Strong CF et al. (2000), Xbra3 induces mesoderm and neural tissue in Xen...

XB-ART-10911

Dev Biol 2000 Jun 15;2222:405-19. doi: 10.1006/dbio.2000.9710.

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Xbra3 induces mesoderm and neural tissue in Xenopus laevis.

Strong CF , Barnett MW , Hartman D , Jones EA , Stott D .

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Homologues of the murine Brachyury gene have been shown to be involved in mesoderm formation in several vertebrate species. In frogs, the Xenopus Brachyury homologue, Xbra, is required for normal formation of posterior mesoderm. We report the characterisation of a second Brachyury homologue from Xenopus, Xbra3, which has levels of identity with mouse Brachyury similar to those of Xbra. Xbra3 encodes a nuclear protein expressed in mesoderm in a temporal and spatial manner distinct from that observed for Xbra. Xbra3 expression is induced by mesoderm-inducing factors and overexpression of Xbra3 can induce mesoderm formation in animal caps. In contrast to Xbra, Xbra3 is also able to cause the formation of neural tissue in animal caps. Xbra3 overexpression induces both geminin and Xngnr-1, suggesting that Xbra3 can play a role in the earliest stages of neural induction. Xbra3 induces posterior nervous tissue by an FGF-dependent pathway; a complete switch to anterior neural tissue can be effected by the inhibition of FGF signalling. Neither noggin, chordin, follistatin, nor Xnr3 is induced by Xbra3 to an extent different from their induction by Xbra nor is BMP4 expression differentially affected.

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Species referenced: Xenopus laevis
Genes referenced: actc1 actl6a bmp4 chrd eomes fgf4 fst gmnn ncam1 neurog2 nodal3.1 nodal3.2 nog not otx2 tbx2 tbx3 tbx5 tbxt tbxt.2 trg vegt

