Role of TAK1 and TAB1 in BMP signaling in early Xenopus development
1998; Springer Nature; Volume: 17; Issue: 4 Linguagem: Inglês
10.1093/emboj/17.4.1019
ISSN1460-2075
Autores Tópico(s)Cancer-related gene regulation
ResumoArticle15 February 1998free access Role of TAK1 and TAB1 in BMP signaling in early Xenopus development Hiroshi Shibuya Corresponding Author Hiroshi Shibuya Division of Morphogenesis, Department of Developmental Biology, National Institute for Basic Biology, Okazaki, 444 Japan Precursory Research for Embryonic Science and Technology, Japan Science and Technology Corporation, Hikari-dai, Kyoto, 619-02 Japan Search for more papers by this author Hiroshi Iwata Hiroshi Iwata Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, 060 Japan Search for more papers by this author Norihisa Masuyama Norihisa Masuyama Department of Genetics and Molecular Biology, Institute for Virus Research, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto, 606-01 Japan Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto, 606-01 Japan Search for more papers by this author Yukiko Gotoh Yukiko Gotoh Department of Genetics and Molecular Biology, Institute for Virus Research, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto, 606-01 Japan Search for more papers by this author Kyoko Yamaguchi Kyoko Yamaguchi Department of Molecular Biology, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya, 464-01 Japan Search for more papers by this author Kenji Irie Kenji Irie Department of Molecular Biology, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya, 464-01 Japan Search for more papers by this author Kunihiro Matsumoto Kunihiro Matsumoto Department of Molecular Biology, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya, 464-01 Japan Search for more papers by this author Eisuke Nishida Eisuke Nishida Department of Genetics and Molecular Biology, Institute for Virus Research, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto, 606-01 Japan Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto, 606-01 Japan Search for more papers by this author Naoto Ueno Naoto Ueno Division of Morphogenesis, Department of Developmental Biology, National Institute for Basic Biology, Okazaki, 444 Japan Search for more papers by this author Hiroshi Shibuya Corresponding Author Hiroshi Shibuya Division of Morphogenesis, Department of Developmental Biology, National Institute for Basic Biology, Okazaki, 444 Japan Precursory Research for Embryonic Science and Technology, Japan Science and Technology Corporation, Hikari-dai, Kyoto, 619-02 Japan Search for more papers by this author Hiroshi Iwata Hiroshi Iwata Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, 060 Japan Search for more papers by this author Norihisa Masuyama Norihisa Masuyama Department of Genetics and Molecular Biology, Institute for Virus Research, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto, 606-01 Japan Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto, 606-01 Japan Search for more papers by this author Yukiko Gotoh Yukiko Gotoh Department of Genetics and Molecular Biology, Institute for Virus Research, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto, 606-01 Japan Search for more papers by this author Kyoko Yamaguchi Kyoko Yamaguchi Department of Molecular Biology, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya, 464-01 Japan Search for more papers by this author Kenji Irie Kenji Irie Department of Molecular Biology, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya, 464-01 Japan Search for more papers by this author Kunihiro Matsumoto Kunihiro Matsumoto Department of Molecular Biology, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya, 464-01 Japan Search for more papers by this author Eisuke Nishida Eisuke Nishida Department of Genetics and Molecular Biology, Institute for Virus Research, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto, 606-01 Japan Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto, 606-01 Japan Search for more papers by this author Naoto Ueno Naoto Ueno Division of Morphogenesis, Department of Developmental Biology, National Institute for Basic Biology, Okazaki, 444 Japan Search for more papers by