A microRNA-mRNA expression network during oral siphon regeneration in Ciona

Abstract

Here we present a parallel study of mRNA and microRNA expression during oral siphon (OS) regeneration in Ciona robusta, and the derived network of their interactions. In the process of identifying 248 mRNAs and 15 microRNAs as differentially expressed, we also identified 57 novel microRNAs, several of which are among the most highly differentially expressed. Analysis of functional categories identified enriched transcripts related to stress responses and apoptosis at the wound healing stage, signaling pathways including Wnt and TGFβ during early regrowth, and negative regulation of extracellular proteases in late stage regeneration. Consistent with the expression results, we found that inhibition of TGFβ signaling blocked OS regeneration. A correlation network was subsequently inferred for all predicted microRNA-mRNA target pairs expressed during regeneration. Network-based clustering associated transcripts into 22 non-overlapping groups, the functional analysis of which showed enrichment of stress response, signaling pathway and extracellular protease categories that could be related to specific microRNAs. Predicted targets of the miR-9 cluster suggest a role in regulating differentiation and the proliferative state of neural progenitors through regulation of the cytoskeleton and cell cycle.

Keywords: Ciona; Correlation network; MicroRNA; RNAseq; Regeneration.

Fig. 1.

Stages of oral siphon regeneration. (Top) The two reference stages used to quantify relative expression levels at subsequent stages. Left image shows an unamputated adult oral siphon (OS) of C. robusta; the right image is immediately post-amputation. (Bottom) Three time points (1, 3 and 8 D) selected to represent three stages of appendage regeneration (wound healing, transition and redevelopment, respectively). The red dashed line indicates the original amputation plane, and the yellow dashed lines indicate the proximal limit of tissue collected for expression profiling. Scale bars: 5 mm.

Fig. 2.

Experimental validation of differential expression. The mean log₂ fold change of transcript expression levels relative to D0 estimated by RNAseq (DESeq2) and qRT-PCR. Three biological replicates comprising three technical replicates each were used to calculate qRT-PCR statistics; error bars indicate s.e.m. for RNAseq and 95% confidence intervals for qRT-PCR. (A) Differentially expressed (DE) mRNAs significant in both RNAseq and qRT-PCR experiments arranged into preselected groups. Ensembl transcript identifiers matching each transcript name are listed in Table S8. (B) DE miRNAs significant in both RNAseq and qRT-PCR data.

Fig. 3.

Standardized z-scores of enriched (z≥1.96) functional categories. z-scores, plotted as heatmaps, were calculated relative to D0 then standardized across post-amputation stages D1, D3 or D8. Dendrograms to the left of each heatmap indicate the results of hierarchical clustering of the indicated GO categories (A-C) or KEGG pathways (D).

Fig. 4.

SB431542 treatment of C. robusta juveniles. (A) Approximately 1-month-old animals were treated with 10 μM SB431542 for the durations indicated. (B) Images of representative animals from the 48 h treatment cohorts (orange in A). DMSO, vehicle control. See also Table 2.

Fig. 5.

miRNA-mRNA correlation network. miRNAs and mRNAs are represented by triangles and circles, respectively. Transcripts are joined by a line when both ρ≤−0.9 and a binding interaction was predicted by TargetScanS. ModuLand clusters are shown in different colors and the miRNA ID that defines each cluster is indicated (black text). Summary interpretations of enriched GO categories and KEGG pathways in each cluster are indicated in matching colors adjacent to the respective cluster. The full set of correlations, assignment of nodes to clusters and functional enrichments for clusters are listed in Table S12.