Morphologies and differentiation dynamics of cerebellar granule cell precursors

• Main Author: Hanzel, Michalina
• Other Authors: Basson, Michiel Albertus ; Wingate, Richard James Tobias
• Published: King's College London (University of London) 2017
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.733399

Cerebellar granule cell precursors (GCPs) are born at the rhombic lip and migrate dorsally across the cerebellar anlagen to form a secondary germinative epithelium, the external germinal layer (EGL). Here, the precursors undergo a period of transit amplification during which they proliferate extensively to produce the most numerous cell type in the brain. The morphological sequence of events that characterizes the differentiation of GCPs in the EGL is well established. However, no research has correlated GCP morphologies with their differentiation status. In this project, I examine the morphological features and transitions of GCPs in the chicken cerebellum. I label a subset of GCPs with a stable genomic expression of green fluorescent transgene and follow their development within the EGL in static images and using time-lapse imaging. I use immunohistochemistry to observe cellular morphologies of proliferating and differentiating GCPs to better understand their differentiation dynamics. Results reveal that mitotic activities of GCPs are more complex and dynamic than currently appreciated. While most GCPs divide in the outer and middle EGL, some are capable of division in the inner EGL. Some GCPs remain mitotically active during neurite extension and tangential migration and retract their processes prior to each cell division. The mitotically active precursors can also express differentiation markers such as TAG1 and NeuroD1. Therefore, I explore the expression of NeuroD1 on a cellular level in GCPs using its conserved non-coding element and conclude that the levels of NeuroD1 expression can differ between neighbouring GCPs. Further, I explore the function of NeuroD1 in cerebellar development by overexpressing the protein at the rhombic lip at different developmental stages. Results suggest that misexpression of NeuroD1 promotes context-dependent differentiation and can alter cellular behaviour. When misexpressed in GCPs, NeuroD1 leads to premature differentiation, defects in migration and reduced cerebellar size and foliation. Overall, this thesis provides the first characterisation of individual morphologies of mitotically active cerebellar GCPs in ovo and explores in detail the expression and role of the differentiation factor NeuroD1 on the development of rhombic lip derivatives.