Abstract
The survival of complex organisms depends on their ability to interact with their environment through precise control of motor behaviours. This is achieved via specialised synapses called neuromuscular junctions (NMJs) found between motor neurons (MNs), projecting from the hindbrain and spinal cord, and skeletal muscle in the periphery. Current evidence suggests that pathological changes at the NMJ precede many neuromuscular disorders. Studying neuromuscular circuits is therefore critical to improving our understanding of this system and advancing therapies for many currently incurable diseases. One key strategy for discovering new treatments are pharmacological screens, however performing these on NMJs in vivo is challenging, with limited opportunities for experimental manipulation or long-term observation.Recent advances in stem cell biology present an exciting opportunity to produce in vitro models of neuromuscular circuits, with improved accessibility, reproducibility and scalability. This thesis describes and validates a model for neuromuscular circuit development and NMJ formation and maturation. Mouse ESC lines were generated to enable the production of spinal MNs and skeletal muscle. Patch-clamp experiments revealed that murine embryonic stem cell-derived motor neurons (ESC-MNs) mature electrically over a period of 3 weeks, progressing from an immature, non-spiking character to a mature phenotype capable of firing high frequency trains of action potentials. This behaviour was recapitulated via photostimulation using a stably integrated Channelrhodopsin-2 (ChR2) transgene. To investigate the functional properties of ESC-MNs, an ESC line was established expressing a doxycycline-inducible Myod1 transgene.
Following Myod1 induction these cells form multinucleated skeletal myotubes in vitro. Co-cultures of ESC-MNs and myotubes show immature but functional synapses, with contractile activity directed by light stimulation via ChR2. Long-term in vitro culture was assessed using an alternative muscle target from the chick model system. Co-culture of ESC-MNs with chick primary skeletal muscle leads to maturation of NMJs, and spontaneous as well as light-evoked muscle contractions. These co-cultures represent an accessible model for studying NMJ development and function, as well as providing a potential assay to screen genetic or pharmacological therapies for muscular and MN diseases.
| Date of Award | 2016 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Juan Burrone (Supervisor) & Ivo Lieberam (Supervisor) |