Xom: a Xenopus homeobox gene that mediates ... - Semantic Scholar

2385

Development 122, 2385-2394 (1996) Printed in Great Britain © The Company of Biologists Limited 1996 DEV1065

Xom: a Xenopus homeobox gene that mediates the early effects of BMP-4 R. Ladher, T. J. Mohun, J. C. Smith and A. M. Snape Division of Developmental Biology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK

SUMMARY Bone morphogenetic protein-4 (BMP-4) is thought to play an important role in early Xenopus development by acting as a ‘ventralizing factor’ and as an epidermal determinant: local inhibition of BMP-4 function in whole embryos causes the formation of an additional dorsal axis, and inhibition of BMP-4 function in isolated ectodermal cells causes the formation of neural tissue. In this paper we describe a homeobox-containing gene whose expression pattern is

similar to that of BMP-4, whose expression requires BMP4 signalling and which, when over-expressed, causes a phenotype similar to that caused by over-expression of BMP-4. We suggest that this gene, which we call Xom, acts downstream of BMP-4 to mediate its effects.

INTRODUCTION

MATERIALS AND METHODS

Many factors are believed to be involved in inducing and patterning the mesoderm of Xenopus laevis. Among the strongest candidates are members of the transforming growth factor type β (TGFβ) family, such as Vg1 (Dale et al., 1993; Thomsen and Melton, 1993), activin (Asashima et al., 1990; Smith et al., 1990; Thomsen et al., 1990; van den Eijnden Van Raaij et al., 1990), the nodal-related genes Xnr1-3 (Jones et al., 1995; Smith et al., 1995), and bone morphogenetic protein 4 (BMP4) (Dale et al., 1992; Jones et al., 1992; Graff et al., 1994; Fainsod et al., 1995; Sasai et al., 1995; Schmidt et al., 1995; Wilson and Hemmati-Brivanlou, 1995). Of these factors, BMP-4 is unusual in that it causes ‘ventralization’ of the embryo. Thus, injection of RNA encoding BMP-4 promotes formation of ventral mesoderm in Xenopus embryos (Dale et al., 1992; Jones et al., 1992, 1996), and injection of RNA encoding a truncated BMP-4 receptor causes loss of ventral structures and an increase in dorsal tissue (Graff et al., 1994; Maeno et al., 1994; Suzuki et al., 1994). A role for BMP-4 in ventralization is supported by the observation that BMP-4 is expressed in the ventral and lateral marginal zones of the early gastrula and is absent from the organizer (Fainsod et al., 1995; Schmidt et al., 1995). BMP-4 is also expressed in the animal hemisphere of Xenopus, and recent work suggests that it acts here to induce epidermal differentiation at the expense of neural structures (Wilson and Hemmati-Brivanlou, 1995). These observations suggest that BMP-4 plays an important role in the early development of Xenopus. Little is known, however, about the way it exerts its effects. In this paper we describe a homeobox-containing gene whose expression pattern is similar to that of BMP-4, whose expression requires BMP-4 signalling and which, when over-expressed, causes a phenotype similar to that caused by over-expression of BMP4. We suggest that this gene, which we call Xom, acts downstream of BMP-4 to cause ventralization of the embryo and perhaps to specify epidermal differentiation.

Xenopus embryos and mesoderm-inducing factors Xenopus embryos were obtained by in vitro fertilisation (Smith and Slack, 1983). They were maintained in 10% Normal Amphibian Medium (NAM; Slack, 1984) and staged according to Nieuwkoop and Faber (1975). Dissection of animal pole regions was carried out in 75% NAM, and they were cultured in 75% NAM containing 0.1% bovine serum albumin. Recombinant human bone morphogenetic protein 4 (BMP-4) was a gift from Genetics Institute Inc. (Cambridge, Massachusetts). Xenopus FGF-2 was prepared by Jeremy Green using an expression plasmid provided by David Kimelman and Marc Kirschner. Partially purified human activin A was prepared from the conditioned medium of COS cells transfected with a human inhibin βA cDNA. The cells were the gift of Dr Gordon Wong (Genetics Institute Inc.). pCSKAXwnt-8 (Christian and Moon, 1993) is described by Cunliffe and Smith (1994). RNA encoding noggin was transcribed from the plasmid noggin∆5′ (Smith and Harland, 1992) and RNA encoding BMP-4 was transcribed from pSP64T-XBMP-4+ (Dale et al., 1992).

Key words: Xenopus, activin, BMP-4, homeobox, Xom

RNA Isolation RNA was isolated using the acid phenol/guanidinium isothiocyanate procedure (Chomczynski, 1993). Poly(A)+ RNA was isolated from total RNA using the poly ATtract system (Promega). cDNA library construction Poly(A)+ RNA was derived from 1000 animal pole regions, which had been treated with 100 units/ml (about 20 ng/ml) FGF-2 from stage 8 (mid-blastula) to stage 10 (early gastrula). A unit of mesoderminducing activity is defined as the minimum amount of factor in a volume of 1 ml that will induce mesoderm formation (Cooke et al., 1987). This material was used to make a directional cDNA library in the vector λZAP-II™ (Stratagene). The primary library contained 1.5×106 independent clones with an average insert size of approximately 1.2 kb. It was excised into pBluescript® SK(−) according to the manufacturer’s instructions. Polymerase chain reaction The plasmid form of the FGF-treated cDNA library was used as

2386 R. Ladher and others template in a polymerase chain reaction (PCR) designed to amplify sequences containing homeobox motifs. The 5′ primer was based on the conserved sequence QIKIWFQ, which is present in helix 3 of the homeodomain. The 3′ primer corresponds to the T7 promoter present in the pBluescript® SK(−) vector. The nucleotide sequences of the primers are below; underlining in the 5′ primer indicates an EcoRI site: 5′ primer: 5’ GGAATTCCAAATCAAGATTTGGTTTCA 3’ 3′ primer:

G T A C C 5’ GTAATACGACTCACTCACTATAGG

A 20 40 60 AAGAACACAA GGACTAATAC AGACAAGATG ACTAAAGCTT TCTCCTCGGT TGAATGGCTT GCTCAAAGCA M T K A F S S V E W L A Q S> 80 100 120 140 GCCGCAGATC TCACAGAGAG CAGCCAAGCA AAGTGGATCA GAGATATTCA CCGTACCCCA GCCCATCCCT S R R S H R E Q P S K V D Q R Y S P Y P S P S L> 160 180 200 GCCTTCCTGG AACAGTGATG TGTCCCCTTC TTCATGGAAC AGCCAACTAT CTCCAGATCC AGACAGTGCC P S W N S D V S P S S W N S Q L S P D P D S A> 220 240 260 280 CAAGTCTCAC CATGCCCTGC GAGTGCACAA GTATCTCCAT ATTCCTCAGA CAGCGAAATA TCACTGTATT Q V S P C P A S A Q V S P Y S S D S E I S L Y>

