Proceedings of The Physiological Society
King's College London (2011) Proc Physiol Soc 22, PC37
Mapping the dynamics of oculomotor nerve projections to the extraocular muscles in the zebrafish and the role of ??2-chimaerin
C. Clark1, M. Meyer1, S. Guthrie1
1. King's College London, London, United Kingdom.
In vertebrates, eye movements are controlled by six extraocular muscles, innervated by three cranial nerves - the oculomotor, the trochlear and the abducens. Studies in the chick embryo have shown that the oculomotor nerve (OMN) undergoes a stereotyped pattern of outgrowth and branching to its four muscle targets(1). Perturbations of this wiring pattern in humans give rise to congenital eye movement disorders such as Duane’s Retraction Syndrome (DRS), which can arise due to mutations in the RacGAP signalling molecule α2-chimaerin(2). However, the dynamics of axon behaviour which govern topographic axon projections in the ocular motor system have not been characterised, nor has the role of α2-chimaerin been elucidated. We have therefore used the zebrafish model system to study the developmental dynamics of axon guidance to the extraocular muscles, and the role of α2-chimaerin in this process. We have used two-photon time-lapse imaging of the Isl1-GFP transgenic zebrafish line, between 24 and 96 hours post fertilisation, to map the normal development of the OMN and to describe its dynamics at key time-points, e.g. branching decisions. Here we show that the OMN first projects filopodia over a wide area, before restricting protrusions to particular areas of the environment corresponding with muscle anlage. Together with immunostaining of embryos at fixed time points, these movies have also revealed a hierarchical order of appearance of oculomotor axon segments. Mosaic expression techniques to image single GFP-expressing neurons have revealed that axons project from individual OMN subnuclei to muscle targets. This suggests that neuromuscular connectivity is generated by direct axon projections from subnuclei to muscles, rather than axon branching to multiple muscles and subsequent pruning. We have also found that single OMN neurons which express α2-chimaerin harbouring human mutations display extensive filopodial extension and exploratory behaviour as for wild-type axons. However, axons expressing mutant α2-chimaerin do not restrict their protrusions to select a particular muscle target, resulting in a cell-autonomous stalling phenotype. We are currently creating stable transgenic zebrafish lines expressing mutant forms of α2-chimaerin. This will allow us to use time-lapse imaging to model the human DRS phenotype and to investigate the effects of α2-chimaerin mutations on the entirety of cranial nerve projections to the extraocular muscles.
Where applicable, experiments conform with Society ethical requirements