1 Division of Developmental Biology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK and Wellcome Trust/Cancer Research UK Institute and Department of Zoology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
2 Whitehead Institute and Massachusetts Institute of Technology, Nine Cambridge Center, Cambridge, MA 02142-1479, USA
* Present address: Merck & Co. Inc., PO Box 4, West Point, PA 19486, USA
Author for correspondence (e-mail: jim{at}welc.cam.ac.uk)
Accepted 2 July 2002
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SUMMARY |
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Key words: Xenopus, Mesoderm induction, derrière, TGFß family, T-box family, VegT, Smad, Fast
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INTRODUCTION |
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Although these factors play important roles in the early embryo, rather little is known about the transcriptional regulation of the nodal-related genes or of derrière. One of the most significant observations in this respect concerns the requirement for VegT in their activation. VegT is a maternally expressed member of the T-box family whose transcripts are restricted to the vegetal hemisphere of the Xenopus egg and early embryo (Lustig et al., 1996; Stennard et al., 1996
; Zhang and King, 1996
; Horb and Thomsen, 1997
). Ablation of maternal VegT transcripts causes loss of endodermal and mesodermal structures, and expression of the nodal-related genes and of derrière does not occur (Zhang et al., 1998
; Kofron et al., 1999
; Takahashi et al., 2000
). VegT is a transcriptional activator, and it is possible that the nodal-related genes and derrière number among its direct targets. However, only Xnr1 has been shown to contain T-box binding sites in its promoter, and these sites appear not to be required for the vegetal expression of a reporter gene driven by the Xnr1 promoter (Hyde and Old, 2000
).
We have investigated the transcriptional regulation of derrière. This gene has been little-studied compared with the nodal-related genes, yet is a strong candidate for an endogenous inducing agent: it is expressed in the right cells, inhibition of its function impairs mesoderm development (Sun et al., 1999), and in this paper we show that derrière, unlike, for example, Xnr2, is able to exert long-range effects in the developing embryo. Our results show that derrière is subject to complex regulation involving not only VegT but also members of the TGFß family and perhaps FGF family members. The combined effects of these different signals, acting in a series of autoregulative loops, may help ensure the rapid activation of mesoderm-inducing agents at the mid-blastula transition.
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MATERIALS AND METHODS |
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Animal cap recombinants were performed as described previously (Jones et al., 1996). Conjugates were fixed when sibling embryos reached stage 10.5 and expression of Xbra (Smith et al., 1991
) was analysed by in situ hybridisation.
Constructs
pSP64T.VegT-GR was produced by fusing the ligand-binding domain of the human glucocorticoid receptor (hGR) to the C terminus of VegT, thereby creating a construct similar to the previously published Xbra-GR (Tada et al., 1997). Details are available on request. A mouse activin A cDNA (Albano et al., 1993
) was cloned into pSP64T to create pSP64T.mactivinA. pCS2-derrière (Sun et al., 1999
), pSP64T.Xnr2 (Jones et al., 1995
) p
XAR (Hemmati-Brivanlou and Melton, 1992
), pXFD/Xss (Amaya et al., 1991
), pd50 (Amaya et al., 1991
) and placZ (Kolm et al., 1997
) were as described. Capped RNAs were synthesised using SP6 RNA polymerase.
To obtain derrière promoter sequence, a Xenopus genomic library, prepared in the vector FIXII (Stratagene), was screened using a probe corresponding to the first 356 base pairs of the derrière open reading frame. Restriction digestion and Southern blotting identified a 2 kb XbaI fragment, which was sub-cloned into pBluescriptII (SK) and sequenced. This fragment (d1.2.1) consists of the 247 base pair exon 1 of derrière preceded by 850 base pairs of upstream sequence and 844 base pairs of intron (see Fig. 2A). The transcription start site was mapped by RNAase protection and is located approximately 30 base pairs downstream of a TATA box.