3’ 300 320 340 CACATGAAGA AGAAGCCTCA TTCTATGGAA TGGACCTTAA TACATCATCA TCCCCTGGAG ACAATGGATT S H E E E A S F Y G M D L N T S S S P G D N G L>

Amplification conditions were: 94°C for 30 seconds, 50°C for 1.5 minutes and 72°C for 2.5 minutes for 5 cycles, and then 94°C for 30 seconds, 65°C for 1.5 minutes and 72°C for 2.5 minutes for 25 cycles. The reaction was then digested with EcoRI and KpnI and ligated into M13. Recombinants were selected using blue/white colour selection and 96 were picked and grown as described by Sambrook et al. (1989). Single-stranded DNA was prepared and the 96 clones were sorted into classes by running the ‘T’ samples from a dideoxy sequencing reaction on a 6% denaturing acrylamide gel. A representative of each type of clone was then sequenced using a deaza T7 sequencing kit (Pharmacia) and the sequences were analysed for existing homologies by database searching. Clones that warranted further study were subcloned into pBluescript® KS(+), and a fulllength cDNA corresponding to one of them (Xom) was obtained by screening the original λZAP-II™ cDNA library. The longest cDNA was sequenced using a deaza T7 sequencing kit (Pharmacia).

360 380 400 420 GCTACACTCT GAAATGGTTT CAGTGCCAGA TAATATTCCC AGAGCCAGTT CCGATGAAGA TGCTGCTAAG L H S E M V S V P D N I P R A S S D E D A A K> 440 460 480 TCTGCCTACA GCACTAGCAC TGACTCAGGC TATGAAAGTG AAACAAGTTG CTCCAGCTCT ACAGCCCCTG S A Y S T S T D S G Y E S E T S C S S S T A P> 500 520 540 560 AAGGAGATGC CATATCTCTG AGTCCCAATG ATACCTCAGA TGAAGAGGGC AAGATGGGTC GAAGGTTGAG E G D A I S L S P N D T S D E E G K M G R R L R> 580 600 620 GACGGCTTTC ACCAGTGATC AGATCTCCAC TCTGGAGAAG ACTTTTCAGA AACACAGATA CCTTGGGGCG T A F T S D Q I S T L E K T F Q K H R Y L G A> 640 660 680 700 TCTGAAAGAC AGAAACTCGC AGCCAAACTC CAGCTTTCTG AAGTCCAGAT TAAAACCTGG TTCCAGAACC S E R Q K L A A K L Q L S E V Q I K T W F Q N> 720 740 760 GCAGGATGAA ATACAAACGG GAAATCCAAG ATGGCAGACC AGACTCATAC CACCCAGCCC AGTTCTTTGG R R M K Y K R E I Q D G R P D S Y H P A Q F F G> 780 800 820 840 TGTCTACGGC TATGCACAGC AGCCCACTCC TGTATTCCAG CATGCAGTCC AACATCCCTA CCCAGGTTAT V Y G Y A Q Q P T P V F Q H A V Q H P Y P G Y> 860 880 900 AACCCACTAA TGGAAACCCT GCCTGGTACC ATGCCCTATA CCATGCATCC TCCTGCCATG GACTCTATGA N P L M E T L P G T M P Y T M H P P A M D S M>

RNAase protection RNAase protection assays were performed as described by Green et al. (1990). Samples were analysed with probes specific for EF-1α (Sargent and Bennett, 1990), ornithine decarboxylase (ODC) (Bassez et al., 1990), cardiac actin (Mohun et al., 1988), Xbra (Smith et al., 1991), αT4 globin (Walmsley et al., 1994) and Xom. For Xom, the PCR fragment described above was used as an RNAase protection probe. This Fig. 1. (A) Xom cDNA sequence and deduced amino acid sequence. Amino acids in bold represent the homeodomain. The threonine at position 47 in this homeodomain is underlined, as is the polyadenylation signal at nucleotides 1226-1231. (B) Comparison of the homeodomain of Xom with those of members of the same homeodomain class. The threonine at position 47 is again underlined. (C) Structure of Xom. The acidic domain (amino acids 41 to 172; red) is defined as a region in which the average charge over 20 amino acids is consistently negative. In the middle of this domain is a region rich in serine and threonine (diagonal stripes). The homeodomain is contained within amino acids 173-233 (blue). The proline-rich domain (amino acids 255-320) contains 14 proline residues in a stretch of 66 amino acids (yellow).

920 940 960 980 CTCCCTTCAA CTCTCAACCT TTTCAGATGC TCTACCTGCC CCAACAGCAC CTTGGGCAAC CTCTGACCTA T P F N S Q P F Q M L Y L P Q Q H L G Q P L T Y> 1000 1020 1040 TCAGGAAGAA AGGCCATTTG TTAGATATTA ATATGAAGCC AATGCAAAGG ACTATACTAA ATACTGGACT Q E E R P F V R Y 1060 1080 1100 1120 TTTTCAGAAA CTTCTGTCTT CGAATATTAG CACTAAACAG TGGCAAAGTG TCCACAAAGT GACCTTTTTG 1140 1160 1180 TATTGGGTCG TGTTTTATGT ATTTGCATTA TTTTTATTCA CATTGAATAG TTAATTTTAT AGATGTTTAA 1200 1220 1240 1260 ATTAACTTTA AAGATGTATT TCTGTTAAAT TAATAATAAA TGTGACTTAT ATTTTAACAG CAAACAAAAA 1280 AAAAAAAAAA AAAAAAAAAA AA

B Xom

GRRLRTAFTSDQISTLEKTFQKHRYLGASERQKLAAKLQLSEVQIKTWFQNRRMKYKRE

Om(1D)

Q-ka-----DH-lQ----s-ErQk--SvQ---E--H--D--dC-v---y----T-wM-Q

75

BarH1

Q-ka-----DH-lQ----s-ErQk--SvQd-ME--N--E--dC-v---y----T-w--Q

75

100

Hox 11

KkkP--S--RL--CE---R-HrQk--aSA--AA--KA-KMtdA-v--------T-wR-Q

65

Prh

RkGgQVRysN--TIE---K-ETQk--SPP--KR--KM-----R-v--------A-wR-L

63

Consensus Homeobox

R--K---y-RY-lLE---E-HFN---TRRR-IE--KS-N-t-R-v-I--------w-k-

65

C

Acidic region 1

41

AAA AAA

Homeodomain

133 152 172173

Serine/threonine rich domain

233

Proline rich region 255

320 326

Xom mediates BMP-4 signalling in Xenopus 2387 consists of a 195 bp (base pair) fragment from positions 677 to 871 in the cDNA. The probe was made by linearising the clone with NotI and transcribing with T3 RNA polymerase. RNA blots RNA blots were prepared as described by Sambrook et al. (1989). They were probed for expression of Xom using a random-primed probe prepared from the 195 bp PCR clone, and for expression of ODC using a 300 bp probe prepared from the 3′ end of the cDNA. After hybridisation overnight, blots were washed twice for 15 minutes each with 2×SSC/0.1% SDS and three times for 30 minutes each with 0.2×SSC/0.1% SDS. Washes were done at 65°C.

