Fig. 1. Nmyc mRNA transcripts (N-myc in figure) are upregulated in Shh-treated CGNPs. (A) Total RNA was prepared from CGNPs (85% pure cultures) treated with Shh (3 µg/ml) for 3 hours±the protein synthesis inhibitor cycloheximide (10 µg/ml), as indicated above the lanes. Northern blot analysis was carried out using cDNA probes for candidate direct targets of Shh signaling. Representative autoradiographs for Gli1, Myc (c-myc in figure), Lmyc (L-myc in figure) and Nmyc are shown. Gapdh signal (bottom) shows equivalent loading. (B) Shh-mediated induction of Nmyc occurs in cultures enriched for CGNPs (>90% pure cultures). Cerebellar homogenates were enriched for CGNPs as described (Materials and Methods), then assayed as above for protein synthesis-independent up-regulation of Nmyc mRNA by Shh. A representative northern blot is shown (top). Gapdh signal (bottom) shows equivalent loading. (C) Nmyc expression is sustained for the duration of Shh exposure (top). The hedgehog signaling pathway is activated, as indicated by Gli1 mRNA expression (middle). Shh signaling maintains a population of immature CGNPs, as indicated by Math1 (Atoh1 — Mouse Genome Informatics) expression (bottom). Representative northern analysis autoradiographs are shown. Ethidium bromide staining indicates equivalent lane loading.