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Repression of XMyoD expression and myogenesis by Xhairy-1 in Xenopus early embryo
Mechanisms of Development 109 (2001) 61–68 www.elsevier.com/locate/modo
Repression of XMyoD expression and myogenesis by Xhairy-1 in Xenopus early embryo Muriel Umbhauer, Jean-Claude Boucaut, De-Li Shi* Groupe de Biologie Expe´rimentale, Laboratoire de Biologie du De´veloppement, CNRS UMR 7622, Universite´ Pierre et Marie Curie, 9 quai Saint-Bernard, 75005 Paris, France Received 20 February 2001; received in revised form 9 July 2001; accepted 3 August 2001
Abstract Activated Notch-Delta signalling was shown to inhibit myogenesis, but whether and how it regulates myogenic gene expression is not clear. We analyzed the implication of Xenopus hairy-1 (Xhairy-1), a member of the hairy and enhancer-of-split (E(spl)) family that may function as nuclear effector of Notch signalling pathway, in regulating XMyoD gene expression at the initial step of myogenesis. Xhairy-1 transcripts are expressed soon after mid-blastula transition and exhibits overlapping expression with Notch pathway genes such as Delta-1 in the posterior somitic mesoderm. We show that overexpression of Xhairy-1 blocks the expression of XMyoD in early gastrula ectodermal cells treated with the mesoderm-inducing factor activin, and in the mesoderm tissues of early embryos. It inhibits myogenesis and produces trunk defects at later stages. Xhairy-1 also inhibits the expression of the pan-mesodermal marker Xbra, but expression of other early mesoderm markers such as goosecoid and chordin is not affected. These effects require the basic helix-loop-helix (bHLH) domain, as well as a synergy between the central Orange domain and the C-terminus WRPW-Groucho-interacting domain. Furthermore, overexpression in ectodermal cells of Xhairy-1/VP16, in which Xhairy-1 repressor domain is replaced by the activator domain of the viral protein VP16, induces the expression of XMyoD in the absence of protein synthesis. Interestingly, Xhairy-1/VP16 does not induce the expression of Xbra and XMyf5 in the same condition. During neurulation, the expression of XMyoD induced by Xhairy-1/VP16 declines and the expression of muscle actin gene was never detected. These results suggest that Notch signalling through hairy-related genes may specifically regulate XMyoD expression at the initial step of myogenesis in vertebrates. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: MyoD; Myf5; Myogenic factor; Myogenesis; Mesoderm; Notch signalling; Hairy; Repressor; Xenopus
1. Introduction The formation of skeletal muscle involves the specification and commitment of myogenic precursor cells. One of the major discoveries has been the identification of the myogenic bHLH transcription factors capable of converting non-muscle cells to skeletal muscle cells in culture. Among these factors, Myf5 and MyoD, which belong to a subgroup of the bHLH class of transcription factors, have early functions in the determination process that commits multipotential mesodermal cells to the myogenic lineage (reviewed by Weintraub et al., 1991). Expression and regionalization of these genes during embryogenesis depend on both positive and negative signals from surrounding tissues. In Xenopus, a low level of transcription of XMyoD genes occurs transiently in the whole embryo at the time of mid-blastula transition. In early gastrula, expression of both XMyf5 and * Corresponding author. Tel.: 133-1-44-272-772; fax: 133-1-44-273451. E-mail address: [email protected] (D.-L. Shi).
XMyoD transcripts is preferentially localized to the lateral and ventral mesoderm. As development proceeds, they are progressively restricted to the forming somites (Hopwood et al., 1989, 1991; Frank and Harland, 1991; Rupp and Weintraub, 1991; Dosch et al., 1997). The early expression of XMyoD may be an immediate response to mesoderm-inducing signals such as activin (Steinbach et al., 1998). Mesoderm induction may also help to stabilize the expression of myogenic genes in the marginal zone mesoderm. Notch signalling components have been shown as regulators of myogenesis in both vertebrates and invertebrates. They may be involved in maintaining the myogenic precursor cells in an undifferentiated state. In the early gastrula of Xenopus embryo, Delta, the Notch ligand, exhibits overlapping expression with XMyoD. In addition, XMyoD was shown to stimulate Notch signalling in the Xenopus gastrula, establishing a feed-back regulatory loop between differentiation promoting bHLH myogenic factors and the Notch pathway (Wittenberger et al., 1999). In vertebrate embryo, hairy-related genes and other genes encoding
0925-4773/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(01)00517-2
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components of Notch pathway exhibit a periodic expression in the presomitic mesoderm. This periodic sequence is reiterated once during the formation of each somite (Palmeirim et al., 1997; Jiang et al., 2000; reviewed by McGrew and Pourquie´ , 1998). Notch signalling is thought to act in the presomitic mesoderm at the time when cells form somitomeres and is required for subdividing the paraxial mesoderm into somites. Interfering with the expression and activity of Notch signalling pathway components disrupts this periodic expression of hairy-1 and somite segmentation (Jen et al., 1997, 1999; Jouve et al., 2000). Consistent with these observations, several lines of evidence suggest that activated Notch signalling inhibits myogenesis in different cultured cell lines (Sasai et al., 1992; Nye et al., 1994; Kopan et al., 1994; Jarriault et al., 1995; Lindsell et al., 1995; Shawber et al., 1996; Notfziger et al., 1999). In addition, ectopic expression of the intracellular domain of mouse Notch was also found to suppress somite formation in Xenopus embryo (Kopan et al., 1996). These lines of evidence implicate Notch signalling pathway in regulating segmental patterning and the expression of segmentation genes, but its function in regulating myogenic gene expression at the initial step of myogenesis remains to be determined. Cell fate determination by Notch signalling pathway plays a pivotal role in pattern formation during development of multicellular organisms. Notch is a large transmembrane receptor whose intracellular region undergoes proteolytic cleavages and interacts with the DNA-binding protein RBP-J/suppressor of hairless (Artavanis-Tsakonas et al., 1999). This complex then mediates the transcriptional activation of the enhancer-of-split (E(spl)) complex genes (Lecourtois and Schweisguth, 1995). It has been shown that activation of Notch signalling leads to the induction of mouse HES1 reporter gene as well as the up-regulation of HES1 mRNA (Jarriault et al., 1995; Kuroda et al., 1999). This signalling likely defines an evolutionarily conserved mechanism, which controls cell fate choices during neurogenesis and myogenesis in vertebrates and invertebrates. Members of the hairy/(E(spl) (HES) family of bHLH proteins function as transcriptional repressors and are nuclear effectors of the Notch pathway (Jarriault et al., 1995). Transcriptional repression is an important feature of developmental processes, it regulates the choice in cell fate (reviewed by Fisher and Caudy, 1998). In Drosophila, hairy-related genes function in the establishment and restriction of muscle precursors (Corbin et al., 1991; Bate et al., 1993; Carmena et al., 1995). However, it is not clear whether they regulate myogenic gene expression and myogenesis in vertebrates. Here we analyzed whether and how hairy-1, a HES family member, influences myogenic gene expression in Xenopus early embryos. We show that overexpression of Xenopus hairy-1 (Xhairy-1) blocks the expression of XMyoD in vivo and in vitro. Conversely, we found that expression of Xhairy-1/VP16 in gastrula ectodermal cells specifically induces the expression of XMyoD, but
not XMyf5. These results suggest that Notch signalling mediated by hairy-related genes may specifically regulate XMyoD expression at the initial step of myogenesis in vertebrates.