Fig. 2. RNAase protection analysis of Xom expression. Xom transcripts are first detectable at stage 9 and expression declines after stage 17. Ornithine decarboxylase (ODC) is used as a loading control.

RNA synthesis and microinjection Xom cDNA was cloned into the BglII site of pSP64T (Krieg and Melton, 1984) in the sense orientation. Capped RNA was transcribed according to Smith (1993). The RNA was translated using a rabbit reticulocyte lysate system (Promega). [35S]methionine-labelled protein products were analysed by polyacrylamide gel electrophoresis. Xenopus embryos at the 1- to 4-cell stage, or the 32-cell stage, were injected with RNA as described by Smith (1993). As a control for RNA injections, a truncated version of Xom, lacking most of the homeodomain, was constructed by excising a BglII fragment from the cDNA. This construct, ∆Xom, was cloned into pSP64T. RNA encoding nucβ-gal was transcribed from pSP6nucβGal (Smith and Harland, 1991). β-gal staining was carried out essentially as described by Whiting et al. (1991). Whole-mount in situ hybridisation and antibody staining Whole-mount in situ hybridisation was carried out essentially as described by Harland (1991). Digoxygenin-labelled riboprobes were prepared from a pBluescript SK(−) plasmid containing the entire Xom cDNA. Transcripts were revealed using BM-purple substrate (Boehringer). Whole-mount immunocytochemistry with monoclonal

Fig. 3. Whole-mount in situ analysis of Xom expression. (A) Vegetal view of a Xenopus embryo at stage 9 shows asymmetric expression of Xom. It is likely that Xom is most highly expressed in ventral cells, because by stage 10.5 transcripts are clearly excluded from the organizer (B). Apparent lack of expression in vegetal tissue is probably due to poor probe penetration. (C) Dorsal view of an embryo at stage 12.5; expression is excluded from the anterior neural plate and the dorsal midline. (D) Dorsal view of an embryo at stage 14. Expression is becoming restricted to two domains, one anterior and one posterior. The posterior domain surrounds the lateral and ventral regions of the embryo. (E) Lateral view of an embryo at stage 20. The anterior domain has resolved to mark the dorsal region of the eye. The posterior domain includes the ventral region of the embryo and (out of the plane of the photograph) the dorsal midline in the trunk. (F) Lateral view of an embryo at stage 32 shows expression of Xom in the dorsal part of the eye, in the proctodeum and in the tailbud. Scattered expression is also visible in the dorsal midline, which may be due to migrating neural crest cells.

Fig. 4. Sections of embryos processed for whole-mount in situ hybridization using a probe specific for Xom. (A) Section of stage10.5 embryo. Transcripts are absent from the organizer, but are present in ventral and lateral regions of the embryo in all three germ layers. The punctate staining in vegetal tissue may be due to poor probe penetration into this yolky tissue. (B) Section of a stage-19 embryo at the level of the head. Transcripts are present in the presumptive eye. (C) Section of the embryo shown in B in the trunk region. Staining is visible in all three germ layers of the ventral and lateral regions of the embryo, and in the dorsal cells of the roof plate of the neural tube. Bars, in A and B are 50 µm; in C is 100 µm.

2388 R. Ladher and others antibodies MZ15 (Smith and Watt, 1985) and 12/101 (Kintner and Brockes, 1984), specific for notochord and muscle, respectively, was performed as described by Smith (1993).

RESULTS Isolation and sequence of Xom Xom was isolated during a PCR screen for homeobox genes that are activated by FGF treatment of Xenopus animal caps. Of the seven classes of clone that were identified by ‘Ttracking’, three contained a homeodomain. Of these, goosecoid (Cho et al., 1991) was represented five times and X-ANF1 (Zaraisky et al., 1992, 1995) was represented twice. The presence of goosecoid in the FGF-treated animal caps library was surprising because its expression is not induced by FGF, and we speculate that it derives from maternal transcripts. The same may be true of X-ANF1. The third homeodomain-containing gene was novel and was represented 21 times; this was chosen for further study. A full-length version of the novel clone was obtained by screening the FGF-treated animal pole region cDNA library. Of 800 000 clones screened, 125 showed strong hybridisation. 20 of these were picked and plaque-purified. The longest cDNA was approximately 1.3 kb, which corresponds to the length predicted by RNA blotting (not shown). To verify that the cDNA was full length, a gastrula-stage cDNA library (gift of Bruce Blumberg) was also screened. Of the 800 000 plaques screened, 247 were positive. Each of the 24 plaques that was further analysed showed an insert size of approximately 1.3 kb. The sequence of the longest cDNA revealed a single open reading frame. Conceptual translation predicted a homeodomain-containing protein of 326 amino acids with a molecular mass of 36.7×103 (Fig. 1A). Sequence comparison revealed that the homeodomain was most closely related to that of the Drosophila annasae gene Om1D and the Drosophila melanogaster gene BarH1 (Fig. 1B); some residues were also conserved in the regions immediately flanking the homeodomain. Om1D and BarH1 are both required for photoreceptor development (Tanda and Corces, 1991; Higashijima et al., 1992a) and BarH1 determines a subtype of neuron in the peripheral nervous system (Higashijima et al., 1992b). The similarities in the homeodomains of the Xenopus and Drosophila genes, and in their expression patterns (see below), inspired us to call the Xenopus gene ‘Xom’. Like Om1D and BarH1, Xom has an unusual homeodomain, which contains a threonine at position 47 rather than the more common valine or isoleucine (Fig. 1B). This substitution changes the DNA binding specificity of Hox 11, another member of this class (Kennedy et al., 1991; Dear et al., 1993), and is likely also to affect the specificity of Xom and other members of this group such as prh, a gene involved in haematopoiesis (Crompton et al., 1992). The amino acid sequence of Xom contains elements in addition to the homeodomain that are consistent with the protein acting as a transcription factor. In particular, there is a highly acidic domain, rich in serines and threonines, and a proline-rich domain (Fig. 1C). It is possible that these are involved in transcription activation (Ma and Ptashne, 1987; Mermod et al., 1989).

