指導教授：黃銓珍臺灣大學：生化科學研究所韓皓偉Han, Hao-WeiHao-WeiHan2014-11-262018-07-062014-11-262018-07-062013http://ntur.lib.ntu.edu.tw//handle/246246/261670The fish lateral line (LL) is a mechanosensory system closely related to the hearing system of higher vertebrates, and it is composed of several neuromasts located on the surface of the fish. These neuromasts can detect changes in external water flow, to assist fish in maintaining a stationary position in a stream. In the present study, we identified a novel function of Nogo/Nogo receptor signaling in the formation of zebrafish neuromasts. Nogo signaling in zebrafish, like that in mammals, involves three ligands and four receptors, as well as three co-receptors (TROY, p75, and LINGO-1). We first demonstrated that Nogo-C2, NgRH1a, p75, and TROY are able to form a Nogo-C2 complex, and that disintegration of this complex causes defective neuromast formation in zebrafish. Time-lapse recording of the CldnB::lynEGFP transgenic line revealed that functional obstruction of the Nogo-C2 complex causes disordered morphogenesis, and reduces rosette formation in the posterior LL (PLL) primordium during migration. Consistent with these findings, hair-cell progenitors were lost from the PLL primordium in p75, TROY, and Nogo-C2/NgRH1a morphants. Notably, the expression levels of pea3, a downstream marker of Fgf signaling, and dkk1b, a Wnt signaling inhibitor, were both decreased in p75, TROY, and Nogo-C2/NgRH1a morphants; moreover, dkk1b mRNA injection could rescue the defects in neuromast formation resulting from knockdown of p75 or TROY. We thus suggest that a novel Nogo-C2 complex, consisting of Nogo-C2, NgRH1a, p75, and TROY, regulates Fgf signaling and dkk1b expression, thereby ensuring stable organization of the PLL primordium. In addition, we also demonstrated that the neuromast formation was abolished in embryos treated with HDAC inhibitors, OSU-HDAC42 and trichostatin A, and the respective expression of Nogo-C2, NgRH1a, p75 and TROY was down-regulated in these embryos.Content i 中文摘要 iv Abstract v Abbreviations vi Introduction 1 Development of the zebrafish lateral line 1 Signaling pathways regulating zebrafish lateral line development 2 Nogo and Nogo receptor signaling in mammals 3 Nogo and Nogo receptor signaling in zebrafish 5 The roles of histone deacetylase in zebrafish development 6 Zebrafish as an experimental model 7 Specific aims 9 Materials and methods 11 Zebrafish care 11 Total RNA extraction, first-strand cDNA synthesis and RT-PCR 11 Isolation of full-length p75, LINGO-1, and TROY from zebrafish 11 Whole-mount in situ hybridization 12 Injection of morpholinos and capped mRNA 12 Neuromast staining 13 Immunoprecipitation 13 Expression plasmid construction 14 Alkaline phosphatase (AP)-binding assay 14 Capped mRNA synthesis 15 BrdU incorporation assay and whole-mount immunostaining 15 TUNEL assay 15 Time-lapse recording and confocal images 16 Treatment of HDAC inhibitors 16 Results 17 Isolation of three Nogo co-receptors from zebrafish 17 Expression profiles of Nogo co-receptors at different developmental stages, as revealed by whole-mount in situ hybridization 18 Morpholino knockdown of the Nogo co-receptors, p75 and TROY, resulted in significant loss of neuromasts in the zebrafish LL 18 Knockdown of Rtn4-m/Nogo-C2 and Nogo receptor, NgRH1a, caused defects in zebrafish neuromast formation 20 Association of TROY with p75 20 Zebrafish Rtn4-l/Nogo-B binds to NgR, while Rtn4-m/Nogo-C2 binds to the NgRH1a and NgRH1b receptors 21 Cell proliferation in the PLL primordium is not mediated by Nogo/Nogo receptor signaling during zebrafish LL development 22 The Rtn4-m/Nogo-C2 complex regulates primordium organization and rosette formation during PLL development 24 Disruption of the Rtn4-m/Nogo-C2 complex results in reduced expression of downstream markers of Fgf signaling in the PLL primordium 25 Nogo/Nogo receptor signaling regulates neuromast formation by mediating expression of dkk1b in zebrafish 27 HDAC-mediated epigenetic regulation is critical for the formation of Nogo/Nogo receptor complex and neuromast in zebrafish embryos 27 Discussion 29 Conclusion and perspective 35 References 37 Figures 45 Table 71 Appendix 726426555 bytesapplication/pdf論文公開時間：2014/01/27論文使用權限：同意有償授權(權利金給回饋學校)Nogo接受器複合體神經丘側線原基質側線斑馬魚thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/261670/1/ntu-102-D96b46003-1.pdf