The mechanisms of nerve guidance...

Growing nerves must navigate precisely to their destinations in order to form correct connections. Our research group studies the mechanisms of nerve guidance during development. We focus on cranial motor neurons, which project axons to muscles in the head and neck.

One major current research area in our lab is the development of the ocular motor system – the arrangement of three nerves and six muscles which control eye movements. We are studying the guidance molecules involved in these axon projections using chick and zebrafish model systems. When the ocular motor system develops incorrectly, this can lead to eye movement disorders.

For example, the genetic condition Duane Retraction Syndrome (DRS) affects 1 in 1000 individuals, and results in limited horizontal eye movements. Some DRS patients carry mutations in the signalling molecule α2-chimaerin, an important cytoskeletal regulator (Miyake et al., 2008). We have shown that expression of mutant α2-chimaerin in the oculomotor nerve in the chick embryo gives rise to striking stalling defects and a failure of the nerve to reach its muscle targets (Figure).

We are investigating the role of α2-chimaerin in normal and abnormal development and Duane syndrome, including the identification of guidance ligands and receptors which signal upstream of α2-chimaerin. We are also exploring the mechanisms by which α2-chimaerin controls the cytoskeleton to regulate axon growth and branching. This research will reveal fundamentally important aspects of axon guidance, and is clinically relevant to understanding cranial nerve disorders in humans, as well as specific and general features of motor neuron development and disease.

Important roles of diffusible repellent and attractant molecules

Our previous work has revealed important roles of diffusible repellent and attractant molecules in shaping cranial motor axon pathways. The repellents Netrin-1, Slit and Semaphorin3A ensure that motor axons do not cross the midline (Varela-Echavarria et al., 1997; Hammond et al., 2005; Murray et al., 2010). By contrast, the chemoattractants HGF and SDF-1 attract motor axons, including oculomotor axons, to their muscle targets (Caton et al., 2000; Lerner et al., 2010). Semaphorin 3s and their receptors are expressed within the oculomotor system (Chilton and Guthrie, 2003), raising the possibility that these repellent molecules might function in the development of the oculomotor projection. Determining how such chemorepellents and chemoattractants might collaborate in shaping stereotyped axon projections such as those of the oculomotor nerve is a key goal of our research programme. Discovering the mediators of signalling downstream of these guidance molecules is also important. Such components include RhoA, ROCK and myosin II (Murray et al., 2010) as well as α2-chimaerin.

In vivo and in vitro analysis of the role of axon guidance molecules in the Oculomotor system – Juan Ferrario and Pranetha Baskaran

To determine the role of candidate molecules that may mediate the correct growth of the oculomotor nerve, we are performing in ovo electroporation into the chick midbrain (Miyake et al., 2008). This targets the developing oculomotor nucleus with plasmids which encode different molecules tagged with a fluorescent reporter. The general strategy allows analysis of the effects of gain-of-function forms of the molecule under study or loss-of-function (RNA interference). Embryos are reincubated for several days and the oculomotor nerve (OMN) nerve and extraocular muscles are visualised by whole-mount immunostaining. An alteration of normal axon projections is manifest by (for example) nerve defasciculation, aberrant nerve branching and muscle overshooting. In parallel, we are studying the potential role of guidance molecules using primary cultures of chick oculomotor neurons. These neurons are transfected with gain or loss of functions plasmids (as above), and their effects on neuron behaviour in response to applied guidance molecules such as Ephrins, Semaphorins or SDF-1 are evaluated. In this in vitro system we can then study cell morphology, perform growth cone collapse assays, as well as biochemical and molecular analysis.

Mapping the dynamics of oculomotor nerve projections in the zebrafish and the role of α2-chimaerin – Christopher Clark

We are using 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 to map the normal development of the OMN, or of single oculomotor axons and to describe axon dynamics at key time-points, e.g. branching decisions. These movies have revealed a hierarchical order of appearance of oculomotor axon segments, and that the OMN first projects filopodia over a wide area, before restricting protrusions to particular areas of the environment corresponding with muscle anlage. Single OMN neurons which express α2-chimaerin harbouring human mutations display distinct changes in this pattern of dynamics, growth and projection to muscle targets. 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.

The chick embryo as a toxicity screen for genes in amyotrophic lateral sclerosis – Vineeta Tripathi (collaboration with Prof. Chris Shaw)

Amyotrophic lateral sclerosis is a devastating neurodegenerative disease , with no effective treatment. Mutations in two RNA-processing proteins, TDP43 and FUS have recently been shown to be causative in ALS. Our aim is to use primary motor neurons and the chick in vivo system to model the effects of TDP43 and FUS mutations on motor neuron growth, guidance and survival. For example, we have used the chick embryo to test the toxicity of mutant TDP43 detected in a linkage study of ALS. TDP43WT (wild-type), and the mutants, TDP43Q331K and TDP43M337V, were transfected into the spinal cords of early stage chick embryos (Sreedharan et al., 2008). Embryos expressing TDP43Q331K and TDP43M337V showed a dramatic reduction in maturation compared to embryos expressing TDP43WT. Chick embryo development proceeded normally over 48 hours with TDP43WT, however TUNEL staining of the embryos demonstrated a significant increase in the number of apoptotic nuclei in embryos expressing either of the mutant TDP43 variants when compared to wild-type. These initial results suggest a toxic gain of function or dominant negative effect of mutant TDP43. We therefore have exciting preliminary data supporting the chick embryo as an effective model for ALS and as a toxicity screen for new putative ALS genes.