Temporal expression pattern of Xom The expression pattern of Xom was first studied by RNA blotting (not shown) and by RNAase protection analysis. Maternal transcripts were not detected by either technique, but RNA blotting revealed that a single 1.3 kb transcript first appeared at stage 9 (late blastula). Transcript levels remained high until stage 17 (late neurula), after which time they declined. The same temporal expression profile was observed using the more sensitive technique of RNAase protection (Fig. 2). Spatial expression pattern of Xom Dissection of embryos at stage 10 suggested that Xom is expressed ubiquitously at the early gastrula stage, with higher levels of expression on the ventral side of the embryo (not shown). Whole-mount in situ hybridisation confirmed that even at late stage 8 and stage 9 (Fig. 3A), transcript levels are higher on one side of the embryo than the other. By stage 10.5 it is clear that Xom is expressed in ventral and lateral regions of the embryo (Fig. 3B), and in the animal cap (see Fig. 7), but that the gene is not expressed in Spemann’s organiser (Fig. 3B). Sections through such embryos (Fig. 4A) reveal that Xom transcripts are present in both ectoderm and mesoderm. Expression is also seen in the endoderm. This early expression pattern of Xom is very similar to that of BMP-4 (Fainsod et al., 1995; Schmidt et al., 1995). By stage 12 (late gastrula/early neurula), Xom is absent only from the dorsal midline and anterior neural plate of the embryo (Fig. 3C). Its expression pattern is essentially the reciprocal of those of chordin (Sasai et al., 1995) and noggin (Smith and Harland, 1992). After this stage, expression declines gradually in all regions of the embryo except those destined to form the tip of the tail and the dorsal half of the retina. This is clear from stage 19 (Fig. 3D), when the future eye first becomes delineated, and stage 24, when the pattern has refined further (Fig. 3E). Sections through embryos at stage 24 confirm that transcripts are present in the eye (Fig. 4B) and show that Xom RNA is detectable in all three germ layers in the ventral part of the trunk, with low levels of expression also detectable in trunk regions in the roof plate of the neural tube (Fig. 4C). By stage 32 expression is almost completely restricted to the dorsal half of the retina, the tail bud, the dorsal midline and the proctodeum (Fig. 3F). At this stage too, the expression patterns of Xom and BMP-4 are very similar (Fainsod et al., 1995; Schmidt et al., 1995). Regulation of Xom expression: the roles of BMP-4 and noggin The regulation of Xom expression was studied in isolated animal pole regions and in intact Xenopus embryos. In the first series of experiments, animal caps were dissected from stage8 (mid-blastula) embryos and cultured to stage 10.5 (early gastrula). Analysis of experiments of this sort is complicated by the fact that there are low levels of Xom expression in untreated animal caps. Nevertheless, these experiments (not shown) revealed that the endogenous level of Xom expression is slightly but persistently increased by treatment of animal caps with activin or FGF and it is also elevated in animal caps derived from embryos injected with pCSKA-Xwnt-8, which directs expression of Xwnt-8 during gastrula stages, or with RNA encoding BMP-4. Animal caps derived from embryos

Xom mediates BMP-4 signalling in Xenopus 2389

Fig. 5. Expression of Xom is prevented by dispersion of Xenopus embryos. Addition of activin or BMP-4, but not FGF, restores expression. Xenopus embryos were transferred to calcium- and magnesium-free medium at stage 7.5, their vitelline membranes were removed, and the blastomeres were kept dispersed by passing a gentle stream of medium over the cells from a Pasteur pipette. Dispersed blastomeres were cultured to stage 10.5 in the presence of the indicated factors and analysed for expression of Xom by RNAase protection. EF-1α was used as a loading control. Lane 1, intact control embryos; lane 2, dispersed blastomeres with no additional factors; lane 3, dispersed blastomeres plus 50 ng/ml FGF-2; lane 4, dispersed blastomeres plus 100 ng/ml BMP-4; lane 5, dispersed blastomeres plus 8 units/ml activin.

injected with RNA encoding noggin do not express Xom to detectable levels. Expression of Xom in intact animal caps, and perhaps in the whole embryo, might occur in response to endogenous inducing signals. To investigate this possibility, Xenopus embryos were dissociated into single cells at stage 7.5 and cultured to stage 10.5. This treatment inhibited expression of Xom (Fig. 5), suggesting that expression of the gene does require intercellular signalling. Expression of Xom in dispersed embryos was restored by addition of activin or BMP-4, but not by addition of FGF-2 (Fig. 5). This induction of Xom expression was not inhibited by cycloheximide, indicating that Xom is an immediate-early target of BMP-4 and activin (Fig. 6). The inability of FGF to induce expression of Xom in dispersed cells suggests that its action in intact animal caps is indirect. The expression pattern of Xom at the early gastrula stage resembles that of BMP-4 and is the reciprocal of noggin, suggesting that these two gene products are involved in the control of Xom expression in vivo. To investigate this possibility, RNA encoding noggin was injected into one blastomere of Xenopus embryos at the 2-cell stage. This treatment inhibited expression of Xom in whole embryos (Fig. 7), as it did in animal caps. The role of BMP-4 in regulating expression of Xom was investigated by injecting Xenopus embryos with RNA encoding a truncated BMP-2/4 receptor (Suzuki et al., 1994). RNAase protection analysis at stages 8 and 9 (mid- and late blastula stages) revealed that this treatment caused a dramatic decrease in levels of expression of Xom (Fig. 8A). Expression recovered by stage 11 (Fig. 8A), but we do not yet know whether this is due to decreasing levels of truncated BMP-2/4

Fig. 6. Activation of Xom expression by activin and BMP-4 is not inhibited by cycloheximide. Xenopus embryos were transferred to calcium- and magnesium-free medium at stage 7.5, their vitelline membranes were removed, and the blastomeres were kept dispersed by passing a gentle stream of medium over the cells from a Pasteur pipette. Dispersed blastomeres were cultured to stage 10.5 in the absence of additional factors (lanes 1 and 2) or in the presence of FGF-2 (lanes 3 and 4), BMP-4 (lanes 5 and 6) or activin (lanes 7 and 8). Samples in even-numbered lanes were cultured in the continual presence of 7.5 µg ml-1 cycloheximide. This was sufficient to reduce incorporation of [35S]methionine into trichloroacetic acid-insoluble material by over 94%. Expression of Xom was analysed by RNAase protection. EF-1α was used as a loading control.