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Electrophoretic mobility shift assays
Electrophoretic mobility shift assays (EMSAs) were performed as described by Trindade and colleagues (Trindade et al., 1999). Proteins used in EMSAs were produced by in vitro translation of synthetic RNA. pSP64TBX.VegT-HA (Conlon et al., 2001
) was linearised with SalI and transcribed with SP6 RNA polymerase and pFTX9-XlFast1 (Howell et al., 1999
) was linearised with XbaI and transcribed with T7 RNA polymerase.
Luciferase assays
Luciferase assays were performed using the Promega Dual-Luciferase assay kit. Embryos were injected with 20 pg pGL3-basic containing the appropriate promoter fragment, 20 pg pRL-SV40/TK, and an appropriate amount of RNA encoding either ß-galactosidase or the inducing agent being tested. Animals caps were dissected at stage 8.5 and cultured in 75% NAM for the desired period. They were then suspended in 10 µl of 1x Passive Lysis Buffer per cap and after centrifugation 5 µl was taken for assay. Oocytes were suspended in 20 µl 1x Passive Lysis Buffer per oocyte and 20 µl was taken for assay. All values are expressed as Relative Luciferase Units (Firefly luciferase activity/Renilla luciferase activity).
Transgenesis
Transgenic Xenopus embryos were created as described by Sparrow and colleagues (Sparrow et al., 2000), itself a modified version of the original protocol of Kroll and Amaya (Kroll and Amaya, 1996
).
Whole-mount in situ hybridisation
In situ hybridisations were carried out essentially as described previously (Harland, 1994), except that BM purple was used as a substrate. derrière (Sun et al., 1999
) and VegT (Zhang and King, 1996
) probes were as described. A GFP construct (mgfp5) (Zernicka-Goetz et al., 1996
) in pBluescriptII(SK) was linearised with NcoI and a Xenopus Brachyury construct (pXT1) (Smith et al., 1991
) was linearised with BglII. Both were transcribed with T7 RNA polymerase in the presence of digoxigenin-11-UTP (Roche). ß-galactosidase staining was performed as described previously (Kolm and Sive, 1995
).
RNA isolation and RNAase protection assays
RNA was prepared from pooled animal caps using the acid guanidinium thiocyanate-phenol-chloroform method (Chomczynski and Sacchi, 1987). RNAase protection analysis was carried out essentially as described by Jones and colleagues (Jones et al., 1992
), using RNAase T1 alone for all probes. A derrière probe was made by cloning a PCR fragment of derrière (nucleotides 775-939) into pBluescriptII, linearising with NotI and transcribing with T7 RNA polymerase. Probes for Bix4 (Casey et al., 1999
) and ornithine decarboxylase (Isaacs et al., 1992
) were as described.
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RESULTS |
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The signalling range of derrière was tested by the technique employed by Jones and colleagues (Jones et al., 1996). Animal pole regions derived from embryos injected with RNA encoding derrière were juxtaposed with animal caps dissected from embryos injected with the lineage label fluorescein-lysine-dextran (FLDx). As controls, caps were derived from embryos injected with RNA encoding activin, which is known to exert long-range effects, or with RNA encoding Xnr2, which in this assay acts essentially cell-autonomously (Jones et al., 1996
). The conjugates were cultured for 3 hours and then fixed and analysed for expression of Xbra by in situ hybridisation. Fig. 2 shows that derrière, like activin and unlike Xnr2, can activate expression of Xbra in FLDx-labelled cells, indicating that it can exert long-range effects.
