Fig. 9. Hedgehog-dependent development of OLPs in neocortical cultures. (A) Dissociated cells from E15.5 wild-type rat neocortex were cultured in defined medium for up to 8DIV in the presence or absence of cyclopamine (cyc, 1 µM). The cultures were immunolabelled with polyclonal anti-NG2 to visualize OLPs and the number of NG2⁺ cells per 18 randomly chosen fields (x63 microscope objective) was determined after different times in culture. OLPs did not develop in E15.5 rat cortical cultures until 4-6DIV (see Fig. 8), but after that the number of OLPs increased, peaking after 7DIV. (B) Production of OLPs was inhibited strongly by cyclopamine (A) in a dose-dependent manner. (C) Transcripts encoding SHH and IHH could be detected by RT-PCR as soon as 1 hour after cell dissociation (lane 0), and throughout the culture period up to 8DIV. mRNA coding for DHH was also detected weakly after dissociation and after 8DIV but not at intermediate times. Sizes of PCR products were as predicted (see Fig. 7).