Conlon FL et al. (1996), Inhibition of Xbra transcription activation cau...

XB-ART-17920

Development 1996 Aug 01;1228:2427-35. doi: 10.1242/dev.122.8.2427.

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Inhibition of Xbra transcription activation causes defects in mesodermal patterning and reveals autoregulation of Xbra in dorsal mesoderm.

Conlon FL , Sedgwick SG , Weston KM , Smith JC .

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The Brachyury (T) gene is required for formation of posterior mesoderm and for axial development in both mouse and zebrafish embryos. In this paper, we first show that the Xenopus homologue of Brachyury, Xbra, and the zebrafish homologue, no tail (ntl), both function as transcription activators. The activation domains of both proteins map to their carboxy terminal regions, and we note that the activation domain is absent in two zebrafish Brachyury mutations, suggesting that it is required for gene function. A dominant-interfering Xbra construct was generated by replacing the activation domain of Xbra with the repressor domain of the Drosophila engrailed protein. Microinjection of RNA encoding this fusion protein allowed us to generate Xenopus and zebrafish embryos which show striking similarities to genetic mutants in mouse and fish. These results indicate that the function of Brachyury during vertebrate gastrulation is to activate transcription of mesoderm-specific genes. Additional experiments show that Xbra transcription activation is required for regulation of Xbra itself in dorsal, but not ventral, mesoderm. The approach described in this paper, in which the DNA-binding domain of a transcription activator is fused to the engrailed repressor domain, should assist in the analysis of other Xenopus and zebrafish transcription factors.

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Species referenced: Xenopus
Genes referenced: cat.2 foxa4 lgals4.2 sst.1 tbx2 tbxt