receptor or to a second phase of Xom expression that is independent of BMP signalling. These results were confirmed by whole-mount in situ hybridization of injected embryos at stage 10.5. Injection of RNA encoding a truncated BMP-2/4 receptor into Xenopus embryos at the 1-cell stage resulted in loss of Xom expression in ventral regions of the embryo in 55% of cases (12/23) (Fig. 8B). The presence of Xom transcripts in lateral regions of the embryo suggests that recovery of expression occurs here before it occurs in ventral regions. It seems likely that recovery was complete in the 45% of embryos in which Xom expression appeared normal. Injection of RNA encoding BMP-4 caused an expansion of Xom expression, with transcripts present throughout the marginal zone, including dorsal tissues (Fig. 8C). The time of onset of expression of Xom is similar to that of BMP-4 (Fig. 9), but the appearance of Xom transcripts slightly precedes zygotic expression of BMP-4, suggesting that maternal levels of BMP-4 may be sufficient to initiate expression of Xom or, alternatively, that BMP-2 plays a role; there are high maternal levels of BMP-2 RNA (Clements et al., 1995). This possibility is supported by the observation that cycloheximide treatment of whole embryos from stage 7.5 does not prevent activation of Xom (not shown). This suggests that maternally provided protein is sufficient to induce expression of the gene. Functional analysis of Xom Over-expression of BMP-4 in Xenopus causes severe ventralization of the embryo (Dale et al., 1992; Jones et al., 1992, 1996; Maeno et al., 1994; Suzuki et al., 1994; Fainsod et al., 1995; Schmidt et al., 1995). It is possible that this ventralization occurs through up-regulation of Xom expression, and to investigate this possibility, RNA encoding Xom (4 ng) was injected into the dorsal side of Xenopus embryos at the 4-cell stage. This treatment caused the formation of embryos with anterior/dorsal deficiencies, including the reduction or complete absence of head structures and the absence of notochord (Fig. 10; Table 1). Injection of a truncated version

2390 R. Ladher and others

Fig. 7. Injection of RNA encoding noggin into one blastomere of Xenopus embryos at the 2-cell stage inhibits expression of Xom. Noggin RNA (1 ng) was injected into one blastomere of Xenopus embryos at the 2-cell stage and the embryos were allowed to develop to the early gastrula stage (stage 10), when they were analysed by whole-mount in situ hybridization. (A) Noggininjected embryo viewed from the animal hemisphere. Note downregulation of Xom expression in half the embryo (arrow). (B) Control embryo viewed from the animal hemisphere.

Fig. 8. Expression of a truncated BMP-4 receptor blocks early expression of Xom, and over-expression of BMP-4 causes ectopic expression. (A) RNA encoding a truncated BMP-2/4 receptor (tBR) was injected into Xenopus embryos at the 1-cell stage and the embryos were allowed to develop to the indicated stages before being analysed by RNAase protection using a probe specific for Xom. At stages 8 and 9 there is a dramatic down-regulation of Xom compared to uninjected controls, but this has recovered by stage 11. EF1α was used as a loading control. (B) In situ hybridization analysis of embryos injected with a truncated BMP-2/4 at stage 10 also reveals inhibition of Xom expression. (C) Overexpression of BMP-4 causes expression of Xom to occur in the dorsal marginal zone as well as in ventral tissues.

Fig. 9. Comparison of the time of onset of expression of Xom and of BMP-4. Xenopus embryos at the indicated times after fertilisation (hours) were analysed by RNAase protection simultaneously for the expression of BMP-4, Xom and EF-1α. Low maternal levels of BMP-4 RNA are visible and zygotic expression of Xom slightly precedes that of BMP-4.

Fig. 10. Injection of RNA encoding Xom into dorsal blastomeres of Xenopus embryos causes loss of anterior structures and of the notochord. Xom RNA (4 ng total) was injected into the two dorsal blastomeres of Xenopus embryos at the 4-cell stage, and the embryos were allowed to develop to stage 32. Control embryos received injections of RNA encoding ∆Xom, which encodes a truncated version of Xom. (A) Embryos expressing ∆Xom develop normally. (B) Embryos injected with RNA encoding Xom lack anterior structures. (C) Whole-mount antibody staining using the monoclonal antibody MZ15 demonstrates that embryos injected with ∆Xom RNA form a notochord. (D) Embryos injected with RNA encoding Xom lack a notochord.

of Xom had no effect on development. In these respects, the effects of over-expression of Xom mimic those of overexpression of BMP-4, although the effects of Xom are less dramatic (see Discussion). Further experiments demonstrated directly that overexpression of Xom changes the fates of dorsal cells of the Xenopus embryo. Dorsal equatorial cells of the 32-cell stage Xenopus embryo were injected with RNA encoding either Xom or ∆Xom, together with RNA encoding β-galactosidase as a lineage tracer. Cells injected with RNA encoding ∆Xom populated the notochord, their normal fate; those injected with RNA encoding Xom populated predominantly the somites (Fig. 11; Table 2). BMP-4 may cause ventralization of Xenopus embryos by inhibiting the response to dorsal mesoderm-inducing factors such as activin: animal pole regions derived from embryos that had received injections of RNA encoding BMP-4 do not

➝

Xom mediates BMP-4 signalling in Xenopus 2391

Fig. 11. Xom, but not ∆Xom, changes the fate of prospective dorsal mesodermal cells. RNA (2 ng) encoding ∆Xom (A, B), or Xom (C, D) was injected into blastomere C1 of Xenopus embryos at the 32-cell stage along with RNA (100 pg) encoding βgalactosidase, which acts as a lineage marker. The embryos were allowed to develop to stage 42, when they were fixed and processed for β-galactosidase expression. Note that blastomeres injected with RNA encoding ∆Xom make a major contribution to notochord (A, B), while those expressing Xom (C, D) preferentially populate the somites.

Table 1. Effects of over-expression of Xom in dorsal blastomeres of Xenopus embryos RNA

Anterior deficiencies (%)

Notochord absent (%)

n

Xom ∆Xom

65 1

48 0

108 125

The two dorsal blastomeres of Xenopus embryos at the 4-cell stage were injected with the indicated RNAs (4 ng total), and embryos were allowed to develop to stage 36. The presence of notochord was established by wholemount antibody staining using the monoclonal antibody MZ15.

