December 1, 1997;
The Xenopus Brachyury promoter is activated by FGF and low concentrations of activin and suppressed by high concentrations of activin and by paired-type homeodomain proteins.
of Xenopus laevis arises through an inductive interaction in which signals from the vegetal hemisphere of the embryo
act on overlying equatorial cells. One candidate for an endogenous mesoderm
-inducing factor is activin, a member of the TGFbeta
superfamily. Activin is of particular interest because it induces different mesodermal cell types in a concentration-dependent manner, suggesting that it acts as a morphogen. These concentration-dependent effects are exemplified by the response of Xbra
, expression of which is induced in ectodermal tissue
by low concentrations of activin but not by high concentrations. Xbra
therefore offers an excellent paradigm for studying the way in which a morphogen gradient is interpreted in vertebrate embryos. In this paper we examine the trancriptional regulation of Xbra2, a pseudoallele of Xbra
that shows an identical response to activin. Our results indicate that 381 bp 5'' of the Xbra2 transcription start site are sufficient to confer responsiveness both to FGF and, in a concentration-dependent manner, to activin. We present evidence that the suppression of Xbra
expression at high concentrations of activin is mediated by paired-type homeobox genes such as goosecoid
, Mix.1, and Xotx2
[+] show captions
Sequence of exon 1 of Xbra2 (boldface type) together with 1.2 kb of 5′-flanking sequence. The first 82 bases of intron 1 are also included. Transcription initiation site is indicated by +1 and is also the first nucleotide in boldface type. ATTA sequences are double-underlined, and Antennapedia- and Bicoid-specific sites are marked Ant and Bcd,respectively. This sequence has been submitted to the EMBL database, accession number AJ001528.
Mesoderm-specific expression of reporter gene constructs containing 2.1 kb of Xbra2 5′-flanking sequence. (A) Xbra2.pGL2 or the promoterless plasmid pGL2Basic was coinjected with pSV.β-galactosidase into a single blastomere of either tier A (prospective ectoderm) or tier C (prospective mesoderm) of 32-cell stage embryos (see inset). Embryos were collected for luciferase and β-galactosidase activity assays at stage 12.5. (B) Luciferase activity in extracts of embryos at stage 8, 11, or 14 injected with Xbra2.pGL2 into a single blastomere of either tier A or tier C at the 32-cell stage. (Inset) RNase protection assay for expression ofXbra2.βg in embryos injected into a single blastomere of tier A or tier C and collected for assay at stage 8 or stage 13. EF-1α was used as a loading control. This experiment has been performed four times, with similar results each time. (C,D)Xbra2.GFP was injected equatorially into all four blastomeres of four-cell stage embryos, and distribution of GFP transcripts at stage 10.5 was determined by whole-mount in situ hybridization. Arrows point to the blastopore lip. (E,F) Xbra2.GFP was used to generate transgenic embryos that were analyzed as in C andD at gastrula (E) or tailbud (F; anterior to the left) stage.
FGF and activin activate expression of a reporter gene construct containing 2.1 kb of Xbra2 5′-flanking sequence. Sequences upstream of −381 in the Xbra25′-flanking sequence are dispensable for response of Xbra2to FGF (25 ng/ml) and activin. Embryos were injected with the indicated constructs and reference plasmid and, in some cases, with 1 pg of activin mRNA, as described in Materials and Methods. Normalized firefly luciferase activity from stage 12.5 caps is expressed as fold activation over untreated caps. (n) Number of experiments.
Activation by FGF and components of the MAP kinase pathway of a reporter gene construct containing 2.1 kb ofXbra2 5′-flanking sequence. Activation of the same reporter in response to activin, and of a reporter containing 381 bp of flanking sequence, is dose dependent. (A) Normalized luciferase activity in animal caps injected with Xbra2.pGL2. One group of caps was also injected with v-Ras mRNA, a second with MEK1S217E/S221E, and a third was treated with FGF-2. Analysis was performed at control stage 12.5. This experiment has been performed 3 times with MEK1S217E/S221Ealone, 10 times with v-Ras alone, and once with MEK1S217E/S221E and v-Ras in the same experiment. Similar results were obtained each time. (B) Normalized luciferase activity in animal caps treated with FGF-2 or injected with different amounts of activin mRNA (in this experiment “low” represents 1 pg, “medium” represents 5 pg, and “high” represents 50 pg). Analysis was at control stage 12.5. Half of each group of caps was assayed for luciferase and β-galactosidase activities, and the other half was assayed for expression of endogenous Xbra2 by RNase protection. Note that high levels of activin suppress expression of both endogenousXbra2 and the reporter construct. This experiment has been performed out three times, with similar results each time. (C) A similar experiment to that in B using −381Xbra2.pGL2 (in this experiment “low” represents 1 pg and “high” represents 100 pg activin RNA).
