Mechanism of motoneuron network formation

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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.