The role of mitogenic sonic hedgehog and insulin-like growth factor signaling in the developing cerebellum

Post-natal cerebellum development represents an elegantly coordinated system that combines precursor proliferation with neuronal migration to establish distinct neuronal layers. Cerebellar granule neuron precursor (CGNP) expansion requires strict control of mitogenic, survival and growth signals as generation of these cells occurs within a relatively short time period and uncontrolled growth results in medulloblastoma, the most common solid pediatric malignancy. During post-natal development CGNPs require sonic hedgehog (Shh) and insulin-like growth factor (IGF) signaling for proliferation and survival. The work presented here investigates how these two signaling pathways communicate to promote CGNP proliferation.;Through receptor binding insulin receptor substrate (IRS) proteins propagate signals induced by IGF and in CGNPs mediate proliferation through a mechanism that does not involve increased canonical IGF signaling. Specifically Shh signaling increases IRS1 protein levels without increasing Irs1 transcripts; rather Shh signaling stabilizes IRS1 protein by interfering with mTOR-dependent IRS1 turnover. CGNPs require IRS1 as knockdown experiments in primary cell or slice culture experiments show decreased proliferation rates. Furthermore IRS1 overexpression drives CGNP proliferation independent of Shh signaling and is elevated in mouse Shh-induced medulloblastoma. These findings indicate a novel interaction between the Shh and IGF pathway as well as identify a non-classical mechanism by which Shh signaling regulates protein levels.;IGF signaling activates downstream components involved in regulating mRNA translation, specifically mTOR. Surprisingly, proliferating CGNPs activate mTOR substrates preferentially as a means to inhibit growth while promoting translation of proliferation-associated mRNAs. Shh signaling induces eIF4E mRNA and protein levels independent of mTOR signaling. eIF4E is required for continued cell cycle progression while eIF4E overexpression maintains CGNP proliferation independent of Shh signaling. In vivo analysis of S6 kinase suggests that this mTOR effector plays a role in transitioning CGNPs from proliferation-competent to post-mitotic states. Indeed S6 kinase overexpression drove CGNPs to exit the cell cycle. To mechanistically determine how proliferating CGNPs inhibit growth signals from S6 kinase, PP2A activity was investigated. Shh signaling upregulated the B56gamma regulatory subunit and the PP2A-B56gamma complex specifically targeted S6 kinase for inactivation. These findings identify unique developmental functions for eIF4E and S6 kinase wherein their activity is uncoupled by Shh signaling.