Deciphering cellular mechanisms regulating neuromuscular development

运动神经元在胚胎发育过程中以非常精确的方式支配其适当的肌肉靶点。为了完成这个任务，生长锥必须在向正确目标生长的过程中做出许多寻路决策。大多数决定可以被描述为朝着一个期望的指导线索或远离一个排斥的线索发展。所有发育中的运动轴突都有一个共同的方向性决定，那就是将轴突伸出脊髓。最后一个决定是更精确的，包括确定是否与快速或缓慢收缩的肌肉纤维突触。最终，所有的引导线索激活不同的细胞内信号通路，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）是包含运动轴突、肌纤维和末端许旺细胞（tSC）的三部分突触。虽然NMJ已经被广泛研究了半个多世纪，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的困难之一是缺乏一个能准确反映内源性NMJ生理学的体外模型系统，本实验室开发了一种新的模型系统，利用小鼠胚胎干细胞（ES）来源的运动神经元研究运动轴突导向和突触形成。在本建议将建立在我们以前的成功，使用ES细胞衍生的运动神经元和ES/鸡嵌合体，以确定：1）如何缓慢运动神经元选择性地支配缓慢的肌肉纤维在发展过程中，2）开发一个功能性的三分NMJ的体外模型系统，和3）确定什么样的细胞内信号传导途径的运动轴突的定向生长出脊髓，并进入周边。

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.