Accessory Kinesin-2 Motors in Cerebellar Development

Abstract

Since its initial discovery in Drosophila nearly forty years ago, the Hedgehog (HH) signaling pathway has been demonstrated to directs certain aspects of development and maintenance of nearly every organ system across invertebrate, vertebrate, and mammalian animal models. One of the HH ligands, Sonic Hedgehog (SHH) promotes proliferation of cerebellar granule neural progenitors (CGNPs), which gives rise to cerebellar granule neurons (CGNs), the most abundant cell type in the central nervous system. A key organelle that regulates HH signaling is the primary cilium, a microtubule-based projection from the cell membrane that serves as signaling centers for multiple pathways, including the HH pathway. Several HH pathway components localize to the primary cilia, and cilia are required for proper GLI processing. Kinesin-2 motor proteins are responsible for anterograde transport of cargo through primary cilia. There are three motor complexes in the kinesin-2 family: heterodimeric motor KIF3A/KIF3B, homodimeric KIF17, and heterodimeric KIF3A/KIF3C. In mice, KIF3A/KIF3B is required for ciliogenesis and therefore proper HH signaling. Kif3a deletion results in an inability to respond to SHH ligand, leading to a reduction in cerebellar granule neural progenitors (CGNP) proliferation. KIF17 and KIF3C do not have clear roles in mammalian embryogenesis or ciliogenesis; these motors are known as accessory kinesin-2 motors. Furthermore, the role(s) of accessory kinesin-2 motors in HH signaling transduction or cerebellar development are unknown. The goal of this dissertation is to investigate the contribution of accessory kinesin-2 motors in HH-dependent cerebellar development. In chapter 2, I investigate the role of homodimeric KIF17 in cerebellar development. Kif17 expression was detected in SHH-producing Purkinje cells and HH-responsive CGNPs. Deletion of Kif17 in Purkinje cells phenocopies germline Kif17 deletion – reduced EGL thickness due to decreased CGNP proliferation and reduced HH target gene expression. Reduced levels of SHH protein are observed within Purkinje cells in Kif17-/¬- cerebella. These data suggest reduced SHH protein levels in Kif17-/- cerebella results in reduced HH signaling levels and decreased CGNP proliferation, resulting in cerebellar hypoplasia. On the contrary, CGNP-specific Kif17 deletion increased HH target gene expression and EGL thickness due to increased CGNP proliferation. Levels of GLI3 repressor are significantly reduced with Kif17 deletion, suggesting KIF17 additionally restricts CGNP proliferation in a cell autonomous fashion. This work identifies dual and opposing roles for KIF17 in HH-dependent cerebellar development– first, as a positive regulator of HH signaling through regulation of SHH protein levels within Purkinje cells, and second, as a negative regulator of HH signaling through regulation of GLI transcription factors in CGNPs. In chapter 3, I explored the contribution of KIF3C to the postnatal cerebellum. Differing from Kif17, Kif3c expression was detected ubiquitously in the cerebellum. Germline Kif3c mutants displayed cerebellar hypoplasia, albeit less severe than Kif17 deletion animals. Notably, even with reduced CGNP proliferation, HH signaling remains intact in Kif3c-/- cerebella. In addition to decreased expression of Notch target, Hes1, we observed abnormal patterning of Bergmann glia in Kif3c mutants. Collectively, these data demonstrate KIF3C’s requirement in the cerebellum and suggest a novel role in regulating Notch signaling during development. Collectively, this dissertation demonstrates essential roles for both KIF17 and KIF3C in cerebellar development. First, I identified dual and opposing roles for KIF17 in HH signaling at the level of SHH ligand and GLI processing, and second, I explored a role for KIF3C in Bergmann glia patterning.