this author Author Information Hiroshi Shibuya 1,2, Hiroshi Iwata3, Norihisa Masuyama4,5, Yukiko Gotoh4, Kyoko Yamaguchi6, Kenji Irie6, Kunihiro Matsumoto6, Eisuke Nishida4,5 and Naoto Ueno1 1Division of Morphogenesis, Department of Developmental Biology, National Institute for Basic Biology, Okazaki, 444 Japan 2Precursory Research for Embryonic Science and Technology, Japan Science and Technology Corporation, Hikari-dai, Kyoto, 619-02 Japan 3Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, 060 Japan 4Department of Genetics and Molecular Biology, Institute for Virus Research, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto, 606-01 Japan 5Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto, 606-01 Japan 6Department of Molecular Biology, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya, 464-01 Japan *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:1019-1028https://doi.org/10.1093/emboj/17.4.1019 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Transforming growth factor-β (TGF-β) superfamily members elicit signals through stimulation of serine/threonine kinase receptors. Recent studies of this signaling pathway have identified two types of novel mediating molecules, the Smads and TGF-β activated kinase 1 (TAK1). Smads were shown to mimic the effects of bone morphogenetic protein (BMP), activin and TGF-β. TAK1 and TAB1 were identified as a MAPKKK and its activator, respectively, which might be involved in the up-regulation of TGF-β superfamily-induced gene expression, but their biological role is poorly understood. Here, we have examined the role of TAK1 and TAB1 in the dorsoventral patterning of early Xenopus embryos. Ectopic expression of Xenopus TAK1 (xTAK1) in early embryos induced cell death. Interestingly, however, concomitant overexpression of bcl-2 with the activated form of xTAK1 or both xTAK1 and xTAB1 in dorsal blastomeres not only rescued the cells but also caused the ventralization of the embryos. In addition, a kinase-negative form of xTAK1 (xTAK1KN) which is known to inhibit endogenous signaling could partially rescue phenotypes generated by the expression of a constitutively active BMP-2/4 type IA receptor (BMPR-IA). Moreover, xTAK1KN could block the expression of ventral mesoderm marker genes induced by Smad1 or 5. These results thus suggest that xTAK1 and xTAB1 function in the BMP signal transduction pathway in Xenopus embryos in a cooperative manner. Introduction Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β (TGF-β) superfamily, which have been implicated in the patterning of the mesoderm and ectoderm of Xenopus embryos. Both BMP-2 and BMP-4 are capable of inducing ventral mesoderm and re-specifying prospective dorsal mesoderm to differentiate into ventral tissues in Xenopus embryos (Dale et al., 1992; Jones et al., 1992; Clement et al., 1995). TGF-β elicits signals through a heteromeric complex consisting of the transmembrane type I and type II TGF-β receptors, which contain a cytoplasmic ser/thr-specific kinase domain (Wrana et al., 1994). Constitutively active type I receptors can signal biological responses in the absence of either ligand, type II receptors or both (Wieser et al., 1995). Three structurally related type I receptors, BMPR-IA [also known as ALK3 (ten Dijke et al., 1993)], BMPR-IB [also known as ALK6 (ten Dijke et al., 1994)] and ActRIA [also known as ALK2 (ten Dijke et al., 1993)], and one type II receptor, BMPR-II (Liu et al., 1995; Nohno et al., 1995), have been identified as the receptors for BMPs. It is known that a truncated form of BMPR-IA lacking its intracellular ser/thr kinase domain acts as a dominant-negative inhibitor of the cellular responses to BMP-2 and BMP-4 ligands in Xenopus embryos (Graff et al., 1994; Suzuki et al., 1994). Overexpression of this dominant-negative BMPR-IA in the ventral side of Xenopus embryos causes prospective ventral mesoderm to differentiate into dorsal mesoderm, and induces the expression of a neural marker, neural cell adhesion molecule (N-CAM), in the ectoderm. Recently, several molecules which are associated with TGF-β family receptors have been cloned. Several groups have reported that the immunophilin FKBP12 