1 Department of Developmental Biology, National Institute for Basic Biology, 38 Nishigonaka, Myodaiji, Okazaki, 444-8585, Japan
2 Department of Molecular Biomechanics, School of Life Science, The Graduate University for Advanced Studies, Okazaki, 444-8585, Japan
*Author for correspondence (e-mail: nueno{at}nibb.ac.jp)
Accepted April 25, 2001
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SUMMARY |
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Key words: Head formation, Homeobox, BMP, MSX, Anterior endomesoderm, nodal, FAST, Xenopus laevis
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INTRODUCTION |
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Ectopic activation of the activin/nodal/Vg1 signal or inhibition of the BMP signal induces a partial dorsal axis (Fagotto et al., 1997; Lustig et al., 1996; Suzuki et al., 1994; Thomsen et al., 1990), suggesting that the actions of these two classes of growth factors are reciprocal. The induction of a partial secondary axis is considered as a consequence of their intracellular signal transduction and target gene activation. Thus, this experimental system has been useful for studying the in vivo molecular mechanisms of dorsalization and ventralization in the early Xenopus embryo. In contrast, head formation has been thought of as a related but distinct molecular event from trunk formation, in which only TGF-ß family ligands are involved (Niehrs, 1999). The role of BMP-2 and BMP-4 has been believed to be limited to suppressing the trunk formation, because inhibiting them results in a partial dorsal axis with no head (Suzuki et al., 1994). Thus, the complete dorsal body axis has been believed to be patterned by two independent activities the head organizer and the trunk organizer. In contrast to the action of BMP as a trunk repressor, the Wnt family of proteins has long been implicated in head formation. One of the Wnt family members, Xwnt-8, was initially reported to induce an ectopic secondary dorsal axis with a complete head when its mRNA was injected into ventral blastomeres (Christian et al., 1991). It was later found, however, that Xwnt-8 is normally expressed in the ventrolateral mesoderm during the early gastrula stage and that dorsal overexpression by DNA injection ventralized the embryo, suggesting that endogenous Xwnt-8 acts as a ventralizing agent (Christian and Moon, 1993). This interpretation implied that the Wnt ligand is involved in head repression rather than head induction. Recent studies have further demonstrated that in addition to inhibition of the BMP signal, inhibition of the Wnt signal is necessary for head induction, suggesting that the default, inhibited, state of both of these signals is required for head induction (Glinka et al., 1997). Recently however, cerberus, a multiple binding protein for Wnt, nodal and BMP, was reported to be responsible for head induction (Piccolo et al., 1999). Because the late overexpression of nodal, resulting from DNA injection into the dorsal side, causes repression of the head, inhibition of nodal by cerberus may be essential for head formation. In addition, a more recent study showed that the inhibition of Wnt ligands in ventral blastomeres by ECD8, an extracellular domain of the Wnt receptor Frizzled, is sufficient to induce an ectopic head (Itoh and Sokol, 1999). Therefore, the precise sequence of gene activation and protein-protein interaction events that promote head development in vivo is still unclear.
Although many of the experiments on head formation in Xenopus have involved looking at the effects of extracellular ligands, in this study, we approached the problem using an inhibitory version of the homeobox protein Xmsx-1. Here we show that Xmsx-1 has an essential role in head repression that is elicited at the level of the transcriptional complex induced by nodal signaling.
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MATERIALS AND METHODS |
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Manipulation of embryos and microinjection of synthetic mRNA
In vitro fertilization of Xenopus eggs was performed as described previously (Suzuki et al., 1995). The fertilized embryos were dejellied using 3% cysteine hydrochloride and washed with water several times. Four-cell-stage embryos were microinjected with capped mRNAs, which were synthesized using the mMESSAGE mMACHINE sp6 kit (Ambion) and then purified by passing through a Sephadex G-50 column (Amersham Pharmacia Biotech). The injected embryos were cultured in 3% Ficoll/0.1x Steinbergs solution until the appropriate stage for each experiment. Embryos were staged according to Nieuwkoop and Faber (Nieuwkoop and Faber, 1967). The ventral marginal zone (VMZ), dorsal marginal zone (DMZ), ventral vegetal quarter, and dorsal vegetal quarter were dissected at stage 10 and cultured in 0.1% bovine serum albumin (BSA)/1x Steinbergs solution (Asashima et al., 1990).
Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted from the marginal zone explants and the embryos using TRIzol reagent (GIBCO/BRL) according to the manufacturers instructions. Extracted RNA was subjected to reverse transcription with random hexameric primers. The expression of each molecular marker was detected by PCR using the following specific primers: anterior endomesodermal markers, cerberus, upstream, 5'-ATTCACTTAACAGCAGAGGT-3' and downstream, 5'-CTTCTA-GAACCATTGTAAGC-3'; Xotx-2, upstream, 5'-GGAGGCCAAAACAAAGTG-3' and downstream, 5'-TCATGGGGTAGGACCTCT-3'; Xhex, upstream, 5'-AAGAGCCAAATGGAGGCGTC-3' and downstream, 5'-GCAAGCTGAATAGAGGTCCA-3'; Xdkk-1, upstream, 5'-CTCTACAGTTGCACGGAAGA-3' and downstream, 5'-CCAGAATGGTTTCTTCCAGG-3'; a pan mesodermal marker, Xbra, upstream, 5'-GGATCATCTTCTCAGCGCTGTGGA-3' and downstream, 5'-GTTGTCGGCTGCCACAAAGTCCA-3'; nodal related gene markers, Xnr-1, upstream, 5'-AACCTCCCAA-GCCTACTGGA-3' and downstream, 5'-TTGTGTGATGGTT-CAGTCTC-3'; Xnr-2, upstream, 5'-GTCTTCTATATCCAGCA-GCAAT-3' and downstream, 5'- TTGATGGAGATAATA- CTGGAGC-3'; Xnr-3, upstream, 5'-GCCTCCCTTCTTTTAGAAAG-3' and downstream, 5'-CATCGTATCTACATTTTCTG-3'; Xnr-4, upstream, 5'-ACTTGGCTGCTCTACCTC-3' and downstream, 5'-CAGCAAGTTGATGTTCTTCC-3'; HoxB9, upstream, 5'-TACTT-ACGGGCTTGGCTGGA-3' and downstream, 5'-AGCGTGTAA-CCAGTTGGCTG-3'; eFGF, upstream, 5'-TTACTGCAATGTGGG- CATCG-3' and downstream, 5'-GCAGAAGCGTCTCTTTGAAT-3'. The primer sequences for Xwnt-8, Xvent-1, Xvent-2, ventrolateral markers, and histone H4, an internal input control, were as previously described (Yamamoto et al., 2000).
Whole-mount in situ hybridization, immunostaining and lineage tracing
Embryos coinjected with HI-Xmsx-1 and ß-galactosidase mRNA were fixed in 4% paraformaldehyde in 0.1 M potassium phosphate buffer (pH 7.8), washed with PBS, and stained with 6-chloro-3-indolyl-ß-D-galactoside (Nacalai tesque) for lineage labeling. Stained embryos were then refixed in MEMFA [0.1 M Mops (pH 7.4), 2 mM EGTA, 1 mM MgSO4, 3.7% formaldehyde] and stored in methanol at -30°C before being used for whole-mount in situ hybridization. Whole-mount in situ hybridization was performed as described previously (Harland, 1991; Hemmati-Brivanlou et al., 1990) using BM Purple (Boehringer) for the color reaction. To visualize the staining in the endomesoderm the embryos were cut with a razor blade along the dorsoventral axis.
Whole-mount immunostaining with anti-phosphoSmad1 antibody (Faure et al., 2000) and anti-MSX-1 (Babco) were performed as follows: embryos were fixed in MEMFA for 1 hour at room temperature, then washed with PBS and stored in methanol at -30°C before being used. The fixed embryos were rehydrated in PBS, permeabilized in PBSTw (0.1% Tween 20 in PBS), blocked in 15% fetal calf serum and incubated in primary antibody solution (1/500 and 1/2,000 dilution for anti-phosphoSmad1 and anti-MSX-1 antibody, respectively) overnight at 4°C. After washing with PBSTw several times, the secondary antibody incubation with HRP-conjugated goat anti-rabbit IgG (1/1,000 dilution) was carried out for 1 hour at room temperature and followed by washes with PBSTw. Immunoreactivity was visualized with diaminobenzidine. Immunostaining with the monoclonal antibody MZ15 was performed as described (Klymkowsky and Hanken, 1991).
