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First published online August 25, 2006
doi: 10.1242/10.1242/dev.02517

Development 133, 3723-3732 (2006)
Published by The Company of Biologists 2006
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The Wnt/ß-catenin pathway regulates Gli-mediated Myf5 expression during somitogenesis

Ugo Borello1,2,*,{dagger}, Barbara Berarducci1,2, Paula Murphy3, Lola Bajard4, Viviana Buffa2, Stefano Piccolo5, Margaret Buckingham4 and Giulio Cossu1,6,{dagger}

1 Stem Cell Research Institute, Dibit, H. San Raffaele, Via Olgettina 58, 20132 Milan, Italy.
2 Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Istologia ed Embriologia Medica, Universita di Roma `La Sapienza', Via A. Scarpa 14, 00161 Rome, Italy.
3 Zoology Department, Trinity College, Dublin 2, Ireland.
4 Department of Developmental Biology, CNRS URA 2578, Pasteur Institute, 25 Rue du Dr Roux, 75724 Paris Cedex 15, France.
5 Department of Histology, Microbiology and Medical Biotechnology, University of Padua, 35131 Padua, Italy.
6 Department of Biology, University of Milan, Via Celoria, 20133 Milan, Italy.


Figure 1
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  		Fig. 1. Dominant-negative frizzled receptor mutants inhibit Myf5 expression. (A) Quantitative analysis of Myf5 activation in explants. PSM and the first somites (I-IV) of Myf5nlacZ/+ targeted mice (Tajbakhsh et al., 1996Go) were co-cultured for 3 days with neural tube and C3H10T1/2 cells expressing Frizzled1{Delta}N (Fz1{Delta}N), Frizzled6{Delta}N (Fz6{Delta}N), Frizzled7{Delta}N (Fz7{Delta}N) or Sfrp3, or with control cells (C). Independent experiments (n=6) were performed in triplicate and averaged. (B) Schematic representation of the frizzled receptor structure compared with the frizzled {Delta}N mutants used in this assay and Sfrp molecules. CRD, cystein-rich domain; NLD, netrin-like domain; red bands, transmembrane domains.

 

Figure 2
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  		Fig. 2. Expression patterns of Tcf/Lef family members, ß-catenin and Gli1 in the PSM and newly formed somites at E9.5. (A) Lateral view of a lightly stained Lef1 whole-mount in situ hybridized embryo after OPT scanning and 3D reconstructions. (B,C) Optical sections through the resulting reconstruction: B is through the tail region, in the same plane of view as A, C is a transverse section, as indicated by the line in A. (D) An embryo hybridized to reveal Tcf3 expression in the newly formed somites. (E) Transverse section through the newly formed somites shown in D. (F) A similar section to C, through a Tcf1 hybridized embryo. (G) ß-catenin WHISH; arrowheads indicate expression in the lateral mesoderm. (H,I) BAT-gal transgenic reporter mice: (H) ß-galactosidase staining, (I) lacZ WHISH. (J,K) Transverse sections through newly formed somites in I and H, respectively. (L) A similar section to in J and K, immunostained for ß-catenin. (M) Gli1 WHISH. (N,O) Transverse sections through PSM (N) and newly formed somites (O) in M.

 

Figure 3
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  		Fig. 3. ß-catenin activation of Myf5 expression in PSM and somite explants. (A-F) PSM explants (A-C) and somite explants (D-F) infected with a lentiviral vector carrying the cDNA for a stabilized form of ß-catenin (Dp). (A,D) Hoechst staining of the nuclei. Infected cells were visualized by GFP fluorescence (B,E), and Myf5nlacZ/+-expressing cells were detected by immunofluorescence with an anti-ß-galactosidase monoclonal antibody (C,F). (G,H) Quantitative analysis of Myf5nlacZ activation in infected PSM (G) and somite (H) explants. Explants were infected with Dp or {Delta}C ß-catenin lentiviruses and co-cultured with or without 2 nM N-Shh recombinant protein (Shh). Explants co-cultured with neural tube (NT) were used as a positive control; explants infected with lentiviruses carrying GFP cDNA only were used as a negative control (C). Independent experiments (n=8) were performed in triplicate and averaged.