interacts with TGF-β family type I receptors (Wang et al., 1994, 1996b). FKBP12 binding to type I receptors may function to suppress spurious signaling resulting from the innate tendency of TβR-I and TβR-II to interact with one another (Chen et al., 1997). The α subunit of farnesyl-protein transferase (FT-α) was also isolated as a TβR-I-associated molecule (Kawabata et al., 1995; Wang et al., 1996a). Farnesyltransferase is known to play a critical role in the activation of p21RAS by attaching a farnesyl group, and aiding in its membrane association. Although FT-α was shown to be phosphorylated by TβR-I, the functional role of this modification is not clear. More recently, a group of proteins, the Smads, have been identified as important components of the TGF-β superfamily signal transduction pathway in a variety of species (Baker and Harland, 1996; Eppert et al., 1996; Graff et al., 1996; Hoodless et al., 1996; F.Liu et al., 1996; Thomsen, 1996). Smad1 has been shown to mimic the effects of BMP-2/4 in Xenopus as well as in mammalian cells. Smad2 and its close isoform Smad3 mediate signaling elicited by TGF-β or activin. Smad4/DPC4, originally isolated as a tumor-suppressor gene on chromosome 18q21 (Hahn et al., 1996), cooperates with Smad1, Smad2 and Smad3 to act as a common mediator of signaling by members of the TGF-β superfamily (Lagna et al., 1996). Smad5 is a closely related gene product of Smad1 (Riggins et al., 1996) and mediates a BMP-like signal in Xenopus (Suzuki et al., 1997). Although Smads have been shown to be critical for TGF-β superfamily signaling, it is not clear whether the involvement of other factors is required for signal transduction. Previously, we identified a novel mouse protein kinase designated TAK1 (TGF-β activated kinase 1) that functions as a MAPKKK (Yamaguchi et al., 1995). TAK1 was found to be involved in at least one TGF-β-induced gene response, and its kinase activity was stimulated in response to TGF-β1 or BMP-4. Furthermore, we identified a human protein (TAB1) that interacts with TAK1 using a yeast interaction screen, and provided data indicating that TAB1 functions as an activator for TAK1 in TGF-β signal transduction (Shibuya et al., 1996). We report here that Xenopus TAK1 (xTAK1) and xTAB1 induce ventral mesoderm. A kinase-negative form of xTAK1 (xTAK1KN) reverts the ventralization caused by constitutively active BMPR-IA and Smad1/5. These results suggest that xTAK1 and xTAB1 are important for the BMP signaling pathways involved in mesoderm induction and patterning in early Xenopus embryo development. Results Isolation of the Xenopus TAK1 and TAB1 cDNAs and their expression patterns in Xenopus embryos To analyze the function of TAK1 and TAB1 in embryonic development, cDNAs encoding Xenopus TAK1 and TAB1 (xTAK1 and xTAB1) were isolated from an oocyte cDNA library under low stringency conditions using the mouse TAK1 (mTAK1) and human TAB1 (hTAB1) cDNAs as probes, respectively. The longest xTAK1 and xTAB1 cDNAs recovered were ∼2.3 and 2.5 kb, respectively. Sequence analysis of the longest cDNAs for xTAK1 and xTAB1 each revealed a single open reading frame. The nucleotide sequences were predicted to encode a protein of 616 and 498 amino acids with a molecular mass of 68 and 54 kDa for TAK1 and TAB1, respectively. The amino acid sequence comparison between xTAK1 and mTAK1, or xTAB1 and hTAB1, shown in Figure 1, reveals that they are 76 or 70% identical, respectively. Figure 1.Protein sequence comparison of Xenopus TAK1 and TAB1 with mammalian homologs. (A) xTAK1 sequence and comparison with mTAK1. Alignment of the predicted amino acid sequences of Xenopus and mouse TAK1. The xTAK1 cDNA encodes a predicted protein of 616 amino acids. The xTAK1 protein is 76% identical to mTAK1 at the amino acid level. Residues conserved in both sequences are indicated by vertical bars, and the amino acid residues are numbered on the right. Note the high degree of conservation in the catalytic (98%) and C-terminal (99%) portions of the proteins. The GenBank accession No. for xTAK1 is U92030. (B) xTAB1 sequences and comparison with human TAB1. The xTAB1 protein is 70% identical to hTAB1 at the amino acid level. The