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	FIG. 3. Spatial distribution of Xbra and Xbra3 mRNA. Embryos were hybridised with antisense RNA probes representing regions of the two transcripts which differed sufficiently to ensure specificity for the two gene products (see text). At stage 12, Xbra3 (A, arrowed) is detected weakly around the blastopore in a pattern similar to that seen with Xbra (B). While Xbra message levels decline at stages 25 (C, arrowed) and 35 (D), Xbra3 is maintained at these stages in notochord and tail tip (C, D, arrowed). Hybridisation with an Xbra3 sense transcript gave no signal (C, D, control). A specific antiserum which recognises Xbra3 and not Xbra protein stains the nuclei of notochord cells at stage 35 (E, arrowed). No staining was seen with preimmune serum (not shown).
	FIG. 1. Comparison of Xbra3 with other T-domain proteins. (A) Alignment of the predicted polypeptide sequence of Xbra3 with Xbra (Smith et al., 1991), mouse Brachyury (Mo T; Herrmann et al., 1990), human T (Hu T; Edwards et al., 1996), chick T (Ch T; Kispert et al., 1995), chick T-box T (Ch TbxT; Knezevic et al., 1997) and zebrafish no-tail (Zf ntl; Schulte-Merker et al., 1994). Dashes have been inserted to introduce gaps to maximise the alignment. An antiserum specific for Xbra3 was raised against a peptide representing residues 420–431 (overlined). (B) Dendrogram of T-box genes produced by the PHYLIP program. Sequences and SwissProt accession numbers (except where indicated) are Brachyury (P20293), human T (Hu-T; translated from GenBank; AJ001699), chicken T-box T (TbxT; P79778), Halocynthia T (As-T; P56158), chicken T (Ch-T; P79777), Drosophila T-related gene (Trg; P55965), Xenopus Brachyury (Xbra; P24781), Xenopus Brachyury 3 (Xbra3; this work), Hemicentrotus T (Hp-T; Q25113), zebrafish T (Zf-T; Q07998), Xenopus Eomesodermin (Eomes; P79944), Xenopus Xombi (Xombi; translated from GenBank; S83518), Xenopus Brat (Brat; P87377), chicken T-box L (TbxL; P79779), human T-box 5 (Hu-Tbx5; Q99593), human T-box 3 (Hu-Tbx3; translated from GenBank; AF002228) and Ascidian T (As-T2; O01409; also from Hemicentrotus).
	FIG. 2. RT-PCR analysis of the temporal expression patterns of Xbra and Xbra3. (A) RNA was extracted from embryos at the stages shown and analysed for the abundance of Xbra and Xbra3 mRNA. (B) Analysis of Xbra and Xbra3 expression in RNA extracted from isolated notochords at stages 12 and 24. All assays were controlled for linearity of the amplification (data not shown).
	FIG. 3. Spatial distribution of Xbra and Xbra3 mRNA. Embryos were hybridised with antisense RNA probes representing regions of the two transcripts which differed sufficiently to ensure specificity for the two gene products (see text). At stage 12, Xbra3 (A, arrowed) is detected weakly around the blastopore in a pattern similar to that seen with Xbra (B). While Xbra message levels decline at stages 25 (C, arrowed) and 35 (D), Xbra3 is maintained at these stages in notochord and tail tip (C, D, arrowed). Hybridisation with an Xbra3 sense transcript gave no signal (C, D, control). A specific antiserum which recognises Xbra3 and not Xbra protein stains the nuclei of notochord cells at stage 35 (E, arrowed). No staining was seen with preimmune serum (not shown).
	FIG. 4. Induction of Xbra and Xbra3 in isolated animal caps by mesoderm-inducing factors. Caps were isolated at stage 9 and treated with either activin or FGF at concentrations from 50 to 3 z 125 ng/ml. RNA was extracted and analysed by RT-PCR at the stages shown. All assays were controlled for linearity of the amplification. Analysis at stage 12 shows that both Xbra and Xbra3 are induced by activin and FGF, with Xbra showing a lower threshold for induction by FGF. At stage 25, a clear difference is seen between Xbra and Xbra3 in the response to both growth factors. Expression of Xbra3 is clearly maintained at high levels, whereas Xbra transcripts are barely detectable.
	FIG. 5. Mesoderm-inducing activity of Xbra3. Embryos were injected with Xbra3 message at the two-cell stage and RNA was extracted from caps isolated at the stages indicated and analysed by RT-PCR. (A) Xbra3 induces mesoderm in an FGF-dependent manner. Xbra is induced after injection of Xbra3 mRNA or treatment with FGF in the absence but not in the presence of XFD. (B) Xbra3 induces markers of dorsal mesoderm and neural tissue. While both Xbra and Xbra3 induce eFGF at stages 12 and 25, only Xbra3 induces cardiac actin and Xnot at stage 25. Induction of NCAM is seen at stages 12 and 25 only in response to Xbra3. (C) Xbra3 induces the early neurogenic genes geminin and Xngnr-1 in animal caps isolated from Xbra3-injected embryos at stage 12, whereas Xbra does not. The cDNA preparations analysed in C are the same as those analysed in Fig. 7.
	FIG. 6. Xbra3 induces posterior neural markers in an FGFdependent manner. Embryos were injected at the two-cell stage with the mRNAs indicated and analysed for markers of neural tissue at stage 25 by RT-PCR. NCAM is induced by Xbra3 in the presence or absence of XFD. The posterior marker Hoxb-9 is induced after injection of Xbra3 alone whereas co-injection of XFD abolishes expression of Hoxb-9 and allows expression of the anterior marker Xotx2.
	FIG. 7. Xbra3 induces early neural genes by a mechanism independent of known neural inducers. Embryos were injected with either Xbra or Xbra3 message at the two-cell stage and caps analysed for known neural-inducing molecules at stage 12 by RT-PCR. Chordin and Xnr3 are not induced by either Xbra or Xbra3. Follistatin and noggin mRNAs are induced by both Xbra and Xbra3 to similar levels at the stages tested. BMP4 expression is not inhibited by the expression of either Xbra or Xbra3. The cDNA preparations analysed are the same as those used in Fig. 5C.
	FIG. 8. Xbra3 can induce Hoxb-9 expression in a non-cellautonomous manner. A partial cDNA representing a Hoxb-9 mRNA was cloned from X. borealis and primers which amplified specifically borealis Hoxb-9 were designed. Caps were isolated from embryos injected with Xbra3 mRNA and cultured to stage 25 by which time Hoxb-9 expression was detected in both species (A). Caps from X. laevis embryos injected with Xbra or Xbra3 RNA were cultured in apposition with caps from uninjected borealis embryos (see B), and RNA from the total sandwiches was extracted and amplified using primers specific for X. borealis Hoxb-9. X. borealis Hoxb-9 was found to be expressed in sandwiches containing laevis caps expressing Xbra3, but not in sandwiches with Xbra-expressing caps (C). In (A) and (C), cDNA input to the RT-PCR was equalised with respect to [32P]GTP incorporation during cDNA synthesis (see Materials and Methods).