Table 2. Effects of over-expression of Xom in blastomere C1 of the 32-cell Xenopus embryo RNA

Notochord

Number of embryos

Xom ∆Xom

1 14

28 31

A C1 blastomere of the 32-cell stage Xenopus embryo was injected with 2 ng RNA encoding Xom or ∆Xom together with 100 pg RNA encoding βgalactosidase. The embryos were allowed to develop to stage 30 to 40, depending on the experiment, and they were then fixed and stained for βgalactosidase expression. This experiment was carried out three times, with similar results each time; the result of one representative experiment is shown here. In embryos injected with RNA encoding ∆Xom, blastomere C1 gave rise predominantly to notochord in approximately half the cases; in the remainder C1 populated head structures. In embryos injected with RNA encoding Xom, C1 gave rise only rarely to notochord and contributed predominantly to the somites.

elongate in response to activin, and formation of dorsal tissues such as muscle is inhibited (Dale et al., 1992; Jones et al., 1992). The same is true of animal caps derived from embryos that had received injections of RNA encoding Xom: elongation of animal caps is inhibited (Fig. 12) and expression of musclespecific actin is greatly reduced (Fig. 13A). The effect of Xom is again less marked than that of BMP-4 and it can be overcome by treatment with high concentrations of activin (not shown). We note that Xom does not induce ventral mesoderm and that its inhibitory effects are specific to dorsal mesoderm induction. Thus, Xom does not induce expression of the ventral marker αT4 globin, and nor of the general mesodermal marker Xbra; furthermore, it does not inhibit expression of Xbra in response to activin (Fig. 13B).

Fig. 12. Animal caps derived from embryos injected with Xom RNA do not elongate in response to activin. Animal caps were dissected from control embryos or from embryos injected with RNA encoding Xom or ∆Xom. They were left untreated or were exposed to 4 units/ml activin. Caps were photographed at the equivalent of stage 17. (A) Control animal caps remain spherical. (B) Activin treatment of animal caps derived from uninjected embryos causes dramatic elongation. (C) Animal caps derived from embryos injected with RNA encoding ∆Xom remain spherical. (D) Activin treatment of animal caps derived from embryos injected with RNA encoding ∆Xom causes elongation. (E) Animal caps derived from embryos injected with RNA encoding Xom remain spherical. (F) Activin treatment of animal caps derived from embryos injected with RNA encoding Xom does not cause elongation.

Finally, we have investigated whether Xom also inhibits the dorsalizing effects of noggin (Smith and Harland, 1992; Smith et al., 1993). In these experiments, RNA encoding Xom and RNA encoding noggin were injected simultaneously into fertilized eggs, and ventral marginal zone tissue was isolated at the early gastrula stage and cultured to tailbud stages. Muscle differentiation was assessed by whole-mount antibody staining using the monoclonal antibody 12/101. In control experiments, ventral marginal zone tissue derived from uninjected embryos formed little muscle, while that derived from embryos injected with RNA encoding noggin formed large amounts. Co-

2392 R. Ladher and others of the Drosophila annasae gene Om1D (Tanda and Corces, 1991) and the Drosophila melanogaster gene BarH1 (Higashijima et al., 1992a,b). This similarity, together with the similarities in expression pattern (all three genes are expressed in the eye and in peripheral neurons) inspired us to call the Xenopus gene Xom. All three genes have an unusual homeodomain, which contains a threonine at position 47 rather than the more common valine or isoleucine. This substitution changes the DNA binding specificity of Hox 11, another member of this class of gene, from TAAT to TAAC (Dear et al., 1993); we plan to investigate whether this is also true of Xom. We also note that Xom contains two additional domains: there is a highly acidic region, rich in serines and threonines, and a proline-rich domain (Fig. 1C). These may be involved in transcription activation (Ma and Ptashne, 1987; Mermod et al., 1989), although we do not yet have data that address this point directly.

Fig. 13. Xom inhibits induction of dorsal mesoderm by activin but does not affect activation of Xbra or induce expression of αT4 globin. Animal caps derived from embryos injected with RNA encoding Xom or ∆Xom were exposed, where appropriate, to activin (4 units/ml) and cultured to the equivalent of stage 25 for analysis of cardiac actin expression, to stage 10.5 for analysis of Xbra and to stage 25 for analysis of αT4 globin. (A) Induction of cardiac (muscle-specific) actin is inhibited by Xom RNA but not by ∆Xom. (B) Expression of Xbra is not affected by Xom and Xom does not induce expression of αT4 globin, even though activin-induced elongation in this experiment was blocked (not shown). Expression of EF-1α was used as a loading control.

injection of RNA encoding Xom caused a slight reduction in the amount of muscle induced by noggin, but this inhibition was far from complete (data not shown).

DISCUSSION In this paper we describe a novel homeobox-containing gene which appears to mediate the effects of BMP-4 during early Xenopus development. Here we discuss its sequence, its control of expression and its embryological function. Xom Xom contains a homeodomain that is most homologous to that

Control of Xom expression Expression of Xom first occurs at the mid-blastula stage, and as soon as transcripts can be detected it is apparent that the highest expression occurs on the ventral side of the embryo. This is clearest at the early gastrula stage, when transcripts are absent from the dorsal blastopore lip but are abundant elsewhere in the embryo. By the late gastrula/early neurula stage Xom is expressed throughout the embryo, with the striking exception of the dorsal midline and the anterior neural plate. Subsequently, expression declines, with transcripts remaining only in the tip of the tail and the dorsal half of the retina. The early expression pattern of Xom is similar to that of BMP-4 (Fainsod et al., 1995; Schmidt et al., 1995) and is the reciprocal of those of noggin (Smith and Harland, 1992) and chordin (Sasai et al., 1994). Consistent with these observations, our results show that interference with the function of BMP-4 blocks Xom expression and over-expression of BMP-4 causes ectopic expression of the gene. Indeed, expression of Xom is an immediate-early response to BMP-4. Similarly, overexpression of noggin causes a marked down-regulation of Xom. The time of onset of Xom expression is similar to that of BMP-4 and may even precede it, raising the possibility that the initial stages of Xom expression depend on maternally provided BMP-4, for which low levels of maternal transcripts can be detected (Dale et al., 1992) (and Fig. 9), or perhaps on BMP2, which has high levels of maternal transcripts (Clements et al., 1995). At neurula stages, the expression patterns of Xom and BMP4 differ; in particular, Xom transcripts are more widespread (see Fainsod et al., 1995; Schmidt et al., 1995). It is possible, however, that expression of Xom still depends on BMP-4 signalling, because at tailbud stages their expression patterns are again similar; the more widespread expression of Xom at neurula stages may reflect a longer mRNA half-life, or there may be expression of BMP-4 that is too low to be detected by in situ hybridization. Function of Xom Over-expression of Xom in dorsal cells of the early Xenopus embryo causes partial loss of anterior and dorsal structures, and specific expression of the gene in cells normally fated to form notochord causes them to populate more ventral structures such