GenBank accession No. for xTAB1 is U92031. Download figure Download PowerPoint The xTAK1 and xTAB1 transcripts are 3.0 and 3.2 kb in size, respectively. Northern blot analysis of mRNA obtained during early embryogenesis revealed that these transcripts are stored maternally and expressed throughout development (Figure 2A). The xTAK1 and xTAB1 mRNAs are not localized to any specific region in the early gastrula stages, as determined by whole-mount in situ hybridization (Figure 2B), and this ubiquitous expression was confirmed by RT–PCR amplification of RNA from dissected embryonic regions (data not shown). However, whole-mount in situ hybridization of embryos showed that xTAK1 and xTAB1 transcripts become localized to the nervous system at the end of neurulation, and are restricted to the central nervous system, eye and head neural crest cell populations by the early tadpole stages (Figure 2C). xTAK1 and xTAB1 transcripts are roughly co-localized, supporting the previous data indicating that these molecules function together in TGF-β family signaling. Figure 2.Developmental expression of Xenopus TAK1 and TAB1. (A) Northern blot of total embryonic RNAs shows that xTAK1 and xTAB1 are maternal and expressed at all stages of early development. The mRNAs for xTAK1 and xTAB1 are 3.0 and 3.2 kb long, respectively. Lane numbers correspond to developmental stages: 0, egg; 9, blastula; 10.5, 12.5, gastrula; 19, neurula; 24, tailbud tadpole; 34, swimming tadpole. (B) Expression of xTAK1 and xTAB1 genes in the early gastrula stages. Whole-mount in situ hybridization shows that the xTAK1 and xTAB1 transcripts are uniformly distributed in the early gastrula stages. Views of the vegetal pole are shown and dorsal is up. (C) Whole-mount in situ hybridization revealed the localization of the xTAK1 and xTAB1 transcripts. At the tailbud tadpole stage, xTAK1 and xTAB1 expression is high in the central nervous system and head. Download figure Download PowerPoint xTAK1 induces ventral mesoderm in Xenopus embryos To investigate the possible role of xTAK1 in embryonic development, xTAK1 mRNA was injected into the ventral or dorsal sides of Xenopus four-cell stage embryos. Injection of 0.2–1 ng of xTAK1 mRNA caused almost all of embryos to die at stage 10.5 as revealed by staining with acridine orange (Figure 3A). To confirm whether the cell death observed is programed cell death, we utilized the TUNEL method (Gavrieli et al., 1992), which revealed that cells were dying by apoptosis, as shown in Figure 3B. Apoptosis induced by xTAK1 was significantly inhibited by co-injection of human bcl-2 mRNA, resulting in normal embryos or those with only minor defects in head formation (Figure 4A). These results suggest that xTAK1, when overexpressed, can induce apoptosis. Figure 3.xTAK1 induces apoptosis. (A) Detection of xTAK1-induced cell death in early gastrula stages. Dorsal injection of xTAK1 mRNA (500 pg) in the four-cell stage caused the cell death at early gastrula stage. Left side: whole embryo without staining (vegetal view with dorsal on the top). Right side: whole embryo with acridine orange staining (lateral view with dorsal on the top). At least 10 embryos were examined and the data are representative of three separate experiments. (B) Detection of apoptotic cells by the TUNEL method in Xenopus embryos. Synthetic mRNAs containing xTAK1 (500 pg) were injected into the equatorial region of blastomeres at the two-cell stage. Paraffin sections at stage 9 were prepared and the TUNEL method for detecting fragmented DNAs diagnostic of apoptosis was performed. (a and b) Phase contrast micrographs; (c and d) fluorescence micrographs. Yellow–green signal indicates labeled apoptotic cells in sections of the control embryo (c) and the xTAK1 mRNA-injected embryo (d) in the early gastrula stage. The signal in the control embryo shows the background level. The scale bar indicates 10 μm. At least 10 embryos were examined in an experiment and the data are representative of three separate experiments. Download figure Download PowerPoint Figure 4.Effect of xTAK1 on Xenopus embryonic development. (A) Dorsal injection of bcl-2 mRNA (250 pg) resulted in the development