Dynamics of Wnt/β-catenin signalling during cerebellum development

Abstract

Medulloblastomas are tumours of cerebellar origin and are thought to arise from the malignant transformation of progenitor cells in the developing cerebellum. A number of developmental signalling pathways are required for the precise cell specification, proliferation, migration and differentiation involved in forming the mature cerebellum and it is the dysregulation of these processes that can lead to the eventual formation of a tumour. Genes encoding components of the canonical Wnt/β-catenin signaling pathway are mutated in around 15% of medulloblastomas and germline mutations that activate this pathway are known to predispose to medulloblastoma. Despite this, the contribution of Wnt/β-catenin signaling to normal cerebellum development is not yet well understood and the developmental origins of medulloblastoma arising from activation of this pathway are only beginning to be revealed. Therefore, the aims of this thesis were to characterise the spatio-temporal nature of Wnt/β-catenin signalling during cerebellum development and to investigate its function, with the broad goal of informing our understanding of how medulloblastoma arises from oncogenic activation of Wnt/β-catenin signalling. To address the first aim I utilised a LacZ expressing Wnt/β-catenin signalling reporter mouse to characterize the spatio-temporal pattern of Wnt/β-catenin pathway activation during cerebellum development. Analysis of LacZ reporter expression revealed a pattern of transient Wnt/β-catenin activity in discrete cell populations throughout cerebellum development. I found that Wnt/β-catenin activity is present during the early specification of granule cells at the cerebellar rhombic lip but not during the expansion of this cell population at later stages. During perinatal development Wnt/β-catenin activity shifts to the cerebellar ventricular zone, a known germinal centre for GABAergic interneurons and glia, and was observed in cells radiating out from this region. By early postnatal development the expression of the Wnt/β-catenin reporter became progressively restricted to the developing Bergmann glia population. To investigate the function of Wnt/β-catenin in these cell lineages and how its dysregulation could contribute to medulloblastoma, I used a combination of ex vivo organotypic culture, in utero electroporation and tissue-specific gene targeting to manipulate components of the pathway. Culturing slices of E18.5 cerebellum in the presence of small molecule activators of the Wnt/β-catenin pathway revealed a reduction in the expression of glial markers Sox9 and GFAP. In addition, interneuron lineage marker Pax2 was also reduced, supporting the conclusion that dysregulation of Wnt/β-catenin signalling affects the generation of cell lineages from the ventricular zone. To investigate this hypothesis further, I constitutively activated the Wnt/β-catenin signalling pathway in the developing cerebellum using Cre-Lox gene targeting to knock out Apc, a negative regulator of the pathway, in ventricular zone derived lineages. Cre-induced recombination of Apc resulted in nuclear accumulation of β-catenin, a sign that the pathway had become ectopically activated. Furthermore, a reduction in the expression of Sox9 and Pax2 was also observed in these mutant cells. From these data, I conclude a potential role for Wnt/β-catenin signaling in the regulation of glial/interneuron progenitors. Combined, these data support a model where Wnt/β-catenin signalling could perform multiple functions in specification of the granule lineage, regulation of glial/interneuron progenitors and in glial differentiation/maturation. Importantly, dysregulation of progenitor self-renewal and differentiation is widely acknowledged to promote tumourigenesis. Thus, the data in this thesis support a potential mechanism for the development of medulloblastoma from the dysregulation of ventricular zone progenitors.