Xom mediates BMP-4 signalling in Xenopus 2393 as the somites. Furthermore, over-expression of Xom in animal caps prevents at least some of the effects of activin treatment: gastrulation-like movements (Symes and Smith, 1987) are blocked and differentiation of dorsal cell types such as muscle is inhibited. By contrast, expression of the general mesodermal marker Xbra (Smith et al., 1991) is unaffected. These effects of Xom resemble those of over-expression of BMP-4, suggesting that Xom mediates, at least in part, the effects of this peptide growth factor. The effects of Xom are not as marked as those of BMP-4, however, and it is possible that we are not able to express the gene to a sufficiently high level, or that other genes are required for the full ventralization of the Xenopus embryo that is caused by BMP-4. The observation that Xom is not able completely to reverse the dorsalizing effects of noggin is consistent with both these suggestions, and this issue is now under investigation. Finally, it is surprising that Xom, a gene that causes ventralization, is induced in animal caps by the dorsal mesoderminducing factor activin. This suggests that activin also induces the expression of genes that counteract the effects of Xom, and it raises the possibility that if the function of Xom could be inhibited, lower levels of activin might be sufficient to induce dorsal mesoderm. This too is under investigation. This work is supported by the Medical Research Council. J.C.S. is an International Scholar of the Howard Hughes Medical Institute. A.M.S. is supported by an EC BioTech grant. We thank Norma Towers and Surendra Kotecha for technical assistance, and members of our laboratory, especially Mike Jones, for advice and discussions. We are also grateful to Atasushi Suzuki and Naoto Ueno for the truncated BMP-4 receptor and to Genetics Institute for rhBMP-4.

REFERENCES Asashima, M., Nakano, H., Shimada, K., Kinoshita, K., Ishii, K., Shibai, H. and Ueno, N. (1990). Mesodermal induction in early amphibian embryos by activin A (erythroid differentiation factor). Roux’s Arch. Dev. Biol. 198, 3305. Bassez, T., Paris, J., Omilli, F., Dorel, C. and Osbourne, H. B. (1990). Posttranscriptional regulation of ornithine decarboxylase in Xenopus laevis oocytes. Development 110, 955-962. Cho, K. W. Y., Blumberg, B., Steinbeisser, H. and De Robertis, E. M. (1991). Molecular nature of Spemann’s organizer: the role of the Xenopus homeobox gene goosecoid. Cell 67, 1111-1120. Chomczynski, P. (1993). A reagent for the single step isolation of RNA, DNA and proteins from cell and tissue samples. BioTechniques 15, 532-537. Christian, J. L. and Moon, R. T. (1993). Interactions between Xwnt-8 and Spemann organizer signaling pathways generate dorsoventral pattern in the embryonic mesoderm of Xenopus. Genes Dev. 7, 13-28. Clements, J. H., Fettes, P., Knöchel, S., Lef, J. and Knöchel, W. (1995). Bone morphogenetic protein 2 in the early development of Xenopus laevis. Mech. Dev. 52, 357-370. Cooke, J., Smith, J. C., Smith, E. J. and Yaqoob, M. (1987). The organization of mesodermal pattern in Xenopus laevis: experiments using a Xenopus mesoderm-inducing factor. Development 101, 893-908. Crompton, M. R., Bartlett, T. J., MacGregor, A. D., Manfioletti, G., Buratti, E., Giancotti, V. and Goodwin, G. H. (1992). Identification of a novel vertebrate homeobox gene expressed in haematopoietic cells. Nucl. Acids Res. 20, 5661-5667. Cunliffe, V. and Smith, J. C. (1994). Specification of mesodermal pattern in Xenopus laevis by interactions between Brachyury, noggin and Xwnt-8. EMBO J. 13, 349-359. Dale, L., Howes, G., Price, B. M. and Smith, J. C. (1992). Bone morphogenetic protein 4: a ventralizing factor in early Xenopus development. Development 115, 573-85. Dale, L., Matthews, G. and Colman, A. (1993). Secretion and mesoderm-

inducing activity of the TGF-beta related domain of Xenopus Vg1. EMBO J. 12, 4471-4480. Dear, T. N., Sanchez-Garcia, I. and Rabbitts, T. H. (1993). The Hox 11 gene encodes a DNA-binding nuclear transcription factor belonging to a distinct family of homeobox genes. Proc. natn Acad. Sci. USA 90, 4431-4435. Fainsod, A., Steinbeisser, H. and De Robertis, E. M. (1995). On the function of BMP-4 in patterning the marginal zone of the Xenopus embryo. EMBO J. 13, 5015-5025. Graff, J. M., Thies, R. S., Song, J. J., Celeste, A. J. and Melton, D. A. (1994). Studies with a Xenopus BMP receptor suggest that ventral mesoderminducing signals override dorsal signals in vivo. Cell 79, 169-179. Green, J. B. A., Howes, G., Symes, K., Cooke, J. and Smith, J. C. (1990). The biological effects of XTC-MIF: quantitative comparison with Xenopus bFGF. Development 108, 229-38. Harland, R. M. (1991). In situ hybridization: an improved whole mount method for Xenopus embryos. Meth. Enzymol. 36, 675-685. Higashijima, S., Michiue, T., Emori, Y. and Saigo, K. (1992a). Dual Bar homeobox genes of Drosophila are required in two photoreceptor cells, R1 and R6, for normal eye development. Genes Dev. 6, 50-60. Higashijima, S., Michiue, T., Emori, Y. and Saigo, K. (1992b). Subtype determination of Drosophila embryonic external sensory organs by redundant homeobox genes BarH1 and BarH2. Genes Dev. 6, 1005-1018. Jones, C. M., Dale, L., Hogan, B. L. M., Wright, C. V. E. and Smith, J. C. (1996). Bone morphogenetic protein-4 (BMP-4) exerts its ventralizing effects on embryonic mesoderm during gastrula stages in Xenopus development. Development 122, 1545-1554. Jones, C. M., Kuehn, M. R., Hogan, B. L. M., Smith, J. C. and Wright, C. V. E. (1995). Nodal-related signals induce axial mesoderm and dorsalize mesoderm during gastrulation. Development 121, 3651-3662. Jones, C. M., Lyons, K. M., Lapan, P. M., Wright, C. V. E. and Hogan, B. L. M. (1992). DVR-4 (Bone morphogenetic protein-4) as a posteriorventralizing factor in Xenopus mesoderm induction. Development 115, 639647. Kennedy, M. A., Gozalez-Sarmiento, R., Kees, U. R., Lampert, F., Dear, T. M., Boehm, T. and Rabbitts, T. H. (1991). Hox 11, a homeobox-containing T cell oncogene found on human chromosome 10q24. Proc. natn Acad. Sci. USA 88, 8900-8904. Kintner, C. R. and Brockes, J. P. (1984). Monoclonal antibodies recognise blastemal cells derived from differentiating muscle in newt limb regeneration. Nature 308, 67-69. Krieg, P. A. and Melton, D. A. (1984). Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNA. Nucl. Acids Res. 12, 7057-7070. Ma, J. and Ptashne, M. (1987). Deletion analysis of GAL4 defines two transcriptional activating segments. Cell 48, 847-853. Maeno, M., Ong, R. C., Suzuki, A., Ueno, N. and Kung, H. F. (1994). A truncated bone morphogenetic protein 4 receptor alters the fate of ventral mesoderm to dorsal mesoderm: roles of animal pole tissue in the development of ventral mesoderm. Proc. natn Acad. Sci. USA 91, 10260-10264. Mermod, N., O’Neill, E. A., Kelley, T. J. and Tjian, R. (1989). The proline rich transcriptional activator of CTF/NF-1 is distinct from the replication and DNA binding domain. Cell 58, 741-753. Mohun, T. J., Garrett, N., Stutz, F. and Spohr, G. (1988). A third striated muscle actin gene is expressed during early development in the amphibian Xenopus laevis. J Mol. Biol. 202, 67-76. Nieuwkoop, P. D. and Faber, J. (1975). Normal Table of Xenopus laevis (Daudin) (Amsterdam: North Holland). Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press. Sargent, M. G. and Bennett, M. F. (1990). Identification in Xenopus of a structural homologue of the Drosophila gene Snail. Development 109, 967973. Sasai, Y., Lu, B., Steinbeisser, H. and De Robertis, E. M. (1995). Regulation of neural induction by the Chd and Bmp-4 antagonistic patterning signals in Xenopus. Nature 376, 333-336. Sasai, Y., Lu, B., Steinbeisser, H., Geisssert, D., Gont, L. K. and De Robertis, E. M. (1994). Xenopus chordin: a novel dorsalizing factor activated by organizer-specific homeobox genes. Cell 79, 779-790. Schmidt, J. E., Suzuki, A., Ueno, N. and Kimelman, D. (1995). Localized BMP-4 mediates dorsal/ventral patterning in the early Xenopus embryo. Dev. Biol. 169, 37-50. Slack, J. M. W. (1984). Regional biosynthetic markers in the early amphibian embryo. J. Embryol. exp. Morph 80, 289-319. Smith, J. C. (1993). Purifying and assaying mesoderm-inducing factors from

