Analysis of Competence and of Brachyury Autoinduction by Use of Hormone- Inducible Xbra

Development 124, 2225-2234 (1997) 2225 Printed in Great Britain © The Company of Biologists Limited 1997 DEV3510

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

M. Tada, M.-A. J. O’Reilly and J. C. Smith* Division of Developmental Biology, NIMR, The Ridgeway, Mill Hill, London NW7 1AA, UK *Author for correspondence (e-mail: [email protected])

SUMMARY

Analysis of gene function in Xenopus development fre- Addition of dexamethasone, which binds to the glucocorti- quently involves over-expression experiments, in which coid receptor and releases Xbra, causes formation of RNA encoding the protein of interest is microinjected into mesoderm. We have used this approach to investigate the the early embryo. By taking advantage of the fate map of competence of animal pole explants to respond to Xbra- Xenopus, it is possible to direct expression of the protein to GR, and have found that competence persists until late particular regions of the embryo, but it has not been gastrula stages, even though by this time animal caps have possible to exert control over the timing of expression; the lost the ability to respond to mesoderm-inducing factors protein is translated immediately after injection. To such as activin and FGF. In a second series of experiments, overcome this problem in our analysis of the role of we demonstrate that Xbra is capable of inducing its own Brachyury in Xenopus development, we have, like Kolm and expression, but that this auto-induction requires intercel- Sive (1995; Dev. Biol. 171, 267-272), explored the use of lular signals and FGF signalling. Finally, we suggest that hormone-inducible constructs. Animal pole regions derived the use of inducible constructs may assist in the search for from embryos expressing a fusion protein (Xbra-GR) in target genes of Brachyury. 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 Key words: Xenopus, mesoderm, Brachyury, steroid receptor, sequestered by the heat-shock apparatus of the cell. competence, target gene, FGF

