Functional identification of zebrafish Nogo-related genes in vitro and in vivo

Nogo-A為哺乳類抑制受損中樞神經再生的重要抑制物之一，相關研究持續增加中，不過有關斑馬魚Nogo基因的研究則未見報導。在斑馬魚中我們發現三個與RTN同源的基因，分別為zNogo-B、zNogo-C1和zNogo-C2，而如同在哺乳類動物中，這些Nogo基因的異構型來自於另類剪接和不同啟動子的利用。目前魚類是唯一未發現Nogo-A相似基因的脊椎動物，而我們在斑馬魚中也未發現Nogo-A。 本研究中發現，zNogo-B會分佈在COS-1細胞內的內質網，但若利用神經細胞表現，則會以顆粒狀的型態分佈在細胞質和其所延展出的神經突的區域；相對地zNogo-C1和zNogo-C2則在細胞株都會表現在細胞的高爾基氏體的位置；這樣的情形也跟我們在斑馬魚胚胎的神經或神經膠質細胞中表現Nogo基因的分佈相同。而當我們抑制zNogo-B在斑馬魚胚胎內的表現時，可以觀察到斑馬魚胚胎的神經突增加以及神經元細胞的增多；因此，可推論斑馬魚的Nogo-B基因跟神經胚胎發育有關。 已知Nogo-A的訊息傳遞是經由Nogo受體往下游傳遞，而zNogo-B又跟它具有相似的弁遄A且在2003年已發現斑馬魚有四種可能的Nogo受體。當在斑馬魚胚胎中抑制兩種位於Nogo作用體下游的Nogo受體時，發現可以得到相似於抑制Nogo-B基因在斑馬魚胚胎時期的表現型，推測其可能為zNogo-B的受體。 在本研究中，我們瞭解到了斑馬魚的Nogo蛋白在細胞內，以及在斑馬魚的神經或神經膠質細胞中的分佈狀況，經由抑制這些蛋白在斑馬魚胚胎內的表現可以發現zNogo-B跟神經發育的調控有關。當進一步抑制斑馬魚Nogo受體在胚胎時期的表現時，有兩者可以得到跟Nogo-B morphant相似的情形，所以這兩種Nogo受體也跟調控神經細胞的發育生長有關，或可能為zNogo-B的受體。In mammal, Nogo is a myelin-associated inhibitor of axon regrowth, which is involved in the regeneration of injured axons in central nervous system (CNS). Despite papers regarding the function and regulation of mammalian Nogo proteins increased rapidly, no report about Nogo gene in zebrafish has been proposed. Here we reported three homologous genes to RTN family in zebrafish, termed z-Nogo-B, -C1, and –C2. As in mammal, these transcripts come from the alternative splicing and promoter usage. So far, fish is the only vertebrate lacking of Nogo-A expression. Our results indicated that cellular localization of zNogo-B was restricted in ER, while zNogo-C1 and zNogo-C2 in Golgi complex. When expressed zNogo-B in neuroblastoma cell line, the cellular localization of zNogo-B will switch to the ctyoplasm including the extending neurite. Knockdown of zNogo-B with morpholino antisense oligonucleotides caused neurite outgrowth, neuronal cell survive and somatic abnormalities. These results provide the functional evidence of z-Nogo-B and zNogo-C2 in zebrafish neuronal development. The inhibitory pathway of Nogo-A is mediated by the binding with Nogo receptor. There were four likely Nogo receptors in zebrafish. To knockdown two of the four likely Nogo receptors in zebrafish, we could obtain the similar morphant as zNogo-B-Mo. They might be the receptor of the zNogo-B. Our data revealed that the distribution of zNogo proteins in cell line and zebrafish embryos’ neuronal or glial system. By knocking down the zNogo-B expression could promote neurite outgrowth and neuronal cell survive in zebrafish embryos. Furthermore, while we knock down 2 of 4 Nogo receptors, we could obtain the similar phenotypes to Nogo-B morphant. Suggesting both 2 receptors might influence the neuron development in zebrafish embryos or act as receptors to zNogo-B.目錄……………………………………………I 圖表目次………………………………………II 縮寫表…………………………………………III 中文摘要………………………………………IV 英文摘要………………………………………V 序言……………………………………………1 材料與方法……………………………………11 結果……………………………………………20 討論……………………………………………24 參考文獻………………………………………2