Fig. 4. Progressive specification of Nkx2.1+/Msx1/2+/Th+ DA neurons. (A,F,K) Schematic of stage 4-6 embryos, showing regions dissected. Prospective hypothalamic (pHyp) regions (green) and anterior (blue) or posterior (purple) lateral neural plate are indicated. (B-E,G-J,L) pHyp explants, cultured for 2-3 days and analysed by immunofluorescent labelling. Co-expression of Shh and Lim1 is detected in HH stage 5 explants (H'). Nkx2.1 expression is detected only in stage 6 explants and is exclusive to Pax6 (L). From HH stage 6, Nkx2.1+/Lim1+, Nkx2.1+/Nkx2.2+, Nkx2.1+/Shh+, Nkx2.1+/Msx1/2+ cells are specified, as in vivo (M-O; see Fig. 1, Fig. 2L). (P-R) Immunofluorescent analysis of stage 6 pHyp explants cultured for 6-7 days. Many Nkx2.1+/Msx1/2+/Th+ neurons differentiate (arrows in P; inset in P). Th+ neurons (arrowheads) do not co-express En1 (Q) or Pax6 (R). (S) Widespread expression of Six 3 is detected within stage 6 pHyp explants after 40 hours culture. Scale bars: 40 µm.