INTRODUCTION liberated the fusion protein and caused the formation of ectopic muscle. Analysis of gene function in Xenopus laevis is frequently Mesoderm induction in amphibian development is believed carried out by means of over-expression experiments, in which to involve members of the FGF and TGF-β families (see Slack, RNA encoding the protein of interest is injected into the early 1994). One of the direct targets of these mesoderm-inducing embryo. It is possible, in this way, to exert control over which factors is Xenopus Brachyury (Xbra) (Smith et al., 1991), a cells express the protein by taking advantage of the accurate gene expressed throughout the presumptive mesoderm at the fate map of Xenopus (Dale and Slack, 1987; Moody, 1987). early gastrula stage, with expression later becoming restricted One difficulty with this approach, however, is that injected to notochord and posterior tissues (Herrmann et al., 1990; RNA is translated immediately (Snape and Smith, 1996) and Wilkinson et al., 1990; Smith et al., 1991; Schulte-Merker et this may lead to difficulties in interpreting the results of exper- al., 1992). Ectopic expression of Xbra in animal pole explants iments of this sort. For example, expression of Xwnt-8 by RNA is sufficient to induce mesoderm formation in a concentration- injection mimics the dorsalising action of the Spemann dependent manner. Low concentrations cause the formation of organizer, while expression at the early gastrula stage, under ventral mesoderm, including mesothelium and mesenchyme, the control of the cytoskeletal actin promoter, causes formation while higher concentrations induce dorsal mesoderm including of ventral mesoderm (Christian and Moon, 1993). In order to somitic muscle (Cunliffe and Smith, 1992, 1994). Expression exert control over when exogenous proteins become functional, of Xbra alone cannot induce notochord, but co-expression with and thereby overcome this problem, Kolm and Sive (1995) either noggin or Pintallavis, each of which is expressed in used techniques borrowed from work in cell culture (Hollen- dorsal mesoderm, does cause formation of notochord (Cunliffe berg et al., 1993) and microinjected Xenopus embryos with and Smith, 1994; O’Reilly et al., 1995). RNA encoding a fusion protein in which MyoD is fused to the Brachyury encodes a nuclear DNA-binding protein (Kispert hormone-binding domain of the glucocorticoid receptor. In the and Herrmann, 1993; Cunliffe and Smith, 1994) and functions absence of hormone, the MyoD fusion protein was sequestered as a transcription activator (Kispert et al., 1995; Conlon et al., by the heat-shock apparatus of the cell, and the embryo 1996). The mechanism by which Brachyury induces mesoderm developed normally. Addition of dexamethasone, however, is, however, poorly understood, and nothing is known about 2226 M. Tada, M.-A. J. O’Reilly and J. C. Smith potential target genes. In this paper, we begin to investigate unit of activin activity is defined by Cooke et al. (1987). Dexametha- these questions using an inducible version of Xbra in which sone (Sigma) was dissolved in ethanol at 2 mM and then diluted to a the protein is fused to the hormone-binding domain of human final concentration of 1 µM in 75% NAM for animal caps or in 10% glucocorticoid receptor, thus generating Xbra-GR. The activity NAM for whole embryos. of Xbra-GR is tightly regulated by dexamethasone (DEX) both Cell dispersal and protein synthesis inhibition in animal pole explants and in whole embryos. We first use this experiments construct to study the competence of animal pole tissue to form Cell dispersal and protein synthesis inhibition experiments were mesoderm in response to Xbra. Our results show that com- performed as described by Smith et al. (1991), except that cyclohex- petence persists well into gastrulation, when animal caps can imide was applied continuously at a concentration of 10 µM. no longer respond to activin or FGF. The second group of experiments addresses the question of target genes, where we In vitro transcription, in vitro translation and investigate the mechanism of Brachyury autoinduction. DEX- immunoprecipitation induced transactivation of the endogenous Xbra gene in animal All pSP64T constructs were linearised with SalI and RNA was syn- pole explants expressing Xbra-GR is inhibited by co- thesized according to Smith (1993). RNA encoding nucβ-gal was tran- β expression of a truncated FGF receptor, and does not occur in scribed from pSP6nuc Gal (Smith and Harland, 1991) and RNA dissociated animal pole blastomeres. These results indicate that encoding the dominant negative FGF receptor, XFD, was transcribed from pXFD/xss (Amaya et al., 1991). In vitro translations of HA- the direct interaction of Xbra with its own promoter is not suf- tagged constructs was carried out in rabbit reticulocyte lysate in the ficient to drive transcription, and that Xbra autoinduction presence of [35S]methionine, using 200 ng of synthetic RNA, requires intercellular signals, presumably through secretion of according to the manufacturer’s recommendations (Promega). a member of the FGF family. In future work we hope to use Immunoprecipitation was performed as described by Cunliffe and this system to isolate genes that are direct targets of Xbra. Smith (1994) with the use of anti-HA monoclonal antibody 12CA5. Western blotting Extraction of protein from animal caps was as described by Cunliffe MATERIALS AND METHODS and Smith (1994). Protein extracted from the equivalent of two animal caps was analysed on a 12% acrylamide gel by SDS-PAGE and then Construction of inducible versions of Brachyury blotted to a PVDF membrane (Immobilon, Millipore) by wet elec- The Xbra-GR construct was generated by fusing the coding region trophoretic transfer. The membrane was reacted with anti-HA mono- (amino acids 1-432) of pSP64T-Xbra (Cunliffe and Smith, 1992) to a clonal antibody 12CA5 and subsequently with anti-mouse IgG con- fragment encoding the hormone-binding domain (amino acids 512- jugated with alkaline phosphatase (Sigma) followed by detection with 777) of the human glucocorticoid receptor (hGR) derived from NBT and BCIP. plasmid p64T-MyoD-GR (Hollenberg et al., 1993) by in vitro muta- genesis and PCR. The GR construct was generated by addition of an Histology, immunocytochemistry and β-gal staining initiation methionine at the amino-terminus of the hormone-binding For histological analysis, specimens were fixed, sectioned and stained domain of hGR using PCR. Fragments encoding Xbra-GR and GR as described by Smith (1993). Whole-mount and tissue section were cloned into the vector pSP64T (Krieg and Melton, 1984) and immunocytochemistry with monoclonal antibodies MZ15 (Smith and designated pSP64T-Xbra-GR and pSP64T-GR. Two copies of the HA Watt, 1985) or 12/101 (Kintner and Brockes, 1984), specific for epitope (YPYDVPDYA) (Field et al., 1988), which can be recognized notochord and muscle, respectively, was performed as described by by the monoclonal antibody 12CA5 (Boehringer Mannheim), were Smith (1993) and Cunliffe and Smith (1994). β-gal staining was introduced at the carboxy-terminus of these proteins to generate carried out essentially as described by Whiting et al. (1991). Embryos pSP64T-Xbra-HA, pSP64T-Xbra-GR-HA and pSP64T-GR-HA. were fixed in 1% paraformaldehyde, 0.2% glutaraldehyde, 2 mM TM An Xbra-ER construct was generated by replacing the region MgCl2, 5 mM EGTA, and 0.02% NP-40 in PBS at 4¡C for 1 hour. encoding the hormone-binding domain of Xbra-GR with a fragment They were then washed in PBS containing 0.02% NP-40 and stained encoding a modified ligand-binding domain of the murine estrogen in 1 mg/ml X-gal, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM receptor. This domain is unable to bind estrogen but has high affinity MgCl2, 0.01% sodium deoxycholate, and 0.02% NP-40 at room tem- for the synthetic ligand 4-hydroxytamoxifen (4-OHT) (Littlewood et perature. al., 1995). A preliminary series of experiments revealed that this construct induced ectopic mesoderm even in the absence of 4-OHT. Whole-mount in situ hybridization The protocol of Harland (1991) was used with minor modifications, Xenopus embryos, microinjection, dissection and including the use of BM purple as a substrate and without treatment mesoderm-inducing factors of RNAase. An unhydrolysed Xbra probe was prepared by linearis- Xenopus embryos were obtained by in vitro fertilization (Smith and ing pXT1 (Smith et al., 1991) with StuI, followed by transcribing with Slack, 1983). They were maintained in 10% Normal Amphibian T7 RNA polymerase in the presence of digoxigenin (DIG)-labelled Medium (NAM; Slack, 1984) and staged according to Nieuwkoop and UTP. The probe corresponds to the 3′-untranslated region of Xbra to Faber (1967). Xenopus embryos at the one- to two-cell stage and the avoid cross-hybridisation with RNA encoding Xbra-GR. 32-cell stage were injected with RNA dissolved in 10 nl and 1 nl water, respectively, as described by Smith (1993). For animal cap RNA isolation and RNAase protection assays assays, embryos were dissected and cultured in 75% NAM or 75% RNA was extracted from animal caps by the acid guanidinium thio- NAM containing 0.1% bovine serum albumin when mesoderm- cyanate-phenol-chloroform method (Chomczynski and Sacchi, 1987) inducing factors were included in the culture medium. Xenopus bFGF followed by lithium precipitation. RNAase protection analysis was was prepared using an expression plasmid provided by David performed essentially as described by Jones at al. (1992). RNAase Kimelman and Marc Kirschner. A crude preparation of recombinant digestion was with RNAase T1 alone for all probes. Samples were human activin A was prepared from the conditioned medium of COS analysed with probes specific for EF-1α (Sargent and Bennett, 1990), cells transfected with a human inhibin βA cDNA. The cells were a ornithine decarboxylase (ODC) (Isaacs et al., 1992), cardiac actin gift from Dr Gordon Wong (Genetics Institute Inc, Massachusetts). A (Mohun et al., 1984), Xbra (Smith et al., 1991), Xhox3 (Ruiz i Altaba Hormone-inducible Brachyury 2227

