Deciphering cellular mechanisms regulating neuromuscular development

在胚胎发育过程中，运动神经元以惊人的精度支配它们合适的肌肉靶点。为了完成这项任务，生长锥体必须在生长到正确的目标时做出许多寻路决定。大多数决策可以被描述为朝着理想的指导线索发展，或者远离令人厌恶的指导线索。所有发育中的运动轴突都有一个共同的方向性决定，那就是将一个轴突延伸到脊髓外。最后一个决定更为精细，涉及确定是与快速收缩的肌肉纤维突触还是与缓慢收缩的肌肉纤维突触。最终，所有的引导线索都会激活不同的细胞内信号通路，从而触发生长锥的不对称变化，使其转向或远离线索。 神经肌肉接头(NMJ)是一个由运动轴突、肌纤维和终末雪旺细胞(TSC)组成的三部分突触。尽管半个多世纪以来对NMJ进行了广泛的研究，但TSC的作用在很大程度上被忽视了。这种情况正在改变，因为越来越明显的是，TSCs调节神经传递，它们的异常功能可能是某些运动神经元疾病的基础。研究TSCS的困难之一是缺乏准确反映内源性NMJ生理的体外模型系统。 我的实验室开发了一种新的模型系统，利用来自小鼠胚胎干细胞的运动神经元来研究运动轴突导引和突触形成。在目前的研究方案中，我们将利用ES细胞衍生的运动神经元和ES/KICK嵌合体来确定：1)慢运动神经元在发育过程中如何选择性地支配慢肌纤维；2)建立一个具有功能的NMJ的体外模型系统；以及3)确定什么细胞内信号通路支持运动轴突出脊髓和进入外周的定向生长。

Motoneurons innervate their appropriate muscle targets with remarkable precision during embryogenesis. To accomplish this task, the growth cone must make numerous pathfinding decisions as it grows to its correct target. Most decisions can be described as growing towards a desired guidance cue or away from a repulsive one. A directional decision shared by all developing motor axons is that to extend an axon out of the spinal cord. The last decision is more refined and involves determining whether to synapse with a fast or slow contracting muscle fibre. Ultimately, all guidance cues activate distinct intracellular signaling pathways that trigger asymmetrical changes in the growth cone causing it to turn towards or away from a cue. The neuromuscular junction (NMJ) is a tripartite synapse containing the motor axon, muscle fibre and terminal Schwann cell (tSC). While the NMJ has been extensively studied for over half a century, the role of the tSC has been largely ignored. This is changing as it is becoming increasingly evident that tSCs modulate neurotransmission and their abnormal function may underlie certain motor neuron diseases. One of the difficulties in studying tSCs is the lack of an in vitro model system that accurately reflects the physiology of the endogenous NMJ. My lab has developed a novel model system to study motor axon guidance and synapse formation using motoneurons derived from mouse embryonic stem (ES) cells. In the present proposal will build on our previous success using ES cell-derived motoneurons and ES/chick chimeras to determine: 1) how slow motoneurons selectively innervate slow muscle fibres during development, 2) develop an in vitro model system of a functional tripartite NMJ, and 3) determine what intracellular signaling pathway underlie the directed growth of motor axons out of the spinal cord and into the periphery.