黃銓珍Huang, Chang-Jen臺灣大學:生化科學研究所朱珍瑩Chu, Cheng-YingCheng-YingChu2010-05-042018-07-062010-05-042018-07-062008U0001-0701200813391500http://ntur.lib.ntu.edu.tw//handle/246246/178791哺乳類在中樞神經受損後，其神經再生會受到抑制，目前的相關研究指出Nogo-A /Rtn4是神經膠質細胞所分泌來抑制中樞神經再生的重要抑制物之一；相對於哺乳類，低等脊椎動物在中樞神經受損後則會進行神經再生。為了了解Nogo基因在魚類中所扮演的角色，我們從斑馬魚找到三個與RTN同源的異構型，分別是zNogo-B、zNogo-C1及zNogo-C2，這些異構型是由於使用不同的啟動子及另類剪接所造成的；我們並選殖出這三個基因的啟動子，發現其在細胞及斑馬魚體內都具有功能及表現，在經由基因體資料庫比對後，證實斑馬魚並不會表現Nogo-A。 在本研究中，我們建立了新的分析系統來研究神經元細胞及神經膠質細胞的相互關係。離體實驗中，利用大量表現斑馬魚Nogo基因的大鼠神經膠質瘤穩定細胞株，來和表現紅色螢光基因的大鼠嗜鉻細胞瘤穩定細胞株相接觸，發現只有zNogo-C2此基因會抑制由神經生長因子(Nerve growth factor, NGF)所誘發的神經突觸生長，且處理重組的紅血球生成素(Erythropoietin, EPO)會讓zNogo-C2蛋白的抑制作用消失。在斑馬魚的活體實驗中，已經發現由神經細胞專一性啟動子所驅動的綠色螢光蛋白會受到神經生長因子或肝素神經促進生長因子(Heparin-binding neurotrophic factor, HBNF)的誘導而產生神經外生長，同時表現由神經膠質細胞專一性啟動子所驅動的zNogo-C2蛋白則發現有神經突觸退縮的現象；再者，同時表現斑馬魚的紅血球生成素會回復神經外生的功能，證明紅血球生成素在離體及活體實驗中皆可促進神經纖維生長。利用配體與受體的結合分析，我們發現zNogo-C2會經由N端片段及Nogo-66片段與斑馬魚的受體zNgRH1b相結合，經由全覆式原位雜交技術也發現zNogo-C2及zNgRH1b基因皆會表現在斑馬魚的腦部。總而言之，在本研究中發現斑馬魚Nogo-C2擔任類似哺乳類Nogo-A的角色來抑制神經突觸生長。 另一方面，我們選殖出斑馬魚的紅血球生成素三種異構型，分別為zEPO-L1、zEPO-L2及zEPO-S，是經由使用不同的啟動子及另類剪接所造成，此三種異構型只有N端訊號序列的差異，表現的成熟蛋白則完全相同，且具有可被醣基化修飾的位置；利用西方墨點法及免疫細胞化學染色法，我們發現zEPO-L1和zEPO-L2都是分泌型的醣蛋白，但zEPO-S則因為缺乏訊號序列而無法被分泌到細胞外。此外，我們利用全覆式原位雜交技術及反轉錄PCR來研究其表現區域及時間點，發現這三種異構型在受精後12小時便都有表現，在成體組織內的表現三者在腦部、心臟、鰓及眼部均有表現，但在其他部位則有表現量的差異。我們進一步利用反義寡核苷酸(morpholino)來研究紅血球生成素蛋白對斑馬魚血球生成的影響，發現在zEPO-L2被基因抑制的斑馬魚胚胎有嚴重的貧血及死亡發生，且下游和造血作用相關的部分基因表現也受到影響。In mammals, the Nogo family consists of Nogo-A, Nogo-B and Nogo-C, and Nogo-A is the longest variant that interferes with axon regrowth after spinal cord injury. In contrast to mammals, we cloned three zebrafish Nogo-related transcripts, termed nogo-B, nogo-C1, and nogo-C2, which are generated by alternative promoter usage and alternative RNA splicing. In addition to the common C-terminal region, the N-terminal regions of Nogo-C1, Nogo-C2 and Nogo-B contain 9, 25 and 132 distinct amino acid residues, respectively. We also isolated the 5’-upstream region of each gene from a BAC clone and demonstrated that these promoter regions are functional in cultured cells and in zebrafish embryos. We developed both in vivo and in vitro glia-neuron interaction assay systems to explore the role of zebrafish Nogo-related proteins in NGF-induced neurite outgrowth. Our results showed that only Nogo-C was able to induce neurite retraction in zebrafish embryos and in co-cultures of Nogo-transfected glioma C6 cells and PC-12 cells, and such neurite retraction could be rescued by Erythropoietin (EPO) administration. Ligand and receptor binding assays also demonstrated that zebrafish Nogo-C2 binds to the zebrafish Nogo receptor NgRH1b via its unique N-terminal of 25 amino acid residues and the Nogo-66 domain. These results indicate that zebrafish Nogo-C2 has a similar function to mammalian Nogo-A in the induction of neurite retraction both in vitro and in vivo. We also cloned three zebrafish EPO-related transcripts, termed epo-L1, epo-L2 and epo-S, which are generated by alternative promoter usage and alternative RNA splicing. Both zEPO-L1 and zEPO-L2 contain two N-glycosylation sites and they were efficiently secreted by COS-1 cells into the culture medium as a glycoprotein. Through RT-PCR and whole-mount in situ hybridization, the expressions of various zepo transcripts were investigated that all three transcripts are detected in all developmental stages and adult brain, heart, gill and eye. Using morpholino approach, we showed that zepo-L2 morphants displayed severe anemia leading to high mortality during development. Furthermore, in the absence of functional zEPO-L2, the expression of erythroid specific markers, such as adult globin genes and the embryonicAbstract (Chinese)… ibstract (English)… iiibbreviation… vntroduction… 1he discovery of Nogo… 1he nogo gene and Nogo proteins…2he Reticulon (RTN) family… 4he functions of Nogo proteins…4he Nogo-66 receptor (NgR) and its ligands..