Fig. 6. The Wnt/ β-catenin signaling pathway negatively regulates specification and differentiation of cells from the SpM and CPM in chick embryos. (A) Schematic representation of the experimental procedure showing the electroporation of the construct into a St. 3c embryo, which was analyzed 16 hours later, at St. 8. (B-D) Fluorescent ISH of the St. 8 electroporated embryos for the markers indicated. As a result of Wnt3a activity, the expression levels of Tbx5 (B), Nkx2.5(C) and Gata4 (D) transcripts were reduced on the electroporated side (arrows; n=14). (E) Representation of the experimental procedure showing the electroporation of the pCIG-Wnt3a-IRES-GFP construct into a St. 8 embryo, and its analysis at St. 12 (note a reverse cardiac looping). (F,G) St. 12 embryos stained for Isl1 (F) and Nkx2.5 (G) following electroporation with pCAGG-GFP (arrows). The normal expression of both proteins was not affected (arrowheads). (F',G') St. 12 embryos, electroporated with pCIG-Wnt3a-IRES-GFP marked by the GFP expression in the surface ectoderm. Both Isl1 (F') and Nkx2.5 (G') levels in the SpM of the AHF (arrowheads) were reduced on the electroporated side (n=17). (H-I') Whole-mount ISH for Tbx1 and capsulin in St. 13 embryos electroporated with either pCAGG-GFP or pCIG-Wnt3a-IRES-GFP. Both Tbx1 (H') and capsulin (I') levels in the BA were reduced (arrows) on the Wnt3a-electroporated side (n=9). nt, neural tube; hrt, heart; ph, pharynx.