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. 2004 Nov 15;18(22):2712-7.
doi: 10.1101/gad.1248604.

WNT signaling, in synergy with T/TBX6, controls Notch signaling by regulating Dll1 expression in the presomitic mesoderm of mouse embryos

Michael Hofmann  1 , Karin Schuster-Gossler, Masami Watabe-Rudolph, Alexander Aulehla, Bernhard G Herrmann, Achim Gossler
Affiliations

WNT signaling, in synergy with T/TBX6, controls Notch signaling by regulating Dll1 expression in the presomitic mesoderm of mouse embryos

Michael Hofmann et al. Genes Dev. .

Abstract

Notch signaling in the presomitic mesoderm (psm) is critical for somite formation and patterning. Here, we show that WNT signals regulate transcription of the Notch ligand Dll1 in the tailbud and psm. LEF/TCF factors cooperate with TBX6 to activate transcription from the Dll1 promoter in vitro. Mutating either T or LEF/TCF sites in the Dll1 promoter abolishes reporter gene expression in vitro as well as in the tail bud and psm of transgenic embryos. Our results indicate that WNT activity, in synergy with TBX6, regulates Dll1 transcription and thereby controls Notch activity, somite formation, and patterning.

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Figures

Figure 1.
Figure 1.
Expression of Dll1 or Tbx6 in T and Tbx6 mutant embryos. In situ hybridization of E8 (a,b,e,f) and E8.5 (c,d,g–j) wild-type (a,c,e,g,i) and homozygous T (f,h,j) and Tbx6 (b,d) mutant embryos with Dll1 (a–h) and Tbx6 (i,j) probes. Dll1 expression was significantly down-regulated in E8 and not detected in E8.5 T and Tbx6 mutant embryos. In E8.5 T mutant embryos Tbx6 expression was significantly reduced.
Figure 2.
Figure 2.
T-box and LEF/TCF sites in the Dll1 promoter and reporter constructs. (A) Schematic representation of the Dll1 promoter region extending 4.3 kb upstream of the ATG. The position of potential LEF/TCF sites (triangles) and T sites (circles) is indicated. H1 and H2 refer to the regions of homology detected between the zebrafish deltaD and mouse Dll1 promoters, which direct gene expression into the central nervous system (Beckers et al. 2000a). msd indicates the region directing gene expression in the paraxial mesoderm (Beckers et al. 2000a). (B) Sequences of the potential T and LEF/TCF sites in the Dll1 4.3-kb promoter region. Arrows indicate the orientation of the sites, and the consensus binding sites (cons) for LEF/TCF (Giese et al. 1992; van de Wetering and Clevers 1992) and T-box (Kispert et al. 1995; Kusch et al. 2002) factors are shown below. (C) Schematic representation of mutant reporter gene constructs used in this study. The mutated sites are marked and indicated above the constructs.
Figure 3.
Figure 3.
Synergistic activation of transcription from the msd promoter fragment by TBX6 and LEF/TCF. Relative luciferase activity in COS7 cells transfected with reporter gene plasmids and combinations of expression vectors as indicated. Luciferase activity was normalized to activity obtained with the wild-type msd reporter plasmid alone. (A) TBX6VP16 and TCF1E activate the msd promoter synergistically. (B) Synergism between TBX6VP16 and LEF1 depends on the β-catenin-binding domain of LEF1. (C) Both T and LEF/TCF sites are critical for synergistic activation. For comparison of luciferase activity between different reporter constructs, activity of the mutant promoter constructs was related to the activity obtained with the msd wild-type construct, whose activity was set to one.
Figure 4.
Figure 4.
Reporter gene expression in transgenic embryos. Representative examples of β-galactosidase-stained transgenic embryos obtained after DNA microinjection. (A) Staining patterns in embryos carrying the wild-type (panel a) and mutant (panels b–d) 4.3-kb promoter constructs. Note that mutations in either T1 (panel b) or T5 (panel c) abolished β-galactosidase expression specifically in the tailbud (arrowheads). Arrows point to the most recently formed somites. Insets show higher-magnification pictures of the tails. (B) Staining patterns in embryos carrying the wild-type (panel a) and mutant (panels b–g) msd promoter constructs. Mutation of either T sites (panels b,e), or LEF/TCF sites (panels c,f), or both (panels d,g) completely abolished β-galactosidase in the presomitic mesoderm, which expresses high levels of β-galactosidase in wild-type msd–lacZ transgenic embryos.
Figure 5.
Figure 5.
Proposed regulatory network directing Dll1 expression in the tailbud and presomitic mesoderm. WNT signals induce mesoderm formation and Brachyury expression, whose function is essential for migration of mesodermal cells through the primitive streak. T acts upstream of Tbx6, potentially as direct activator, and Tbx6 is required to maintain paraxial mesoderm. Both T and Tbx6 regulate Dll1 in the tailbud and in the presomitic mesoderm, respectively. WNT signals acting through the canonical Wnt pathway control Dll1 expression in the paraxial mesoderm synergistically with TBX6. (Bold arrows) Proven direct regulation; (dashed arrows) genetically upstream.
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