… 5he co-receptors of NgR and their intracellular messengers…. 8euronal regeneration in fish…10he role of Erythropoietin in the nervous system… 11he Erythropoietin of zebrafish… 13ebrafish as an experimental model… 14ebrafish as a model for reverse genetic study…14pecific aims…16aterials and methods… 19aterials…19ish… 19ell cultures..… 19loning of the P1, P2 and P3 promoter regions of Nogo-related genes.… 20ransactivation assay…. 20otal RNA isolation and first-stranded cDNA synthesis…21loning of the full-length cDNAs encoding Nogo-B, Nogo-C1, Nogo-C2 and Nogo receptors from zebrafish and rat…. 21loning of the full-length cDNAs encoding zEPO and hEPO... 23T-PCR analysis of zebrafish erythropoietin transcripts… 24onstruction of expression plasmids… 25o-culture of PC-12-RFP cells and different C6 stable lines... 27estern blot and immunocytochemistry assay… 27lkaline phosphatase binding assay… 29icroinjection of expression plasmids into zebrafish embryos.…30orpholino injection… 30orpholino rescue experiment by mRNA injection… 31-dianisidine staining… 31hole-mount in situ hybridization… 31esults… 33hree Nogo-related transcripts, Nogo-B, Nogo-C1 and Nogo-C2, were generated by alternative promoter usage and alternative RNA splicing…33xpression profiles of zebrafish Nogo-related genes at different developmental stages and in different adult tissues… 34nalysis of the P1 promoter activity in cultured cells and zebrafish embryos…34nalysis of the P2 and P3 promoter activities in cultured cells and zebrafish embryos…….. 35ellular localization of zebrafish Nogo-related proteins in COS-1 and C6 giloma cells… 36xpression and cellular localization of receptors of zebrafish Nogo-related proteins in COS-1 cells… 37ifferential expression of all four Nogo receptor mRNAs during embryogenesis… 38ebrafish Nogo-C2 caused neurite retraction in the co-culture of C6 glioma and PC-12 cells which could be rescued by EPO…39nly Nogo-C2 is able to elicit retraction of NGF-induced neurite outgrowth in zebrafish embryos…41ebrafish Nogo-C2 binds to Nogo receptor NgRH1b via its unique N-terminal 25 amino acid residues and the common Nogo-66 domain…42loning of the erythropoietin gene from zebrafish… 44ecretion of zebrafish EPO in COS-1 cells… 44etraction of NGF-induced neurite outgrowth by zNogo-C2 can be rescued by zEPO in zebrafish embryos… 45nockdown of zepo resulted in strong suppression of hemoglobin production in zebrafish embryos and reduction of erythroid-specific gene expression…. 46hree EPO-related transcripts, EPO-L1, EPO-L2 and EPO-S, were generated by alternative RNA splicing……47xpression profiles of EPO-realted genes in different developmental stages and adult tissues in zebrafish….. 48ecretion of zEPO-L1, zEPO-L2 and zEPO-S in COS-1 cells…49iscussion…51onclusion and perspective…61igures…. 63eference… 113ppendix…. 127ublications..… 129application/pdf6525855 bytesapplication/pdfen-US脊髓受損神經突觸退縮神經生長因子紅血球生成素反義寡核苷酸醣蛋白斑馬魚Spinal cord injuryNogoNeurite retractionNGFEPOMorpholino oligonucleotideGlycoproteinZebrafishhttp://ntur.lib.ntu.edu.tw/bitstream/246246/178791/1/ntu-97-F90242004-1.pdf