Interestingly, the pattern of Xbra activation in derrière-expressing conjugates differs from that in activin-expressing conjugates. In the former, Xbra is activated throughout the derrière-expressing animal cap and appears to spread from there into the FLDx-labelled tissue. In the latter, and as described by others, Xbra is most strongly induced in the FLDx-labelled animal cap, in a domain that presumably corresponds to a particular concentration of activin. We are now investigating whether this difference is due to different diffusion properties of the two factors (see Ohkawara et al., 2002) or to their different concentration-dependent effects. Whatever the explanation, the results show clearly, at least in this over-expression system, that derrière is capable of exerting long-range effects in the Xenopus embryo.
derrière is an immediate-early target of VegT
Ablation of mRNA encoding the vegetally localised transcription factor VegT prevents zygotic expression of derrière in the Xenopus embryo, indicating that VegT is essential for expression of derrière during late gastrula stages of development (Kofron et al., 1999). Is derrière a direct target of VegT? To answer this question we constructed a hormone-inducible version of VegT termed VegT-GR, in which the ligand-binding domain of the glucocorticoid receptor is fused to the C terminus of VegT. This construct is inactive unless the steroid hormone dexamethasone (DEX) is added to the embryo culture medium (data not shown). In combination with the protein synthesis inhibitor cycloheximide (CHX), this construct allows one to examine the ability of VegT to activate derrière directly.
RNA encoding VegT-GR was injected into Xenopus embryos at the 1-cell stage. Animal pole regions were then dissected at the late blastula stage, and groups of 15-20 animal caps were incubated in the absence of factors, in DEX or CHX alone, or in both reagents. Derrière proved to be activated by dexamethasone even in the presence of cycloheximide (Fig. 3), indicating that the effects of VegT do not require intervening protein synthesis. This observation is consistent with the suggestion that VegT acts directly to induce expression of derrière. We note, however, that the level of activation of derrière by VegT-GR is reduced by cycloheximide, and this does not occur with the induction of Bix4 (Fig. 3). As we discuss below, one explanation of this observation is that optimal activation of derrière by VegT involves some indirect effects.
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The derrière promoter responds to TGFß signals through Fast sites
VegT activates the expression of the nodal-related genes Xnr1-4 as well as that of derrière itself (Clements et al., 1999; Kofron et al., 1999
; Sun et al., 1999
; Yasuo and Lemaire, 1999
). One possibility, therefore, is that the indirect induction of derrière reporter constructs by VegT occurs through activation of TGFß family members such as these. Consistent with this idea we have identified two potential Fast sites in the derrière 5' regulatory region (Fig. 4), and electrophoretic mobility shift assays confirm that these do indeed bind Fast-1 (Fig. 8).
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DISCUSSION |
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One noteworthy feature of derrière is that it is able to exert long-range effects in our animal cap assay, whereas Xnr2, for example, is not. In this regard, we note that although the N-terminal region of derrière contains a group of four basic amino acids (KKRR), this is not as extensive a domain as the basic region in BMP4 that interacts with heparan sulphate proteoglycans and thereby restricts its signalling range (Ohkawara et al., 2002). The ability of derrière to exert long-range effects in the Xenopus embryo, together with results obtained following inhibition of its activity (see above) mark this protein out as a strong candidate for an endogenous mesoderm-inducing factor, and makes analysis of its regulation all the more significant.
derrière is a direct target of VegT
Experiments using a hormone-inducible version of VegT and cycloheximide suggest that derrière is a direct target of VegT (Fig. 3). Our results indicate, however, that cycloheximide does cause some reduction in the level of activation of derrière by VegT-GR, a phenomenon we do not observe with induction of Bix4 (Fig. 3). This reduction is consistent with the conclusion of this paper that the activation of derrière by VegT also involves indirect effects, mediated in part by the induction of TGFß family members.
The direct action of VegT on the 5' regulatory region of derrière is likely to occur through the T box sites highlighted in Fig. 4. These sites are required for the early activation of a derrière reporter construct in isolated animal pole regions (Fig. 10A) and for the activation of such constructs in Xenopus oocytes, in which indirect activation does not occur (Fig. 10B). Consistent with binding site selection experiments (Kispert and Herrmann, 1993; Conlon et al., 2001
), the site designated Tbs1 (TGACACCT) proved to interact more strongly with VegT in electrophoretic mobility shift assays than did Tbs2 (AGACACCT) (Fig. 6). We note that the nucleotide that differs between the two sites (the first T in Tbs1 becomes an A in Tbs2) is directly contacted by Xbra in the crystal structure of that protein (Muller and Herrmann, 1997
).
