Temporal and spatial controls of cell fate specification in the cerebellum

The domain of the developing cerebellum in dorsal rhombomere 1 is specified by signals from two adjacent organising centres, the midbrain-hindbrain boundary (isthmus) and the roof plate of the fourth ventricle. Two major precursor pools give rise to all the cells of the cerebellum: the ventricular zone and the cerebellar rhombic lip. The rhombic lip comprises a dorsal progenitor population, defined by the expression of the transcription factor Atoh1, which gives rise to different cell types contributing to both extra-cerebellar and cerebellar populations. In chick embryos, I have investigated the timing, morphology and gene expression of successive cohorts of rhombic lip derivatives using a combination of in ovo electroporation and in situ hybridisation. Using this approach, I first derived an accurate spatiotemporal map of cell production within rhombomere 1, which helps illuminate the evolutionary mechanisms generating diversity in the structure and function of the cerebellum. I then explored how signals from the roof plate and isthmus specify distinct neuronal derivatives of the cerebellar rhombic lip at different time points. In these experiments, I show that the isthmus abuts an expanded region of Atoh1 expression from which early, extra-cerebellar, Lhx9-expressing rhombic lip derivatives are born. Through surgical manipulation of the isthmus in cultured explants and through induction of an ectopic isthmic organiser through in ovo electroporation of early genetic regional determinants (Otx2), I demonstrate that this expanded region of Atoh1 is dependent upon the isthmus for its induction and maintenance. I also demonstrate the role of the isthmic gene, Fgf8, in the maintenance of the expanded domain through in ovo electroporation of plasmids encoding Fgf8 and a dominant-negative FGF receptor. This FGF-dependent domain of Atoh1 is regulated independently from the specification of rhombic lip derivatives, suggesting a novel role for both Atoh1 and FGF signalling in rhombomere1. Analysis of triple knockout FGF receptor mutants and FGF hypomorph mice confirmed the results of dominant negative overexpression in chick. To investigate temporal factors influencing the specification of temporal cell fate, I examined the role of thyroid hormone, which is synthesised in the fourth ventricle choroid plexus adjacent to the rhombic lip. I show that pharmacological manipulations of thyroid signalling can specifically disrupt the expression of markers of late-born rhombic lip derivatives, pointing to an important role for steroid hormone receptor signalling in specifying the fate of specific temporal cohorts in the rhombic lip lineage.

Name dc:type.qualificationname

Doctor of Philosophy

Level dc:type.qualificationlevel

Doctoral Thesis

Grantor dc:publisher.institution

King's College London

Year dc:date.issued

2012

Author dc:creator

• Green, Mary

Advisors dc:contributor.advisor

• Wingate, Richard James Tobias
• Chambers, David

Language dc:language

eng

Identifier

oai:kclpure.kcl.ac.uk:studenttheses/511aa247-85db-4d31-90a7-2f8f1ba95b93

OAI identifier oai:identifier

oai:kclpure.kcl.ac.uk:studenttheses/511aa247-85db-4d31-90a7-2f8f1ba95b93