Investigation of the developmental mechanisms underlying cerebellum diversity

While the ground plan for the development of the central nervous system is similar throughout all vertebrates, the final shape and size of its adult components can be highly variable. Perhaps the most variable of all central nervous system structures is the cerebellum, a centre of unconscious movement control in mammals, which shows an enormous range of scale and form. A paradox that intrigued early neuroanatomists is that the internal organisation of the cerebellum, in terms of the different types of neurons and their connections, is more-or-less constant. At its core, the synapse between the granule cell (which channels the majority of inputs to the cerebellum) and the Purkinje cell (its output) is invariable. This puzzle has remained unresolved since the study of the comparative neuroanatomy of the cerebellum entered a decline in the 1970's. However, new molecular approaches to the analysis of gene function during development in a variety of model organisms have led to a radical revision and dramatic simplification of the general model for the development of this region. We wish to test the hypothesis that variability between vertebrate cerebellums is caused by subtle changes in the organisation of cell division within one highly defined strip of cells in the developing brain, the rhombic lip. The cells that the rhombic lip produces are responsible for channeling both inputs to the cerebellum and the output of its Purkinje cells. However, most significantly, the rhombic lip produces one of the most unusual populations of cells in the brain, a migratory, dividing cell which will generate a single product, the cerebellar granule neuron. This highly motile precursor population, which collectively organises itself into a temporary layer of rapidly proliferating cells on the surface of the cerebellum, is the brain's foremost example of 'transit amplification', the process by which the products of stem cells become multiplied into vast numbers. Understanding the origins of transit amplification will help us to understand how stem cells contribute to normal development. Granule cell precursors are also the prime suspects in causing medulloblastoma, the major childhood brain cancer and the molecules that contribute to their uniqueness may help to understand this disease. In the broadest sense, this research will also help to illuminate the function of the cerebellum whose beguilingly simple cellular structure masks its participation in a vast range of neural processes from simple eye movement control to learning and complex cognition. Various conditions such as dyslexia and autism, which are interpreted as defects of relatively complex neural behaviour, have neuroanatomical deficits associated with the cerebellum - an organ characterised as principally mediating subconscious motor reflexes. In this proposal, the survey of the genes and processes at the rhombic lip in the spotted dogfish, the Mississippi paddlefish, zebrafish, frog, and chick will shed vital insights into how cerebellar function and development.

The size and connections of the adult cerebellum vary widely amongst vertebrates and yet in all species, the central functional synapse between granule cell and Purkinje cell remains constant. Understanding the paradox of how such a stereotyped simple circuit could be presented in so many diverse forms constituted a substantial challenge to comparative neuroanatomists of the last century, who consequently interpreted cerebellar evolution in terms of the adaptive topography of its inputs. However, recent experimental insights allow us to speculate that variability in cerebellar form resides in changing patterns of neurogenesis in a thin strip of neuroepithelium bordering the roofplate of the fourth ventricle; the rhombic lip. The upper rhombic lip gives rise to a sequence of populations that culminates in the generation of the unique migratory precursor cell that forms the external granule cell layer (EGL) of the cerebellum. The EGL gives rise to over half of the neuronal complement of the brain, but our preliminary studies suggest that this transient transit amplification population is absent in lower vertebrates. In this proposed research, we will look for evidence of the emergence of the EGL in a range of vertebrates that display widely varying cerebellar structure (dogfish, paddlefish, zebrafish, Xenopus) using immunohistochemisty and in situ hybridisation to look for hallmarks of secondary proliferation. We will also use the photoconvertible protein, Kaede, to fate-map dividing cells and we will confirm the identity of neuronal populations by axon tracing. We will use similar techniques to investigate the patterning of deep cerebellar and pontine nuclei, rhombic lip derivatives which are prominent features in mammals but largely absent in other vertebrates. In particular, we will investigate the functional role of Pax6 in generating pontine neurons using in ovo electroporation in chick to manipulate their genesis in the lower rhombic lip.