2. Results and discussion 2.1. Xhairy-1 suppresses the expression of a subset of early mesodermal genes Xenopus hairy-1 (Xhairy-1) has been identified previously (Dawson et al., 1995). We found that Xhairy-1 is a zygotic mRNA expressed soon after mid-blastula transition. At tail-bud stage, in situ hybridization analysis revealed that its expression pattern in the posterior somitic mesoderm overlaps that of Notch pathway genes such as Delta-1 (data not shown). Since Notch signalling regulates myogenesis and hairy-related genes may function as nuclear effectors of this pathway, we examined whether Xhairy-1 regulates XMyoD expression at the initial stage of myogenesis. We first injected 100 pg Xhairy-1 mRNA into the animal pole region of the 2-cell stage embryos. Animal cap explants from uninjected and injected embryos were dissected at the mid-blastula stage and treated with activin, which activates the expression of different mesodermal genes including XMyoD. It was shown that XMyoD expression is an early response to activin induction (Hopwood et al., 1989; Steinbach et al., 1998). RT-PCR analysis revealed that Xhairy-1 strongly inhibited XMyoD transcription activated by activin at the early gastrula stage. It had no effect on the expression of goosecoid and chordin, but also strongly inhibited the expression of the pan-mesodermal gene Xbra (Fig. 1A). This indicates that Xhairy-1 does not act as a general repressor of early mesodermal genes, it only represses a subset of these genes. At tail-bud stage, expression of muscle actin gene was not detected in activin-treated explants expressing Xhairy-1 (Fig. 1A), suggesting that Xhairy-1 inhibits muscle differentiation. Since Xbra is involved in mesoderm induction (Cunliffe and Smith, 1992), the inhibition of XMyoD expression by Xhairy-1 in activin-treated animal caps may be an indirect consequence. To test this possibility, we overexpressed Xbra in animal caps alone or co-expressed with Xhairy-1 and analyzed the expression of XMyoD by RT-PCR. The result indicates that Xhairy-1 was able to block the expression of XMyoD induced by Xbra (Fig. 1B). Thus, we conclude that Xhairy-1 inhibits XMyoD expression downstream of Xbra. This in vitro analysis therefore suggests that Xhairy-1 may inhibit XMyoD transcriptional activation at the early step of mesoderm induction. In early gastrula, induction-dependent XMyoD expression is restricted to lateral and ventral mesoderm (Hopwood et al., 1989; Frank and Harland, 1991; Rupp and Weintraub, 1991). To test if overexpression of Xhairy-1 inhibits this early XMyoD induction in vivo, synthetic Xhairy-1 mRNA
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(100 pg) was injected at the 4-cell stage into the marginal zone of one dorsal blastomere and XMyoD expression was analyzed by whole-mount in situ hybridization. In control stage 11 gastrula, XMyoD expression was detected in lateral and ventral marginal zones (Fig. 2A). At the end of gastrulation (stage 12.5–13), expression of XMyoD is localized to the trunk and posterior mesoderm (Fig. 2C). Injection of
Fig. 1. Xhairy-1 suppresses early XMyoD expression and myogenesis in vitro. (A) Animal cap explants expressing Xhairy-1 were treated with activin and RT-PCR analysis of different mesodermal genes was performed when control embryos reached the early gastrula stage (10.5) and the tailbud stage. ODC (ornithine decarboxylase) was used as a loading control. RT-, whole embryo control sample without reverse transcriptase. At stage 10.5, Xhairy-1 suppresses the expression of XMyoD and Xbra induced by activin, while the expression of goosecoid and chordin is not affected. At stage 25, Xhairy-1 suppresses myogenesis induced by activin, no muscle actin gene expression can be detected. (B) Two-cell stage embryos were injected with 500 pg Xbra mRNA either alone or co-injected with 100 pg Xhairy-1 mRNA. Animal caps were dissected at stage 8 and cultured to stage 11 for RT-PCR. Coinjection of Xhairy-1 mRNA blocks XMyoD expression induced by Xbra.
Fig. 2. Xhairy-1 inhibits XMyoD expression and myogenesis in whole embryo. (A–D) Embryos at 4-cell stage were injected with 100 pg Xhairy-1 mRNA into one blastomere at the dorsal equatorial region. They were fixed at different stages for in situ hybridization. (A) Expression of XMyoD in control embryos at stage 11. (B) Injection of Xhairy-1 mRNA suppresses the expression of XMyoD on the injected side (arrows). (C) Expression of XMyoD in control stage 12.5 late gastrula. (D) Reduced expression of XMyoD on the injected side (arrows) at the same stage. (E,F) Dorsal injection of 100 pg Xhairy-1 mRNA does not affect the expression of the Spemann organizer marker chordin. No significant difference can be observed between control (E) and injected (F) embryos. (G,H) Dorsal injection of 100 pg Xhairy-1 mRNA inhibits myogenesis and disrupts trunk development at tail-bud stage. (G) Control embryo at stage 28 showing the expression of myosin light chain gene in somites. (H) A Xhairy-1-injected embryo at the same stage exhibits reduced expression of this gene.
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Xhairy-1 completely blocked XMyoD expression on the injected site (Fig. 2B). As gastrulation proceeds, injected embryos exhibited a delayed gastrulation compared to control uninjected embryos, and XMyoD expression was always absent or reduced on the injected side (Fig. 2D). Although Xhairy-1 blocked XMyoD expression, its dorsal overexpression did not affect the expression and localization
of the Spemann organizer marker chordin (Fig. 2E,F). At larval stage, Xhairy-1-injected embryos exhibit trunk defects but with well developed head structures including eye and cement gland. In situ hybridization analysis of the expression of the myosin light chain gene indicated that Xhairy-1-injected embryos exhibit reduced and disorganized somites (Fig. 2G,H). This result is consistent with the observation that ectopic expression of the intracellular domain of mouse Notch suppresses somite formation in Xenopus embryo (Kopan et al., 1996). Since members of the HES family are nuclear effectors of the Notch signalling pathways and Delta, a Notch ligand, has been shown to colocalize with XMyoD in the early gastrula (Wittenberger et al., 1999), our results raise the possibility that Notch signalling may regulate early XMyoD expression through hairy-related genes. Further, it was recently shown that activated Notch signalling inhibits myogenesis and up-regulates HES1 mRNA in cultured C2C12 muscle progenitor (Kuroda et al., 1999). This result is therefore extended and confirmed by the present study. Our observation also suggests that Xhairy-1 may maintain myogenic precursor cells in an undifferentiated state by repressing XMyoD expression. 2.2. The effect of Xhairy-1 requires the DNA-binding and the repressor domains Xhairy-1 belongs to a subfamily of bHLH proteins that function as DNA-binding transcriptional repressors (Fisher and Caudy, 1998). Besides the DNA-binding domain, Xhairy-1 contains a central Orange domain and an C-terminus WRPW domain which may interact with corepressors such as the Groucho protein (Dawson et al., 1995). To identify the functional domains involved in regulating XMyoD expression, we generated different Xhairy-1 constructs (Fig. 3A). They include truncated Xhairy-1 proteins with deleted bHLH (Xhairy-1/DbHLH), Orange (Xhairy-1/DOrange) and the WRPW (Xhairy-1/DWRPW) domains, as well as a truncated Xhairy-1 protein that retains only the bHLH domain (Xhairy-1/bHLH). Synthetic mRNAs (100 pg) encoding these truncated proteins were injected radially into the marginal zone at 4-cell stage, and RT-PCR was performed to examine XMyoD expression in whole early Fig. 3. Repression of XMyoD expression by different Xhairy-1 deletion mutants. (A) Schematic representation of Xhairy-1 deletion mutants. (B) Synthetic mRNAs (100 pg) encoding different Xhairy-1 deletion mutants were radially injected in the marginal zone at 4-cell stage and RT-PCR was performed at stage 10 early gastrula. Xhairy-1/DbHLH with the DNA-binding domain deleted and Xhairy-1/bHLH with only the DNA-binding domain do not suppress XMyoD expression. Xhairy-1/DWRPW with the C-terminus repressor domain deleted and Xhairy-1/DOrange with the central Orange domain deleted suppress XMyoD expression less efficiently. (C) Relative abundance of XMyoD transcripts in embryos expressing various Xhairy-1 mutants. The relative abundance of the PCR products was quantified with a phosphorimager system, by subtracting background signals from control samples without reverse transcriptase and normalizing to ODC as an internal control of RNA input. The result represents the mean from two independent experiments.
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gastrula. As in activin-treated animal cap explants, wildtype Xhairy-1 strongly inhibited the expression of XMyoD (Fig. 3B,C). However, injection of 100 pg Xhairy-1/DbHLH mRNA had no effect on XMyoD expression when compared with uninjected embryos (Fig. 3B,C). This suggests that the DNA-binding domain is required for its repressor activity. In contrast to Xhairy-1/DbHLH, injection of 100 pg mRNA encoding Xhairy-1/DOrange or Xhairy-1/DWRPW inhibited XMyoD expression, albeit to a lesser extent than the wild-type Xhairy-1 (Fig. 3B,C). Furthermore, Xhairy-1/ bHLH with the central Orange domain and the WRPW motif deleted lost the activity to inhibit XMyoD expression. These results suggest that repression of XMyoD expression by Xhairy-1 requires its bHLH, as well as a cooperation between the central Orange and the C-terminus WRPW domains. They are also consistent with the results obtained in Drosophila, which show that the bHLH, Orange and WRPW domains are all required for hairy function (Dawson et al., 1995). Wild-type and mutant Xhairy-1 proteins had no significant effect on the expression of goosecoid and chordin. These analyses indicate that XMyoD may be one of the specific targets of Xhairy-1 regulation at the initial step of myogenesis.