Luciferase reporter assay
Four-cell-stage embryos were injected marginally with 50 pg of the -226gsc/Luc (Watabe et al., 1995) construct alone or in combination with the indicated capped synthesized mRNAs as described above. At stage 10.25, or 10.5, five DMZs or VMZs were isolated and lysed with 30 µl of cell lysis buffer [25 mM Tris-phosphate (pH 7.8), 2 mM dithiothreitol, 2 mM 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, 10% glycerol, 1% Triton X-100]. For the luciferase assay, 100 µl of Luciferase Assay Substrate (Promega) were added to each extract and luciferase activity was measured using a Luminescencer-PSN AB-2200 (ATTO).
Immunoprecipitation and GST pull-down analysis
293T cells were transiently transfected with the indicated constructs by the calcium phosphate method. Forty hours after transfection, the cells were lysed in lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 50 mM NaF] in the presence of protease inhibitors. Immunoprecipitation and the GST pull-down analysis were performed by incubating the extracts with the M2 Flag monoclonal antibody (Sigma) coupled to protein A Sepharose CL 4B and with glutathione Sepharose 4B (Pharmacia), respectively, at 4°C for 1 hour. The precipitates were then washed with lysis buffer and subjected to western blot analysis using an anti-HA antibody (Santa Cruz Biotechnology).
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RESULTS |
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Xmsx-1 inhibits the transcriptional complex formation of Smad2/4 and FAST-1
How does Xmsx-1 inhibit nodal signaling? It has been shown that, following the activation of the nodal receptors ActRIA/IB and ActRII, and an EGF-CFC coreceptor (one eyed pinhead, and crypto/criptic for zebrafish and mouse, respectively), formation of a transcriptional complex is essential to activate nodal target genes. This complex includes Smad2, Smad4 and a winged helix transcription factor FAST, a protein whose binding site is the essential minimal enhancer sequence in the ARE (Watanabe and Whitman, 1999). This led us to speculate that Xmsx-1 may physically interact with the transcriptional complex triggered by nodal signaling. We tested whether Xmsx-1 protein binds the pathway-restricted Smads, Xsmad1, Xsmad2 (Baker and Harland, 1996; Graff et al., 1996), or the common Smads Xsmad4 and Xsmad4ß (Howell et al., 1999; Masuyama et al., 1999). We expressed Flag-tagged Xmsx-1 protein and HA-tagged Smad proteins transiently in 293T cells, and the cell extracts were subjected to immunoprecipitation with anti-Flag antibodies, followed by western blot analysis with anti-HA antibodies. Interestingly, all of the Smads were coprecipitated with Xmsx-1, although the efficacy varied (Fig. 7A). Among them, Xsmad4ß was most efficiently immunoprecipitated with Xmsx-1. This may be due to the fact that Smad4ß is localized exclusively to nucleus at a higher concentration, while the other Smads are present in both the cytoplasm and the nucleus. To investigate the possibility that the binding of Xmsx-1 to Smad4 replaces the activin/nodal-regulated Smad2, we examined whether increasing doses of Xmsx-1 could change Smad2 level in the complex (Fig. 7B). However, the level of Smad2 in the complex did not change as a result of Xmsx-1 expression. This indicates that Xmsx-1 is likely to bind to the Smad2/4 complex additively. Furthermore, in this context we tested whether the incorporation of Xmsx-1 into a Smad2/4 complex could exclude the transcription factor FAST. GST-tagged xFAST-1, HA-tagged Xsmad2 and increasing dose of HA-tagged Xmsx-1 were expressed in 293T cell in the presence or absence of Xsmad4ß and subjected to GST pull down analysis (Fig. 7C). The results showed that Xmsx-1 did not bind to xFAST-1 (Fig. 7C, lane 8) but clearly inhibited xFAST-1 to bind Xsmad2 in a dose-dependent manner (Fig. 7C, lanes 2-5). Interestingly, this competitive inhibition depended on the presence of Xsmad4ß (Fig. 7C, lanes 4 and 6). These results suggest that the binding of Xmsx-1 to Smad4 may exclude FAST from the Smad2/4 complex and this mechanism may explain how the transcriptional activity of the complex is negatively regulated.