of normal embryos (top). Dorsal injection of both xTAK1 (100 pg) and bcl-2 mRNAs caused weak ventralization (middle). Dorsal injection of xTAK1ΔN (100 pg) and bcl-2 mRNAs caused severe ventralization (bottom). Synthetic mRNAs containing xTAK1 or bcl-2 sequences were injected into the equatorial region of two dorsal or two ventral blastomeres at the four-cell stage, and phenotypes were scored at tadpole stage 36. The average dorso-anterior index (DAI; Kao and Elinson, 1989), a measure of the degree of dorsal and anterior mesodermal patterning, for each group was: bcl-2, average DAI = 5.0 (n = 21); xTAK1, average DAI = 4.1 (n = 21); xTAK1ΔN, average DAI = 2.6 (n = 22). (B) RT–PCR analysis of mesodermal marker gene expression in animal caps. Synthetic mRNAs containing xTAK1 or bcl-2 sequences were injected into the equatorial region of blastomeres at the two-cell stage. Animal caps injected with xTAK1ΔN (100 pg) and bcl-2 (250 pg), or bcl-2 mRNA, were cultured until gastrula stage 11 (early) or tadpole stage 38 (late), and total RNA was harvested. RNA was analyzed by RT–PCR for the presence of the indicated transcripts: lane 1, whole embryo; 2, uninjected; 3, xTAK1ΔN and bcl-2; 4, bcl-2, 5, constitutively active MEKK, 6, −RT. xTAK1 induces the expression of the ventral mesodermal marker globin but not the dorsal marker α-actin. Histone, ubiquitously expressed, was used as a loading control. Xhox-3 is a marker of ventral and posterior mesoderm. Xwnt-8 is a marker of ventral and lateral mesoderm. The globin is a definitive ventral marker. The α-actin is a marker of dorsal mesoderm. Download figure Download PowerPoint It has been shown previously that expression of an activated form of mammalian TAK1 (TAK1ΔN), a truncation lacking the NH2-terminal 20 amino acids, is able to enhance transcription from a reporter gene driven by the plasminogen activator inhibitor-1 (PAI-1) gene promoter (Yamaguchi et al., 1995). We therefore generated an mRNA encoding a similar truncated form of xTAK1 (xTAK1ΔN) and examined its effects in embryos. The injection of the xTAK1ΔN mRNA also induced cell death, but at doses lower than that required for the intact xTAK1 mRNA (data not shown). When xTAK1ΔN mRNA and human bcl-2 mRNA were co-injected into the dorsal marginal zone of four-cell embryos, cell death was inhibited and the embryos were significantly ventralized (Figure 4A, bottom). On the other hand, injection of both xTAK1ΔN and bcl-2 mRNAs into the ventral side had no effect on the development of the embryos (data not shown). Furthermore, injection of xTAK1ΔN and bcl-2 mRNA induced ventral posterior mesoderm in animal caps. As shown in Figure 4B, ventral (Xwnt-8 and globin) and posterior (Xhox-3) mesoderm markers, but not a dorsal mesoderm marker (α-actin), were induced in embryos co-injected with xTAK1ΔN and bcl-2. No mesodermal marker gene was induced by bcl-2 alone. Moreover, the injection of the cDNA encoding the activated form of another MAPKKK, MEKK1 (an activator of MKK3 and 4), along with bcl-2 did not induce any mesodermal marker genes (Figure 4B, lane 5), although injection of the activated form of MEKK1 mRNA alone caused apoptosis (data not shown). These results indicate that the induction of ventral mesoderm appears to be an event specifically induced by TAK1ΔN. The pattern of mesoderm induction by xTAK1 is similar to that induced by BMP-4, suggesting that TAK1 may function in the BMP-4 signaling pathway. xTAB1 enhances xTAK1-inducing mesodermal patterning It has been shown previously that expression of mammalian TAK1 with TAB1 is able to enhance transcription from a reporter gene driven by the PAI-1 gene promoter (Shibuya et al., 1996). We next examined the effects of expression of both xTAK1 and xTAB1 in embryos. At lower doses of xTAK1 (50 pg) and xTAB1 (1 ng), the injection of both mRNAs with or without bcl-2 mRNA into the dorsal marginal zone resulted in no change from the normal phenotypes (Figure 5A, data not shown). At higher doses of xTAK1 (100 pg) and xTAB1 (1 ng), the injection of both mRNAs also induced cell death (data not shown). When xTAK1 (100 pg) and xTAB1 (1 ng) mRNAs and human bcl-2 mRNA were co-injected into the dorsal