2.3. An anti-morphic Xhairy-1 activates the expression of XMyoD, but not XMyf5 and Xbra To analyze further the regulation of XMyoD transcription by Xhairy-1, we generated a fusion protein (Xhairy-1/ VP16) in which the C-terminus WRPW repressor domain was replaced by the activator domain of the viral protein VP16. This will allow us to analyze more precisely how Xhairy-1 regulates the expression of early mesodermal genes. Embryos at 2-cell stage were injected with 100 pg Xhairy-1/VP16 mRNA at the animal pole region and animal caps were dissected at stage 8 and cultured to various stages. RT-PCR analysis of a panel of mesodermal and neural markers indicated that Xhairy-1/VP16 induced the early expression of XMyoD at stage 10, but not the expression of the pan-mesodermal marker Xbra (Fig. 4). The expression level of XMyoD induced by Xhairy-1/VP16 peaks at stage 12 (late gastrula), then it declines at the early neurula stage (stage 13) to a barely detectable level by the end of neurulation (stage 20). Accordingly, the expression of muscle actin gene was never detected (Fig. 4). However, the expression of the neural marker N-CAM was induced from neurula stage onwards, concomitant with the decline of XMyoD expression level (Fig. 4). Interestingly, Xhairy-1/ VP16 did not induce the expression of XMyf5 at any stages (Fig. 4). This suggests that XMyoD gene may be a direct target of Xhairy-1 regulation. This result is consistent with recent data obtained in chick embryo showing that Notch signalling down-regulates MyoD expression without affecting the expression of Myf5 (Delfini et al., 2000; Hirsinger et al., 2001). It is also consistent with different studies in mice,
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which show that Myf5 acts upstream of MyoD during somite and limb development (Tajbakhsh et al., 1997). It is unlikely that Xhairy-1/VP16 activates the expression or activity of mesoderm-inducing factors which in turn induce the expression of XMyoD, because Xbra expression, which is an immediate early response to mesoderm induction (Smith et al., 1991), was not detected in animal caps expressing Xhairy-1/VP16 (Fig. 4). In addition, using a hormone-inducible form of Xhairy-1/VP16 (Xhairy-1/ VP16-GR), we also found that the expression of XMyoD induced by Xhairy-1/VP16 was not blocked by treatment with the protein synthesis inhibitor cycloheximide (Fig. 5A,B). These results further suggest that Xhairy-1 may directly regulate the expression of XMyoD. The observation that Xhairy-1 regulates the expression of XMyoD during gastrulation while it is involved in neurogenesis at later stages is consistent with its expression in the neural tissues during late development (data not shown). In Drosophila, neurogenic genes exhibit dual function in neurogenesis and myogenesis (Corbin et al., 1991; Bate et al., 1993; Carmena et al., 1995), the dual effect of Xhairy-1/VP16 suggests a conserved function of hairy-related genes in vertebrates. We have shown that Xhairy-1 inhibits the early expression of XMyoD both in vivo and in activin-treated ectodermal cells. Conversely, Xhairy-1/VP16 specifically induces XMyoD expression in native ectodermal explants. Overexpression of Xhairy-1 in the myogenic progenitors disrupts somitogenesis and results in embryos with trunk defects. These results suggest that Notch signalling through HES
Fig. 4. Xhairy-1/VP16 induces the early expression of XMyoD and the late expression of N-CAM in animal cap explants. Following injection of Xhairy-1/VP16 mRNA (100 pg) in the animal pole region at 2-cell stage, animal cap explants were dissected at mid-blastula stage and cultured to different stages for RT-PCR analysis. Xhairy-1/VP16 induces the expression of XMyoD at the early gastrula stage and the level of XMyoD expression peaks at stage 12. There is a decline in XMyoD expression induced by Xhairy-1/VP16 from the end of gastrulation (stage 13) onward, which correlates with the onset of N-CAM expression. Xhairy-1/VP16 does not induce the expression of muscle actin (Ms-Act) and XMyf5 at any stages.
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proteins function to maintain myogenic precursor cells in an undifferentiated state. Furthermore, we observed that Xhairy-1 exhibits a relatively ubiquitous expression in the early gastrula (not shown), thus, it may also function to suppress XMyoD expression in non-muscle cells like ectoderm and endoderm. In the presumptive mesoderm, XMyoD expression is regulated by both positive (mesoderm-inducing factors) and negative signals (Notch signalling; Wittenberger et al., 1999). Our results provide evidence that the effect of Notch signalling in regulating myogenic gene expression and myogenesis is likely mediated by hairyrelated genes.
3. Experimental procedures 3.1. Plasmid constructs
Fig. 5. Induction of XMyoD expression by Xhairy-1/VP16 in the absence of protein synthesis. Animal cap explants injected with Xhairy-1/VP16-GR mRNA (100 pg) were dissected at stage 11 and incubated in cycloheximide (CHX) followed by incubation in dexamethasone (DEX) for 2 h (see Section 3). (A) RT-PCR analysis of XMyoD expression. (B) Relative abundance of XMyoD transcripts in explants expressing Xhairy-1/VP16-GR and treated by CHX and DEX. The result represents the mean from two independent experiments. CHX does not block XMyoD expression induced by Xhairy-1/VP16-GR.
proteins may be directly involved in the regulation of myogenic gene expression at the early step of myogenesis. Recently, a molecular clock of somitogenesis was identified based on the periodic expression of chicken hairy (c-hairy) mRNA in the presomitic mesoderm (Palmeirim et al., 1997; McGrew and Pourquie´ , 1998). The periodic expression of the Notch pathway genes regulates segmental identity during somitomere formation (Conlon et al., 1995; Jen et al., 1997, 1999; Jouve et al., 2000). In the mesoderm of Xenopus gastrula, XMyoD triggers Notch signalling through transcriptional activation of the Notch ligand, Delta-1 (Wittenberger et al., 1999). Taken together, these analyses suggest that a feed-back regulatory loop, involving activator and repressor genes and operating at the transcriptional level, plays an important role in muscle specification. They are consistent with the model that suggests that bHLH repressor proteins are potential regulators of MyoD gene expression and myogenesis (Cossu et al., 1996). These
The full-length Xhairy-1 coding sequence (Dawson et al., 1995) was PCR amplified from a gastrula expression library in pRN3 vector (Lemaire et al., 1995) using the Xhairy-1specific antisense primer (5 0 -GCATCAGAATTCTCACTACCG-3 0 ) including an Eco RI site and the T3 primer. The PCR product was digested by Eco RI and Bgl II which is present in the pRN3 polylinker sequence, and cloned into the Bam HI and Eco RI sites of the pCS2 1 vector (Turner and Weintraub, 1994) to generate pCS2Xhairy-1. This construct was then used to generate all other constructs used in this study. Xhairy-1/DWRPW was PCR-amplified using SP6 primer and a Xhairy-1 antisense primer (5 0 -AGTTCTAGACAGAATCAATTGTGACTG-3 0 ) which introduces a stop codon before the WRPW motif and contains a Xba I site. Xhairy-1/bHLH was obtained by digesting pCS2-Xhairy-1 with Nco I and Xba I followed by blunt-end ligation. To generate Xhairy-1/DOrange, cDNA fragment encoding the repressor domain was amplified by PCR from the pCS2-Xhairy-1 plasmid using T7 primer and Xhairy-1 sense primer (5 0 -AGGAGCAGCTCTGCAGAGCAGCC-3 0 ) with a substituted nucleotide to introduce a Pst I site. The PCR product was subcloned in-frame into an internal Pst I site just after the bHLH coding sequence and the Xba I site of the pCS2 1 vector. The same principle was used to obtain Xhairy-1/ DbHLH, but in two steps. In the first step, an intermediate construct was obtained by PCR amplification of the cDNA fragment upstream of the bHLH domain using SP6 primer and Xhairy-1 antisense primer (5 0 -TTACTCGAGGCAGTTTCCCTTTAT-3 0 ) with a Xho I site. The PCR product was subcloned into the Cla I and Xho I sites of the pCS2 1 vector. In a second step, the cDNA fragment downstream of bHLH domain was amplified by PCR using T7 primer and a sense primer (5 0 -CTGCACTCGAGTACAGACC-3 0 ) with a substituted nucleotide to introduce a Xho I site, and was subcloned into the intermediate construct. pCS2-Xhairy-1/VP16 was obtained by PCR amplification of the VP16 sequence in pSP64T vector with sense (5 0 -
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GTCCCATGGCCCCCCCGACCGAT-3 0 ) and antisense (5 0 -GGCTCTAGATCAGTTCAGGTCG-3 0 ) primers including a Nco I and Xba I site, respectively. The PCR product was cloned into the corresponding sites of pCS2-Xhairy-1 in-frame with the DNA-binding and Orange domains of Xhairy-1. To obtain pSP64T-Xhairy-1/VP16-GR, Xhairy1/VP16 was PCR amplified using sense primer (5 0 GAAGATATCCGGCTGATGTG-3 0 ) with an EcoR V site and the T7 primer. The PCR product was cloned in-frame with the glucocorticoid receptor cDNA in pSP64T vector. All constructs were checked by sequencing and were found identical to the original sequence. The pSP64T-Xbra plasmid was provided by Dr J. Smith (Cunliffe and Smith, 1992).
