Fig. 5. NG2 can mimic Shh and Smo^* induction of targets in presomitic mesoderm. (A) PSM explants were cultured alone (–Shh), with Shh-N (+Shh) or infected with adenoviruses carrying EGFP, Smo or Smo^* for 24 hours and analyzed for induction of Shh target genes by RT-PCR. Shh-N induces target genes, while infection with EGFP virus does not. Expression of Smo^* induces all Shh targets tested (n=8). (B) PSM explants were infected with adenoviruses carrying either EGFP, full-length Gli1, Gli2, Gli3 or NG2 for 24 hours and analyzed for induction of Shh targets by RT-PCR. Gli1 induced Ptch, Hhip and moderate levels of Ccnd2. Gli2 induced Ccnd2, and moderate levels of Ptch. Gli3 did not induce any Shh targets, while NG2 activated all targets tested (n=8). (C) PSM was cultured for 24 hours in the absence (–) or presence (+) of Shh-N and adenovirus as indicated. Expression of Ptch and Pax1, was assessed by RT-PCR. Infection with EGFP and Gli1 does not affect induction of target genes by Shh, while infection with Gli2 does seem to have a moderate inhibitory affect specifically on Pax1. Infection with Gli3 acts to repress Shh induction of both Ptch and Pax1 (n=3). (D) PSM infected with Gli2 and NG2 induce Gli1 at 24 hours (n=8). (E) PSM explants cultured alone, with Shh-N, with 1 µg/ml cycloheximide (CHX) or the combination for 9 hours were analyzed for Gli1, Ccnd2 and Hhip by RT-PCR. In the presence of CHX, Shh activates only Ccnd2 and Gli1, but not Hhip (n=2). CHX (1 µg/ml) effectively inhibits over 90% of new protein synthesis under similar culture conditions (Fu et al., 2002).