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As predicted, BMP signaling represented by the anti-phosphoSmad1 staining was detected in the ventral endoderm region but not in the anterior endoderm (Fig. 8A). Consistent with this, Xmsx-1 was also detected immunohistochemically in the overlapping region with its specific antibodies (Fig. 8B). These results further support the idea that BMP/Xmsx-1 activities are necessary in the ventral endoderm to repress head formation and that both activities are absent in the corresponding dorsal region, where the head organizer is formed.
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DISCUSSION |
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Previously, the ectopic overexpression of nodal was reported to induce only a partial secondary dorsal axis without a head, and it was shown to induce a head only when cooverexpressed with noggin (Lustig et al., 1996). However, we have clearly demonstrated here that nodal alone can induce a complete secondary axis with a head when it is ventrally overexpressed. Therefore, the former explanation is consistent with our functional analysis of nodal in the early Xenopus embryo. It is also known that, later in development, nodal must be inhibited dorsally. This has been proposed to occur indirectly through the induction of cerberus (Piccolo et al., 1999). In addition, head induction by chordin and frz-b was also rescued by the coinjection of Xnr-1 DNA. Therefore, cerberus expressed ectopically by DNA injection may serve as an inhibitor of nodal in later stages, fulfilling the requirement for head formation. When HI-Xmsx-1 was dorsally injected, defects in head formation were observed (data not shown). This may be due to the hyperactivation of nodal signaling, and is consistent with the above speculation.
The latter possibility, involving the down-regulation of Xwnt-8, is also likely. The ventral mesoderm marker, Xwnt-8 was significantly down-regulated as a result of the inhibition of Xmsx-1 activity. It is believed that the BMP activity, most likely through its induction of Xmsx-1 activity (Takeda et al., 2000), is necessary for the onset and maintenance of the ventral expression of Xwnt-8 at the early gastrula stage. Our results further confirmed that termination of the BMP signaling cascade with HI-Xmsx-1 leads to the loss of Xwnt-8 expression. Despite the similar inhibition of both BMP and Xwnt-8 activity, the mechanism of head induction by Dkk-1 and tBR (Glinka et al., 1998) may be slightly different because the inhibition of Wnts and BMP by Dkk-1 and tBR is thought to be solely an extracellular event, while HI-Xmsx-1 inhibits BMP signals intracellularly and Xwnt-8 at the transcriptional level.
To investigate the above possibilities, we expressed HI-Xmsx-1 using a DNA vector, with the intention that HI-Xmsx-1 be expressed later than it would be by mRNA injection. Interestingly, ectopic induction of the head was not observed (data not shown), indicating that early action of HI-Xmsx-1 is essential for ectopic head induction, which would support the former possibility. Also it was previously demonstrated that the blockage of BMP and anti-dorsalizing morphogenetic protein (ADMP), a member of TGF-ß superfamily expressed in the Xenopus trunk organizer, induces head formation (Dosch and Niehrs, 2000). To address whether the ectopic head induction by HI-Xmsx-1 was due to the inhibition of ADMP, we tested the effect of HI-Xmsx-1 on the ventralizing phenotype by ADMP. No antagonizing ability of HI-Xmsx-1 was observed against ADMP (data not shown). Furthermore, ADMP expression level was not repressed in DMZ, while it was induced in VMZ by HI-Xmsx-1 (data not shown). These results suggest that Xmsx-1-induced ectopic head production does not involve ADMP inhibition.
As shown in Fig. 9, we demonstrated that inhibition of an extensive spectrum of BMP activities using multiple truncated BMP receptors or three extracellular BMP antagonists, so-called organizer factors, could produce an ectopic head structure, often with eyes and a cement gland, suggesting that the incomplete secondary axis formation observed in previous studies was due to residual endogenous BMP signals. Therefore it appears that BMP/Xmsx-1 signaling play a role, not only as a trunk repressor, but also as a head repressor in vivo.
