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Wnt and Bmp signalling cooperatively regulate graded Emx2 expression in the dorsal telencephalon

Institute for Animal Developmental and Molecular Biology, Heinrich-Heine-University, D-40225 Düsseldorf, Germany

View larger version (9K): [in a new window]   Fig. 1. Transgenic analysis of Emx2 regulation in the forebrain. The diagram depicts a restriction map upstream of the Emx2-coding region (the translational start (ATG) is indicated). The restriction fragments, which are cloned into a vector containing lacZ and a minimal promoter are represented by bars. For each construct, the numbers of transgenic embryos that showed ß-galactosidase staining in the telencephalon (tel) or diencephalon (dien) and the total number of transgenic embryos (tg) are indicated. B, BamHI; E, EcoRI; G, BglII; H, HindIII; N, NdeI; Ns, NsiI; S, SacII; X, XhoI.

View larger version (91K): [in a new window]   Fig. 2. Characterisation of the Emx2 enhancer activity in the forebrain. (A) In situ hybridisation analysis of Emx2 expression in the telencephalon of E10.5 wild-type mice. (B) lacZ staining of a transgenic embryo carrying the mouse Emx2 enhancer (construct 2). (C) Emx2 enhancer activity in the forebrain of an homozygous XtJ embryo. lacZ expression in the dorsomedial telencephalon (t) is abolished (arrow), while expression in the cortical neuroepithelium occurs at lower levels than in wild-type embryos. lacZ expression in the diencephalon (d) remains unaffected. (D-F) Time course analysis of enhancer activity in the forebrain at (D) the 12-somite stage, (E) E10.5 and (F) E11.5. (D) Start of lacZ expression in the dorsal forebrain. (E) Expression of the reporter gene is observed in the dorsal part of the telencephalic vesicles with highest expression levels in the caudal telencephalon. (F) At E11.5, the reporter gene is expressed in a gradient with highest expression levels in the medial-caudal telencephalon (arrows). lacZ expression in the developing heart as seen in B-F is specific for the transgenic line shown.

View larger version (89K): [in a new window]   Fig. 3. In situ hybridisation analysis of Wnt gene expression in the forebrains of E10.5 embryos lacking Gli3 function. (A,B) Wnt3a expression in wild-type embryos covers the dorsomedial telencephalon, while XtJ/XtJ embryos completely lack Wnt3a mRNA in this tissue. (C,D) Wnt8b expression in homozygous XtJ embryos is disrupted, except for a narrow band of cells in the caudal telencephalon (arrowhead) (E) Wnt7b is expressed in the dorsal telencephalon of wild-type embryos with highest expression levels in the dorsomedial part (arrows). (F) Wnt3a mRNA is specifically absent from the dorsomedial telencephalon of XtJ/XtJ embryos (arrow), but still present in the dorsolateral telencephalic neuroepithelium.

View larger version (30K): [in a new window]   Fig. 4. The DT1 element of the Emx2 enhancer contains adjacent Tcf- and Smad-binding sites (A) Nucleotide sequence of DT1. The canonical Tcf- and Smad-binding sites (underlined) are indicated. For mutational analysis, the underlined nucleotides were replaced by GC and C, respectively. (B) The binding capacity of the Emx2 enhancer was tested in electrophoretic mobility shift assays with recombinant Lef1 and Smad1MH1 protein. Lef1 and Smad1 bind to an Emx2 enhancer oligonucleotide (lanes 2 and 3, respectively). Binding is potentiated in the presence of both proteins (lane 4) and an additional slower migrating, Lef1/Smad1 complex is observed. Complex formation is competed by a molar excess (100x) of the Emx2 enhancer oligonucleotide but not by an oligonucleotide containing mutations in the Tcf- and Smad-binding sites (lanes 5-8).

View larger version (82K): [in a new window]   Fig. 5. The Tcf- and Smad-binding sites are required for dorsal telencephalic enhancer activity. (A) Embryo transgenic for the 4.6 kb enhancer (construct 2) shows staining in the dorsal telencephalon and ventral diencephalon. (B,C) Mutations of the Tcf- and Smad-binding sites lead to weak enhancer activity in the ventral diencephalon. lacZ staining in the dorsal telencephalon is completely abolished (B) or drastically reduced (arrowhead) (C). (D) Staining in embryos with a transgene carrying a mutation of the Tcf-binding site is restricted to the dorsomedial telencephalon. (E) Embryo carrying a transgene with a mutated Smad-binding site shows weak ß-galactosidase activity throughout the cortical neuroepithelium with higher expression levels in the dorsomedial telencephalon (arrowheads).

View larger version (73K): [in a new window]   Fig. 6. Ectopic stimulation of the Emx2 enhancer through ectopic activation of Bmp and Wnt signalling. E11.5 mesencephalic tissue was electroporated with the indicated constructs and processed for immunohistochemistry (A,B) or stained for lacZ expression (C-F). (A) Electroporation of a GFP expression vector leads to robust GFP expression in dissected tissue. (B) Co-transfection of the GFP expression vector with a plasmid encoding a Myc-tagged Bmp receptor (ALK3 or Bmpr1a). Expression of both proteins, GFP and Myc-ALK3, is observed in the same cells (arrows). GFP expression was detected by its intrinsic green fluorescence while Myc-Alk3 protein expression was detected by immunofluorescent labelling with an anti-Myc antibody. (C) Electroporation of the reporter plasmid alone does not result in enhancer activity. Co-electroporation of the reporter gene construct together with either constitutive active ß-catenin (D) or activated ALK3 (E) leads to an induction of enhancer activity in few cells (arrowheads). The inserts shown in D,E represent higher magnifications of the boxed areas. (F) The Emx2 enhancer is heavily stimulated after activation of both Bmp and Wnt signalling.