Study of zebrafish Nogo-related proteins and their receptors: expression pattern analysis, binding properties and EPO-mediated neuritogenesis
Date Issued
2008
Date
2008
Author(s)
Chu, Cheng-Ying
Abstract
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 embryonic
Subjects
Spinal cord injury
Nogo
Neurite retraction
NGF
EPO
Morpholino oligonucleotide
Glycoprotein
Zebrafish
File(s)![Thumbnail Image]()
Loading...
Name
ntu-97-F90242004-1.pdf
Size
23.32 KB
Format
Adobe PDF
Checksum
(MD5):9301fcc567f9239d8a06faf4d4a5739a