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Acknowledgements We thank Drs J. Smith, R. Dosch and T. Mohun for providing the reagents used in this study; A. Pascal and A. Bourdelas for excellent technical assistance, P. Nguyen for illustrations and R. Schwartzmann for phosphorimager analysis. This work was supported by grants from Centre National de la Recherche Scientifique (CNRS), Ministe`re de la Recherche (ACI program), Association pour la Recherche sur le Cancer (ARC), Association Franc¸ aise contre la Myopathie (AFM) and Ligue National Contre le Cancer (LNCC).
References 3.2. Xenopus embryos and mRNA microinjections Xenopus eggs were obtained from females injected with 500 IU of human chorionic gonadotropin (Sigma), and artificially fertilized. Eggs were dejellied with 2% cysteine hydrochloride (pH 7.8) and embryos were staged according to Nieuwkoop and Faber (1967). Capped mRNAs were synthesized from linearized plasmids using SP6 RNA polymerase (Boehringer Mannheim). Microinjection of embryos was performed as previously described (Djiane et al., 2000).
3.3. RT-PCR and in situ hybridization Animal cap explants from control and injected embryos were dissected at mid-blastula stage (stage 8) and incubated for 1 h in 1 £ MBS containing 10 units/ml recombinant Xenopus activin A (provided by Dr J.C. Smith). For cycloheximide (CHX) and dexamethasone (DEX) treatment, animal cap explants injected with 100 pg Xhairy-1/VP16GR mRNA were first incubated in 10 mg/ml CHX (Sigma) for 30 min, they were then incubated in 10 mM DEX (Sigma) for 2 h in the presence of CHX. Extraction of RNA and RT-PCR were as described previously (Djiane et al., 2000). PCR primers were as described (Wittenberger et al., 1999; Djiane et al., 2000) except for muscle actin (5 0 GCTGACAGAATGCAGAAG-3 0 and 5 0 -TTGCTTGGAGGAGTGTGT-3 0 ), XMyf5 (5 0 -CTATTCAGAATGGAGATGGT-3 0 and 5 0 -GTCTTGGAGACTCTCAATA-3 0 ), and NCAM (5 0 -CACAGTTCCACCAAATGC-3 0 and 5 0 -GGAATCAAGCGGTACAGA-3 0 ). Each experiment was performed on triplicate and analyzed using a phosphorimager system (Biorad). Whole-mount in situ hybridization was performed according to standard protocol (Harland, 1991) except that chromogenic reaction was done using BM purple as substrate (Boehringer Mannheim). Probes for chordin and XMyoD were as described (Dosch et al., 1997; Djiane et al., 2000). The Xenopus myosin light chain cDNA was kindly provided by Dr T. Mohun.
Artavanis-Tsakonas, S., Rand, M.D., Lake, R.J., 1999. Notch signaling: cell fate control and signal integration in development. Science 284, 770– 776. Bate, M., Rushton, E., Frasch, M., 1993. A dual requirement for neurogenic genes in Drosophila myogenesis. Development (Suppl.), 149–161. Carmena, A., Bate, M., Jimenez, F., 1995. Lethal of scute, a proneural gene, participates in the specification of muscle progenitors during Drosophila embryogenesis. Genes Dev. 9, 2373–2383. Conlon, R.A., Reaume, A.G., Rossant, J., 1995. Notch1 is required for the coordinate segmentation of somites. Development 121, 1533–1545. Corbin, V., Michelson, A.M., Abmayr, S.M., Neel, V., Alcamo, E., Maniatis, T., Young, M.W., 1991. A role for the Drosophila neurogenic genes in mesoderm differentiation. Cell 67, 311–323. Cossu, G., Tajbakhsh, S., Buckingham, M., 1996. How is myogenesis initiated in the embryo? Trends Genet. 12, 218–223. Cunliffe, V., Smith, J.C., 1992. Ectopic mesoderm induction in Xenopus embryos caused by widespread expression of a Brachyury homologue. Nature 358, 427–430. Dawson, S.R., Turner, D.L., Weintraub, H., Parkhurst, S.M., 1995. Specificity for the Hairy/Enhancer of split basic Helix-Loop-Helix (bHLH) proteins maps outside the bHLH domain and suggest two separate modes of transcriptional repression. Mol. Cell Biol. 15, 6923–6931. Delfini, M.C., Hirsinger, E., Pourquie´ , O., Duprez, D., 2000. Delta 1-activated Notch inhibits muscle differentiation without affecting Myf5 and Pax3 expression in chick myogenesis. Development 127, 5213–5224. Djiane, A., Riou, J.F., Umbhauer, M., Boucaut, J.C., Shi, D.L., 2000. Role of frizzled 7 in the regulation of convergent extension movements during gastrulation in Xenopus laevis. Development 127, 3091–3400. Dosch, R., Gawantka, V., Delius, H., Blumenstock, C., Niehrs, C., 1997. Bmp-4 acts as a morphogen in dorsoventral mesoderm patterning in Xenopus. Development 124, 2325–2334. Fisher, A., Caudy, M., 1998. The function of hairy-related bHLH repressor proteins in cell fate decisions. BioEssays 20, 298–306. Frank, D., Harland, R.M., 1991. Transient expression of XMyoD in nonsomitic mesoderm of Xenopus gastrulae. Development 113, 1387– 1393. Harland, R.M., 1991. In situ hybridization: an improved whole mount method for Xenopus embryos. In: Kay, B.K., Peng, H.B. (Eds.). Methods in Cell Biology, Vol. 36. Academic Press, San Diego, CA, pp. 685– 695. Hirsinger, E., Malapert, P., Dubrulle, J., Delfini, M.C., Duprez, D., Henrique, D., Ish-Horowicz, D., Pourquie´ , O., 2001. Notch signalling acts in postmitotic avian myogenic cells to control MyoD activation. Development 128, 107–116. Hopwood, N.D., Pluck, A., Gurdon, J.B., 1989. MyoD expression in the forming somites is an early response to mesoderm induction in Xenopus embryos. EMBO J 8, 3409–3417. Hopwood, N.D., Pluck, A., Gurdon, J.B., 1991. Xenopus Myf5 marks early
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muscle cells and can activate muscle genes ectopically in early embryo. Development 111, 551–560. Jen, W.C., Wettstein, D., Turner, D., Chitnis, A., Kintner, C., 1997. The Notch ligand, X-Delta-2, mediates segmentation of the paraxial mesoderm in Xenopus embryos. Development 124, 1169–1178. Jen, W.C., Gawantka, V., Pollet, N., Niehrs, C., Kintner, C., 1999. Periodic repression of Notch pathway genes governs the segmentation of Xenopus embryos. Genes Dev. 13, 1486–1499. Jarriault, S., Brou, C., Logeat, F., Schroeter, E.H., Kopan, R., Israel, A., 1995. Signalling downstream of activated mammalian Notch. Nature 377, 355–358. Jiang, Y.J., Aerne, B.L., Smithers, L., Haddon, C., Ish-Horowicz, D., Lewis, J., 2000. Notch signalling and the synchronization of the somite segmentation clock. Nature 408, 475–479. Jouve, C., Palmeirim, I., Henrique, D., Beckers, J., Gossler, A., Ish-Horowicz, D., Pourquie´ , O., 2000. Notch signalling is required for cyclic expression of the hairy-like gene HES1 in the presomitic mesoderm. Development 127, 1421–1429. Kopan, R., Nye, S.J., Weintraub, H., 1994. The intracellular domain of mouse Notch: a constitutively activated repressor of myogenesis directed at the basic helix–loop–helix region of MyoD. Development 120, 2385–2396. Kopan, R., Schroeter, E.H., Weintraub, H., Nye, J.S., 1996. Signal transduction by activated mNotch: Importance of proteolytic