FGF signalling. (A) Mesoderm formation in response to Xbra requires FGF signalling. Embryos at the one-cell stage were injected with 1 ng of RNA encoding a dominantnegative FGF receptor (XFD) and/or 100 pg of RNA encoding Xbra- GR. Animal caps were dissected at stage 8, treated as appropriate with 10-6 M DEX and cultured to stage 23, when they were analysed for expression of actin genes by RNAase protection. Inhibition of FGF signalling blocks induction of mesoderm by Xbra-GR. Induction of cardiac actin gene expression by bFGF (50 ng/ml) in control animal caps was abolished by injection of RNA encoding XFD (1 ng) under the same conditions. (B) Xbra autoinduction requires FGF signalling. Embryos at the one-cell stage were injected with 25 pg of RNA encoding Xbra-GR and/or 1 ng of RNA encoding XFD. Animal caps were dissected at stage 8, treated as appropriate with 10-6 M DEX and cultured to the equivalent of stage 10.5, when they were analysed for expression of Xbra by RNAase protection. Inhibition of FGF signalling prevented autoinduction of Xbra. Expression of Xbra induced by bFGF treatment (100 ng/ml) in control caps was abolished by injection of RNA encoding XFD (1 ng) under the same conditions. ODC serves as a control for loading in samples in these experiments.