Transcriptional profiling of the Sonic hedgehog response: a critical role for N-myc in proliferation of neuronal precursors

Abstract

Cerebellar granule cells are the most abundant neurons in the brain, and granule cell precursors (GCPs) are a common target of transformation in the pediatric brain tumor medulloblastoma. Proliferation of GCPs is regulated by the secreted signaling molecule Sonic hedgehog (Shh), but the mechanisms by which Shh controls proliferation of GCPs remain inadequately understood. We used DNA microarrays to identify targets of Shh in these cells and found that Shh activates a program of transcription that promotes cell cycle entry and DNA replication. Among the genes most robustly induced by Shh are cyclin D1 and N-myc. N-myc transcription is induced in the presence of the protein synthesis inhibitor cycloheximide, so it appears to be a direct target of Shh. Retroviral transduction of N-myc into GCPs induces expression of cyclin D1, E2F1, and E2F2, and promotes proliferation. Moreover, dominant-negative N-myc substantially reduces Shh-induced proliferation, indicating that N-myc is required for the Shh response. Finally, cyclin D1 and N-myc are overexpressed in murine medulloblastoma. These findings suggest that cyclin D1 and N-myc are important mediators of Shh-induced proliferation and tumorigenesis.

Fig. 1.

Induction of D-cyclins and N-myc by Shh. GCPs were cultured in serum-free media containing no stimulus (-) or 3 μg/ml Shh-N (+) for the indicated times. Total RNA was separated by electrophoresis and transferred onto a nylon membrane. The membrane was hybridized with ³²P-labeled cDNA probes for cyclin D1, cyclin D2, N-myc, or c-myc, and then exposed to a PhosphorImager. Images were processed by using photoshop (Adobe Systems, Mountain View, CA).

Fig. 2.

N-myc induction does not require protein synthesis. GCPs were cultured for 6 h with no stimulus (-) or 3 μg/ml Shh-N (+), in the presence or absence of 10 μg/ml cycloheximide (CHX). RNA was separated by electrophoresis, transferred onto membranes, and hybridized with probes for cyclin D1 or N-myc. Membranes were exposed to a PhosphorImager, and images were processed by using photoshop.

Fig. 3.

Retroviral expression of N-myc promotes cell cycle entry. GCPs were infected with retroviruses carrying no cDNA (Control), N-myc, or dominant-negative N-myc (ΔMBII) and cultured in the presence or absence of Shh-N for 48 h. Cells were labeled with BrdUrd for 16 h and then stained with anti-BrdUrd antibodies. Representative images for control and N-myc-infected cells are shown in A and B. For each condition, the percentage of virus-infected cells (green staining) that had incorporated BrdUrd (red/yellow staining) was determined. Data in C represent mean ± SEM for four fields. Similar results were obtained in three independent experiments.

Fig. 4.

N-myc promotes expression of cell cycle genes. GCPs were infected with retroviruses carrying no cDNA (control) or wild-type N-myc (A), or cultured in medium containing no stimulus (control) or 3 μg/ml Shh-N (B). After 24 h, cells were harvested (and for virus-infected cultures, cells were fluorescence-activated cell sorted to isolate GFP-positive cells), and total RNA was isolated and reverse transcribed. Equal amounts of cDNA were analyzed by real-time PCR with primers for the indicated genes. Data represent mean ± SEM for experiments performed in triplicate.

Fig. 5.

cyclin D1 and N-myc are expressed in medulloblastomas from patched1 mutant mice. Total RNA from GCPs, tumor cells from patched1 mutant mice, and normal adult cerebellum were separated by electrophoresis, transferred onto membranes, hybridized with probes specific for cyclin D1 or N-myc, and then exposed to a PhosphorImager screen. Images were processed by using photoshop.