Comparative transcriptomic profiling of peripheral efferent and afferent nerve fibres at different developmental stages in mice

Abstract

Peripheral nerve injury impairs motor and sensory function in humans, and its functional recovery largely depends on the axonal outgrowth required for the accurate reinnervation of appropriate targets. To better understand how motor and sensory nerve fibres select their terminal pathways, an unbiased cDNA microarray analysis was conducted to examine differential gene expression patterns in peripheral efferent and afferent fibres at different developmental stages in mice. Gene ontology (GO) and Kyoto Enrichment of Genes and Genomes (KEGG) analyses revealed common and distinct features of enrichment for differentially expressed genes during motor and sensory nerve fibre development. Ingenuity Pathway Analysis (IPA) further indicated that the key differentially expressed genes were associated with trans-synaptic neurexin-neuroligin signalling components and a variety of gamma-aminobutyric acid (GABA) receptors. The aim of this study was to generate a framework of gene networks regulated during motor and sensory neuron differentiation/maturation. These data may provide new clues regarding the underlying cellular and molecular mechanisms that determine the intrinsic capacity of neurons to regenerate after peripheral nerve injury. Our findings may thus facilitate further development of a potential intervention to manipulate the therapeutic efficiency of peripheral nerve repair in the clinic.

Figure 1

Schematic diagram illustrating tissue collection and microarray data quality assessment. Tissues were dissected from the areas highlighted using circles in the diagram, which contain peripheral nerve fibres extending from the spinal cord (A). Sample correlation matrices (B) and three-dimensional principal component analysis plot (C) demonstrating a reasonable clustering of the 24 samples (8 groups in triplicate).

Figure 2

General overview of differentially expressed genes in efferent and afferent nerve fibres during development. Histogram (A) showing a comparison of differentially expressed genes between efferent and afferent nerve fibres at different developmental stages (postnatal day 0 [P0], 1 week [1 W], 3 weeks [3 W], and 5 weeks [5 W]). Histogram (B) showing the number of genes differentially expressed in the efferent or afferent nerve fibres throughout development (up-regulated in red, down-regulated in green, and total number in blue). Five distinct expression patterns in the efferent and afferent nerve fibres during development are shown for comparison (C).

Figure 3

Heat map displaying the hierarchical clustering of differentially expressed genes in efferent and afferent nerve fibres during development. The relative expression level of each gene is indicated by colour intensity. Genes that were generally down-regulated are highlighted in green rectangles, while genes that were generally up-regulated are highlighted in red rectangles.

Figure 4

Enriched gene ontology analysis of differentially expressed genes. Comparison of genes differentially expressed between efferent and afferent nerve fibres at different developmental stages (postnatal day 0 [P0], 1 week [1 W], 3 weeks [3 W], and 5 weeks [5 W]), categorized using Biological Processes (A), Cellular Components (B), and Molecular Functions (C). Square with p < 0.05 are labelled in blue, while those with p > 0.05 are labelled in white.

Figure 5

Enriched KEGG pathways for the differentially expressed genes. Comparison of genes differentially expressed between efferent and afferent nerve fibres (A) in different developmental processes, and Z-scores for the six critical Kyoto Enrichment of Genes and Genomes (KEGG) pathways (B). The differentially expressed genes in efferent (C) and afferent (D) nerve fibres at postnatal developmental stages (1 week [1 W], 3 weeks [3 W], and 5 weeks [5 W]) were compared with those at postnatal day 0 (P0). The colour intensity of the dots represents the p value, and the size of the dots represents the ratio of differentially expressed genes in the specific KEGG pathway.

Figure 6

Cascade regulation of genes differentially expressed between efferent and afferent nerve fibres during development. The regulatory network exhibits the comparison of genes differentially expressed between efferent and afferent nerve fibres at different developmental stages (postnatal day 0 [P0], 1 week [1 W], 3 weeks [3 W], and 5 weeks [5 W]). The up-regulated genes are shown in red, while the down-regulated genes are shown in green, and the colour intensity indicates the fold change in differential expression. The key genes are highlighted with blue asterisks.

Figure 7

Dynamic changes in differentially expressed genes associated with efferent and afferent nerve fibre development. The gene network diagrams show the interactions and correlations of differentially expressed genes during efferent and afferent nerve fibre development at different postnatal stages (1 week [1 W], 3 weeks [3 W], and 5 weeks [5 W], when compared with postnatal day 0 [P0]). Red colour indicates the fold change in up-regulation, while the green colour indicates the fold change in down-regulation. Colour intensity indicates the relative fold change values. The key genes are highlighted with blue asterisks.

Figure 8

Real-time quantitative polymerase chain reaction-based validation of key genes associated with efferent and afferent nerve fibre development. Heat map clustering displaying the relative mRNA expression levels of 9 differentially expressed genes selected from all 24 samples (8 groups in triplicate). The orange colour indicates the genes that are upregulated, and the blue colour indicates the genes that are downregulated. The values were calculated using comparative 2^−ΔΔCt method, with Gapdh levels as the internal control (efferent nerve fibres are shown in yellow and afferent nerve fibres are shown in purple). Error bars represent standard deviations from the mean. Data were obtained in three independent experiments. ^{*, #,§}p < 0.05; ^{**, ##, §§}p < 0.01; ^*in efferent nerve fibre development, ^#in afferent nerve fibre development, ^§difference between efferent and afferent nerve fibre expression.