Figure 2

Characterization of dI2 enhancers. The EdI2/V1 and EdI1/2 enhancer elements were cloned upstream of Cre recombinase or Gal4 and electroporated with (A) a conditional alternating mCherry/GFP plasmid (CAGG-loxP-mCherry-loxP-GFP), (B-E) a conditional nuclear GFP (nGFP) plasmid (CAGG-loxP-STOP-loxP-nGFP) and (F-H) a double conditional GFP plasmid (UAS-loxP-STOP-loxP-GFP). Chick embryos were electroporated at stage 16 and fixed at stage 23 (B-H) or stage 26 (A). (A) Embryos were co-electroporated with a EdI2/V1::Cre plasmid and a conditional alternating mCherry/GFP plasmid. Most of the cells, along the entire dorsal/ventral levels, express mCherry, while a subpopulation of dorsally and medial-laterally located cells expresses GFP. (B-E) Embryos were co-electroporated with a EdI2/V1::Cre plasmid and a conditional nGFP plasmid. Cross-sections of electroporated neural tube were stained with cell fate markers. nGFP-expressing neurons at the dorsal neural tube are dI2 neurons, as indicated by the expression of Lhx1/5⁺/Lhx2/9^- (B), Lhx2/9^-/Isl1^- (C), and Lhx1/5⁺/Pax2^- (D); at the medial neural tube, nGFP-expressing neurons are V1 neurons, as indicated by the co-expression of Lhx1/5⁺/Pax2⁺ (arrowhead in (D)) and En1⁺/Pax2⁺ (E). (F-H) Expression of GFP in neurons that co-express the EdI2/V1 and EdI1/2 enhancers. Embryos were electroporated with three plasmids: EdI2/V1::Gal4, EdI1/2::Cre and the double conditional GFP plasmid. Note that GFP is expressed in the cytoplasm, axons and dendrites. The arrows point to the center of the neurons. GFP-expressing neurons are Lhx1/5⁺/Pax^- (F), Pax^-/En1^- (G) and Lhx2/9^-/Isl1^- (H). (I) A table showing the specificity of the antibodies used in Figures 1 and 2. Boxed areas in the panels are represented as enlargements in their different channels at the bottom of each. The arrows point to the nGFP- and GFP-expressing cells. Scale bars: 50 μm.