Understanding the rules of life

Bioscience for an integrated understanding of health

Category: Standard Studentships

Alpha2-chimaerin signalling in motor neuron development and axon guidance

Project No.2225

Primary Supervisor

Prof Sarah Guthrie – University of Sussex

Co-Supervisor(s)

Dr Frank Schubert – University of Portsmouth

 

Summary

Our research focusses on the development of the ocular motor system, the arrangement of three nerves and six muscles that control eye movements.

We have previously shown that the signalling protein alpha2-chimaerin is a key orchestrator of axon guidance in this system, controlling signalling cascades that regulate the cytoskeleton in growing motor neurons. Mutations in alpha2-chimaerin lead to axon wiring defects of the ocular motor system and cause eye movement disorders in humans (squint).

This project will unravel the signalling pathways from alpha2-chimaerin to the cytoskeleton, focussing on stathmins 1 and 2 – molecules that we recently identified in a proteomics screen for alpha2-chimaerin-interacting molecules. Stathmin1/2 control the supply of tubulin subunits to the microtubule cytoskeleton. We have shown that they colocalise with alpha2-chimaerin in neurons, and demonstrated that knocking down stathmins in zebrafish larva in vivo cause axon pathfinding defects, consistent with their involvement in alpha2-chimaerin signalling pathways. The student will knock out stathmins in the zebrafish using CRISPR/Cas9 and assess visual behaviour, neuroanatomy and cytoskeletal phenotypes. They will use zebrafish mutant lines harbouring alpha2-chimaerin mutations (loss of function and gain of function lines; already generated), to cross with lines mutant for stathmins to analyse genetic interactions. Live imaging will be used to visualise the effects of manipulations on ocular motor axons, including visualisation of microtubule dynamics using labelled tubulins or EB1/3 (microtubule plus tips proteins). The mechanism whereby stathmins control microtubules and axon guidance will be determined using constitutively active (unphosphorylatable) and dominant negative (phosphorylated) isoforms which we have already generated. They will be expressed using the Gal4:UAS system in zebrafish, or by electroporation into the chicken embryo for transient transfection. Neuroanatomy and axon dynamics will be analysed using live imaging (fish), immunostaining of nerves and muscles (fish and chick) and 3D reconstruction of axon pathways.