processing and its regulation by the extracellular domain. Proc. Natl. Acad. Sci. USA 93, 1683–1688. Kuroda, K., Tani, S., Tamura, K., Minogushi, S., Kurooka, H., Honjo, T., 1999. Delta-induced Notch signaling mediated by RBP-J inhibits MyoD expression and myogenesis. J. Biol. Chem. 274, 7238–7244. Lecourtois, M., Schweisguth, F., 1995. The neurogenic suppressor of hairless DNA-binding protein mediates the transcriptional activation of the enhancer of split complex genes triggered by Notch signaling. Genes Dev. 9, 2598–2608. Lemaire, P., Garrett, N., Gurdon, J.B., 1995. Expression cloning of Siamois, a Xenopus homeobox gene expressed in dorsal-vegetal cells of blastula and able to induce a complete secondary axis. Cell 81, 85–94. Lindsell, C.E., Shawber, C.J., Boulter, J., Weinmaster, G., 1995. Jagged, a mammalian ligand that activates Notch1. Cell 80, 909–917. McGrew, M.J., Pourquie´ , O., 1998. Somitogenesis: segmenting a vertebrate. Curr. Opin. Genet. Dev. 8, 487–493. Nieuwkoop, P.D., Faber, J., 1967. Normal Table of Xenopus laevis
(Daudin), 2nd edn. North-Holland Publishing Company, Amsterdam, The Netherlands. Notfziger, D., Miyamoto, A., Lyons, K.M., Weinmaster, G., 1999. Notch signaling imposes two distinct blocks in the differentiation of C2C12 myoblasts. Development 126, 1689–1702. Nye, J.S., Kopan, R., Axel, R., 1994. An activated Notch suppresses neurogenesis and myogenesis but not gliogenesis in mammalian cells. Development 120, 2421–2430. Palmeirim, I., Henrique, D., Ish-Horowicz, D., Pourquie, O., 1997. Avian hairy gene expression identifies a molecular clock linked to vertebrate segmentation and somitogenesis. Cell 91, 639–648. Rupp, R.A.W., Weintraub, H., 1991. Ubiquitous MyoD transcription at the midblastula transition precedes induction-dependent expression in the presumptive mesoderm. Cell 65, 927–937. Sasai, Y., Kageyama, R., Tagawa, Y., Shigemoto, R., Nakanishi, S., 1992. Two mammalian helix-loop-helix factors structurally related to Drosophila hairy and Enhancer of split. Genes Dev. 6, 2620–2634. Shawber, C., Nofziger, D., Hsieh, J.D., Lindsell, C., Bo¨ gler, O., Hayward, D., Weinmaster, G., 1996. Notch signaling inhibits muscle cell differentiation through a CBF1-independent pathway. Development 122, 3765–3773. Smith, J.C., Price, B.M., Green, J.B.A., Weigel, D., Herrmann, B., 1991. Expression of a Xenopus homolog of Brachyury (T) is an immediateearly response to mesoderm induction. Cell 67, 79–87. Steinbach, O.C., Ulsho¨ fer, A., Authaler, A., Rupp, R.A.W., 1998. Temporal restriction of MyoD induction and autocatalysis during Xenopus mesoderm formation. Dev. Biol. 202, 280–292. Tajbakhsh, S., Rocancourt, D., Cossu, G., Buckingham, M., 1997. Redefining the genetic hierarchies controlling skeletal myogenesis: Pax-3 and Myf-5 act upstream of MyoD. Cell 89, 127–138. Turner, D.L., Weintraub, H., 1994. Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to neural fate. Genes Dev. 8, 1434–1447. Weintraub, H., Davis, R., Tapscott, S.J., Thayer, M., Krause, M., Benezra, R., Blackwell, T.K., Turner, D., Rupp, R., Hollenberg, S., Zhuang, Y., Lassar, A., 1991. The myoD gene family: nodal point during specification of the muscle cell ligneage. Science 251, 761–766. Wittenberger, T., Steinbach, O.C., Authaler, A., Kopan, R., Rupp, R.A.W., 1999. MyoD stimulates Delta-1 transcription and triggers Notch signaling in the Xenopus gastrula. EMBO J. 18, 1915–1922.
Repression of XMyoD expression and myogenesis by Xhairy-1 in Xenopus early embryo Muriel Umbhauer, Jean-Claude Boucaut, De-Li Shi* Groupe de Biologie Expe´rimentale, Laboratoire de Biologie du De´veloppement, CNRS UMR 7622, Universite´ Pierre et Marie Curie, 9 quai Saint-Bernard, 75005 Paris, France Received 20 February 2001; received in revised form 9 July 2001; accepted 3 August 2001
Abstract Activated Notch-Delta signalling was shown to inhibit myogenesis, but whether and how it regulates myogenic gene expression is not clear. We analyzed the implication of Xenopus hairy-1 (Xhairy-1), a member of the hairy and enhancer-of-split (E(spl)) family that may function as nuclear effector of Notch signalling pathway, in regulating XMyoD gene expression at the initial step of myogenesis. Xhairy-1 transcripts are expressed soon after mid-blastula transition and exhibits overlapping expression with Notch pathway genes such as Delta-1 in the posterior somitic mesoderm. We show that overexpression of Xhairy-1 blocks the expression of XMyoD in early gastrula ectodermal cells treated with the mesoderm-inducing factor activin, and in the mesoderm tissues of early embryos. It inhibits myogenesis and produces trunk defects at later stages. Xhairy-1 also inhibits the expression of the pan-mesodermal marker Xbra, but expression of other early mesoderm markers such as goosecoid and chordin is not affected. These effects require the basic helix-loop-helix (bHLH) domain, as well as a synergy between the central Orange domain and the C-terminus WRPW-Groucho-interacting domain. Furthermore, overexpression in ectodermal cells of Xhairy-1/VP16, in which Xhairy-1 repressor domain is replaced by the activator domain of the viral protein VP16, induces the expression of XMyoD in the absence of protein synthesis. Interestingly, Xhairy-1/VP16 does not induce the expression of Xbra and XMyf5 in the same condition. During neurulation, the expression of XMyoD induced by Xhairy-1/VP16 declines and the expression of muscle actin gene was never detected. These results suggest that Notch signalling through hairy-related genes may specifically regulate XMyoD expression at the initial step of myogenesis in vertebrates. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: MyoD; Myf5; Myogenic factor; Myogenesis; Mesoderm; Notch signalling; Hairy; Repressor; Xenopus
1. Introduction The formation of skeletal muscle involves the specification and commitment of myogenic precursor cells. One of the major discoveries has been the identification of the myogenic bHLH transcription factors capable of converting non-muscle cells to skeletal muscle cells in culture. Among these factors, Myf5 and MyoD, which belong to a subgroup of the bHLH class of transcription factors, have early functions in the determination process that commits multipotential mesodermal cells to the myogenic lineage (reviewed by Weintraub et al., 1991). Expression and regionalization of these genes during embryogenesis depend on both positive and negative signals from surrounding tissues. In Xenopus, a low level of transcription of XMyoD genes occurs transiently in the whole embryo at the time of mid-blastula transition. In early gastrula, expression of both XMyf5 and * Corresponding author. Tel.: 133-1-44-272-772; fax: 133-1-44-273451. E-mail address: [email protected] (D.-L. Shi).
