Fig. 3. Induction of Nkx2.1+/Shh+ cells and Nkx2.1+/Msx/2+/Th+ DA neurons can be triggered by prechordal mesoderm in a contact-independent manner. (A-D) Stage 4-5 LNP explants (blue box, Fig. 4F) cultured alone express Lim1/2, Pax6 and Pax7 but not Nkx2.1 or Shh. (E-H) In stage 4-5 LNP explants co-cultured in contact with stage 5+ prechordal mesoderm, Nkx2.1+/Foxa2+/Bmp7+ ventral tuberal hypothalamic cells are detected (white brackets) at the boundary with prechordal mesoderm (broken line). Nkx2.1+/Shh+/Lim1+ lateral tuberal hypothalamic progenitor cells are detected adjacent to these. (I-L) In stage 5 LNP explants co-cultured at a distance from HH stage 5+ prechordal mesoderm, Nkx2.1+/Shh+/Lim1+ lateral tuberal hypothalamic cells are detected, but no Nkx2.1+/Foxa2+/Bmp7+ ventral tuberal cells are found. (M,N) Th+ neurons, but not Nkx2.1+/Th+ neurons, differentiate in a stage 5 pHyp explant (green box, Fig. 4F) cultured alone (M). Nkx2.1+/Msx1/2+/Th+ neurons differentiate in stage 5 pHyp explant co-cultured with prechordal mesoderm (N). (O) Transverse section of stage 10 embryo showing expression of Bmp7 in the ventral tuberal hypothalamus (arrow) and prechordal mesoderm (arrowhead). Scale bar: 40 µm in A-L; 30 µm in M,N.