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.

Dynamic expression pattern of distinct genes in the presomitic and somitic mesoderm during Xenopus development.

Whole-mount and sectioned in situ hybridization was used to identify genes with restricted expression pattern in the presomitic and somitic mesoderm during Xenopus early development. Here we report the dynamic expression pattern of six distinct genes differentially expressed in these regions. These include Xenopus homologues of purine nucleoside phosphorylase, acetylcholine receptor, aspartate aminotransferase, glycine amidinotransferase and brain and muscle isoform creatine kinases. Purine nucleoside phosphorylase was initially expressed in the marginal zone at gastrula stage and then localized to the tail bud. Although the other genes showed no significantly localized expression at gastrula or neurula stages, they are progressively restricted in the somitic mesoderm as development proceeds. A common feature for these genes is that their deficiency in humans leads to an impairment of several tissue or cell functions. An analysis of their expression pattern could provide information regarding their implication in early development.

FGF8, Wnt8 and Myf5 are target genes of Tbx6 during anteroposterior specification in Xenopus embryo.

The T-box transcription factor Tbx6 is required for somite formation and loss-of-function or reduced activity of Tbx6 result in absence of posterior paraxial mesoderm or disorganized somites, but how it is involved in a regulatory hierarchy during Xenopus early development is not clear. We show here that Tbx6 is expressed in the lateral and ventral mesoderm of early gastrula, and it is necessary and sufficient to directly and indirectly regulate the expression of a subset of early mesodermal and endodermal genes. Ectopic expression of Tbx6 inhibits early neuroectodermal gene expression by strongly inducing the expression of posterior mesodermal genes, and expands the mesoderm territory at the expense of neuroectoderm. Conversely, overexpression of a dominant negative Tbx6 mutant in the ventral mesoderm inhibits the expression of several mesodermal genes and results in neural induction in a dose-dependent manner. Using a hormone-inducible form of Tbx6, we have identified FGF8, Xwnt8 and XMyf5 as immediate early responsive genes of Tbx6, and the induction of these genes by Tbx6 is independent of Xbra and VegT. These target genes act downstream and mediate the function of Tbx6 in anteroposterior specification. Our results therefore identify a regulatory cascade governed by Tbx6 in the specification of posterior mesoderm during Xenopus early development.

Antagonistic interaction between IGF and Wnt/JNK signaling in convergent extension in Xenopus embryo.

The homeobox gene Otx2 is expressed during gastrulation in the anterior domain of the vertebrate embryo and is involved in neural and head induction during Xenopus early development. It also prevents convergent extension movements in trunk and posterior mesoderm. Insulin-like growth factors (IGFs) were shown to have similar function. However, whether they interact and the mechanism by which they affect convergent extension remain unclear. We show that IGF pathway specifically induces the expression of Otx2 in the early gastrula and blocks convergent extension of neuroectoderm and mesoderm through the transcriptional activation of Otx2 gene. Otx2 represses the expression of Xbra and Xwnt-11, and the effects of IGF on gastrulation movements can be partially rescued by antisense Otx2 morpholino oligonucleotide. These indicate that IGF pathway interacts with Otx2 to restrict Xbra and Xwnt-11 expression in the trunk and posterior regions. Consistent with this, we show that inhibition of IGF signaling or Otx2 function induces Xbra and Xwnt11 expression and convergent extension in ectodermal cells. Furthermore, the blockade of convergent extension by IGF-I and Otx2 can be rescued by coexpression of Xwnt-11 or a constitutively active Jun N-terminal kinase (JNK). Because Xbra and Xwnt-11 are required for convergent extension movements and Xwnt-11 activates the non-canonical Wnt-11/JNK pathway, our results reveal a mutually exclusive function between IGF and Wnt-11/JNK pathways in regulating cell behaviours during vertebrate head and trunk development.

Identification of distinct genes with restricted expression in the somitic mesoderm in Xenopus embryo.

We have used whole-mount in situ hybridisation to identify genes expressed in the somitic mesoderm during Xenopus early development. We report here the analysis of eight genes whose expression pattern has not been described previously. They include the Xenopus homologues of eukaryotic initiation factor 2beta, methionine adenosyltransferase II, serine dehydratase, alpha-adducin, oxoglutarate dehydrogenase, fragile X mental retardation syndrome related protein 1, monocarboxylate transporter and voltage-dependent anion channel 1. Interestingly, these genes exhibit very dynamic expression pattern during early development. At early gastrula stages several genes do not show localised expression pattern, while other genes are expressed in the marginal mesoderm or in ectoderm. As development proceeds, the expression of these genes is gradually restricted to different compartments of somite. This study thus reveals an unexpected dynamic expression pattern for various genes with distinct function in vertebrates.

Direct binding of the PDZ domain of Dishevelled to a conserved internal sequence in the C-terminal region of Frizzled.

The cytoplasmic protein Dishevelled (Dvl) and the associated membrane-bound receptor Frizzled (Fz) are essential in canonical and noncanonical Wnt signaling pathways. However, the molecular mechanisms underlying this signaling are not well understood. By using NMR spectroscopy, we determined that an internal sequence of Fz binds to the conventional peptide binding site in the PDZ domain of Dvl; this type of site typically binds to C-terminal binding motifs. The C-terminal region of the Dvl inhibitor Dapper (Dpr) and Frodo bound to the same site. In Xenopus, Dvl binding peptides of Fz and Dpr/Frodo inhibited canonical Wnt signaling and blocked Wnt-induced secondary axis formation in a dose-dependent manner, but did not block noncanonical Wnt signaling mediated by the DEP domain. Together, our results identify a missing molecular connection within the Wnt pathway. Differences in the binding affinity of the Dvl PDZ domain and its binding partners may be important in regulating signal transduction by Dvl.

Zygotic Wnt/beta-catenin signaling preferentially regulates the expression of Myf5 gene in the mesoderm of Xenopus.

Zygotic Wnt signaling has been shown to be involved in dorsoventral mesodermal patterning in Xenopus embryos, but how it regulates different myogenic gene expression in the lateral mesodermal domains is not clear. Here, we use transient exposure of embryos or explants to lithium, which mimics Wnt/beta-catenin signaling, as a tool to regulate the activation of this pathway at different times and places during early development. We show that activation of Wnt/beta-catenin signaling at the early gastrula stage rapidly induces ectopic expression of XMyf5 in both the dorsal and ventral mesoderm. In situ hybridization analysis reveals that the induction of ectopic XMyf5 expression in the dorsal mesoderm occurs within 45 min and is not blocked by the protein synthesis inhibitor cycloheximide. By contrast, the induction of XMyoD is observed after 2 h of lithium treatment and the normal expression pattern of XMyoD is blocked by cycloheximide. Analysis by RT-PCR of ectodermal explants isolated soon after midblastula transition indicates that lithium also specifically induces XMyf5 expression, which takes place 30 min following lithium treatment and is not blocked by cycloheximide, arguing strongly for an immediate-early response. In the early gastrula, inhibition of Wnt/beta-catenin signaling blocks the expression of XMyf5 and XMyoD, but not of Xbra. We further show that zygotic Wnt/beta-catenin signaling interacts specifically with bFGF and eFGF to promote XMyf5 expression in ectodermal cells. These results suggest that Wnt/beta-catenin pathway is required for regulating myogenic gene expression in the presumptive mesoderm. In particular, it may directly activate the expression of the XMyf5 gene in the muscle precursor cells.