The Regulation of Cerebellar Granule Neuron Development by Suppressor of Fused

Cerebellar granule cell (GC) development relies on precise regulation of the Sonic Hedgehog (Shh) signaling pathway. Mutations that impair the pathway cause reduced GCP proliferation and cerebellar hypoplasia, underlying multiple motor dysfunction disorders. In contrast, aberrantly activated signalling is associated with medulloblastoma, a debilitating pediatric malignant brain tumour. However, intracellular mechanisms that establish the correct level of pathway activity within GCPs have not been defined. The pathway regulator, Suppressor of Fused (Sufu) controls early cerebellar development and is mutated in human and mouse medulloblastomas. As it interacts with both Gli activator and repressor (Gli-A/R) proteins, its precise function in Shh pathway regulation is highly context-dependent and remains enigmatic. Here, Math1-Cre-mediated deletion of Sufu from GCPs demonstrates that Sufu is required to restrain postnatal GCP proliferation and promote cell cycle exit, together restricting the GCP proliferative window. This function relies on maintenance of Gli3R levels and, surprisingly, suppression of Gli2, together allowing Sufu to dynamically modulate Shh activity. Genetic overexpression of Gli3R and reduction of Gli2 not only rescued the Sufu-deficient phenotype, but revealed intriguing spatial differences in Sufu function. GCPs in the central cerebellar lobules were more deleteriously impacted by Sufu deficiency, and more strongly dependent on suppression of Gli2. RNA-Sequencing further identified novel Sufu gene targets, including Fgf8. The overproliferation of Sufu-deficient GCPs was suppressed by Fgf8 neutralising antibodies, while exogenous Fgf8 was sufficient to stimulate GCP proliferation, demonstrating a previously unknown mitogenic function of Fgf8. Finally, interrogation of non-Cre targeted cells identified non-cell autonomous effects of GC-localised Sufu on BG differentiation. Collectively, these findings implicate Sufu in the spatial and temporal modulation of Shh signalling, and in mediating cross-talk with other signalling partners, to control GCP proliferation and cerebellar development. Moreover, they provide a platform to interrogate the dysregulation of Shh signalling underlying congenital and pathogenic disorders.Ph.D.2019-11-19 00:00:0