Mutation of Tbs1 did not, however, completely prevent reporter gene activation in response to VegT in Xenopus oocytes (Fig. 10B), indicating that Tbs2 may also play a role in the activation of derrière by VegT. It is possible that the two sites cooperate to ensure the normal regulation of the gene, in a manner resembling that suggested to occur in the regulation of Ci-trop in Ciona intestinalis (Di Gregorio and Levine, 1999). This is under investigation.
The derrière promoter is also subject to indirect regulation
Although the T box sites in the derrière promoter are required for the early response to VegT and for activation in a system such as the Xenopus oocyte where indirect effects do not occur, they are not required for later responses. Thus, mutation of the T box sites does not prevent the vegetal activation of derrière reporter constructs in transgenic Xenopus embryos (Fig. 7C-F), and nor does it prevent induction by VegT of similar constructs in animal pole regions following culture for 3, rather than 2 hours (Fig. 7B). Our results suggest that the activation of derrière reporter constructs under these conditions occurs indirectly, due, at least in part, to the activation of members of the TGFß family such as the nodal-related genes and perhaps even derrière itself. Thus activin, a member of the TGFß family, can induce expression of endogenous derrière and can activate the expression of derrière reporter constructs. This inducing activity is likely to occur through the Fast sites, as shown in Fig. 4; deletion analysis demonstrates that loss of the Fast sites reduces significantly the ability of activin to induce reporter gene expression (Fig. 9A), although there may be indirect effects at work here too, because there remains some residual activity that is substantially abolished by deletion of the T box sites (Fig. 9B). It is possible that while VegT can exert indirect effects through the activation of TGFß family members, TGFß family members can exert indirect effects through the activation of T box genes. These might include Xbra (Smith et al., 1991) and eomesodermin (Ryan et al., 1996
), as well as Antipodean (Stennard et al., 1996
), itself an alternatively spliced isoform of VegT (Stennard et al., 1999
).
Investigation of this indirect pathway will require carefully timed experiments that make use of specific inhibitors of particular members of the TGFß family. Our initial experiments along these lines indicate that a truncated activin receptor does not prevent the initial activation of derrière but does inhibit its maintenance (Fig. 11). Interestingly, a recent report making using of dominant-negative versions of Xnr5 and Xnr6 found no evidence of down-regulation of derrière at any stage, even though the Xnr5 construct also inhibited the functions of Xnr2, Xnr4, Xnr6, derrière itself and BVg1 (Onuma et al., 2002). This suggests that maintenance of derrière expression can occur through Xnr1 or activin signalling.
Other regulatory elements may also play a role in derrière regulation
The data described so far suggest that VegT and members of the TGFß family participate in a network of autoregulatory loops (see Fig. 12). But VegT and members of TGFß family are unlikely to be the only members of this network, and we may not have identified all the regulatory elements in the derrière promoter. For example, expression of our reporter construct persists in involuted mesoderm (Fig. 5D); as discussed above, this might reflect perdurance of GFP RNA, but it may be that our constructs lack an element that is responsible for the down regulation of derrière following involution. Furthermore, we find that the late response to VegT, and the expression of reporter constructs in transgenic embryos, is not abolished (and if anything is slightly elevated) even if both the Fast sites and the T-box sites are mutated (data not shown). This suggests that there may be another gene X that is regulated by VegT and which acts on the derrière promoter to enhance and maintain its expression (Fig. 12). Candidates for such a gene include members of the FGF family; inhibition of FGF function does not inhibit the initial expression of derrière but does prevent its continued expression (Fig. 11).
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ACKNOWLEDGMENTS |
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