XMyoD transcripts is preferentially localized to the lateral and ventral mesoderm. As development proceeds, they are progressively restricted to the forming somites (Hopwood et al., 1989, 1991; Frank and Harland, 1991; Rupp and Weintraub, 1991; Dosch et al., 1997). The early expression of XMyoD may be an immediate response to mesoderm-inducing signals such as activin (Steinbach et al., 1998). Mesoderm induction may also help to stabilize the expression of myogenic genes in the marginal zone mesoderm. Notch signalling components have been shown as regulators of myogenesis in both vertebrates and invertebrates. They may be involved in maintaining the myogenic precursor cells in an undifferentiated state. In the early gastrula of Xenopus embryo, Delta, the Notch ligand, exhibits overlapping expression with XMyoD. In addition, XMyoD was shown to stimulate Notch signalling in the Xenopus gastrula, establishing a feed-back regulatory loop between differentiation promoting bHLH myogenic factors and the Notch pathway (Wittenberger et al., 1999). In vertebrate embryo, hairy-related genes and other genes encoding
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components of Notch pathway exhibit a periodic expression in the presomitic mesoderm. This periodic sequence is reiterated once during the formation of each somite (Palmeirim et al., 1997; Jiang et al., 2000; reviewed by McGrew and Pourquie´ , 1998). Notch signalling is thought to act in the presomitic mesoderm at the time when cells form somitomeres and is required for subdividing the paraxial mesoderm into somites. Interfering with the expression and activity of Notch signalling pathway components disrupts this periodic expression of hairy-1 and somite segmentation (Jen et al., 1997, 1999; Jouve et al., 2000). Consistent with these observations, several lines of evidence suggest that activated Notch signalling inhibits myogenesis in different cultured cell lines (Sasai et al., 1992; Nye et al., 1994; Kopan et al., 1994; Jarriault et al., 1995; Lindsell et al., 1995; Shawber et al., 1996; Notfziger et al., 1999). In addition, ectopic expression of the intracellular domain of mouse Notch was also found to suppress somite formation in Xenopus embryo (Kopan et al., 1996). These lines of evidence implicate Notch signalling pathway in regulating segmental patterning and the expression of segmentation genes, but its function in regulating myogenic gene expression at the initial step of myogenesis remains to be determined. Cell fate determination by Notch signalling pathway plays a pivotal role in pattern formation during development of multicellular organisms. Notch is a large transmembrane receptor whose intracellular region undergoes proteolytic cleavages and interacts with the DNA-binding protein RBP-J/suppressor of hairless (Artavanis-Tsakonas et al., 1999). This complex then mediates the transcriptional activation of the enhancer-of-split (E(spl)) complex genes (Lecourtois and Schweisguth, 1995). It has been shown that activation of Notch signalling leads to the induction of mouse HES1 reporter gene as well as the up-regulation of HES1 mRNA (Jarriault et al., 1995; Kuroda et al., 1999). This signalling likely defines an evolutionarily conserved mechanism, which controls cell fate choices during neurogenesis and myogenesis in vertebrates and invertebrates. Members of the hairy/(E(spl) (HES) family of bHLH proteins function as transcriptional repressors and are nuclear effectors of the Notch pathway (Jarriault et al., 1995). Transcriptional repression is an important feature of developmental processes, it regulates the choice in cell fate (reviewed by Fisher and Caudy, 1998). In Drosophila, hairy-related genes function in the establishment and restriction of muscle precursors (Corbin et al., 1991; Bate et al., 1993; Carmena et al., 1995). However, it is not clear whether they regulate myogenic gene expression and myogenesis in vertebrates. Here we analyzed whether and how hairy-1, a HES family member, influences myogenic gene expression in Xenopus early embryos. We show that overexpression of Xenopus hairy-1 (Xhairy-1) blocks the expression of XMyoD in vivo and in vitro. Conversely, we found that expression of Xhairy-1/VP16 in gastrula ectodermal cells specifically induces the expression of XMyoD, but
not XMyf5. These results suggest that Notch signalling mediated by hairy-related genes may specifically regulate XMyoD expression at the initial step of myogenesis in vertebrates.
2. Results and discussion 2.1. Xhairy-1 suppresses the expression of a subset of early mesodermal genes Xenopus hairy-1 (Xhairy-1) has been identified previously (Dawson et al., 1995). We found that Xhairy-1 is a zygotic mRNA expressed soon after mid-blastula transition. At tail-bud stage, in situ hybridization analysis revealed that its expression pattern in the posterior somitic mesoderm overlaps that of Notch pathway genes such as Delta-1 (data not shown). Since Notch signalling regulates myogenesis and hairy-related genes may function as nuclear effectors of this pathway, we examined whether Xhairy-1 regulates XMyoD expression at the initial stage of myogenesis. We first injected 100 pg Xhairy-1 mRNA into the animal pole region of the 2-cell stage embryos. Animal cap explants from uninjected and injected embryos were dissected at the mid-blastula stage and treated with activin, which activates the expression of different mesodermal genes including XMyoD. It was shown that XMyoD expression is an early response to activin induction (Hopwood et al., 1989; Steinbach et al., 1998). RT-PCR analysis revealed that Xhairy-1 strongly inhibited XMyoD transcription activated by activin at the early gastrula stage. It had no effect on the expression of goosecoid and chordin, but also strongly inhibited the expression of the pan-mesodermal gene Xbra (Fig. 1A). This indicates that Xhairy-1 does not act as a general repressor of early mesodermal genes, it only represses a subset of these genes. At tail-bud stage, expression of muscle actin gene was not detected in activin-treated explants expressing Xhairy-1 (Fig. 1A), suggesting that Xhairy-1 inhibits muscle differentiation. Since Xbra is involved in mesoderm induction (Cunliffe and Smith, 1992), the inhibition of XMyoD expression by Xhairy-1 in activin-treated animal caps may be an indirect consequence. To test this possibility, we overexpressed Xbra in animal caps alone or co-expressed with Xhairy-1 and analyzed the expression of XMyoD by RT-PCR. The result indicates that Xhairy-1 was able to block the expression of XMyoD induced by Xbra (Fig. 1B). Thus, we conclude that Xhairy-1 inhibits XMyoD expression downstream of Xbra. This in vitro analysis therefore suggests that Xhairy-1 may inhibit XMyoD transcriptional activation at the early step of mesoderm induction. In early gastrula, induction-dependent XMyoD expression is restricted to lateral and ventral mesoderm (Hopwood et al., 1989; Frank and Harland, 1991; Rupp and Weintraub, 1991). To test if overexpression of Xhairy-1 inhibits this early XMyoD induction in vivo, synthetic Xhairy-1 mRNA
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(100 pg) was injected at the 4-cell stage into the marginal zone of one dorsal blastomere and XMyoD expression was analyzed by whole-mount in situ hybridization. In control stage 11 gastrula, XMyoD expression was detected in lateral and ventral marginal zones (Fig. 2A). At the end of gastrulation (stage 12.5–13), expression of XMyoD is localized to the trunk and posterior mesoderm (Fig. 2C). Injection of
Fig. 1. Xhairy-1 suppresses early XMyoD expression and myogenesis in vitro. (A) Animal cap explants expressing Xhairy-1 were treated with activin and RT-PCR analysis of different mesodermal genes was performed when control embryos reached the early gastrula stage (10.5) and the tailbud stage. ODC (ornithine decarboxylase) was used as a loading control. RT-, whole embryo control sample without reverse transcriptase. At stage 10.5, Xhairy-1 suppresses the expression of XMyoD and Xbra induced by activin, while the expression of goosecoid and chordin is not affected. At stage 25, Xhairy-1 suppresses myogenesis induced by activin, no muscle actin gene expression can be detected. (B) Two-cell stage embryos were injected with 500 pg Xbra mRNA either alone or co-injected with 100 pg Xhairy-1 mRNA. Animal caps were dissected at stage 8 and cultured to stage 11 for RT-PCR. Coinjection of Xhairy-1 mRNA blocks XMyoD expression induced by Xbra.
Fig. 2. Xhairy-1 inhibits XMyoD expression and myogenesis in whole embryo. (A–D) Embryos at 4-cell stage were injected with 100 pg Xhairy-1 mRNA into one blastomere at the dorsal equatorial region. They were fixed at different stages for in situ hybridization. (A) Expression of XMyoD in control embryos at stage 11. (B) Injection of Xhairy-1 mRNA suppresses the expression of XMyoD on the injected side (arrows). (C) Expression of XMyoD in control stage 12.5 late gastrula. (D) Reduced expression of XMyoD on the injected side (arrows) at the same stage. (E,F) Dorsal injection of 100 pg Xhairy-1 mRNA does not affect the expression of the Spemann organizer marker chordin. No significant difference can be observed between control (E) and injected (F) embryos. (G,H) Dorsal injection of 100 pg Xhairy-1 mRNA inhibits myogenesis and disrupts trunk development at tail-bud stage. (G) Control embryo at stage 28 showing the expression of myosin light chain gene in somites. (H) A Xhairy-1-injected embryo at the same stage exhibits reduced expression of this gene.
