May 1, 2010;
The RNA-binding protein Seb4/RBM24 is a direct target of MyoD and is required for myogenesis during Xenopus early development.
RNA-binding proteins play an important role to post-transcriptionally regulate gene expression. During early development they exhibit temporally and spatially regulated expression pattern. The expression of Xenopus laevis Seb4
gene, also known as RBM24
in other vertebrates, is restricted to the lateral
and ventral mesoderm
during gastrulation and then localized to the somitic mesoderm
, in a similar pattern as XMyoD
gene. Using a hormone-inducible form of MyoD
to identify potential direct MyoD
target genes, we find that Seb4
expression is directly regulated by MyoD
at the gastrula
stage. We further show that a 0.65kb X. tropicalis RBM24
regulatory region contains multiple E boxes (CANNTG), which are potential binding sites for MyoD
and other bHLH proteins. By injecting a RBM24
reporter construct into the animal pole of X. laevis embryos, we find that this reporter gene is indeed specifically activated by MyoD
and repressed by a dominant negative MyoD
mutant. Knockdown of Seb4
produces similar effects as those obtained by the dominant negative MyoD
mutant, indicating that it is required for the expression of myogenic genes and myogenesis in the embryo
. In cultured ectodermal explants, although overexpression of Seb4
has no obvious effect on myogenesis, knockdown of Seb4
inhibits the expression of myogenic genes and myogenesis induced by MyoD
. These results reveal that Seb4
is a target of MyoD
during myogenesis and is required for myogenic gene expression.
[+] show captions
Fig. 1. (A–H) MyoD regulates the expression of Seb4 in whole embryos at different stages analyzed by in situ hybridization. (A and B) Expression of Seb4 (A) and XMyoD (B) in the lateral mesoderm of a stage 11 gastrula. (C) Overexpression of XMyoD induces ectopic Seb4 expression on the injected side of an early gastrula. (D) A dominant negative MyoD mutant, MyoD-ENR, blocks Seb4 expression. (E) Injection of Lac Z mRNA and treatment with DEX do not induce the expression of Seb4 in the early gastrula. (F) An early gastrula previously injected on one side with MyoD-GR and Lac Z mRNAs and treated with DEX for 1.5 h shows ectopic expression of Seb4. (G) Injection of Lac Z mRNA and treatment with DEX for 1.5 h do not induce ectopic Seb4 expression at stage 12. (H) Injection of MyoD-GR and Lac Z mRNAs and treatment with DEX for 1.5 h strongly induce ectopic Seb4 expression at stage 12. (I) Induction of Seb4 expression in ectodermal explants by MyoD-GR in the absence of protein synthesis. Both uninjected and injected explants were treated with DEX or CHX, or both, for 1.5 h. The expression of Seb4, Xbra and Xwnt8 was analyzed by RT-PCR. Seb4 expression is induced in explants injected with MyoD-GR mRNA and treated with DEX in the presence of CHX, while the expression of Xbra and Xwnt8 is not induced. ODC (ornithine decarboxylase) was used as a loading control. (J) Effect of CHX on the translation of injected Seb4MT mRNA.
Fig. 3. Knockdown of Seb4 inhibits myogenic gene expression at the gastrula stage. Embryos at four-cell stage were injected in the dorso-lateral region with CoMO, Seb4MO or MyoD-ENR mRNA. In situ hybridization was performed at the early gastrula stage using probes indicated on the right side. Injection of CoMO (A) does not affect Xbra expression, while injection of Seb4MO (B) or MyoD-ENR mRNA (C) similarly inhibits, but does not completely block, Xbra expression. (D) Injection of CoMO has no effect on XMyoD expression, while both Seb4MO (E) and MyoD-ENR (F) inhibit XMyoD expression. (G) XMyf5 expression is not affected by CoMO, but Seb4MO (H) and MyoD-ENR (I) inhibit its expression. Xwnt8 expression is not affected by CoMO (J), Seb4MO (K) and MyoD-ENR (L). Similar chordin expression pattern in embryos injected with CoMO (M), Seb4MO (N) and MyoD-ENR mRNA (O). (P) Inhibition of the Myf5 promoter reporter (XTM1-luc) activity by Seb4MO and MyoD-ENR in stage 10.5 early gastrula. The luciferase reporter assay was performed in triplicates. Error bars indicate standard deviation.
Fig. 4. Knockdown of Seb4 inhibits myogenesis. In situ hybridization using indicated probes was performed on embryos injected with CoMO or Seb4MO and cultured to stage 30. (A) Myosin light chain (MLC) expression in the somitic mesoderm of a CoMO-injected embryo. (B) A Seb4MO-injected embryo with reduced and disorganized somitic mesoderm. (C) XMyf5 expression in a CoMO-injected embryo. (D) Injection of Seb4MO inhibits XMyf5 expression in the anterior and trunk region. (E) XMyoD expression in a CoMO-injected embryo. (F) Injection of Seb4MO inhibits XMyoD expression. (G) Tbx6 expression in the tail-bud of a CoMO-injected embryo. (H) Injection of Seb4MO reduces the expression level of Tbx6 in the tail-bud. (I) HoxB9 expression in the spinal cord of a CoMO-injected embryo. (J) Injection of Seb4MO does not significantly affect HoxB9 expression.
Fig. 5. Effectiveness and specificity of Seb4MO. (A) Western blot analysis. Seb4MT (lanes 1 and 2) or mutSeb4MT (lanes 3 and 4) mRNA was coinjected with CoMO (lanes 1 and 3) or Seb4MO (lanes 2 and 4). Xdsh-myc mRNA was coinjected as an unrelated mRNA in each condition and used as a loading control. CoMO has no effect on Seb4MT translation (lane 1), while Seb4MO completely blocks Seb4MT translation (lane 2), but not the coinjected Xdsh-myc mRNA. Neither CoMO (lane 3) nor Seb4MO (lane 4) has any effect on mutSeb4MT translation. (B) Coinjection of mutSeb4MT mRNA rescues Seb4MO-inhibited Xbra and XMyf5 expression in whole embryos at the early gastrula stage, and MO2 similarly inhibits Xbra and XMyf5 expression as Seb4MO.