Zebrafish Embryonic Neurons Transport Messenger RNA to Axons and Growth ConesIn Vivo

Abstract

Although mRNA was once thought to be excluded from the axonal compartment, the existence of protein synthesis in growing or regenerating axons in culture is now generally accepted. However, its extent and functional importance remain a subject of intense investigation. Furthermore, unambiguous evidence of mRNA axonal transport and local translation in vivo, in the context of a whole developing organism is still lacking. Here, we provide direct evidence of the presence of mRNAs of the tubb5, nefma, and stmnb2 genes in several types of axons in the developing zebrafish (Danio rerio) embryo, with frequent accumulation at the growth cone. We further show that axonal localization of mRNA is a specific property of a subset of genes, as mRNAs of the huc and neurod genes, abundantly expressed in neurons, were not found in axons. We set up a reporter system in which the 3Ј untranslated region (UTR) of candidate mRNA, fused to a fluorescent protein coding sequence, was expressed in isolated neurons of the zebrafish embryo. Using this reporter, we identified in the 3ЈUTR of tubb5 mRNA a motif necessary and sufficient for axonal localization. Our work thus establishes the zebrafish as a model system to study axonal transport in a whole developing vertebrate organism, provides an experimental frame to assay this transport in vivo and to study its mechanisms, and identifies a new zipcode involved in axonal mRNA localization.

An in vivo translation-reporter system for the study of protein synthesis in zebrafish embryos

“…3). In agreement with previously observed localisation of mRNA (Baraban et al, 2013), newly synthesised protein frequently accumulated in the axon initial segment (arrowheads in Fig. 3A,B,G,H).…”

“…The TimeStamp cassette comprising the NS3 domain of the hepatitis C virus (HCV) polyprotein, flanked by its NS4A/B specific cleavage sites and followed by an HA epitope coding sequence was obtained from the PSD95 plasmid (Lin et al, 2008). It was cloned in frame with Venus coding sequence in plasmid 5xUAS:myrVenus_3'UTR_chicken_ß-actin (Baraban et al, 2013). Chicken ßactin-∆Zipcode and zebrafish tubulin-ß5 and neuroD 3'UTR sequences as described in (Baraban et al, 2013) were substituted to the original chicken ß-actin 3'UTR to obtain the corresponding plasmids.…”

“…It was cloned in frame with Venus coding sequence in plasmid 5xUAS:myrVenus_3'UTR_chicken_ß-actin (Baraban et al, 2013). Chicken ßactin-∆Zipcode and zebrafish tubulin-ß5 and neuroD 3'UTR sequences as described in (Baraban et al, 2013) were substituted to the original chicken ß-actin 3'UTR to obtain the corresponding plasmids. All the sequences were checked and are available on request.…”

“…Since protein synthesis in axons, formerly presumed not to occur (see Alvarez et al, 2000 for a review), has now attracted considerable attention as a possible mechanism underlying axonal growth, survival, and guidance (recent reviews: Cioni et al, 2018;Riccio, 2018), we particularly scrutinised whether the SPoT transgene allowed detecting translation events in axons. In the SPoT transgenes used above, the SPoT protein is produced from a mRNA containing the 3'UTR sequence of chicken beta-actin (SPoT_3'UTR ß-actin) or tubulin-ß5 (SPoT_3'UTR tubb5) mRNA, both of which allow axonal transport in zebrafish neurons (Baraban et al, 2013). Therefore, we searched whether Danoprevir treatment enabled detecting axonal translation of the reporter.…”

Clemizole Hydrochloride Is a Novel and Potent Inhibitor of Transient Receptor Potential Channel TRPC5

“…Combining clemizole with HCV protease inhibitors proved to be highly synergistic, strongly enhancing the potency of such therapeutics (Einav et al, 2010). Moreover, a recent study of Baraban et al (2013) reported that clemizole inhibits spontaneous seizures in a zebrafish model for Dravet syndrome (scn1Lab mutant), a rare and severe form of monogenic epilepsy, which is mainly caused by mutations in Nav1.1 (SCN1A), a voltage-gated sodium channel (Catterall, 2014). However, high concentrations of 100 mM clemizole were needed to suppress spontaneous hyperactivity.…”