Suppression of Xbra expression in gastrula-stage embryos by paired-type homeodomain proteins. Embryos at the four-cell stage were injected equatorially with 500 pg ofgoosecoid (A), Xotx2 (B), orXotx2 K → E (C) RNA in one blastomere, and the expression pattern of Xbra was analyzed by whole-mount in situ hybridization at the early gastrula stage. Note lack of Xbraexpression in part of the equatorial region of embryos in Aand B. All embryos shown are from the same experiment; that inC is at a slightly later stage than those in A andB. (D) Suppression of FGF-induced Xbra2 mRNA in animal caps by goosecoid, Mix.1, and Xotx2 but not by Xotx2 K → E. Embryos at the one-cell stage were injected with 1 ng of the indicated RNAs, and animal caps were cut at stage 8 and incubated in FGF until stage 12. Xbra2 and EF-1α transcripts were detected by RNase protection. Note that FGF-induced expression of Xbra2 is strongly inhibited bygoosecoid, Mix.1, and Xotx2 but not by Xotx2K → E. (E) Suppression of FGF-inducedXbra2.pGL2 luciferase activity in animal caps bygoosecoid, Mix.1, and Xotx2 but not by Xotx2K → E. Embryos were coinjected with 1 ng of the indicated RNAs, 30 pg of Xbra2.pGL2, and 10 pg of pRL–SV reference plasmid. Animal caps were excised at stage 8 and incubated with FGF until stage 12.5, and firefly and Renilla luciferase activities were determined. Activity of Xbra2.pGL2 is presented as fold activation over uninduced levels. This experiment was repeated twice for all four RNAs and four additional times with goosecoid, with similar results.
goosecoid, Xotx2, and Mix.1 bind to the −174/−152 region of the Xbra2promoter. goosecoid, Xotx2, and Mix.1 were translated in a coupled transcription–translation system; the DNA template used in each reaction is indicated above the brackets. In some cases, binding reactions were preincubated with a 200-fold molar excess of specific competitor (unlabeled −174/−152 oligonucleotide, S) or with a 200-fold molar excess of irrelevant competitor (unlabeled −43/−19 oligonucleotide, I). Complexes were resolved on a 4% polyacrylamide gel. The asterisk (*) indicates the position of a nonspecific DNA-binding complex; this complex varied between lanes and between different experiments.
Suppression of −381Xbra2.luciferase activity by goosecoid requires Antennapedia and Bicoid homeodomain-binding sites. (A) Embryos were injected with 15 pg mutated −381Xbra2.firefly luciferase (see C), 15 pg −381Xbra2.Renilla luciferase, and 30 pg pEF-1α.β-galactosidase; in addition to this combination of DNAs, some embryos were injected with 1 pg of activin mRNA or with a mixture of 1 pg of activin mRNA and 1 ng of goosecoid mRNA. Animal caps were excised at the blastula stage and incubated until stage 12.5, when firefly and Renilla luciferase and β-galactosidase activities were determined. The graph shows fold induction by activin in the presence or absence of goosecoid, as obtained with the wild-type and mutated reporters. Normalized luciferase activities from four independent experiments were used in this figure. Bars showS.D.s. (B) NIH-3T3 cells were cotransfected in duplicate with a combination of CMV overexpression vector (either empty or goosecoid expressing), wild-type or mutatedXbra2/SV40 reporter, and reference plasmid. Luciferase activities were determined, and the results are expressed as the fold repression of reporter activity by goosecoid for each construct. Data from four experiments was used for this graph. Bars show S.D.s. (C) Sequence of the wild-type and the mutant Antennapedia and Bicoid sites.