Proceedings of The Physiological Society
King's College London (2011) Proc Physiol Soc 22, C04
Semaphorins and Plexins signal via alpha2-chimaerin in the normal development of the oculomotor projection and Duane's Retraction Syndrome
J. E. Ferrario1, P. Baskaran1, S. Guthrie1
1. MRC Centre for Developmental Neurobiology, King's College London, London, United Kingdom.
Eye movements in vertebrates depend on the function of six extraocular muscles, which are innervated by three cranial nerves. In humans, incorrect development of this pattern of innervation leads to eye movements disorders such as strabismus, which affects 1% of the population, and may result in amblyopia or partial blindness (1). However the etiology of these syndromes, and the guidance cues and molecular mechanisms involved in the precise axon projections of the ocular motor system are largely unknown. We have recently identified mutations in the α2-chimaerin (α2-chn) [an isoform of the chimaerin-1 (CHN1) gene] which have been shown to be responsible for a congenital form of strabismus, Duane's Retraction Syndrome (DRS) (2). Expression of α2-chn forms harbouring identified human mutations in the oculomotor nerves of chick embryos suggest a role in axon pathfinding (2), placing α2-chn downstream of yet unknown guidance cues. Supported by previous evidence, we hypothesize herein that repellent guidance cues such as Semaphorins might orchestrate topographic axon branching of the oculomotor nerve to its particular extraocular muscle targets. We sought to investigate and dissect the molecular pathways that guide the OMN to its target muscles, as well as their implications in DRS. We used the chick embryo as model, and the main techniques involved in this work include: (i) in situ hybridization from embryos at stages 28 to 31 (5 to 6 days old), (ii) in ovo electroporation of the ventral midbrain at stage 11-12 (2 days of development), and then allowing them to grow to stage 30 (6 days of development), and finally (iii) OMN primary cultures from 5-day-old embryos. We demonstrate that Sema3A/C and Plexin receptors expression patterns are consistent with their possible role in oculomotor axon guidance. We further show that in vivo knock-down of Sema3A/C-Plexin signalling or of α2-chimaerin function by means of shRNA electroporations into the oculomotor neurons of embryos lead to characteristic axon guidance phenotypes, reminiscent of DRS. In primary cultures of OMN neurons, Sema3A induces growth cone collapse, which is blocked or potentiated by the expression of either α2-CHN shRNA or gain-of-function constructs, respectively. Finally, in vivo expression of a combination of plexin loss-of-function and α2-chn gain-of-function constructs rescue the Plexin loss of function phenotype to generate an essentially normal axon projection pattern. This work provides both in vitro and in vivo evidence that α2-chn is critical in the pathfinding of oculomotor neurons as a downstream component of the Sema3A/C-Plexin signalling pathway.
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