Xmsx-1 inhibits nodal signaling
We showed that Xmsx-1 inhibited the activation of an ARE reporter gene in the dorsal blastomeres of Xenopus embryos. We therefore conclude that wild-type Xmsx-1 inhibits intracellular nodal signaling. This result may in turn suggest that HI-Xmsx-1 might have activated ectopic nodal signaling when overexpressed in ventral blastomeres. In other words, potential nodal signaling may be present in the ventral side of the embryo (as it has been shown that nodal transcript is evenly distributed in early embyo) (Jones et al., 1995), but is inactivated by the BMP/Xmsx-1 signal. In fact, endogenous activated Smad2 is detected in the ventral endoderm as well as in the anterior endoderm of the gastrula stage embryo (Faure et al., 2000). In this study, we hypothesized that this inhibition occurs at the intracellular signaling level. We also revealed that an increasing level of Xmsx-1 could efficiently exclude FAST protein from the Smad2/4 complex depending on the presence of Smad4. Taken together, these results suggest that through the binding to Smad4, Xmsx-1 associates with Smad2 and inhibits FAST-induced transcription. However, the exact mechanism in vivo by which nodal signaling is preferentially inhibited by the binding of Xmsx-1 to the complex remains to be investigated.
In this study, we clearly showed that Xmsx-1 inhibits nodal at the intracellular signaling level in the ventral endoderm without apparent change in the level of nodal transcripts. Recently, however, the expression of nodal was shown to be autoinduced by nodal signaling through a FAST-regulated module in the first intron of the gene (Osada et al., 2000). This indicates that the modulation of nodal signaling should lead to change in expression level of the ligand, which was not the case in this study. One possibility to explain this discrepancy is the fact that antivin/lefty, which is a member of TGF-ß superfamily and the antagonist of nodal signaling acting in a negative feedback loop to suppress the maintenance of nodal ligand, is also induced by nodal signaling. By this mechanism, the expression level of nodal may be maintained at a constant level even when Xmsx-1 is overexpressed.
Finally, an important question is, how universal among species is the head repression mechanism proposed here. Targeted disruption of mouse Msx1 and/or Msx2 reveals no anteroposterior patterning defect, although developmental defects were found in several organs of each Msx gene-disrupted mice (Satokata et al., 2000; Satokata and Maas, 1994). In the mouse, three Msx genes were isolated, Msx1, Msx2 and Msx3. Thus, Msx3 or another related homeobox genes may act redundantly in the mutants, as functional redundancy was previously reported for Msx1 and Msx2 (Satokata et al., 2000). Alternatively, the mechanism of head repression by BMP/Msx-1 may be specific to amphibians. We have shown nuclear localization of Xmsx-1 as well as phosphorylated BMP-driven Smads in ventral endoderm but not in anterior endoderm (Fig. 8A,B), supporting the proposed mechanism of head induction (de Souza and Niehrs, 2000). However, expression of mouse Msx proteins in the primitive streak and absence in anterior visceral endoderm (AVE), has not been demonstrated.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Asashima, M., Nakano, H., Shimada, K., Kinoshita, K., Ishii, K., Shibai, H. and Ueno, N. (1990). Mesoderm induction in early amphibian embryos by activin A (erythroid differentiation factor). Rouxs Arch. Dev. Biol. 198, 330-335.
