Fig. 7. Hedgehog-dependent development of OLPs in ventral forebrain cultures of wild-type and Nkx2.1^-/- mice. Dissociated ventral forebrain cells from E13.5 wild-type and Nkx2.1^-/- mice were cultured in defined medium for up to 8DIV in the presence or absence of the HH inhibitor cyclopamine (cyc, 1 µM). The cultures were immunolabelled with polyclonal anti-NG2 to visualize OLPs and the average number of NG2⁺ cells per 20 fields (x63 microscope objective) was determined for different times in culture. (A) Wild-type cultures contained NG2⁺ process-bearing cells from the outset, whereas Nkx2.1 null cultures did not develop OLPs until 4-6DIV. The development of OLPs in Nkx2.1^-/- cultures was strongly inhibited by cyclopamine. Asterisks denote that no NG2⁺ progenitors were detected. (B) Transcripts for SHH and IHH but not DHH could be detected by RT-PCR throughout the culture period. Molecular size markers (M) are 200 bp and 400 bp. The sizes of PCR products were as predicted: Shh, 242 bp; Ihh, 267 bp; Dhh, 311 bp; Gapdh, 382 bp.