Cloning of Zebrafish Neurofilament cDNAs for Plasticin and Gefiltin: Increased mRNA Expression in Ganglion Cells After Optic Nerve Injury

Abstract

During retinal growth and optic axon regeneration, the differential expression of the neuronal intermediate filament proteins, plasticin and gefiltin, in the goldfish visual pathway suggests that these proteins support programmed axonal growth. To investigate plasticin and gefiltin during axonogenesis, we turned to the zebrafish, a system that is more amenable to mutational analysis. As a first step, we demonstrated that the intermediate filament compositions of goldfish and zebrafish are similar. In addition, the cDNAs for zebrafish plasticin and gefiltin were cloned and characterized. Using in situ hybridization in retina, we show increased mRNA levels for these proteins following optic nerve crush. Zebrafish plasticin and gefiltin peak and return to baseline levels of expression more rapidly than in goldfish. Furthermore, in the unoperated eye of experimental fish, there was a moderate increase in the levels of plasticin and gefiltin mRNA, suggesting that soluble factors influence the expression of these proteins. The successive expression of plasticin and gefiltin suggests that these neuronal intermediate filament proteins are integral components of axonogenesis. The cloning and characterization of cDNAs for plasticin and gefiltin permit mutational analyses of these proteins during zebrafish axonogenesis.

Increased expression of multiple neurofilament mRNAs during regeneration of vertebrate central nervous system axons

“…It later disappears from most of the CNS as axons mature (Gervasi et al, 2000;Undamatla and Szaro, 2001). The correlation between peripherin expression and axon outgrowth is especially strong during regeneration, when its expression increases dramatically in regenerating fish optic (Glasgow et al, 1992Fuchs et al, 1994;Hall, 1994;Asch et al, 1998) and mammalian peripheral axons (Oblinger et al, 1989;Wong and Oblinger, 1990;Troy et al, 1990a). Thus, the dramatic return of peripherin expression to Xenopus RGCs during axonal regeneration further underscores the importance of peripherin to growing axons.…”

“…For example, in transected spinal cord axons of lamprey, NF-M expression is initially suppressed and then rises in only those axons that successfully regenerate, while remaining suppressed in those that do not (Jacobs et al, 1997). Similarly, in fish optic nerve expression of the xefiltin ortholog, gefiltin, is also initially suppressed, then rises above normal during the peak period of regeneration, and then falls to normal levels after regeneration is complete Asch et al, 1998). This return to normal depends on signals from the optic tectum (Niloff et al, 1998), and may involve the successful formation of a retinotopic map, since in the lizard Ctenophorus ornatus, where vision fails to return after optic nerve crush despite the regrowth of axons and the reformation of synaptic connections in visual targets (Beazley et al, 1997;Stirling et al, 1999), levels of gefiltin expression remain permanently elevated (Rodger et al, 2001).…”

Partially redundant proneural function reveals the importance of timing during zebrafish olfactory neurogenesis

“…Antisense DIG-labelled probes for neurog1 (Blader et al, 1997), gfp (Blader et al, 2003), neurod4 (Park et al, 2003), deltaA (Haddon et al, 1998), olfactory marker protein (Celik et al, 2002), robo2 (Lee et al, 2001), gefiltin (inab -Zebrafish Information Network) (Asch et al, 1998), olfactomedin 1b (Nakaya and Tomarev, 2007) and insulinoma-associated 1b (Lukowski et al, 2006) were generated using standard procedures. In situ hybridisations were visualised using either BCIP and NBT (Roche) or Fast Red (Roche) as substrates.…”

“…Similarly, the expression of olfactomedin 1b (olfm1b) and gefiltin (gef) is restricted to EONs at 24 hpf in wild-type embryos and is absent from neurog1 mutant olfactory placodes (see Fig. S5A-L in the supplementary material) (Asch et al, 1998;Nakaya and Tomarev, 2007). Thus, although neurons with certain characteristics of EONs can develop in the absence of Neurog1 function, not all aspects of EON specification are apparent.…”

Axonal Regeneration of Fish Optic Nerve after Injury

“…3) Miscellaneous Molecules After ON injury, many proteins or mRNAs are reported to be induced in the retina or ON in fish, such as 2Ј,3Ј-cyclic-nucleotide 3Ј-phosphodiesterase after about 7 d, 62,63) myosine light chain kinase in goldfish RGCs, 64) reggie-1 and reggie-2, cell surface proteins, 65) plasticin and gefilitin, neurofilament proteins, in RGCs 10-20 d after ON injury in zebrafish, 66,67) GAP-43 mRNA in RGCs of zebrafish to 20 times at 5 d, 68) a1-tubulin, 69) upregulation of protein kinase-C in regenerating ON fibers at 2-11 weeks and tectum at 5 weeks in goldfish, 70) b-thymosin, a modulator of the actin cytoskelton, mostly found in growth cone at 5-8 d in retina, and 4 d in ON of zebrafish, 71) increase of NADPH diaphorase activity in RGCs of goldfish at 5-30 d, 72) phosphorylated microtubule associated protein 1B in RGCs and regenerating axon. 73) The membrane guiding molecules, the Eph family of receptor tyrosine kinase and their membrane associated ligands, ephrins, are important for precise retinotectal projections.…”