Baker, J. C. and Harland, R. M. (1996). A novel mesoderm inducer, Madr2, functions in the activin signal transduction pathway. Genes Dev. 10, 1880-1889.[Abstract]
Beddington, R. S. and Robertson, E. J. (1999). Axis development and early asymmetry in mammals. Cell 96, 195-209.[Medline]
Chen, X., Rubock, M. J. and Whitman, M. (1996). A transcriptional partner for MAD proteins in TGF-beta signalling [published erratum appears in Nature (1996) Dec 19-26;384(6610):648]. Nature 383, 691-696.[Medline]
Chen, X., Weisberg, E., Fridmacher, V., Watanabe, M., Naco, G. and Whitman, M. (1997). Smad4 and FAST-1 in the assembly of activin-responsive factor. Nature 389, 85-89.[Medline]
Christian, J. L., McMahon, J. A., McMahon, A. P. and Moon, R. T. (1991). Xwnt-8, a Xenopus Wnt-1/int-1-related gene responsive to mesoderm- inducing growth factors, may play a role in ventral mesodermal patterning during embryogenesis. Development 111, 1045-1055.[Abstract]
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.[Abstract]
Conlon, F. L., Lyons, K. M., Takaesu, N., Barth, K. S., Kispert, A., Herrmann, B. and Robertson, E. J. (1994). A primary requirement for nodal in the formation and maintenance of the primitive streak in the mouse. Development 120, 1919-1928.
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, 573-585.[Abstract]
Dale, L. and Jones, C. M. (1999). BMP signalling in early Xenopus development. BioEssays 21, 751-60.[Medline]
de Souza, F. S. and Niehrs, C. (2000). Anterior endoderm and head induction in early vertebrate embryos. Cell Tissue Res. 300, 207-217.[Medline]
Dosch, R. and Niehrs, C. (2000). Requirement for anti-dorsalizing morphogenetic protein in organizer patterning. Mech. Dev. 90, 195-203.[Medline]
Fagotto, F., Guger, K. and Gumbiner, B. M. (1997). Induction of the primary dorsalizing center in Xenopus by the Wnt/GSK/beta-catenin signaling pathway, but not by Vg1, Activin or Noggin. Development 124, 453-460.
Faure, S., Lee, M. A., Keller, T., ten Dijke, P. and Whitman, M. (2000). Endogenous patterns of TGFß superfamily signaling during early Xenopus development. Development 127, 2917-2931.
Feldman, B., Gates, M. A., Egan, E. S., Dougan, S. T., Rennebeck, G., Sirotkin, H. I., Schier, A. F. and Talbot, W. S. (1998). Zebrafish organizer development and germ-layer formation require nodal- related signals [see comments]. Nature 395, 181-185.[Medline]
Gawantka, V., Delius, H., Hirschfeld, K., Blumenstock, C. and Niehrs, C. (1995). Antagonizing the Spemann organizer: role of the homeobox gene Xvent-1. EMBO J. 14, 6268-6279.[Abstract]
Glinka, A., Wu, W., Delius, H., Monaghan, A. P., Blumenstock, C. and Niehrs, C. (1998). Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction. Nature 391, 357-362.[Medline]
Glinka, A., Wu, W., Onichtchouk, D., Blumenstock, C. and Niehrs, C. (1997). Head induction by simultaneous repression of Bmp and Wnt signalling in Xenopus. Nature 389, 517-519.[Medline]
Graff, J. M., Bansal, A. and Melton, D. A. (1996). Xenopus Mad proteins transduce distinct subsets of signals for the TGF ß superfamily. Cell 85, 479-487.[Medline]
Harland, R. M. (1991). In situ hybridization: an improved whole-mount method for Xenopus embryos. Methods Cell Biol. 36, 685-695.[Medline]
Heasman, J. (1997). Patterning the Xenopus blastula. Development 124, 4179-4191.
Hemmati-Brivanlou, A., Frank, D., Bolce, M. E., Brown, B. D., Sive, H. L. and Harland, R. M. (1990). Localization of specific mRNAs in Xenopus embryos by whole-mount in situ hybridization. Development 110, 325-330.[Abstract]
Hogan, B. L. M. (1996). Bone morphogenetic proteins: multifunctional regulators of vertebrate development. Genes Dev. 10, 1580-1594.[Medline]
Howell, M., Itoh, F., Pierreux, C. E., Valgeirsdottir, S., Itoh, S., ten Dijke, P. and Hill, C. S. (1999). Xenopus Smad4beta is the co-Smad component of developmentally regulated transcription factor complexes responsible for induction of early mesodermal genes. Dev. Biol. 214, 354-69.[Medline]
Iemura, S., Yamamoto, T. S., Takagi, C., Uchiyama, H., Natsume, T., Shimasaki, S., Sugino, H. and Ueno, N. (1998). Direct binding of follistatin to a complex of bone-morphogenetic protein and its receptor inhibits ventral and epidermal cell fates in early Xenopus embryo. Proc. Natl. Acad. Sci. USA 95, 9337-9342.
