Zetser A et al. (2001), MAP kinase converts MyoD into an instructive mu...

XB-ART-7825

Dev Biol 2001 Dec 01;2401:168-81. doi: 10.1006/dbio.2001.0465.

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MAP kinase converts MyoD into an instructive muscle differentiation factor in Xenopus.

Zetser A , Frank D , Bengal E .

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In amphibian development, muscle is specified in the dorsal lateral marginal zone (DLMZ) of the gastrula embryo. Two critical events specify the formation of skeletal muscle: the expression of the myogenic transcription factor, XMyoD, and the secretion of bone morphogenetic protein (BMP) antagonists by the adjacent Spemann organizer. Inhibition of BMP signaling during early gastrula stages converts XMyoD protein into an instructive differentiation factor in the DLMZ. Yet, the intracellular signaling factors connecting BMP antagonism and activation of XMyoD remain unknown. Our data show that BMP antagonism induces the activity of mitogen-activated protein kinase (MAPK), and that the activity of MAPK is necessary for muscle-specific differentiation. Treatment of gastrula-stage DLMZ explants with MAPK pathway inhibitors ventralized mesoderm and prevented muscle differentiation. Expression of XMyoD in ventral mesoderm weakly induced muscle formation; however, the coexpression of a constitutively active MEK1 with XMyoD efficiently induced muscle differentiation. Activation of the MAPK pathway did not induce the transcription of XMyoD, but increased its protein levels and transcriptional activity. Thus, MAPK activation is subsequent to BMP antagonism, and participates in the dorsalization of mesoderm by converting the XMyoD protein into a potent differentiation factor.

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Species referenced: Xenopus
Genes referenced: actl6a cdk20 eef1a2 kit map2k1 mapk1 myod1 tbxt ventx2.2

