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Function of RTN4/Nogo during development and as transgene in adult zebrafish

Function of RTN4/Nogo during development and as transgene in adult zebrafish

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PINZON-OLEJUA, Manuel Alejandro, 2014. Function of RTN4/Nogo during development and as transgene in adult zebrafish [Dissertation]. Konstanz: University of Konstanz

@phdthesis{PinzonOlejua2014Funct-29211, title={Function of RTN4/Nogo during development and as transgene in adult zebrafish}, year={2014}, author={Pinzon-Olejua, Manuel Alejandro}, address={Konstanz}, school={Universität Konstanz} }

terms-of-use In mammals, oligodendrocytes and CNS myelin express neurite growth inhibitors, in particular Nogo-A / RTN4A, which is (among others) responsible for the inability of lesioned axons to regenerate after a lesion. In fish, by contrast, lesioned CNS axons successfully regenerate which correlates with the growth-permissive properties of fish CNS myelin / oligodendrocytes.<br />To address the question whether axon regeneration in fish would be impaired by inhibitors such as Nogo-A / RTN4 in mammals, we generated transgenic zebrafish by Tol2 transposon-mediated transgenesis. This includes the generation of the two effector lines tg(hsp70: LoxPDsRedLoxP-EGFP-T2A-rat-NogoA) and tg(hsp70:LoxPDsRedLoxP-EGFP-T2A-ZFrtn4a) and of a driver line tg(mbp:mCherry-T2-CreERT2). We used the Cre/lox system to achieve tissue-specific transgenic expression. This allows the expression of the most inhibitory region of rat Nogo-A and of zebrafish rtn4 in the Mbp specific domain, thus restricting the expression to oligodendrocytes and CNS myelin. The “tamoxifen inducible-CreERT2” and the “heat shock” promoter regions allow to temporally control recombination and to induce transgene expression in adult animals, which receive an optic nerve transection (as CNS injury). The present work describes in Part II the generation of the effector and driver lines, which are now ready for crosses and expression analyses after CNS lesion.<br />Part I of this study has analyzed the RTN4A / Nogo-A paralogues in zebrafish during embryogenesis. This work shows that the zebrafish genes rtn4a and rtn4b are expressed early in development in the neural tube and many brain regions derived thereof including the retina, optic tectum and motoneurons. Using gene specific morpholino mediated knock down of zebrafish rtn4 genes we show that both are essential for neuronal development and for some specific non-neuronal structures (lower jaw, notochord, heart). Downregulation of rtn4a and rtn4b caused severe CNS malformations, which persist in rtn4b-morphants so that they die at early larval stages. This contrasts to the situation in mammals: the RTN4 / Nogo knock out mice are viable and fertile but show defects in CNS plasticity when the mice grow up.<br />This work thus demonstrates in Part I a major role of zebrafish rtn4a and rtn4b, which is most likely connected to their function as structural components of the ER. Part II is aimed at solving how the inhibitory function of rat Nogo-A in / on zebrafish oligodendrocytes / myelin affects zebrafish axon regeneration, i.e., whether regenerating axons would recognize surface-exposed and myelin-membrane associated Nogo-A as an inhibitor which supposedly would render them unable to regenerate. Function of RTN4/Nogo during development and as transgene in adult zebrafish 2014-11-04T09:27:36Z 2014 2014-11-04T09:27:36Z Pinzon-Olejua, Manuel Alejandro eng Pinzon-Olejua, Manuel Alejandro

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