Itoh, K. and Sokol, S. Y. (1999). Axis determination by inhibition of Wnt signaling in Xenopus. Genes Dev. 13, 2328-2336.
Jones, C. M., Kuehn, M. R., Hogan, B. L., Smith, J. C. and Wright, C. V. (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. and Hogan, B. L. (1992). DVR-4 (bone morphogenetic protein-4) as a posterior-ventralizing factor in Xenopus mesoderm induction. Development 115, 639-647.
Klymkowsky, M. W. and Hanken, J. (1991). Whole-mount staining of Xenopus and other vertebrates. Methods Cell Biol. 36, 4194-41.
Kurata, T., Nakabayashi, J., Yamamoto, T. S., Mochii, M. and Ueno, N. (2000). Visualization of endogenous BMP signaling during Xenopus development. Differentiation 67, 33-40.
Ladher, R., Mohun, T. J., Smith, J. C. and Snape, A. M. (1996). Xom: a Xenopus homeobox gene that mediates the early effects of BMP-4. Development 122, 2385-2394.
Lustig, K. D., Kroll, K., Sun, E., Ramos, R., Elmendorf, H. and Kirschner, M. W. (1996). A Xenopus nodal-related gene that acts in synergy with noggin to induce complete secondary axis and notochord formation. Development 122, 3275-3282.
Masuyama, N., Hanafusa, H., Kusakabe, M., Shibuya, H. and Nishida, E. (1999). Identification of two Smad4 proteins in Xenopus. Their common and distinct properties. J. Biol. Chem. 274, 12163-12170.
McMahon, A. P. and Moon, R. T. (1989). Ectopic expression of the proto-oncogene int-1 in Xenopus embryos leads to duplication of the embryonic axis. Cell 58, 1075-1084.[Medline]
Miyama, K., Yamada, G., Yamamoto, T. S., Takagi, C., Miyado, K., Sakai, M., Ueno, N. and Shibuya, H. (1999). A BMP-inducible gene, dlx5, regulates osteoblast differentiation and mesoderm induction. Dev. Biol. 208, 123-133.[Medline]
Niehrs, C. (1999). Head in the WNT: the molecular nature of Spemanns head organizer. Trends Genet. 15, 314-319.[Medline]
Nieuwkoop, P. D. and Faber, J. (1967). A Normal Table of Xenopus laevis. Amsterdam: North Holland Publishing Co.
Onichtchouk, D., Gawantka, V., Dosch, R., Delius, H., Hirschfeld, K., Blumenstock, C. and Niehrs, C. (1996). The Xvent-2 homeobox gene is part of the BMP-4 signalling pathway controlling dorsoventral patterning of Xenopus mesoderm. Development 122, 3045-3053.
Osada, S. I., Saijoh, Y., Frisch, A., Yeo, C. Y., Adachi, H., Watanabe, M., Whitman, M., Hamada, H. and Wright, C. V. (2000). Activin/nodal responsiveness and asymmetric expression of a Xenopus nodal-related gene converge on a FAST-regulated module in intron 1. Development 127, 2503-2514.