 

Figure 4
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  		Fig. 4. ß-catenin is required for Myf5 activation in somite explants. (A-D) ß-cateninfloxdel/floxed somite explants were infected with a lentiviral vector carrying the cDNA for GFP (A,B) or CRE-IRES-GFP (C,D), and co-cultured with neural tube. Infected cells were visualized by GFP fluorescence (B,D) and Myf5-expressing cells were visualized with an anti-Myf5 monoclonal antibody (A,C). The GFP+ cells were also Cre+ when tested with an anti-Cre antibody (data not shown). (E) The absence of ß-catenin in Cre-infected explants was demonstrated by PCR with specific oligonucleotides discriminating between the floxed and floxdel allele of ß-catenin (Brault et al., 2001Go). (F) Quantification of Myf5 activation. Independent experiments (n=8) were performed in triplicate and averaged.

 

Figure 5
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  		Fig. 5. Analysis of the Tcf/Lef binding sites in the EpExt enhancer regions. (A) The sequence of the EpExt enhancer. The Tcf/Lef binding sites are highlighted in red and the Gli binding site in green. The dashed line underlines a non-consensus Tcf/Lef site. Underlined are the EcoRI (-6.6 kb) and BamHI (-6.0 kb) sites that indicate the boundaries of the originally defined Ep Enhancer. Nucleotide positions (in kb) relative to the Myf5 coding sequence are indicated. (B) EMSAs performed with oligonucleotides of the Tcf/Lef binding sites (TBF1-2-3, TBF4 and TBF5), as indicated on the sequence in A, incubated with the reticulocyte lysate alone (RL) or with Lef1 recombinant protein (Lef1), in the presence of a specific antibody (antiHA) and a 100 molar excess of the mutated (mut.) or specific cold (wt) oligonucleotide.

 

Figure 6
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  		Fig. 6. Quantitative analysis of the transcriptional activity of the Lef/Tcf binding sites in the Ep and EpExt enhancers. (A) NIH3T3 cells were transfected with the reporter constructs indicated and described in Fig. 6B. TK, TK minimal promoter. (B) The deletions of the EpExt enhancer analyzed by luciferase assay. Tcf/Lef binding sites (red circles) and a Gli site (green rectangle) are indicated; numbers indicate nucleotide position in the genomic sequence, as in Fig. 5A. (C-F) NIH3T3 cells were transfected with either the Ep (C,E) or the EpExt (D,F) enhancer driving firefly luciferase and the expression vectors indicated below each bar on the graph. Dp, stabilized ß-catenin; {Delta}C, dominant-negative ß-catenin; Lef1{Delta}N, dominant-negative Lef1. The values of fold induction represent the ratio of the firefly luciferase activity of cells transfected with and without the ß-catenin and Lef1 expression vectors, normalized to the activity of a control renilla luciferase-expressing vector. Independent experiments (n=10) were performed in triplicate and averaged.

 

Figure 7
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  		Fig. 7. Transgenic analysis of the role of the Tcf/Lef and Gli binding sites present in the epaxial enhancer of the Myf5 gene. (A-E) X-gal staining of whole-mount embryos at E9.5, with the following enhancer sequences upstream of the ß-globin promoter and the nlacZ reporter transgene: (A) the early epaxial enhancer (Ep), (B) the extended version of this enhancer (EpE), (C) the Ep enhancer with a 5' and 3' end deletion that removes the TBF4 and TBF5 Tcf/Lef binding sites ({Delta}Ep), (D) the deleted {Delta}Ep enhancer with the Gli site mutated ({Delta}EpGm), and (E) the deleted {Delta}Ep enhancer with the Gli and remaining non-consensus Tcf/Lef binding site mutated ({Delta}EpGT/Lm). (F,H) Transverse sections of the embryo shown in A at two different levels in the interlimb region, showing extensive labelling of cells in the epithelial dermomyotome of somites. (G,I) Transverse sections of the embryo shown in B at two different levels in the interlimb region; labelled cells are restricted to the epaxial region of the dermomyotome, closest to the neural tube (NT). The number of somites (S) is indicated. (J) Schematic representation of the fragments of the epaxial enhancer used in the different transgenic constructs shown in A to I. EpE corresponds to the EpExt sequence and {Delta}Ep to the {Delta}5 sequence presented in Fig. 6B. Red circles indicate the positions of the Tcf/Lef binding sites, the green rectangle indicates the position of the Gli binding site and the blue circle indicates the non-consensus Tcf/Lef binding site. White crosses indicate that the corresponding sites have been mutated.

 




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