Coexpression of Gas1 and Ptch1Δloop2 blocks the Gas1-mediated promotion of Shh-dependent cell fates. HH stage 21–22 chick neural tubes electroporated with Gas1–pCIG and pCIR (A–D,I–L,Q–T) or Gas1–pCIG and Ptch1Δloop2-pCIR (E–H,M–P,U–X). Forelimb-level sections were examined for Nkx6.1 (blue; C,G), Pax7 (blue; K,O), and Pax6 (S,W) expression. Gas1-expressing cells are visualized with GFP (green), while pCIR and Ptch1Δloop2-pCIR-expressing cells are visualized with anti-DsRed antibodies (red). Arrows in A–D indicate Gas1/pCIR-expressing cells that ectopically express Nkx6.1. Arrows in E–H indicate similarly positioned cells that coexpress Gas1/Ptch1Δloop2 that do not express Nkx6.1. Arrows in I–L and Q–T denote Gas1/pCIR-expressing cells that down-regulate Pax7 and Pax6 expression, respectively; arrowheads indicate non-cell-autonomous expansion of Pax7 and Pax6. (M–P,U–X) Cells that coexpress Gas/Ptch1Δloop2 do not inhibit Pax7 and Pax6 expression (arrows). Cell-autonomous expansion of Pax7 and Pax6 is marked by arrowheads in M–P and U–X. The results are representative of six Gas1/pCIR-electroporated embryos and eight Gas1/Ptch1Δloop2 embryos. Bar: A, 50 μm.