4 YEAR PhD IN NEUROSCIENCE
Institute of Child Health
Tyrosine phosphorylation in development
We focus on understanding the molecular mechanisms of embryonic nerve growth and guidance. Specifically, we study receptor tyrosine phosphatases (RPTPs) which have key roles in axon growth and pathfinding. The structure of RPTPs gives them unique abilities to control the motility, turning and target recognition of growth cones. We use molecular genetic and cell biological approaches to investigate RPTP function in the retinotectal projection and spinal nerve projections of the chick embryo. Our tools include custom-designed retroviral vectors and the novel “in ovo electroporation” method, allowing us to express recombinant genes in live neural tissues. The laboratory also benefits from being in a multidisciplinary, EC Research Training Network addressing RPTP functions in both developing and damaged nervous systems.
1) RPTP functions in developing sensory nerves.
Trunk sensory neurons express several RPTP genes. These neurons are derived from the neural crest lineage, which in turn is accessible to electroporation with DNA vectors. The student would construct DNA vectors encoding RPTPs and mutants thereof and electroporate these in ovo. The fate of transgenic neurons will be followed using co-expressed marker genes, and the axonal projections will be analysed using immunohistochemistry and lipophilic dye tracing.
2) PTP function in growth cones.
The aim is to understand the influence of RPTPs over the growth cone cytoskeleton, making use of a novel protein transduction system. Large proteins fused with the HIV TAT peptide can cross membranes and be reactivated inside cells. The student will set up this TAT system with cultured neurons. TAT fusions will be made that should perturb PTP signalling directly. Cultured neurons will be treated with these proteins and then growth cone behaviour will be analysed by digital time lapse microscopy.
Ledig, M., Haj, F., McKinnell, I., Stoker, A. W. and Mueller, B. (1999)
The receptor tyrosine phosphatase CRYPalpha promotes intraretinal axon growth.
J. Cell Biol. 147, 375-388.
Haj, F., McKinnell, I. and Stoker, A. (1999)
Retinotectal Ligands for the Receptor Tyrosine Phosphatase CRYPalpha.
Mol Cell Neurosci 14, 225-240.
Stoker,A. and Dutta, R. (1998)
Protein tyrosine phosphatases and neural development.
Bioessays 20, 463-472.