Transcriptome analysis of embryonic and adult sensory axons reveals changes in mRNA repertoire localization - PubMed (original) (raw)

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

Laura F Gumy et al. RNA. 2011 Jan.

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.

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Figures

FIGURE 1.

FIGURE 1.

Venn diagrams comparing the three different analyses: GCOS, GC-RMA, and RMA in embryonic and adult DRG axons. (A) The 2627 genes present in embryonic axons, (B) the 2924 genes present in adult axons. Numbers in parentheses indicate the number of genes as determined by either the GCOS, RMA, or GCRMA algorithms (see Materials and Methods).

FIGURE 2.

FIGURE 2.

Embryonic and adult sensory axons contain different repertoires of mRNAs. Venn diagram showing overlapping (1445) and uniquely localized mRNAs in embryonic (1182) and adult (1479) DRG axons. Of the overlapping mRNAs, 1272 were up-regulated (enriched) in embryonic axons while 173 were enriched in adult ones.

FIGURE 3.

FIGURE 3.

Tubb3 is locally translated in embryonic DRG axons. (A) Fluorescent micrograph of Tubb3 immunolabeled embryonic DRG neurons cultured in the compartmentalized microfluidic chamber. Cycloheximide treatment (25 μM) for 3 h significantly decreased Tubb3 immunoreactivity in distal axons. CB indicates the cell body compartment. Scale bar = 100 μm. (B) Fluorescent micrograph of Tubb3 immunolabeled adult DRG neurons cultured in the compartmentalized microfluidic chamber. Cycloheximide treatment (25 μM) for 3 h of distal axons had no effect on Tubb3 levels. CB indicates the cell body compartment. Scale bar = 100 μm. (C) Quantification of the raw intensity levels of Tubb3 immunofluorescence in cycloheximide-treated and untreated distal axons proximal axons in the cell body compartment. Tubb3 intensity levels in distal axons decreased significantly following cycloheximide treatment compared to untreated controls (*P = 0.010; two-tailed Student's _t_-test). Error bars indicate SD.

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