Nkx1.2 represses Irx3, Pax6, and RA activity to establish FP competence.

<p>(A–D) Irx3 and Pax6 are ectopically induced when Nkx1.2 activity is inhibited. Embryos were electroporated laterally with plasmids encoding a dominant-negative Nkx1.2^DBD-VP16 (A, C) or control GFP (B, D) at HH stage 8+ and cultured for 12 h (for Pax6) or for 18 h (for Irx3). Whole-mount in situ hybridization was carried out for Pax6 (A, B) and Irx3 (C, D). In both cases, the right side (as viewed) of the embryo is electroporated. Scale bar (A, B, C, D) = 100 µm. (E, e1–e12) Irx3 and Pax6 are ectopically expressed in FP if Nkx1.2 function is inhibited. Either control (e1, e3, e5, e7, e9, e11) or Nkx1.2^DBD-VP16 (e2, e4, e6, e8, e10, e12) were electroporated ventrally at HH stage 8+ and embryos cultured for 12 h. Expression of Pax6 (e1, e2, red in e5, e6) and Irx3 (e7, e8, red in e11, e12) were analyzed by immunohistochemistry. Transfected cells were identified by GFP expression (green in e3–e6, e9–e12). The aberrant expression of Pax6 and Irx3 in the FP is indicated by arrowheads (e2, e4, e6, e8, e10, e12). Scale bar (e1, e2, e7, e8) = 50 µm. (F) A dominant-negative Nkx1.2 aberrantly up-regulates intermediate neural genes. Explants electroporated with control (i, ii) or Nkx1.2^DBD-VP16 (iii) were treated with control media (i) or with the medium containing 5 nM of FGF (ii, iii) for 12 h. The expression of the indicated genes was examined by qRT-PCR. (G–I′) Pax6 represses FP differentiation. HH stage 8 embryos were electroporated with the expression plasmid encoding Pax6 and incubated for 48 h. Immunohistochemistry for Arx, FoxA2, and Nkx2.2 (G, H, I, red in G′, H′, I′) indicated Pax6 represses Arx expression in the FP. The affected area is indicated by a bracket. Scale bar (G) = 50 µm. (J) A dominant-negative RAR (RAR-403) induces Nkx1.2, whereas the dominant-negative Pax6 (Pax6^PD-EnR) and Irx3 (Irx3^DBD-VP16) induce FoxA2. Explants electroporated with the indicated plasmids were incubated for 12 h and analyzed by qRT-PCR for Nkx1.2 and FoxA2 expression (*<i>p</i><0.001; Student's <i>t</i> test). (K–S′) Inhibition of RAR and Irx3 or Pax6 activity, using RAR-403, Pax6^PD-EnR, and Irx3^DBD-VP16, prolongs the competence period of the neural explant cells to differentiate into FP. The indicated combinations of the dominant-negative expression plasmids were electroporated into the HH stage 8 embryos, and embryos were incubated for 48 h (K–N′) or explants (O–S′) were prepared and cultured with a delayed treatment of Shh as indicated in the schema (O). In (K–N′), ectopic expression of each gene is indicated by arrowheads. Scale bar (K) = 50 µm for (K–N′).