Mechanism of motoneuron network formation

我们进行了cDNA筛选的信号序列陷阱方法，以确定运动神经元表达的信号分子。用免疫淘洗法纯化E5鸡胚脊髓运动神经元。我们获得了三个功能未知的cDNA，暂命名为10 B4，273和12 D3，并制备了针对这三个蛋白的单克隆抗体。10 B4和273是跨膜蛋白，并且在其胞外结构域中含有富含亮氨酸的重复序列（LRR）。由于10 B4-AP（碱性磷酸酶）和273-AP的嵌合蛋白不与任何胚胎组织结合，我们推测它们可能是可溶性配体的受体。事实上，发现10 B4是人LINGO-1的同源物，LINGO-1已被报道为Nogo/p75受体复合物的共受体。尽管273在发育的早期在运动神经元细胞体中瞬时表达，但其在远端轴突中持续表达。因此，我们推测273可能对来自肌肉或雪旺细胞的某些因子起受体作用。可溶性蛋白12 D3由顶板细胞产生，迁移并大量沉积在脊髓背外侧基底膜上，为了揭示这些蛋白在卵内的功能，我们采用电穿孔和siRNA方法进行了功能获得和功能丧失分析。然而，我们还没有发现它们的明确功能，并计划使用鸡和斑马鱼系统继续进一步分析这些蛋白质。

We performed cDNA screening by a signal sequence trap method in order to identify signaling molecules expressed by motoneurons. Motoneurons were purified by an immuno-panning method using SC1 monoclonal antibody from E5 chick embryonic spinal cords. We got three function unknown cDNAs, tentatively named 10B4, 273 and 12D3, and produced monoclonal antibodies against all three proteins. 10B4 and 273 are transmembrane proteins and contain leucine rich repeat (LRR) in their extracellular domain. Since the chimeric proteins of 10B4-AP (alkaline phosphatase) and 273-AP did not bind to any embryonic tissues, we expected that they might be receptors of soluble ligands. In fact 10B4 is found to be a homologue of human LINGO-1, which has been reported as a co-receptor of Nogo/p75 receptor complex. Although 273 is transiently expressed in the motoneuron cell body at the early phase of development, it is continuously expressed in the distal axons. Therefore we expect that 273 might play roles of receptor against some factors derived from muscle or Schwann cells. Soluble protein, 12D3, is produced by roof plate cells, migrated and heavily deposited at the dorsolateral basement membrane of the spinal cord.To reveal the functions of these proteins in ovo we performed gain-of-function and loss-of-function analysis by methods of electroporation and siRNA. However, we have not yet found their clear functions, and plan to continue further analysis of these proteins using both chick and zebrafish systems.