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	FIG. 1. BMP antagonism induces phosphorylation of ERK in the ventral marginal zone (VMZ). Dorsalization of the VMZ to muscle by BMP antagonism is dependent on MAPK activity. (A) Extracts from nine DLMZ or VMZ explants were separated by SDS–PAGE, Western blotted, and sequentially probed with a polyclonal antibody specific for phosphorylated ERK and with a polyclonal antibody specific for total ERK protein (New England Biolabs PhosphoPlus p44/p42 MAPK Antibody Kit). Phosphorylated p42 was used as a positive control (lane 9). Lanes are labeled according to the developmental stages (Nieuwkoop and Faber, 1967). (B) Embryos at the one-cell stage were injected with either 0.1 or 0.4 ng of in vitro transcribed Xenopus BMP DN receptor RNA, or with either 2.5 or 5 ng of in vitro transcribed constitutively active form of MEK1 (Act MEK1) RNA. VMZ explants were removed at stage 101–10.25 and cultured until sibling embryos reached stage 12. Protein was isolated from each treatment group (14 explants) for Western blot analysis using the New England Biolabs PhosphoPlus p44/p42 MAPK Antibody Kit. (C) Embryos at the one-cell stage were injected with 0.4 ng of in vitro transcribed Xenopus BMP DN receptor RNA. Thirty VMZ explants were removed from uninjected and injected embryos. Fifteen explants from each group were grown with (lane 4) or without (lane 3) 50 mM PD098059. Total RNA was isolated from pools of 15 neurula stage-15 VMZ explants from each treatment. All VMZ RNA was loaded per well for Northern analysis. One embryo equivalent of RNA was loaded (lane 5). The filter was sequentially hybridized with Xenopus cDNA probes for muscle-specific actin (mActin) and EF1a. EF1a served as a positive control for quantitating RNA levels in the different samples.
	FIG. 2. Prevention of skeletal muscle formation by inhibition of MEK activity during gastrulation. (A) Thirty-six DLMZ explants were removed at stage 101–10.25. Eighteen explants were cultured in the presence of 50 mM PD098059 until sibling embryos reached stage 19 (left) or stage 11 (right). Eighteen untreated explants served as controls. Total RNA was isolated from pools of nine explants from each group. All DLMZ explant RNA and one embryo equivalent of RNA were loaded per well for Northern analysis. The stage-19 filter was sequentially hybridized with Xenopus cDNA probes for mActin and EF1a. The stage-11 filter was sequentially hybridized with Xenopus cDNA probes for Xbra, Vent2, and EF1a. EF1a served as a positive control for quantitating RNA levels in the different samples. (B) Thirty-six DLMZ explants were removed at stage 101–10.25 and cultured in the presence of 20 mM UO126 until sibling embryos reached stage 19 (left) or stage 11 (right). Eighteen untreated explants served as controls. Total RNA was isolated from pools of nine explants from each treatment. All the DLMZ RNA and one embryo equivalent of RNA were loaded per well for Northern analysis. The stage-19 filter was sequentially hybridized with Xenopus cDNA probes for mActin and EF1a. The stage-11 filter was sequentially hybridized with Xenopus cDNA probes for Xbra, Vent2, and EF1a. EF1a served as a positive control for quantitating RNA levels in the different samples. (C) DLMZ explants were removed at stage 101–10.25 and cultured without or in the presence of 20 or 50 mMUO126 until sibling embryos reached stage 18. Explants were examined for elongation. (D) DLMZ explants from albino embryos were cultured until stage 27 without or in the presence of 20 mM UO126; embryos were stained with the muscle-specific 12/101 monoclonal antibody.
	FIG. 3. Ectopic expression of a constitutively active form of MEK1 enhances muscle formation in the VMZ. (A) Embryos at the one-cell stage were injected with 2 ng of in vitro transcribed XMyoD RNA or/and with 2.5 ng of in vitro transcribed constitutively active form of MEK1 (Act MEK1) RNA. VMZ explants removed at stage 101–10.25, and animal cap explants removed at stage 8–9, were cultured until sibling embryos reached stage 20. Total RNA was isolated from each group of explants (15 explants per group) and from whole embryos. All explant RNA and one embryo equivalent of RNA were loaded per well for Northern analysis. The filter was sequentially hybridized with Xenopus cDNA probes for mActin and EF1a. EF1a served as a positive control for quantitating RNA levels in the different samples. (B) Embryos at the one-cell stage were injected with 2 ng of in vitro transcribed XMyoD RNA or/and with 2.5 ng of in vitro transcribed constitutively active form of MEK1 (Act MEK1) RNA. Twenty animal caps were removed from uninjected and injected groups of blastula-stage embryos. Explants from each group were grown to stage 19 and total RNA was isolated. RT-PCR analysis was performed with the markers XMyoD, mActin, and EF1a. EF1a served as a positive control for quantitating RNA levels in the different samples. For controls, RT-PCR and 2RT-PCR were performed on total RNA isolated from normal embryos. (C) Embryos at the one-cell stage were injected with 2 ng of in vitro transcribed XMyoD RNA or/and with 2.5 ng of in vitro transcribed constitutively active form of MEK1 (Act MEK1) RNA. At stage 101–10.25, 16–30 VMZ explants were removed from each injected or uninjected embryo group. Explants were grown until sibling embryos reached stage 18, when they were examined for elongation. (D) The same embryos and VMZ explants (see Fig. 3C) were grown to stage 35 and stained with the muscle-specific 12/101 monoclonal antibody, that recognizes mature skeletal muscle.
	FIG. 4. Activation of MAPK does not induce XMyoD expression in VMZ explants. Embryos at the one-cell stage were injected with 0.4 ng of in vitro transcribed Xenopus BMP DN receptor RNA or with 2.5 ng of the constitutively active form of MEK1 (Act MEK1) RNA. At stage 10.25, 18 VMZ explants were removed from each injected or uninjected embryo group. Explants were grown until sibling embryos reached stage 14, and total RNA was isolated. RT-PCR analysis was performed with the markers XMyoD, mActin, and EF1a. EF1a served as a positive control for quantitating RNA levels in the different samples. For controls, RT-PCR and 2RT-PCR were performed on total RNA isolated from normal embryos.
	FIG. 5. Induction of XMyoD transcriptional activity by MAPK. (A) Embryos at the one-cell stage were injected with 2 ng of in vitro transcribed XMyoD RNA or/and with 2.5 ng of in vitro transcribed constitutively active form of MEK1 (Act MEK1) RNA. All embryos were injected with 50 pg of the E-Boxdependent reporter gene plasmid, 4R-tk-Luc. At stage 101– 10.25, 27 VMZ explants were removed from each injected embryo group. Explants were grown until sibling embryos reached stage 12. Protein extracts were examined for luciferase activity. One representative experiment is shown; experiments were performed four times. (B) Embryos at the one-cell stage were injected with 50 ng of the previously mentioned reporter plasmid (Fig. 5A). Embryos were injected with 2 ng of in vitro transcribed XMyoD RNA. At stage 101–10.25, 27 DLMZ explants were removed from the injected and control embryo groups. Half of the explants were cultured in the presence of 50 mM UO126 until sibling embryos reached stage 12. Protein extracts were examined for luciferase activity. One representative experiment is shown; experiments were performed four times.
	FIG. 6. Activation of MAPK increases XMyoD protein levels. (A) Protein extracts (9 explant equivalents), as described in Fig. 5B, were separated by SDS–PAGE, Western blotted, and sequentially probed with the XMyoD-specific D7F2 monoclonal antibody and with a polyclonal antibody specific for total ERK protein (left). Total ERK protein served as a positive control for quantitating protein levels in the different samples. A portion of the same extract was also used for total RNA preparation. RT-PCR analysis was performed with the markers XMyoD and EF1a (right). EF1a served as a positive control for quantitating RNA levels in the different samples. For controls, RT-PCR and 2RT-PCR were performed on total RNA isolated from normal embryos. (B) Protein extracts (9 explant equivalents), as described in Fig. 5A, were separated by SDS–PAGE, Western blotted, and sequentially probed with the XMyoD-specific D7F2 monoclonal antibody and with a polyclonal antibody specific for total ERK protein (left). Total ERK protein served as a positive control for quantitating protein levels in the different samples. A portion of the same extract was used for total RNA preparation. RT-PCR analysis was performed with the markers XMyoD and EF1a (right). EF1a served as a positive control for quantitating RNA levels in the different samples. For controls, RT-PCR and 2RT-PCR were performed on total RNA isolated from normal embryos.
	FIG. 7. Immunostaining of ectopically expressed MyoD protein following MAPK pathway inhibition. Embryos at the one-cell stage were injected with 2 ng of in vitro transcribed XMyoD RNA. Two DLMZ explants were removed from 36 injected embryos. One explant from each DLMZ pair was grown in the presence of 50 mM UO126, and the other was grown as a control, without treatment.
	FIG. 8. Model of the role of MAPK signaling in muscle specification in the DLMZ. The inhibition of BMP activity induces XMyoD expression and increases MAPK activity in the DLMZ. MAPK activity causes posttranscriptional modification of the XMyoD protein, which converts MyoD into an instructive differentiation factor.