Nogo in fishes: Function and evolution of RTN-4/-6 Nogo domains and their influence on axon regeneration
Final Report Abstract
In mammals, Nogo-A/RTN4 in CNS myelin and oligodendrocytes inhibits axon regeneration. Since RGC axons regenerate in the fish CNS, we have analyzed expression and function of the Nogo-orthologues, Rtn4a and –b in zebrafish (Part I). Moreover, we generated transgenic zebrafish lines and are close to having a line that produces rat Nogo-A in the adult zebrafish CNS (Part II). Part I) Zebrafish Rtn4b, the closest homologue of Nogo-A, was found to be expressed in retinal ganglion cells and to be massively upregulated after optic nerve lesion. In fact, our results after morpholino-mediated downregulation, suggest that Rtn4b acts as an important neuron-intrinsic determinant of axon regeneration. To test the substrate properties of Rtn4b in comparison to the known inhibitory properties of rat Nogo-A, M4 peptides comprising the Nogo-A delta-20 region and the corresponding region of zebrafish Rtn4b were recombinantly expressed and purified, and offered as substrate to growing axons in the stripe assay. In a series of technically optimal experiments, axons grew preferentially on polylysine lanes and avoided the rat M4, whereas they grew across both lanes with zebrafish M4. This suggests that rat M4 is by far less inhibitory than zebrafish M4 which corresponds to earlier data showing no growth inhibition through fish CNS myelin but growth cone collapse with rat CNS myelin and Nogo-A. However, additional experiments are carried out to substantiate our data. Part II) The Tol2 transposon system enabled us to generate three effector lines, as well as a driver line expressing Cre ERT2 in oligodendrocytes and Schwann cells as well as myelin (in collaboration with S. Hans and M. Brand, CRT Dresden). Next, transgenic lines were generated showing CreERT2 expression under the control of the zebrafish mbp (myelin basic protein) promoter. Stable lines (tg (mpb: mCherry T2A CreERT2 )) that express CreERT2 transcripts in CNS and PNS myelin were established. To obtain recombination, the driver lines were crossed with the red-to-green reporter line. 5 d old larvae were treated with tamoxifen (TAM) and heat shock (1h) to induce transgene expression. A strong eGFP signal was detected in oligodendrocytes and Schwann cells. The Cre-lox recombination approach should now allow us to express Nogo-A in a tissuespecific manner. The three effector lines which we established show successful recombination and (over-) expression of the inserted genes (rtn4a; rtn4b; rat rtn4/nogo), after injection of Cre mRNA and heat shock. This will allow us to examine whether rat Nogo-A expressed by fish oligodendrocytes and CNS myelin will inhibit fish RGC axon regeneration.
Publications
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Origin of Nogo-A by domain shuffling in an early jawed vertebrate. Molecular Biology and Evolution, Vol. 28.2011, Issue 4, pp. 1363–1370.
Shypitsyna A., Málaga-Trillo E., Reuter A., Stuermer C.A.O.
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Essential roles of zebrafish rtn4/Nogo paralogues in embryonic development. Neural Development, Vol. 9. 2014: 8.
Pinzon-Olejua A., Welte C., Abdesselem H., Malaga-Trillo E., Stuermer C.A.
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Upregulation of the zebrafish Nogo-A homologue, Rtn4b, in retinal ganglion cells is functionally involved in axon regeneration. Neural Development, Vol. 10. 2015: 6.
Welte C., Engel S., Stuermer C.A.
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Cre-inducible Site-specific Recombination in Zebrafish Oligodendrocytes. Developmental Dynamics, Vol. 246. 2017, Issue 1, pp. 41–49.
Alejandro Pinzon-Olejua, Cornelia Welte, Avinash Chekuru, Viktoria Bosak, Michael Brand, Stefan Hans, Claudia A.O. Stuermer
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Substrate properties of zebrafish Rtn4b/Nogo and axon regeneration in the zebrafish optic nerve. The Journal of Comparative Neurology, published online 2017.
Vsevolod Bodrikov, Cornelia Welte, Marianne Wiechers, Markus Weschenfelder, Gurjot Kaur