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Xhairy-1 completely blocked XMyoD expression on the injected site (Fig. 2B). As gastrulation proceeds, injected embryos exhibited a delayed gastrulation compared to control uninjected embryos, and XMyoD expression was always absent or reduced on the injected side (Fig. 2D). Although Xhairy-1 blocked XMyoD expression, its dorsal overexpression did not affect the expression and localization
of the Spemann organizer marker chordin (Fig. 2E,F). At larval stage, Xhairy-1-injected embryos exhibit trunk defects but with well developed head structures including eye and cement gland. In situ hybridization analysis of the expression of the myosin light chain gene indicated that Xhairy-1-injected embryos exhibit reduced and disorganized somites (Fig. 2G,H). This result is consistent with the observation that ectopic expression of the intracellular domain of mouse Notch suppresses somite formation in Xenopus embryo (Kopan et al., 1996). Since members of the HES family are nuclear effectors of the Notch signalling pathways and Delta, a Notch ligand, has been shown to colocalize with XMyoD in the early gastrula (Wittenberger et al., 1999), our results raise the possibility that Notch signalling may regulate early XMyoD expression through hairy-related genes. Further, it was recently shown that activated Notch signalling inhibits myogenesis and up-regulates HES1 mRNA in cultured C2C12 muscle progenitor (Kuroda et al., 1999). This result is therefore extended and confirmed by the present study. Our observation also suggests that Xhairy-1 may maintain myogenic precursor cells in an undifferentiated state by repressing XMyoD expression. 2.2. The effect of Xhairy-1 requires the DNA-binding and the repressor domains Xhairy-1 belongs to a subfamily of bHLH proteins that function as DNA-binding transcriptional repressors (Fisher and Caudy, 1998). Besides the DNA-binding domain, Xhairy-1 contains a central Orange domain and an C-terminus WRPW domain which may interact with corepressors such as the Groucho protein (Dawson et al., 1995). To identify the functional domains involved in regulating XMyoD expression, we generated different Xhairy-1 constructs (Fig. 3A). They include truncated Xhairy-1 proteins with deleted bHLH (Xhairy-1/DbHLH), Orange (Xhairy-1/DOrange) and the WRPW (Xhairy-1/DWRPW) domains, as well as a truncated Xhairy-1 protein that retains only the bHLH domain (Xhairy-1/bHLH). Synthetic mRNAs (100 pg) encoding these truncated proteins were injected radially into the marginal zone at 4-cell stage, and RT-PCR was performed to examine XMyoD expression in whole early Fig. 3. Repression of XMyoD expression by different Xhairy-1 deletion mutants. (A) Schematic representation of Xhairy-1 deletion mutants. (B) Synthetic mRNAs (100 pg) encoding different Xhairy-1 deletion mutants were radially injected in the marginal zone at 4-cell stage and RT-PCR was performed at stage 10 early gastrula. Xhairy-1/DbHLH with the DNA-binding domain deleted and Xhairy-1/bHLH with only the DNA-binding domain do not suppress XMyoD expression. Xhairy-1/DWRPW with the C-terminus repressor domain deleted and Xhairy-1/DOrange with the central Orange domain deleted suppress XMyoD expression less efficiently. (C) Relative abundance of XMyoD transcripts in embryos expressing various Xhairy-1 mutants. The relative abundance of the PCR products was quantified with a phosphorimager system, by subtracting background signals from control samples without reverse transcriptase and normalizing to ODC as an internal control of RNA input. The result represents the mean from two independent experiments.
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gastrula. As in activin-treated animal cap explants, wildtype Xhairy-1 strongly inhibited the expression of XMyoD (Fig. 3B,C). However, injection of 100 pg Xhairy-1/DbHLH mRNA had no effect on XMyoD expression when compared with uninjected embryos (Fig. 3B,C). This suggests that the DNA-binding domain is required for its repressor activity. In contrast to Xhairy-1/DbHLH, injection of 100 pg mRNA encoding Xhairy-1/DOrange or Xhairy-1/DWRPW inhibited XMyoD expression, albeit to a lesser extent than the wild-type Xhairy-1 (Fig. 3B,C). Furthermore, Xhairy-1/ bHLH with the central Orange domain and the WRPW motif deleted lost the activity to inhibit XMyoD expression. These results suggest that repression of XMyoD expression by Xhairy-1 requires its bHLH, as well as a cooperation between the central Orange and the C-terminus WRPW domains. They are also consistent with the results obtained in Drosophila, which show that the bHLH, Orange and WRPW domains are all required for hairy function (Dawson et al., 1995). Wild-type and mutant Xhairy-1 proteins had no significant effect on the expression of goosecoid and chordin. These analyses indicate that XMyoD may be one of the specific targets of Xhairy-1 regulation at the initial step of myogenesis.
2.3. An anti-morphic Xhairy-1 activates the expression of XMyoD, but not XMyf5 and Xbra To analyze further the regulation of XMyoD transcription by Xhairy-1, we generated a fusion protein (Xhairy-1/ VP16) in which the C-terminus WRPW repressor domain was replaced by the activator domain of the viral protein VP16. This will allow us to analyze more precisely how Xhairy-1 regulates the expression of early mesodermal genes. Embryos at 2-cell stage were injected with 100 pg Xhairy-1/VP16 mRNA at the animal pole region and animal caps were dissected at stage 8 and cultured to various stages. RT-PCR analysis of a panel of mesodermal and neural markers indicated that Xhairy-1/VP16 induced the early expression of XMyoD at stage 10, but not the expression of the pan-mesodermal marker Xbra (Fig. 4). The expression level of XMyoD induced by Xhairy-1/VP16 peaks at stage 12 (late gastrula), then it declines at the early neurula stage (stage 13) to a barely detectable level by the end of neurulation (stage 20). Accordingly, the expression of muscle actin gene was never detected (Fig. 4). However, the expression of the neural marker N-CAM was induced from neurula stage onwards, concomitant with the decline of XMyoD expression level (Fig. 4). Interestingly, Xhairy-1/ VP16 did not induce the expression of XMyf5 at any stages (Fig. 4). This suggests that XMyoD gene may be a direct target of Xhairy-1 regulation. This result is consistent with recent data obtained in chick embryo showing that Notch signalling down-regulates MyoD expression without affecting the expression of Myf5 (Delfini et al., 2000; Hirsinger et al., 2001). It is also consistent with different studies in mice,
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which show that Myf5 acts upstream of MyoD during somite and limb development (Tajbakhsh et al., 1997). It is unlikely that Xhairy-1/VP16 activates the expression or activity of mesoderm-inducing factors which in turn induce the expression of XMyoD, because Xbra expression, which is an immediate early response to mesoderm induction (Smith et al., 1991), was not detected in animal caps expressing Xhairy-1/VP16 (Fig. 4). In addition, using a hormone-inducible form of Xhairy-1/VP16 (Xhairy-1/ VP16-GR), we also found that the expression of XMyoD induced by Xhairy-1/VP16 was not blocked by treatment with the protein synthesis inhibitor cycloheximide (Fig. 5A,B). These results further suggest that Xhairy-1 may directly regulate the expression of XMyoD. The observation that Xhairy-1 regulates the expression of XMyoD during gastrulation while it is involved in neurogenesis at later stages is consistent with its expression in the neural tissues during late development (data not shown). In Drosophila, neurogenic genes exhibit dual function in neurogenesis and myogenesis (Corbin et al., 1991; Bate et al., 1993; Carmena et al., 1995), the dual effect of Xhairy-1/VP16 suggests a conserved function of hairy-related genes in vertebrates. We have shown that Xhairy-1 inhibits the early expression of XMyoD both in vivo and in activin-treated ectodermal cells. Conversely, Xhairy-1/VP16 specifically induces XMyoD expression in native ectodermal explants. Overexpression of Xhairy-1 in the myogenic progenitors disrupts somitogenesis and results in embryos with trunk defects. These results suggest that Notch signalling through HES
Fig. 4. Xhairy-1/VP16 induces the early expression of XMyoD and the late expression of N-CAM in animal cap explants. Following injection of Xhairy-1/VP16 mRNA (100 pg) in the animal pole region at 2-cell stage, animal cap explants were dissected at mid-blastula stage and cultured to different stages for RT-PCR analysis. Xhairy-1/VP16 induces the expression of XMyoD at the early gastrula stage and the level of XMyoD expression peaks at stage 12. There is a decline in XMyoD expression induced by Xhairy-1/VP16 from the end of gastrulation (stage 13) onward, which correlates with the onset of N-CAM expression. Xhairy-1/VP16 does not induce the expression of muscle actin (Ms-Act) and XMyf5 at any stages.