Brachyury GR

DNA-binding Activation Hormone-binding Fig. 1. Construction of Xbra-GR. The hormone binding AAA Xbra-GR domain of the human glucocorticoid receptor (hGR) amino acids 512-777 was fused to the carboxy-terminus 1 AAA432 512 777 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 BglII domains of Xbra are shown by shaded and hatched TCA GTA GAT CTC TCT GAA boxes, respectively (see Kispert and Herrmann, 1993; S V D L S E Conlon et al., 1996). The hormone-binding domain of hGR is shown by a solid box. Xbra hGR and Melton, 1989) and CAT. For CAT, pTriCAT (Ambion) was tran- To investigate whether Xbra-GR activates mesoderm in scribed with T7 RNA polymerase, thus generating a probe of 241 animal caps in a DEX-dependent manner, animal caps derived nucleotides. The protected fragment is 150 nucleotides long. from embryos injected with RNA encoding Xbra-GR were cultured in the presence or absence of DEX and subjected to morphological and histological analyses at stage 42 (Fig. 3). RESULTS In control experiments, addition of DEX to animal caps derived from uninjected embryos, or from embryos injected with RNA Xbra-GR induces mesoderm in animal cap explants encoding GR alone, did not form mesoderm (Fig. 3A-D, M- in a hormone-dependent manner P). Similarly, addition of DEX to animal caps derived from Following Kolm and Sive (1995), we have tested a construct embryos injected with 400 pg RNA encoding wild-type Xbra in which Xbra is fused to the ligand-binding domain of the glu- neither enhanced nor repressed mesoderm formation (Fig. 3E- cocorticoid receptor (Xbra-GR; see Fig. 1). Xbra functions as H). However, injection of the same amount of RNA encoding a transcription activator (Kispert et al., 1995; Conlon et al., Xbra-GR did not cause mesoderm formation unless DEX was 1996) and we ﬁrst asked whether Xbra-GR is capable of acti- added to the medium (Fig. 3I-L). Interestingly, DEX treatment vating expression of a target gene in animal cap explants in a of animal caps expressing Xbra-GR reproducibly caused the hormone-dependent manner. Xenopus embryos were injected formation of large masses of muscle (Fig. 3K,L; see also Fig. with RNA encoding Xbra-GR along with a chloramphenicol 5F), whereas wild-type Xbra usually induced ventral acetyl transferase (CAT) reporter plasmid which contains two mesoderm (Fig. 3E-H). copies of the palindromic Brachyury binding site (Kispert and The mesoderm-inducing activity of Xbra-GR was also Herrmann, 1993) upstream of a thymidine kinase minimal assessed by analysing the expression of various mesodermal promoter (Fig. 2A; Conlon et al., 1996). Animal caps derived markers (Fig. 4). Addition of DEX to animal pole regions from these embryos were cultured for 3 hours in the presence derived from embryos injected with RNA encoding Xbra-GR or absence of DEX and then assayed for expression of CAT induced expression of Xhox3, a ventroposterior marker, but mRNA. DEX caused a marked stimulation of CAT expression not goosecoid, a dorsoanterior marker. In these respects the (Fig. 2B), indicating that Xbra-GR activates a putative target effects of Xbra-GR resemble those of wild-type Xbra (Fig. 4 gene in animal cap explants. and Cunliffe and Smith, 1992). Consistent with the histological data described above, DEX treatment of animal pole regions derived from embryos injected with RNA encoding Xbra-GR caused stronger expression of muscle-speciﬁc actin than did the same amount of RNA encoding wild-type Xbra. Previous work has indicated that different concentrations of