2394 R. Ladher and others vertebrate embryos. In Cellular Interactions in Development – a Practical Approach (ed. D. Hartley), pp. 181-204. Oxford: Oxford University Press. Smith, J. C., Price, B. M. J., Green, J. B. A., Weigel, D. and Herrmann, B. G. (1991). Expression of a Xenopus homolog of Brachyury (T) is an immediate-early response to mesoderm induction. Cell 67, 79-87. Smith, J. C., Price, B. M. J., Van, N. K. and Huylebroeck, D. (1990). Identification of a potent Xenopus mesoderm-inducing factor as a homologue of activin A. Nature 345, 732-4. Smith, J. C. and Slack, J. M. W. (1983). Dorsalization and neural induction: properties of the organizer in Xenopus laevis. J. Embryol. Exp. Morph. 78, 299-317. Smith, J. C. and Watt, F. M. (1985). Biochemical specificity of Xenopus notochord. Differentiation 29, 109-115. Smith, W. C. and Harland, R. M. (1991). Injected Xwnt-8 RNA acts early in Xenopus embryos to promote formation of a vegetal dorsalizing center. Cell 67, 753-765. Smith, W. C. and Harland, R. M. (1992). Expression cloning of noggin, a new dorsalizing factor localized to the Spemann organizer in Xenopus embryos. Cell 70, 829-840. Smith, W. C., Knecht, A. K., Wu, M. and Harland, R. M. (1993). Secreted noggin protein mimics the Spemann organizer in dorsalizing Xenopus mesoderm. Nature 361, 547-549. Smith, W. C., McKendry, R., Ribisi, S. J. and Harland, R. M. (1995). A nodal-related gene defines a physical and functional domain within the Spemann organizer. Cell 82, 37-46. Suzuki, A., Thies, R. S., Yamiji, N., Song, J. J., Wozney, J. M., Murakami, K. and Ueno, N. (1994). A truncated bone morphogenetic protein receptor affects dorso-ventral patterning in the early Xenopus embryo. Proc. natn Acad. Sci. USA 91, 10255-10259. Symes, K. and Smith, J. C. (1987). Gastrulation movements provide an early marker of mesoderm induction in Xenopus. Development 101, 339-349. Tanda, S. and Corces, V. G. (1991). Retrotransposon-induced overexpression

of a homeobox gene causes defects in eye morphogenesis in Drosophila. EMBO J. 10, 407-417. Thomsen, G., Woolf, T., Whitman, M., Sokol, S., Vaughan, J., Vale, W. and Melton, D. A. (1990). Activins are expressed early in Xenopus embryogenesis and can induce axial mesoderm and anterior structures. Cell 63, 485-93. Thomsen, G. H. and Melton, D. A. (1993). Processed Vg1 protein is an axial mesoderm inducer in Xenopus. Cell 74, 433-441. van den Eijnden Van Raaij, A. J., van Zoelent, E. J., van Nimmen, K., Koster, C. H., Snoek, G. T., Durston, A. J. and Huylebroeck, D. (1990). Activin-like factor from a Xenopus laevis cell line responsible for mesoderm induction. Nature 345, 819-22. Walmsley, M. E., Guille, M. J., Bertwistle, D., Smith, J. C., Pizzey, J. A. and Patient, R. K. (1994). Negative control of Xenopus GATA-2 by activin and noggin with eventual expression in precursors of the ventral blood islands. Development 120, 2519-2529. Whiting, J., Marshall, H., Cook, M., Krumlauf, R., Rigby, P. W. J., Stott, D. and Allemann, R. K. (1991). Multiple spatially specific enhancers are required to reconstruct the pattern of Hox-2.6 gene expression. Genes Dev. 5, 2048-2059. Wilson, P. A. and Hemmati-Brivanlou, A. (1995). Induction of epidermis and inhibition of neural fate by Bmp-4. Nature 376, 331-333. Zaraisky, A. G., Ecochard, V., Kazanskaya, O. V., Lukyanov, S. A., Fesenko, I. V. and Duprat, A.-M. (1995). The homeobox-containing gene XANF-1 may control development of the Spemann organizer. Development 121, 3839-3847. Zaraisky, A. G., Lukynov, S. A., Vasiliev, O. L., Smirnov, Y. V., Belyavsky, A. V. and Kazanskaya, O. V. (1992). A novel homeobox gene expressed in the anterior neural plate of the Xenopus embryo. Dev. Biol. 152, 373-382. (Accepted 20 May 1996)