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	Fig. 1. Transcription activation by Xbra and Ntl. Transcription activation by Xbra and Ntl in yeast was demonstrated by induction of the expression of a GAL4UASlacZ reporter gene in the presence of a plasmid encoding the S. cerevisiae GAL4 DNA-binding domain fused to either Xbra or Ntl coding sequences. Top row: transcription activation by full-length GAL4 (positive control) and by fusions of GAL4DBD to Xbra and Ntl, respectively. Bottom row: lack of transcription activation by fusions of GAL4DBD to human lamin C (negative control), to Xbra truncated at amino acid 303, and to Ntl truncated at amino acid 334.
	Fig. 2. Mapping of the Xbra and Ntl transcription activation domains using a modification of the assay described in Fig. 1. The GAL4 DNA-binding domain (DBD) was fused to a 3¢ deletion series of Xbra or Ntl generated by transposition mutagenesis or to 5¢ truncations and internal deletions generated by conventional techniques. Strong activation: ‘+++’; weaker activation: ‘++’; no activation: ‘-’. (A) Representative examples of results obtained with Xbra deletions. The region mapped as the Xbra activation domain is shown at the bottom. This region gives slightly lower activation than that obtained by the full-length protein, and it is likely that a small number of flanking amino acids are required for full induction of transcription. (B) Representative examples of results obtained with Ntl deletions. The region mapped as the Ntl activation domain is shown at the bottom. For both Xbra and Ntl, additional deletions and truncations gave results consistent with those described here. Interruptions of coding sequence due to the ntlb160 and ntlb195 mutations are indicated. In both A and B, the DNA-binding domain, shown in red, is based on Kispert and Herrmann (1993) together with band-shift analyses using Xbra and Ntl deletions (not shown).
	Fig. 3. Transcription activation by Xbra and Ntl and repression of activation by a dominant interfering Xbra-EnR construct. NIH3T3 cells were lipofected with the indicated effector plasmids together with a chloramphenicol acetyl transferase (CAT) reporter plasmid carrying two copies of the Brachyury-binding site (Kispert and Herrmann, 1993) upstream of a minimal promoter. Plasmids encoding wild-type Xbra (lane 2) or wild-type Ntl (lane 4) cause activation of transcription while the Xbra and Ntl alleles lacking the putative activator domain (see Fig. 2) have little or no effect (lanes 3 and 5). A plasmid encoding an Xbra allele in which the activation domain is replaced by the Drosophila engrailed repressor domain (Xbra-EnR) causes no activation (lanes 6 and 8) and it inhibits activation due to Xbra in a dose-dependent manner, with complete inhibition obtained with a two-fold excess of Xbra-EnR over Xbra (lanes 7 and 9). This experiment was carried out three times, with similar results obtained each time. Loading was normalised by reference to levels of b-galactosidase activity derived from the co-transfected MLVlacZ plasmid. Mean levels of stimulation are indicated. Xbra did not cause transcription activation in the absence of a Brachyury-binding site (not shown).
	Fig. 4. Construction of Xbra-EnR. The entire carboxy terminal of Xbra, including the transcription activation domain, was removed by digestion with ClaI and the DNA-binding domain was then fused to amino acids 2-298 of the Drosophila engrailed protein.
	Fig. 5. Xbra-EnR interferes with gastrulation movements in Xenopus. (A,C) Control embryos. (B,D) Embryos injected with RNA encoding Xbra-EnR. Embryos are at stages 10.5 (A,B) and stage 14 (C,D). Note that the blastopore is slow to close in embryos injected with Xbra-EnR.
	Fig. 6. Phenotype of Xenopus embryos injected with RNA encoding Xbra-EnR. (A) Control, uninjected embryo. Embryos injected with RNA encoding EnR alone are indistinguishable from such controls. (B,C) Embryos injected with 0.5 ng RNA encoding Xbra-EnR lack posterior structures. (D) Histological section of an embryo injected with 0.5 ng RNA encoding EnR alone. This embryo is indistinguishable from sections of control embryos. Note notochord (not) and muscle (mus). (E) Histological section of an embryo injected with RNA encoding Xbra-EnR. This embryo lacks posterior structures but has formed notochord (not) and somitic muscle (mus) anteriorly. (F–H) Embryos stained with the notochord-specific antibody MZ15. (F) Control embryo. (G) Embryo injected with RNA encoding Xbra-EnR; notochord is present anteriorly. (H) Embryo injected with RNA encoding Xbra-EnR; notochord is absent. (I,J) Embryos stained with the muscle-specific antibody 12/101. (I) Control embryo. (J) Embryo injected with RNA encoding Xbra-EnR; muscle is present anteriorly. Results similar to these were obtained following injection of 0.2 and 1.0 ng Xbra-EnR RNA (not shown).
	Fig. 7. Inhibition of notochord differentiation by Xbra-EnR revealed using Tor70 staining. (A) Control, uninjected embryo. (B,C) Embryos injected with 0.5 ng RNA encoding Xbra- EnR. Note normal notochord in A, anterior notochord in B and lack of notochord in C.
	Fig. 8. Co-injection of RNA encoding wild-type Xbra brings about partial rescue of the effects of Xbra-EnR. (A) Control embryo. (B) Embryo injected with 1 ng RNA encoding Xbra-EnR. (C) Embryo injected with 1 ng RNA encoding Xbra-EnR together with 2 ng RNA encoding wild-type Xbra. Note that posterior structures are more complete in C than in B, although anterior structures in C are reduced.
	Fig. 9. Phenotype of zebrafish embryos injected with RNA encoding Xbra-EnR. (A) Wild-type zebrafish embryo approximately 24 hours after fertilization. (B) Sibling ntl mutant embryo at the same stage as that in A. (C) Control uninjected zebrafish embryo approximately 24 hours after fertilisation. (D) Sibling zebrafish embryo injected with RNA encoding Xbra-EnR at the same stage as that in C. Posterior tissue is greatly reduced but anterior structures such as the eye (arrow) are present. (E) High-power view of the somites and notochord of a control zebrafish embryo in the trunk region. Note the highly vacuolated cells of the notochord (not) and the chevronshaped somites (som). (F) High-power view of the trunk region of a zebrafish embryo injected with RNA encoding Xbra-EnR. The notochord (not) is poorly differentiated, the somites (som) are only slightly chevron-shaped and they are fused at the ventral midline. In all these respects embryos injected with RNA encoding Xbra-EnR resemble the ntl mutation (Halpern et al., 1993).
	Fig. 10. In situ hybridization analysis of embryos injected with RNA encoding Xbra-EnR. (A–D) Embryos hybridised with an Xbra probe at stage 10 (A,B) and stage 14 (C,D). Control embryos at the early gastrula stage express Xbra throughout the marginal zone (A). Control embryos at the neurula stage express Xbra in the notochord and in posterior cells (C). Embryos injected with RNA encoding Xbra-EnR do not express Xbra in the organizer at the early gastrula stage (B; see left-hand side of embryo) or in the notochord at the neurula stage (D). The greater intensity of staining in the ventral region of the embryo shown in B is not a consistent observation and decreases in endogenous levels of Xbra have been confirmed by RNAase protection (not shown). (E–H) Embryos hybridised with a Pintallavis probe at stage 10 (E,F) and stage 14 (G,H). Expression of Pintallavis in embryos injected with RNA encoding Xbra-EnR (F,H) is similar to that in controls (E,G).