Persson, U., Izumi, H., Souchelnytskyi, S., Itoh, S., Grimsby, S., Engstrom, U., Heldin, C. H., Funa, K. and ten Dijke, P. (1998). The L45 loop in type I receptors for TGF-beta family members is a critical determinant in specifying Smad isoform activation. FEBS Lett. 434, 83-87.[Medline]
Piccolo, S., Agius, E., Leyns, L., Bhattacharyya, S., Grunz, H., Bouwmeester, T. and De Robertis, E. M. (1999). The head inducer Cerberus is a multifunctional antagonist of Nodal, BMP and Wnt signals. Nature 397, 707-710.[Medline]
Piccolo, S., Sasai, Y., Lu, B. and De Robertis, E. M. (1996). Dorsoventral patterning in Xenopus: inhibition of ventral signals by direct binding of chordin to BMP-4. Cell 86, 589-598.[Medline]
Sampath, K., Rubinstein, A. L., Cheng, A. M., Liang, J. O., Fekany, K., Solnica-Krezel, L., Korzh, V., Halpern, M. E. and Wright, C. V. (1998). Induction of the zebrafish ventral brain and floorplate requires cyclops/nodal signalling. Nature 395, 185-189.[Medline]
Sasai, Y. and De Robertis, E. M. (1997). Ectodermal patterning in vertebrate embryos. Dev. Biol. 182, 5-20.[Medline]
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.[Medline]
Satokata, I., Ma, L., Ohshima, H., Bei, M., Woo, I., Nishizawa, K., Maeda, T., Takano, Y., Uchiyama, M., Heaney, S. et al. (2000). Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation. Nat. Genet. 24, 391-395.[Medline]
Satokata, I. and Maas, R. (1994). Msx1 deficient mice exhibit cleft palate and abnormalities of craniofacial and tooth development [see comments]. Nat. Genet. 6, 348-356.[Medline]
Suzuki, A., Shioda, N. and Ueno, N. (1995). Bone morphogenetic protein acts as a ventral mesoderm modifier in early Xenopus embryos. Dev. Growth Differ. 37, 581-588.
Suzuki, A., Thies, R. S., Yamaji, N., Song, J. J., Wozney, J. M., Murakami, K. and Ueno, N. (1994). A truncated bone morphogenetic protein receptor affects dorsal-ventral patterning in the early Xenopus embryo. Proc. Natl. Acad. Sci. USA 91, 10255-10259.
Suzuki, A., Ueno, N. and Hemmati-Brivanlou, A. (1997). Xenopus msx1 mediates epidermal induction and neural inhibition by BMP4. Development 124, 3037-3044.
Takeda, M., Saito, Y., Sekine, R., Onitsuka, I., Maeda, R. and Maeno, M. (2000). Xenopus msx-1 regulates dorso-ventral axis formation by suppressing the expression of organizer genes. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 126, 157-168.[Medline]
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, 485-493.[Medline]
Varlet, I., Collignon, J. and Robertson, E. J. (1997). nodal expression in the primitive endoderm is required for specification of the anterior axis during mouse gastrulation. Development 124, 1033-1044.
Watabe, T., Kim, S., Candia, A., Rothbacher, U., Hashimoto, C., Inoue, K. and Cho, K. W. (1995). Molecular mechanisms of Spemanns organizer formation: conserved growth factor synergy between Xenopus and mouse. Genes Dev. 9, 3038-3050.[Abstract]
Watanabe, M. and Whitman, M. (1999). FAST-1 is a key maternal effector of mesoderm inducers in the early Xenopus embryo. Development 126, 5621-5634.
Weinstein, D. C. and Hemmati-Brivanlou, A. (1999). Neural induction. Annu. Rev. Cell Dev. Biol. 15, 411-433.[Medline]
Wilson, P. A. and Hemmati-Brivanlou, A. (1995). Induction of epidermis and inhibition of neural fate by Bmp-4. Nature 376, 331-333.[Medline]
Yamamoto, T. S., Takagi, C. and Ueno, N. (2000). Requirement of Xmsx-1 in the BMP-triggered ventralization of Xenopus embryos. Mech. Dev. 91, 131-141.[Medline]
Zeng, L., Fagotto, F., Zhang, T., Hsu, W., Vasicek, T. J., Perry, W. L., 3rd, Lee, J. J., Tilghman, S. M., Gumbiner, B. M. and Costantini, F. (1997). The mouse Fused locus encodes Axin, an inhibitor of the Wnt signaling pathway that regulates embryonic axis formation. Cell 90, 181-192.[Medline]
Zimmerman, L. B., De Jesus-Escobar, J. M. and Harland, R. M. (1996). The Spemann organizer signal noggin binds and inactivates bone morphogenetic protein 4. Cell 86, 599-606.[Medline]
Zorn, A. M., Butler, K. and Gurdon, J. B. (1999). Anterior endomesoderm specification in Xenopus by Wnt/beta-catenin and TGF-beta signalling pathways. Dev. Biol. 209, 282-297.[Medline]