marginal zone of four-cell embryos, cell death was inhibited and the embryos were significantly ventralized (Figure 5A). At the same doses, the injection of xTAK1 (100 pg) and bcl-2 mRNAs induced only minor defects in head formation as indicated above, suggesting that TAB1 enhances TAK1 activity. Injection of these mRNAs into the ventral side had no effect on the development of the embryos. Injection of xTAK1, xTAB1 and bcl-2 mRNAs also induced ventral posterior mesoderm in animal caps. As shown in Figure 5B, ventral (Xwnt-8 and globin), posterior (Xhox-3) and pan- (Xbra) mesoderm markers, but not dorsal mesoderm markers (goosecoid and α-actin), were induced in embryos injected with xTAK1, TAB1 and bcl-2. Interestingly, the injection of xTAK1 with bcl-2 mRNAs also induced only the Xwnt-8 and globin marker genes, consistent with the observed weak defect in phenotype. These results suggest that both TAK1 and TAB1 may function in the BMP-4 signaling pathway. Figure 5.Effect of xTAK1 and xTAB1 on Xenopus embryonic development. (A) Dorsal injection of bcl-2 (250 pg), xTAB1 (1 ng) with bcl-2 or xTAB1 and xTAK1 (50 pg) with bcl-2 mRNAs resulted in the development of normal embryos (a–c). Dorsal injection of both xTAK1 (100 pg) and bcl-2 mRNAs caused weak ventralization (d). Dorsal injection of xTAK1 (100 pg), xTAB1 (1 ng) and bcl-2 mRNAs caused severe ventralization (e). Synthetic mRNAs containing xTAK1, xTAB1 or bcl-2 sequences were injected into the equatorial regions of two dorsal or two ventral blastomeres at the four-cell stage, and phenotypes were scored at tadpole stage 36. The average DAI (Kao and Elinson, 1989) for each group was: bcl-2, average DAI = 5.0 (n = 20); xTAK1 and bcl-2, average DAI = 4.1 (n = 20); xTAK1, xTAB1 and bcl-2, average DAI = 2.9 (n = 20). (B) RT–PCR analysis of mesodermal marker gene expression in animal caps. Synthetic mRNAs containing xTAK1, xTAB1 or bcl-2 sequences were injected into the equatorial regions of blastomeres at the two-cell stage. Animal caps injected with xTAK1 (100 pg), xTAB1 (1 ng) and bcl-2 (250 pg), xTAK1 and bcl-2, xTAB1 and bcl-2, or bcl-2 mRNA were cultured until gastrula stage 11 (early) or tadpole stage 38 (late), and total RNA was harvested. RNA was analyzed by RT–PCR for the presence of the indicated transcripts: lane 1, whole embryo; 2, uninjected; 3, xTAK1, xTAB1 and bcl-2; 4, bcl-2; 5, xTAK1 and bcl-2; 6, xTAB1 and bcl-2; 7, −RT. Xbra is a marker of pan-mesoderm. Goosecoid is a marker of dorsal mesoderm. Download figure Download PowerPoint xTAK1 functions downstream of the BMP-2/4 receptor A mutant of BMPR-IA, in which glutamine is replaced by aspartic acid at 233, is constitutively active, independent of binding by BMP-2/4 ligands (Hoodless et al., 1996). We found that overexpression of this constitutively active BMP receptor in the dorsal side of Xenopus embryos caused ventralization (Figure 6A), mimicking the effect of BMP-2/4. If xTAK1 functions downstream of the BMP type I receptor, inhibition of xTAK1 function is expected to rescue the phenotypes generated by expression of the constitutively active BMP type I receptor. Overexpression of TAK1KN has been shown previously to inhibit TGF-β-stimulated transcription from a PAI-1 gene promoter (Yamaguchi et al., 1995). We thus examined the effect of overexpressing this mutant, in which Lys52 of the ATP-binding site of the xTAK1 catalytic domain has been mutated to Arg, on the ventralization of Xenopus embryos caused by the expression of the constitutively active form of the BMP-2/4 type I receptor. We co-injected xTAK1KN mRNA along with an mRNA encoding the constitutively active BMPR-IA mRNA. Expression of xTAK1KN partially reverted the ventralization (Figure 6A) and reversed the inhibition of anterior neural (Xotx-2) and ectodermal (XAG-1) marker expression (Figure 6B) caused by dorsal expression of the constitutively active BMPR-IA. Figure 6.xTAK1 functions downstream of BMPR-IA. (A) Kinase-negative xTAK1 (xTAK1KN) rescues the phenotypes caused by constitutively active BMPR-IA. Synthetic mRNAs were injected into the equatorial regions of two dorsal blastomeres at the four-cell stage, and phenotypes were scored at tadpole