Fig. 2. Xbra-GR activates transcription Table 1. Morphology of animal caps derived from embryos of a synthetic target gene. (A) Schematic representation of the 2T-CAT reporter injected with increasing amounts of Xbra-GR RNA plasmid. The plasmid contains two copies of the palindromic Xbra-GR RNA Ventral Brachyury binding site (Kispert and Herrmann, 1993) upstream of a (pg) Uninduced vesicles Muscle Total thymidine kinase (tk) minimal promoter driving expression of Dex (−) chloramphenicol acetyl transferase (CAT) (Conlon et al., 1996). 0 14 − − 14 (B) Analysis of transcription activation by Xbra-GR in Xenopus 400 10 − − 10 embryos. Xbra-GR RNA (400 pg) and the 2T-CAT reporter plasmid Dex* (+) (20 pg) were coinjected into both cells of Xenopus embryos at the 2- 25 1 11 3 15 cell stage. Animal caps were dissected at stage 8, then treated with 100 2 4 7 13 10−6 M DEX and cultured to stage 10.5, when they were analysed for 400 − − 15 15 expression of CAT by RNAase protection. Addition of DEX causes − activation of CAT transcription. *The concentration used was 10 6 M. 2228 M. Tada, M.-A. J. O’Reilly and J. C. Smith

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).

Xbra induce different types of mesoderm: low concentrations When the protrusions are present in the head, the embryos induce ventral mesoderm containing mesothelium and mes- lack eyes (Fig. 6C) and when they occur near the tail, they enchyme, while higher concentrations induce muscle (O’Reilly contain ﬁn-like structures (Fig. 6D). Exposure to DEX at et al., 1995). This raises the possibility that Xbra-GR induces stages 8 or 12 was less efficient in this assay than treatment large amounts of muscle because it is a more efficient inducer at stage 10, the normal time of onset of Xbra expression of mesoderm than wild-type Xbra. To investigate this, different amounts of Xbra-GR RNA were injected into fertilized Xenopus eggs and animal caps derived from the resulting embryos were exposed to DEX. Animal caps derived from embryos injected with 25 pg Xbra-GR RNA formed ventral mesodermal vesicles containing mesothelium and mesenchyme, similar to those formed in response to 400 pg of Xbra RNA (Table 1; Fig 5B). Injection of 400 pg Xbra-GR RNA caused most explants to form large masses of muscle (Table 1; Fig. 5C,F). Two explants out of 45 even contained a small patch of notochord-like tissue (data not shown). RNAase protection analysis of similar explants conﬁrmed that increasing amounts of Xbra-GR RNA elicit the formation of increasing amounts of muscle (data not shown). Together, these results indicate that Xbra-GR has stronger mesoderm-inducing activity in animal cap explants than wild-type Xbra (see Dis- cussion). Over-expression of Xbra-GR in intact Xenopus embryos Previous work has shown that microinjection of Xbra RNA into fertilized Xenopus eggs results in severe perturbation of gastrulation, due to the formation of ectopic mesoderm in the animal hemisphere of the embryo (Cunliffe and Smith, 1992). Fig. 4. Mesoderm formation by Xbra-GR. RNAase protection These experiments do not reveal, however, when Xbra exerts analysis of mesoderm formation in animal caps derived from uninjected embryos or embryos injected with 200 pg of RNA its effects. To investigate this question we have injected encoding Xbra, Xbra-GR or GR. Animal caps were dissected at stage animal pole blastomeres of the 32-cell stage Xenopus embryo 8, treated with 10−6 M DEX, and cultured to stage 12 for analysis of with 10 pg Xbra-GR RNA and then treated the embryo at goosecoid, to stage 14 for analysis of Xhox3 or to stage 23 for stage 8, 10 or 12 with DEX. Addition of DEX at stage 10 analysis of cardiac actin gene expression. ODC, EF-1α and causes embryos to form tail-like protrusions (Fig. 6B,C). cytoskeletal actin serve as loading controls. Hormone-inducible Brachyury 2229

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 µm for histological analysis or at 10 µm 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 nucβ-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 nucβ-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). 2230 M. Tada, M.-A. J. O’Reilly and J. C. Smith

