LEF1-mediated regulation of Delta-like1 links Wnt and Notch signaling in somitogenesis

Abstract

Wnt signaling, which is mediated by LEF1/TCF transcription factors, has been placed upstream of the Notch pathway in vertebrate somitogenesis. Here, we examine the molecular basis for this presumed hierarchy and show that a targeted mutation of Lef1, which abrogates LEF1 function and impairs the activity of coexpressed TCF factors, affects the patterning of somites and the expression of components of the Notch pathway. LEF1 was found to bind multiple sites in the Dll1 promoter in vitro and in vivo. Moreover, mutations of LEF1-binding sites in the Dll1 promoter impair expression of a Dll1-LacZ transgene in the presomitic mesoderm. Finally, the induced expression of LEF1-beta-catenin activates the expression of endogenous Dll1 in fibroblastic cells. Thus, Wnt signaling can affect the Notch pathway by a LEF1-mediated regulation of Dll1.

Figure 1.

Skeletal malformations in homozygous Lef1-βgal mice. Alcian blue/alizarin red staining of skeletal preparations of wildtype (wt; a,c,e) and homozygous Lef1-βgal (b/b; b,d,f) newborns. (a,b) The vertebral column of b/b mutants shows a reduction in the number of vertebrae with extensive fusions in their bodies and neural arches. (c,d) The ribcage of mutant mice contains fewer ribs that show fusions, bifurcations, and asymmetric attachment to the sternum. (e,f) The forelimbs of homozygous Lef1-βgal newborns are malformed, with a frequent loss of the radius and a consistent reduction in the number of fingers.

Figure 2.

Defects of somite formation in homozygous Lef1-βgal mice. (A) Analysis of Lef1-βgal expression in heterozygous (b/+) and homozygous (b/b) E9.5 embryos by whole-mount staining for LacZ activity. (Panels a,b) Abundant Lef1-βgal expression is observed in forelimb buds (FL), the presomitic mesoderm (PSM), and newly formed somites. (Panels c,d) Cross-sections of the thoracic region show the lack of somites in the mutant embryos. (B) Expression of molecular markers in E9.5 wild-type (wt) and homozygous Lef1-βgal embryos by whole-mount in situ hybridization. (Panels a,b) In mutants, the segmented expression pattern of the sclerotome marker Pax1 is severely impaired. (Panels c,d) Brachyury (T) expression in the anterior region of the PSM is reduced in mutant embryos (b/b), but normal expression is detected in the posterior region of the PSM and notochord. (Panels e,f) Expression of Axin2 in the PSM is reduced in mutant embryos. Arrowheads point to the forelimb buds.

Figure 3.

(A) Defects in the patterning and polarity of somites in homozygous Lef1-βgal (b/b) mice. (Panels a,b) Whole-mount anti-neurofilament staining of E11.5 embryos shows the lack of regularly spaced spinal nerve projections and a misrouting of axons in mutant embryos. (Panels c–f) Whole-mount in situ hybridizations of E9.5 mutant embryos show a caudal loss of expression of the posterior somite marker Uncx4.1 and the abrogation of expression of the anterior somite marker Cerberus (Cer1). Arrowheads indicate somite boundaries. (B) Expression of Notch signaling components in wildtype and homozygous Lef1-βgal mice. (Panels a,b) Expression of Mesp2 in the anterior presomitic mesoderm (PSM) is markedly reduced in mutant embryos. Arrowhead indicates the anterior boundary of the PSM. (Panels c–f) Expression of Notch1 and Dll1 in the PSM is also reduced in mutant embryos but is maintained in neural tissues. (Panels g,h). Expression of the Notch antagonist Lnfg is undetectable in the newly formed somites of mutant embryos.

Figure 4.

LEF1-mediated regulation of Dll1. (A) Schematic presentation of the 5′-flanking region of Dll1, including homology domain (HD)-1 and HD-2, the mesoderm enhancer (msd enh), and promoter (msd prom). The positions of confirmed LEF1/TCF-binding sites are indicated by boxes. Black and gray boxes represent strong and weak binding sites, respectively. (B) Electrophoretic mobility shift assay showing binding of recombinant LEF1 to sites in the msd promoter region. Purified LEF1 (100 or 300 ng) was incubated with radiolabeled oligonucleotides encompassing binding sites IX–XII. The specificity of binding was confirmed by addition of 100-fold molar excess of unlabeled oligonucleotides encompassing a wild-type (w) or mutated (m) LEF1 consensus site from the TCRα enhancer (Galceran et al. 2001). (C) Schematic presentation of Dll1–LacZ gene constructs containing the msd promoter, in which the positions of wild-type or mutated LEF1-binding sites are indicated. (D) Whole-mount staining for LacZ activity of representative E9.5 (top) and E8.5 (bottom) embryos carrying wild-type or mutant transgenes. The wild-type transgene is expressed in the PSM (bar) and somites, whereas the mutant transgenes are expressed only in the somites. (E) Quantitation of the transgenic expression analysis, in which the frequency of expression in PSM and somites (SOM) is presented as the ratio of number of embryos that express (Expr.)/carry a transgene (TG pos.).

Figure 5.

catC–LEF-induced expression of Dll1 and binding of LEF1 to Dll1 regulatory sequences in vivo. (A) Schematic presentation of the catC–LEF1 protein that activates gene expression in the absence of Wnt signals (Hsu et al. 1998). (B) Anti-LEF-immunoblot analysis of NIH-BC29 cells containing a catC–LEF1 construct under the control of ecdysone-response elements. Protein expression can be detected 12 h after treatment of the cells with Ponasterone. (C) Quantitative RT–PCR to detect the expression of the endogenous Axin2 and Dll1 genes in BC29 cells, uninduced and induced for 12 h. The levels of expression were normalized against the expression of actin. (D) Schematic presentation of the msd regulatory regions of the Dll1 gene, in which the positions of the LEF1-binding sites and the regions analyzed by chromatin immunoprecipitations (ChIPs) are indicated. (E) ChIP of uninduced and induced BC29 cells to detect binding of catC–LEF1 to Dll1 sequences in vivo. Semiquantitative PCR was performed with serial dilutions of template DNA. Binding can be detected with anti-LEF antibodies, but not without addition of antibodies. (F) ChIP of PSM from E9.5 embryos to detect binding of endogenous LEF1 to Dll1 regulatory sequences in vivo. Binding can be detected in both msd enhancer and promoter regions. (G) Schematic model for the regulation of Dll1 by LEF1, which links the Wnt signaling pathway with the Notch/Dll pathway in somitogenesis. Negative feedback loops that regulate the cycling expression of genes are indicated (for review, see Pourquié 2003).