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proteins function to maintain myogenic precursor cells in an undifferentiated state. Furthermore, we observed that Xhairy-1 exhibits a relatively ubiquitous expression in the early gastrula (not shown), thus, it may also function to suppress XMyoD expression in non-muscle cells like ectoderm and endoderm. In the presumptive mesoderm, XMyoD expression is regulated by both positive (mesoderm-inducing factors) and negative signals (Notch signalling; Wittenberger et al., 1999). Our results provide evidence that the effect of Notch signalling in regulating myogenic gene expression and myogenesis is likely mediated by hairyrelated genes.
3. Experimental procedures 3.1. Plasmid constructs
Fig. 5. Induction of XMyoD expression by Xhairy-1/VP16 in the absence of protein synthesis. Animal cap explants injected with Xhairy-1/VP16-GR mRNA (100 pg) were dissected at stage 11 and incubated in cycloheximide (CHX) followed by incubation in dexamethasone (DEX) for 2 h (see Section 3). (A) RT-PCR analysis of XMyoD expression. (B) Relative abundance of XMyoD transcripts in explants expressing Xhairy-1/VP16-GR and treated by CHX and DEX. The result represents the mean from two independent experiments. CHX does not block XMyoD expression induced by Xhairy-1/VP16-GR.
proteins may be directly involved in the regulation of myogenic gene expression at the early step of myogenesis. Recently, a molecular clock of somitogenesis was identified based on the periodic expression of chicken hairy (c-hairy) mRNA in the presomitic mesoderm (Palmeirim et al., 1997; McGrew and Pourquie´ , 1998). The periodic expression of the Notch pathway genes regulates segmental identity during somitomere formation (Conlon et al., 1995; Jen et al., 1997, 1999; Jouve et al., 2000). In the mesoderm of Xenopus gastrula, XMyoD triggers Notch signalling through transcriptional activation of the Notch ligand, Delta-1 (Wittenberger et al., 1999). Taken together, these analyses suggest that a feed-back regulatory loop, involving activator and repressor genes and operating at the transcriptional level, plays an important role in muscle specification. They are consistent with the model that suggests that bHLH repressor proteins are potential regulators of MyoD gene expression and myogenesis (Cossu et al., 1996). These
The full-length Xhairy-1 coding sequence (Dawson et al., 1995) was PCR amplified from a gastrula expression library in pRN3 vector (Lemaire et al., 1995) using the Xhairy-1specific antisense primer (5 0 -GCATCAGAATTCTCACTACCG-3 0 ) including an Eco RI site and the T3 primer. The PCR product was digested by Eco RI and Bgl II which is present in the pRN3 polylinker sequence, and cloned into the Bam HI and Eco RI sites of the pCS2 1 vector (Turner and Weintraub, 1994) to generate pCS2Xhairy-1. This construct was then used to generate all other constructs used in this study. Xhairy-1/DWRPW was PCR-amplified using SP6 primer and a Xhairy-1 antisense primer (5 0 -AGTTCTAGACAGAATCAATTGTGACTG-3 0 ) which introduces a stop codon before the WRPW motif and contains a Xba I site. Xhairy-1/bHLH was obtained by digesting pCS2-Xhairy-1 with Nco I and Xba I followed by blunt-end ligation. To generate Xhairy-1/DOrange, cDNA fragment encoding the repressor domain was amplified by PCR from the pCS2-Xhairy-1 plasmid using T7 primer and Xhairy-1 sense primer (5 0 -AGGAGCAGCTCTGCAGAGCAGCC-3 0 ) with a substituted nucleotide to introduce a Pst I site. The PCR product was subcloned in-frame into an internal Pst I site just after the bHLH coding sequence and the Xba I site of the pCS2 1 vector. The same principle was used to obtain Xhairy-1/ DbHLH, but in two steps. In the first step, an intermediate construct was obtained by PCR amplification of the cDNA fragment upstream of the bHLH domain using SP6 primer and Xhairy-1 antisense primer (5 0 -TTACTCGAGGCAGTTTCCCTTTAT-3 0 ) with a Xho I site. The PCR product was subcloned into the Cla I and Xho I sites of the pCS2 1 vector. In a second step, the cDNA fragment downstream of bHLH domain was amplified by PCR using T7 primer and a sense primer (5 0 -CTGCACTCGAGTACAGACC-3 0 ) with a substituted nucleotide to introduce a Xho I site, and was subcloned into the intermediate construct. pCS2-Xhairy-1/VP16 was obtained by PCR amplification of the VP16 sequence in pSP64T vector with sense (5 0 -
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GTCCCATGGCCCCCCCGACCGAT-3 0 ) and antisense (5 0 -GGCTCTAGATCAGTTCAGGTCG-3 0 ) primers including a Nco I and Xba I site, respectively. The PCR product was cloned into the corresponding sites of pCS2-Xhairy-1 in-frame with the DNA-binding and Orange domains of Xhairy-1. To obtain pSP64T-Xhairy-1/VP16-GR, Xhairy1/VP16 was PCR amplified using sense primer (5 0 GAAGATATCCGGCTGATGTG-3 0 ) with an EcoR V site and the T7 primer. The PCR product was cloned in-frame with the glucocorticoid receptor cDNA in pSP64T vector. All constructs were checked by sequencing and were found identical to the original sequence. The pSP64T-Xbra plasmid was provided by Dr J. Smith (Cunliffe and Smith, 1992).
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Acknowledgements We thank Drs J. Smith, R. Dosch and T. Mohun for providing the reagents used in this study; A. Pascal and A. Bourdelas for excellent technical assistance, P. Nguyen for illustrations and R. Schwartzmann for phosphorimager analysis. This work was supported by grants from Centre National de la Recherche Scientifique (CNRS), Ministe`re de la Recherche (ACI program), Association pour la Recherche sur le Cancer (ARC), Association Franc¸ aise contre la Myopathie (AFM) and Ligue National Contre le Cancer (LNCC).
References 3.2. Xenopus embryos and mRNA microinjections Xenopus eggs were obtained from females injected with 500 IU of human chorionic gonadotropin (Sigma), and artificially fertilized. Eggs were dejellied with 2% cysteine hydrochloride (pH 7.8) and embryos were staged according to Nieuwkoop and Faber (1967). Capped mRNAs were synthesized from linearized plasmids using SP6 RNA polymerase (Boehringer Mannheim). Microinjection of embryos was performed as previously described (Djiane et al., 2000).
3.3. RT-PCR and in situ hybridization Animal cap explants from control and injected embryos were dissected at mid-blastula stage (stage 8) and incubated for 1 h in 1 £ MBS containing 10 units/ml recombinant Xenopus activin A (provided by Dr J.C. Smith). For cycloheximide (CHX) and dexamethasone (DEX) treatment, animal cap explants injected with 100 pg Xhairy-1/VP16GR mRNA were first incubated in 10 mg/ml CHX (Sigma) for 30 min, they were then incubated in 10 mM DEX (Sigma) for 2 h in the presence of CHX. Extraction of RNA and RT-PCR were as described previously (Djiane et al., 2000). PCR primers were as described (Wittenberger et al., 1999; Djiane et al., 2000) except for muscle actin (5 0 GCTGACAGAATGCAGAAG-3 0 and 5 0 -TTGCTTGGAGGAGTGTGT-3 0 ), XMyf5 (5 0 -CTATTCAGAATGGAGATGGT-3 0 and 5 0 -GTCTTGGAGACTCTCAATA-3 0 ), and NCAM (5 0 -CACAGTTCCACCAAATGC-3 0 and 5 0 -GGAATCAAGCGGTACAGA-3 0 ). Each experiment was performed on triplicate and analyzed using a phosphorimager system (Biorad). Whole-mount in situ hybridization was performed according to standard protocol (Harland, 1991) except that chromogenic reaction was done using BM purple as substrate (Boehringer Mannheim). Probes for chordin and XMyoD were as described (Dosch et al., 1997; Djiane et al., 2000). The Xenopus myosin light chain cDNA was kindly provided by Dr T. Mohun.
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