Table 2. Targeted injection of Xbra-GR RNA Tail-like Tiny Gastrulation protrusions protrusions defect Unaffected Injection (%) (%) (%) (%) n Experiment 1 Xbra 17 11 2 70 79 Xbra-GR Dex (−) − 1 1 98 78 Dex* (+) 61 16 1 22 83 Experiment 2 Xbra-GR Dex (−) − 7 − 93 15 Dex (+) Stage 8 33 20 13 34 15 Stage 10 80 13 − 7 15 Stage 12 47 33 − 20 15 Fig. 7. Stability of Xbra-GR protein in Xenopus embryos. Experiment 3 (A) Immunoprecipitation analysis of in vitro translated protein Xbra-GR derived from Xbra-HA, Xbra-GR-HA and GR-HA, all of which Dex (−) include repeated nonapeptides from the influenza hemagglutinin Tier A − 4 − 96 22 protein (HA), using anti-HA monoclonal antibody 12CA5 (see Tier C − − 4 96 22 Materials and methods). Immunoprecipitates were analysed by 12% Dex* (+) acrylamide gel electrophoresis followed by fluorography. Positions Tier A 61 28 4 7 197 of molecular mass markers (×10−3) are indicated. (B) Western Tier C − 8 12 80 79 blotting analysis of Xbra-GR protein levels in Xenopus embryos. Embryos were injected in one blastomere (animal tier or marginal tier) at Animal caps from embryos injected with 400 pg of RNA encoding −6 32 cell stage with 400 pg of Xbra RNA or 10 pg of Xbra-GR RNA. The Xbra-GR-HA were dissected at stage 8, treated with 10 M DEX embryos were scored by morphology at stage 34. *Embryos were treated with where appropriate and collected at the indicated stages. Two cap Dex at stage 10. equivalents of protein were analysed by western blotting using the anti-HA antibody after 12% acrylamide gel electrophoresis. Positions of molecular mass markers (×10−3) are indicated. The band (Table 2). Consistent with this, injection of wild-type Xbra corresponding to the protein of Xbra-GR-HA is indicated by an RNA has less activity than Xbra-GR, even when 400 pg RNA arrowhead. Note that Xbra-GR-HA protein levels are stable in the is injected (Table 2). absence of DEX but fall rapidly after its addition. We next asked whether the ectopic tail-like protrusions are produced by Xbra-GR-expressing cells themselves and whether they can form from equatorial blastomeres as well as Competence to respond to Xbra persists during animal pole cells. In this experiment, tier A or tier C (equa- gastrulation torial) blastomeres were injected with Xbra-GR RNA together The competence of animal pole explants to respond to with β-galactosidase RNA. The results are summarized in mesoderm-inducing factors such as activin or FGF is lost by Table 2. In the absence of DEX, injection into a tier A blas- the early gastrula stage (Green et al., 1990). One way this might tomere results in labelled cells being found in the surface occur is through loss of receptors for inducing factors, as ectoderm (Fig. 6E) or in neural tissues, whereas addition of suggested by Gillespie et al. (1989). Alternatively, the signal DEX at stage 10 causes the labelled cells to populate a tail-like transduction pathways for inducing factors might remain protrusion (Fig. 6F). Some of the ectopic tail-like tissue was intact, but cells could become refractory to the effects of genes recruited from uninjected cells. In contrast, addition of DEX such as Xbra that are activated by these pathways. To investi- to embryos receiving injections into tier C had little effect; gate this question, we have used our hormone-inducible labelled cells were restricted to notochord and paraxial meso- construct to examine the competence to respond to Xbra at dermal tissues (compare Fig. 6G and H). These results confirm different developmental stages. that ectopic expression of Xbra can change the fate of injected Preliminary experiments examined the stability of the Xbra- cells, but indicate that it is more difficult to change the fate of GR protein in the Xenopus embryo. Influenza hemagglutinin equatorial cells, which express the endogenous gene to high protein (HA)-tagged Xbra constructs can readily be detected levels. with the anti-HA monoclonal antibody 12CA5 (Fig. 7A). RNA Tucker and Slack (1995) state that a bone fide tail bud encoding Xbra-GR-HA was therefore injected into Xenopus consists of notochord, somite and neural tissues. The tail-like embryos at the one-cell stage and animal pole explants were protrusions induced by injection of RNA encoding Xbra-GR dissected at stage 8. Half were treated with DEX and they were were therefore analysed by whole-mount immunohistochem- then cultured and analysed by western blotting at stage 10, 12 istry using antibodies specific for notochord and for somitic or 14. In the absence of DEX, levels of Xbra-GR-HA remain muscle. Notochord-specific staining was not detected in Xbra- constant at least to stage 14 (Fig. 7B). Addition of DEX, GR-induced tail-like protrusions (Fig. 6J), except in one case however, caused rapid loss of Xbra-GR-HA (Fig. 7B). where the ectopic tail formed close to the host notochord (not Together, these results suggest that Xbra-GR-HA, like MyoD- shown). By contrast, well-organized somitic muscle was con- GR (Kolm and Sive, 1995) is remarkably stable. This is likely sistently observed (Fig. 6L). Thus, expression of Xbra alone is to be due to association with cytoplasmic heat-shock protein sufficient to cause formation of a tail-like protrusion, but 90, because addition of DEX causes rapid degradation. Con- cannot induce a bone fide tail. sistent with this suggestion, Xbra-HA is less stable than the Hormone-inducible Brachyury 2231