stage 36. Injection of constitutively active BMPR-IA mRNA (50 pg) ventralized the embryos with an average DAI of 2.7 (n = 26) (top). Co-expression of xTAK1KN (2 ng) with constitutively active BMPR-IA partially rescued the head structure (average DAI = 3.7, n = 40) (bottom). (B) xTAK1KN reverts the expression of anterior marker genes repressed by expression of constitutively active BMPR-IA. Synthetic mRNAs were injected into the equatorial regions of two dorsal blastomeres at the four-cell stage, and total RNAs were prepared at tadpole stage 36. RNA was analyzed by RT–PCR for anterior marker gene expression. Xotx-2 is an anterior neural marker and XAG-1 is an anterior ectodermal marker. Lane 1, uninjected embryo; 2, constitutively active BMPR-IA; 3, constitutively active BMPR-IA and TAK1KN; 4, −RT. Download figure Download PowerPoint We next examined whether the dominant-negative effect of xTAK1KN is specific to ventral or dorsal signaling. We found that expression of xTAK1KN inhibited the induction of ventral mesoderm markers otherwise caused by expression of xTAK1 and TAB1, BMP-4 or Smad1. In contrast, expression of xTAK1KN did not markedly reverse the induction of dorsalization markers by expression of activin, the constitutively active activin type IB receptor [CA-ActRIB; ALK4 (ten Dijke et al., 1993)] or Smad2. In addition, xTAK1KN barely inhibited the induction of a pan-mesoderm marker (Xbra) induced by basic fibroblast growth factor (FGF) (Figure 7). Injection of activin, CA-ActRIB or Smad2 mRNAs is known to induce dorsal mesoderm in animal caps (Baker and Harland, 1996; Graff et al., 1996; Chang et al., 1997), and we observed that each induced expression of the mesoderm-specific genes, goosecoid and Xbra (Figure 7). We further observed that co-injection of xTAK1KN did not reverse the induction of these markers by activin, CA-ActRIB or Smad2 (Figure 7B). These results suggest that xTAK1 may function in early Xenopus embryos in a pathway downstream of the BMP-2/4 receptor, but not downstream of the activin receptor. Figure 7.Kinase-negative xTAK1 inhibits mesoderm induction caused by BMP, but not activin signaling. Embryos were injected with xTAK1KN (2 ng) together with either xTAK1 (500 pg), xTAB1 (1 ng) and bcl-2 (250 pg), BMP-4 (50 pg), Smad1 (1 ng), activinβB (2 pg), CA-ActRIB (50 pg) or Smad2 (1 ng) mRNAs at the two-cell stage, and animal cap explants were dissected at blastula stages 8 to 9. The caps were either incubated alone or with basic FGF. Total RNA was assayed at gastrula stage 11 by RT–PCR for expression of mesodermal-specific markers. Download figure Download PowerPoint Cooperation of xTAK1 and Smad Smad1 mediates signaling by BMP-2/4 in mammalian cells or Xenopus embryos (Graff et al., 1996; Hoodless et al., 1996; F.Liu et al., 1996; Thomsen, 1996). Smad5, which is closely related to Smad1 (Riggins et al., 1996), also mediates a BMP-like signal in Xenopus (Suzuki et al., 1997). Injection of Smad1 or Smad5 mRNA into the dorsal marginal zone of four-cell embryos resulted in their significant ventralization (data not shown), suggesting that Smad5 also induces ventral mesoderm in Xenopus embryo. To examine the relationship between TAK1 and Smad1 or Smad5, we co-injected xTAK1KN mRNA together with an mRNA encoding either Smad1 or Smad5. Co-injection of xTAK1KN mRNA reversed the expression of ventral mesoderm markers (Xwnt-8, Xvent-1 and globin) induced by Smad1 or Smad5 (Figure 8), whereas it had no effect on dorsal markers (goosecoid, Xbra and actin). These results suggest that normal regulation of the BMP signaling pathway may require the cooperation of TAK1 with Smad1 or Smad5. Figure 8.Cooperation of TAK1 and Smads. xTAK1KN reverts expression of ventral marker genes induced by expression of Smad1 or Smad5. Animal caps co-injected with xTAK1KN (2 ng) and Smad1 (1 ng) or Smad5 (2 ng) mRNA were cultured until gastrula stage 11 (early) or tadpole stage 38 (late), and total RNA was harvested. RNA was analyzed by RT–PCR for the presence of the indicated transcripts: lane 1, whole embryo; 2, uninjected; 3, Smad1; 4, Smad1 and xTAK1KN; 5, Smad1 and β-globin; 6, S
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