Transcriptome analysis of embryonic and adult sensory axons reveals changes in mRNA repertoire localization

Abstract

mRNAs are transported, localized, and translated in axons of sensory neurons. However, little is known about the full repertoire of transcripts present in embryonic and adult sensory axons and how this pool of mRNAs dynamically changes during development. Here, we used a compartmentalized chamber to isolate mRNA from pure embryonic and adult sensory axons devoid of non-neuronal or cell body contamination. Genome-wide microarray analysis reveals that a previously unappreciated number of transcripts are localized in sensory axons and that this repertoire changes during development toward adulthood. Embryonic axons are enriched in transcripts encoding cytoskeletal-related proteins with a role in axonal outgrowth. Surprisingly, adult axons are enriched in mRNAs encoding immune molecules with a role in nociception. Additionally, we show Tubulin-beta3 (Tubb3) mRNA is present only in embryonic axons, with Tubb3 locally synthesized in axons of embryonic, but not adult neurons where it is transported, thus validating our experimental approach. In summary, we provide the first complete catalog of embryonic and adult sensory axonal mRNAs. In addition we show that this pool of axonal mRNAs dynamically changes during development. These data provide an important resource for studies on the role of local protein synthesis in axon regeneration and nociception during neuronal development.

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

“…Building upon the large body of knowledge generated by previous work on cultured neurons, particularly transcriptomics studies aimed at characterizing the full complement of axonal mRNA Taylor et al, 2009;Andreassi et al, 2010;Gumy et al, 2011;Zivraj et al, 2010), our findings provide, for the first time, evidence that mRNAs are transported to developing axons in the zebrafish embryo, a leading model organism for studying early development of the vertebrate nervous system. The axonal mRNA we observe shares several important features with what has been described in culture systems, such as accumulation at the growth cone, signal carried by the 3ЈUTR, dependence on microtubule integrity, and transport of ␤-actin mRNA.…”

“…Because no instance of axonal mRNA transport has yet been reported in the zebrafish, we started our study by selecting candidate genes whose mRNA could be detected in axons in at least one of the large-scale screens conducted on rat Taylor et al, 2009;Gumy et al, 2011), mouse, and Xenopus cultured neurons. Among the top-listed genes, we selected nefm, stathmin-like 2b, and tubulin␤5, which have been repeatedly detected in these screens (Table 1) and whose orthologues showed neuronspecific expression profiles in the early zebrafish embryo, ac- Numbers in the table represent the rank, in the ordered list of axonal mRNA species detected in each study, of the mRNA orthologous to the considered zebrafish gene.…”

“…The newly identified tubb5 zipcode identified in the present work is not only necessary, but also sufficient, for axonal localization. Examination of the zipcode sequence reveals that it is G/U rich and does not contain any of the proposed consensus motifs suggested to mediate binding of Imp1 to its cognate targets (Jønson et al, 2007;Hafner et al, 2010;Patel et al, 2012), implying that it is of a novel kind. However, the function of the tubb5 zipcode may ultimately depend on its secondary/tertiary structure upon folding, precluding comparison with known motifs: such cases have been described for RNA cis-elements in the Drosophila syncytial blastoderm (Bullock et al, 2010), as well as in Aplysia neurites (Meer et al, 2012).…”

“…Zebrafish were raised and maintained as described previously (Kimmel et al, 1995). Embryos of either sex were staged according to the number of hours postfertilization (hpf) or days postfertilization (dpf) at 28°C.…”

Targeting Axonal Protein Synthesis in Neuroregeneration and Degeneration

“…Together, these findings strongly suggest that the ability for protein synthesis persists in postdevelopmental axons in vivo. However, the lower number of mRNAs and ribosomes localized to axons, as well as the changed composition of the mature axonal transcriptome [28], indicates that its function and relevance might be distinct from the developmental period. For example, if one of the chief purposes of intra-axonal translation is the rapid and spatially restricted response to changes in the axon's environment, as is suggested by numerous developmental studies, then axons in the mature and inherently more stable nervous system might simply have a much lower need to synthesize proteins locally unless they are challenged.…”