Xbra-GR-HA, and could not be detected at stage 12, whether Xbra expression was dramatically reduced by cycloheximide, or not DEX was present (data not shown). but induction of Xbra by FGF was also slightly inhibited, pre- The ability of animal cap explants to respond to Xbra-GR, sumably due to inhibition of cell division of animal pole blas- activin or FGF was assessed by morphological criteria at stage tomeres by cycloheximide (data not shown). As an alternative 20 (data not shown) and expression of cardiac actin at stage 23 test for the direct action of Xbra, we therefore asked whether (Fig. 8A). By both criteria, competence to respond to bFGF Xbra autoinduction could occur in dissociated animal pole was lost by stage 10 and responsiveness to activin was lost by blastomeres (Smith et al., 1991). DEX-induced Xbra stage 11. By contrast, explants derived from embryos injected expression was not observed in dissociated animal cap cells, with RNA encoding Xbra-GR were able to respond to DEX while Xbra expression was induced under the same conditions even at stage 11, and further experiments revealed that DEX by bFGF (Fig. 10B). Together with the time-course experi- was still capable of inducing cardiac actin expression at stage ment, these results suggest that Xbra autoinduction requires 12 (Fig. 8B). By stage 13, activation of Xbra-GR did not induce protein synthesis and intercellular signals. cardiac actin, but ventral mesoderm was formed (data not Several lines of evidence suggest that Xbra and eFGF are shown). These experiments indicate that the ability to respond involved in an autoregulatory loop in which Xbra induces to Xbra persists longer than the ability to respond to either FGF eFGF expression, and eFGF is required for maintenance of or activin. Xbra expression (Isaacs et al., 1994; Schulte-Merker and It is possible that low levels of DEX-independent Xbra Smith, 1995). To examine the possibility that autoinduction of activity during blastula and early gastrula stages cause com- Xbra involves FGF signalling, RNA encoding a truncated FGF petence to respond to mesoderm induction to be extended. To receptor, XFD, was injected into Xenopus embryos along with address this point, competence to respond to FGF or activin Xbra-GR RNA. In confirmation of previous work, XFD was assessed in the presence of Xbra-GR but in the absence of blocked mesoderm induction by both FGF and by Xbra (Fig. DEX. In these experiments, as in the absence of Xbra-GR, 11A), and additional experiments demonstrated that Xbra competence to respond to FGF and activin was lost at stages autoinduction also requires FGF signalling (Fig. 11B). 10 and 11, respectively, while competence to respond to Xbra- Analysis of Xbra expression at different times after addition of GR persisted beyond stage 11 (data not shown). DEX reveals that the truncated FGF receptor blocks the initial induction of Xbra, and that the lack of expression seen in Fig. Indirect autoinduction of Brachyury 11B is not due to inhibition of maintenance of expression (data Experiments in mouse, zebrafish and Xenopus embryos not shown). indicate that maintenance of Brachyury expression requires Brachyury function (Herrmann, 1991; Schulte-Merker et al., 1994; Conlon et al., 1996). Consistent with this, Rao has demonstrated that Xbra can activate its own expression in Xenopus animal caps (Rao, 1994), and we have confirmed this using hormone-inducible Xbra both in animal caps (Fig. 9A) and in whole embryos (Fig. 9B,C). These results raise the possibility that one target of Brachyury, a transcription activator, is Brachyury itself, and we have used Xbra-GR to investigate whether interaction of Xbra with its own promoter is sufficient to drive transcription. In the Fig. 8. Competence of animal first experiment we compared the time- caps to respond to mesoderm course of Xbra induction by FGF (which inducers. (A) Embryos at the one-cell stage were left activates Xbra in an immediate-early uninjected or injected with 100 fashion) and by Xbra itself (Fig. 10A). pg of RNA encoding Xbra-GR. Induction of Xbra in animal pole explants Animal caps were dissected at was first detectable 2 hours after exposure stage 8, 9, 10 or 11, then to FGF, while in animal caps derived from treated with 100 ng/ml bFGF, 8 embryos injected with RNA encoding U/ml activin or 10−6 M DEX, Xbra-GR, Xbra transcripts were not and cultured to stage 23, when observed until 3 hours after addition of they were analysed for DEX. expression of actin genes by To assess whether Xbra autoinduction 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 requires protein synthesis, animal pole to respond to Xbra. Embryos at the one-cell stage were injected with 100 pg of RNA explants derived from embryos injected encoding Xbra-GR. Animal caps were dissected at stage 8, treated with 10−6 M DEX at the with Xbra-GR RNA were treated with indicated stages, and cultured to stage 23, when they were analysed for expression of actin DEX in the continuous presence or genes by RNAase protection. Competence of animal caps to respond to Xbra persists absence of cycloheximide. DEX-induced during until the late gastrula stage (stage 12). 2232 M. Tada, M.-A. J. O’Reilly and J. C. Smith

