Tbx6-mediated Notch signaling controls somite-specific Mesp2 expression

Abstract

Mesp2 is a transcription factor that plays fundamental roles in somitogenesis, and its expression is strictly restricted to the anterior presomitic mesoderm just before segment border formation. The transcriptional on-off cycle is linked to the segmentation clock. In our current study, we show that a T-box transcription factor, Tbx6, is essential for Mesp2 expression. Tbx6 directly binds to the Mesp2 gene upstream region and mediates Notch signaling, and subsequent Mesp2 transcription, in the anterior presomitic mesoderm. Our data therefore reveal that a mechanism, via Tbx6-dependent Notch signaling, acts on the transcriptional regulation of Mesp2. This finding uncovers an additional component of the interacting network of various signaling pathways that are involved in somitogenesis.

Fig. 1.

Characterization of the Mesp2 enhancer region. (A) Comparisons between the genomic sequences of mouse Mesp2 and zebrafish mespb (an ortholog of mouse Mesp2) reveal five conserved sites in the 300-bp proximal promoter region. These conserved sites are denoted as A–E. The numbers above the genomic sequences indicate the base count from the ATG transcriptional start site of the Mesp2 ORF. (B) Summary of the transient transgenic assay results with different combinations of conserved sites from the Mesp2 upstream region (sites A–C). Each site was tested either alone or in combination with other sites for somite-specific enhancer activity. The presence (+) or absence (−) of β-gal activity and the incidence of this among transgene-positive embryos is shown schematically on the right of each reporter construct. The combination of site A and site B (shown as A+B) resulted in strong somite-specific enhancer activity (Left).

Fig. 2.

Tbx6 binds to the Mesp2 enhancer and promotes gene expression. (A) Wild-type and mutant sequences of site B and site D. The bases highlighted in lowercase denote the mutation sites. (B) Tbx6 binds to site B and site D. Digoxigenin-labeled oligonucleotide probes containing site B or site D were subjected to EMSA with (+) and without (−) Tbx6. Mutation of the CACAC motif in site B (lane mB1) resulted in the loss of both the wild-type band shifts, whereas mutations in GGGTC (lane mB2) abolished only the upper band. Mutations in the CACAC motif from site D also eliminated the band shift (lane mD). (C) β-Gal reporter expression analysis in transgenic mouse embryos with constructs containing either wild-type or mutated Mesp2 upstream regions. Each of the images is a lateral view with the anterior region toward the top. The numbers of β-gal-positive transgenic embryos are shown in each image (β-gal-positive/transgene-positive).

Fig. 3.

Mesp2 expression is activated by Notch signaling in a Tbx6-dependent manner. For each set of analyses, the luciferase activity was normalized to the values obtained in the absence of an expression vector (None). Error bars represent the standard deviation from six independent experiments. RVP16, RBPJκ-VP16. (A) Tbx6 activates a Mesp2–luciferase reporter gene construct synergistically with the NICD or RBPJκ-VP16. Mutation of site B and site D (denoted as P2EmB1D) eliminates this transactivation. (B) Notch signal activates the Mesp2 reporter construct via site A and site C. The reporter constructs are indicated to the left of the graph. (C) Nucleotide sequences of the possible RBPJκ binding sites in site A (Left) and site C (Right) and the comparison between these regions and the RBPJκ binding consensus sequence (denoted as RBPJk) (27). The nucleotides matching the consensus sequence are shown in red for site A and site C. Nucleotide substitutions in site C (denoted as mC) are indicated in lowercase.

Fig. 4.

Proposed mechanisms underlying the control of Mesp2 expression. Tbx6 and NICD (colored ovals) interact with the conserved upstream sites in the Mesp2 gene, sites A–D (represented by boxes). Tbx6 binds to site B (two molecules) and site D (single molecule). Site A and site C interact with RBPJκ to achieve a significant increase in Mesp2 expression levels in the presence of Notch signals (A). This activation fully depends on the binding of Tbx6 to site B or site D (B). Tbx6 may activate Mesp2 expression without site A and site C, presumably through an RBPJκ-independent Notch signaling pathway and via other signals (C). (D) Schematic representation of a proposed model that may explain developmentally regulated Mesp2 expression in the anterior PSM. (a) NICD is highly accumulated in the anterior PSM and less in the posterior (1, 2) to activate Mesp2 expression (red arrows). There may be a threshold level of NICD accumulation to initiate Mesp2 activation (broken line). (b) Tbx6 protein is distributed in the tailbud and PSM (20) and facilitates Mesp2 activation by NICD. (c) It is possible that the activation of Mesp2 expression in the tailbud and posterior PSM, if any, is repressed by other factor(s), such as Fgf8 (36), via an unknown mechanism. (d) As a result, Mesp2 expression is restricted in the anterior PSM (red box).

Fig. 5.

The expression of Mesp2 is not achieved solely by RBPJκ-dependent Notch signaling. (A) Transgenic analyses reveal that somite-specific reporter expression can still be observed by using the P2EΔAmC construct, which contains a deletion of site A and mutations in site C. The numbers of β-gal-positive embryos are indicated for each image (β-gal-positive/transgene-positive). (B) The expression of a dominant-negative RBPJκ diminishes reporter activation by Tbx6 for both the wild-type (wt) and P2EΔAmC (Tbx6+R218H, purple bars) vectors. Wild-type RBPJκ also strongly suppressed reporter activity driven by Tbx6 (Tbx6+RBPJκ, orange bars). Error bars represent the standard deviation in six independent experiments.