Fig. 9. Autoinduction of Xbra. (A) Autoinduction of Xbra in animal pole explants. Embryos were injected with 25 pg of RNA encoding Fig. 10. Xbra autoinduction is indirect. (A) Time course of Xbra Xbra-GR at the one-cell stage. Animal caps were dissected at stage induction. Embryos were left uninjected or injected at the one-cell −6 stage with 200 pg of RNA encoding Xbra-GR. Animal caps were 8, treated as appropriate with 10 M DEX and cultured to stage −6 10.5, when they were analysed for expression of Xbra by RNAase dissected at stage 8, and treated as appropriate with 10 M DEX or protection. ODC serves as a loading control. (B,C) Autoinduction of with 50 ng/ml bFGF. They were harvested at the indicated times after Xbra in whole embryos. Embryos were injected with 10 pg of RNA treatment and analysed for expression of Xbra by RNAase encoding Xbra-GR into a tier A blastomere at the 32-cell stage. The protection. Expression of Xbra was elevated in response to bFGF embryos were left untreated (B) or exposed to 10−6 M DEX at stage within 2 hours, but the response to Xbra required 3 hours. (B) Xbra 10 (C) and allowed to develop to stage 11.5, when they were autoinduction requires intercellular signals. Embryos at the one-cell analysed by whole-mount in situ hybridisation. Ectopic expression of stage were injected with 25 pg of RNA encoding Xbra-GR or left Xbra is seen in the prospective ectoderm. 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, DISCUSSION when they were analysed for expression of Xbra by RNAase. ODC serves as a control for loading in samples in these experiments. Xbra Hormone-dependent Xbra autoinduction is not observed in dissociated cells, whereas induction In this paper, we demonstrate that a hormone-inducible version by bFGF is unaffected. of Xbra, Xbra-GR, is functional both in animal caps and in whole embryos. Like wild-type Xbra, Xbra-GR induces different mesodermal cell types at different concentrations, Duration of competence to respond to Xbra although we note that less Xbra-GR RNA is required than wild- A particular advantage of using hormone-inducible constructs type RNA for strong induction of muscle. This is likely to be like Xbra-GR is that it allows control of the effective timing of due to the greater stability of Xbra-GR protein in the embryo, expression of genes such as Xbra, MyoD (Kolm and Sive, as is also the case for a MyoD-GR fusion protein (Kolm and 1995) and, most recently, Otx2 (Gammill and Sive, 1997). Sive, 1995). This greater stability is likely to be due to associ- Using this approach we have demonstrated that induction of ation of Xbra-GR with the cytoplasmic protein hsp90, an asso- tail-like structures in whole embryos is most efficient if ciation which blocks the activity of Xbra until DEX is added expression occurs at stage 10, and we have shown that the com- (see Smith and Toft, 1993). It is also possible, however, that the petence of animal pole explants to respond to Xbra persists greater activity of the fusion protein is due to the TAFII activity during gastrulation (Fig. 8), even when the response to of the ligand-binding domain of the glucocorticoid receptor (see mesoderm inducing factors, such as FGF and activin, has been Mattioni et al., 1994) or to enhanced dimerization of the Xbra- lost (Green et al., 1990). GR protein caused by the GR moiety (see Fawell et al., 1990). This latter result impinges on the question of what controls We have used Xbra-GR to study the duration of competence the nature of the response that cells make to inducing factors. to respond to Xbra and to ask whether Xbra activates its own FGF has recently been shown to have two separable effects on transcription directly, by acting on its own promoter, or indi- animal pole explants, depending on the time during develop- rectly. The approach we describe will be applicable to other ment that it is applied. Before the onset of gastrulation it transcription factors, and may help in the identiﬁcation of their functions as a mesoderm-inducing factor, whereas if applied downstream targets. after the onset of gastrulation it induces neural tissue (Kengaku Hormone-inducible Brachyury 2233

et al., 1990; Halpern et al., 1993; Schulte-Merker et al., 1994), and dominant-negative studies in Xenopus (Conlon et al., 1996), suggest that one function of Brachyury is to activate transcription of mesoderm-specific genes which are involved in patterning posterior mesoderm and in differentiation of the notochord. No such target genes have yet been identified, but it may be possible to use Xbra-GR to search for them (see also Gammill and Sive, 1997). In this paper we demonstrate that Xbra is not able directly to activate its own transcription. Thus, induction by DEX of Xbra expression in animal caps derived from embryos injected with Xbra-GR RNA is inhibited by cycloheximide (data not shown), by cell dispersion (Fig. 10B) and by the presence of a truncated FGF receptor (Fig. 11). These results support the idea that Xbra and eFGF are com- ponents of an indirect autoregulatory loop in which Xbra induces expression of eFGF, and eFGF is required for maintenance of Xbra expression (Isaacs et al., 1994; Schulte-Merker and Smith, 1995). They also define three criteria that any putative Xbra target must satisfy: first, that the gene must be activated by DEX in animal caps expressing Xbra-GR even in the presence of cycloheximide (see Gammill and Sive, 1997); second, that it should be activated in dispersed cells; and finally, that the gene should be activated in the presence of a Fig. 11. Xbra function requires FGF signalling. (A) Mesoderm truncated FGF receptor. One potential target of Xbra in formation in response to Xbra requires FGF signalling. Embryos at the Xenopus is eFGF; the expression patterns of the two genes one-cell stage were injected with 1 ng of RNA encoding a dominant- during gastrulation are remarkably similar (Isaacs et al., 1996) negative FGF receptor (XFD) and/or 100 pg of RNA encoding Xbra- and ectopic expression of Xbra induces eFGF expression in GR. Animal caps were dissected at stage 8, treated as appropriate with animal caps (Isaacs et al., 1994; Schulte-Merker and Smith, 10-6 M DEX and cultured to stage 23, when they were analysed for 1995). We are now going on to investigate this question using expression of actin genes by RNAase protection. Inhibition of FGF inducible Xbra. signalling blocks induction of mesoderm by Xbra-GR. Induction of Finally, these experiments also emphasize that FGF family cardiac actin gene expression by bFGF (50 ng/ml) in control animal members play at least two roles in Xenopus mesoderm caps was abolished by injection of RNA encoding XFD (1 ng) under the same conditions. (B) Xbra autoinduction requires FGF signalling. formation. Initially, they are capable of inducing mesoderm Embryos at the one-cell stage were injected with 25 pg of RNA from presumptive ectodermal tissue, and their ability to do this encoding Xbra-GR and/or 1 ng of RNA encoding XFD. Animal caps declines around the beginning of gastrulation (Fig. 11A). were dissected at stage 8, treated as appropriate with 10−6 M DEX and Thereafter, although FGF signalling cannot induce mesoderm cultured to the equivalent of stage 10.5, when they were analysed for from naive ectodermal tissue, it is required in newly induced expression of Xbra by RNAase protection. Inhibition of FGF mesoderm for the autoinduction of Xbra (Fig. 11B), for main- signalling prevented autoinduction of Xbra. Expression of Xbra tenance of expression of Xbra (Isaacs et al., 1994; Schulte- induced by bFGF treatment (100 ng/ml) in control caps was abolished Merker and Smith, 1995), and for formation of mesoderm in by injection of RNA encoding XFD (1 ng) under the same conditions. response to Xbra (Fig. 11A). ODC serves as a control for loading in samples in these experiments.

This work is supported by the Medical Research Council, of which and Okamoto, 1995; Lamb and Harland, 1995). This switch M.-A. O’R. was a Training Fellow. M. T. is supported by the HFSP, and J. C. S. is an International Scholar of the Howard Hughes Medical occurs around the time that cells lose the ability to activate Institute. We thank Lynne Fairclough for advice on western blotting Xbra in response to FGF (Lamb and Harland, 1995), and the and Wendy Hatton for doing the histology. We are also grateful to results described in this paper emphasize how important this Kathy Weston for suggesting we try inducible Xbra constructs and to loss of competence is, because activation of Xbra-GR during Hazel Sive for telling us about her data before publication and for gastrulation causes mesoderm to form (Fig. 8). Before gastru- sending the GR construct, also before publication. lation, FGF activates Xbra, and induces mesoderm, through the MAP kinase pathway (Gotoh et al., 1995; LaBonne et al., 1995; Umbhauer et al., 1995). It will be important to investi- REFERENCES gate the signal transduction pathway that is employed by FGF after gastrulation, one which may not induce transcription of Amaya, E., Musci, T. J. and Kirschner, M. W. (1991). Expression of a Xbra. This might involve different FGF receptors, alterna- dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos. Cell 66, 257-270. tively-spliced forms of the same FGF receptor, or perhaps a Chomczynski, P. and Sacchi, N. (1987). Single-step method of RNA isolation different target for MAP kinase. by acidic guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156-159. Christian, J. L. and Moon, R. T. (1993). Interactions between Xwnt-8 and Downstream targets of Brachyury Spemann organizer signaling pathways generate dorsoventral pattern in the Mutant analyses in mouse and zebraﬁsh embryos (Herrmann embryonic mesoderm of Xenopus. Genes Dev. 7, 13-28. 2234 M. Tada, M.-A. J. O’Reilly and J. C. Smith

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