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. 2016 Jun 29;11(6):e0157106.
doi: 10.1371/journal.pone.0157106. eCollection 2016.

A Conserved MicroRNA Regulatory Circuit Is Differentially Controlled During Limb/Appendage Regeneration

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Free PMC article

A Conserved MicroRNA Regulatory Circuit Is Differentially Controlled During Limb/Appendage Regeneration

Benjamin L King et al. PLoS One. .
Free PMC article

Abstract

Background: Although regenerative capacity is evident throughout the animal kingdom, it is not equally distributed throughout evolution. For instance, complex limb/appendage regeneration is muted in mammals but enhanced in amphibians and teleosts. The defining characteristic of limb/appendage regenerative systems is the formation of a dedifferentiated tissue, termed blastema, which serves as the progenitor reservoir for regenerating tissues. In order to identify a genetic signature that accompanies blastema formation, we employ next-generation sequencing to identify shared, differentially regulated mRNAs and noncoding RNAs in three different, highly regenerative animal systems: zebrafish caudal fins, bichir pectoral fins and axolotl forelimbs.

Results: These studies identified a core group of 5 microRNAs (miRNAs) that were commonly upregulated and 5 miRNAs that were commonly downregulated, as well as 4 novel tRNAs fragments with sequences conserved with humans. To understand the potential function of these miRNAs, we built a network of 1,550 commonly differentially expressed mRNAs that had functional relationships to 11 orthologous blastema-associated genes. As miR-21 was the most highly upregulated and most highly expressed miRNA in all three models, we validated the expression of known target genes, including the tumor suppressor, pdcd4, and TGFβ receptor subunit, tgfbr2 and novel putative target genes such as the anti-apoptotic factor, bcl2l13, Choline kinase alpha, chka and the regulator of G-protein signaling, rgs5.

Conclusions: Our extensive analysis of RNA-seq transcriptome profiling studies in three regenerative animal models, that diverged in evolution ~420 million years ago, reveals a common miRNA-regulated genetic network of blastema genes. These comparative studies extend our current understanding of limb/appendage regeneration by identifying previously unassociated blastema genes and the extensive regulation by miRNAs, which could serve as a foundation for future functional studies to examine the process of natural cellular reprogramming in an injury context.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Vertebrate models used to study limb/appendage regeneration.
(a) Phylogenetic relationship among three vertebrate taxa studied to determine conserved gene expression patterns in regenerating limb/appendages following blastema formation. These animal systems last shared a common ancestor ~420 million years ago. (b) Blastema tissues as shown by hematoxylin and eosin staining on paraffin tissue sections of regenerating zebrafish caudal fin, bichir pectoral fins and axolotl forelimbs. (* = blastema; dpa = days post-amputation).
Fig 2
Fig 2. miRNA expression profiling by small RNA sequencing.
(a) Overview of shared and unique miRNAs detected in regenerating limb/appendages following blastema formation in zebrafish caudal fin, bichir pectoral fins and axolotl forelimbs. (b) Gene expression patterns of differentially expressed zebrafish miRNAs shown as a heat map of log2-transformed read counts per million, in triplicate. (c) Relationship among fold-change and average level of expression for each miRNA in each taxa with miR-21 and miR-133. (dpa = days post-amputation).
Fig 3
Fig 3. Differentially regulated miRNAs shared among zebrafish, bichir and axolotl.
(a, b) qRT-PCR validation of differential expression of commonly upregulated and downregulated miRNAs in regenerating zebrafish caudal fins (0 dpa vs. 4 dpa), bichir pectoral fins (0 dpa vs. 7 dpa) and axolotl forelimbs (0 dpa vs. 6 dpa). At least 4 regenerating tissues per replicate were used, in triplicate. Average values +/- SEM are plotted. (*, **, *** = p-value < 0.01, 0.005 and 0.001 respectively).
Fig 4
Fig 4. Analyses of conserved sequence tags reveal processed tRNA fragments and isomiRs.
(a) Analysis workflow used to identify conserved sequence tags. Two-hundred-forty sequence tags expressed in all zebrafish samples were also expressed in all bichir and axolotl samples. 232 sequence tags were isomiRs including 4 that had substitutions in the seed sequence. Of the eight sequence tags were not derived from miRNAs, 4 mapped to ribosomal RNAs (rRNAs) and 4 were processed tRNA fragments. (b) The 4 processed tRNA fragments expressed in all zebrafish, bichir and axolotl samples. We found these processed tRNAs to also be small RNA sequence data from in chicken, mouse and human tissues [78].
Fig 5
Fig 5. Commonly expressed blastema-associated genes.
(a) qRT-PCR validation of common blastema-associated genes differentially expressed in regenerating zebrafish caudal fins (0 dpa vs. 4 dpa), bichir pectoral fins (0 dpa vs. 7 dpa) and axolotl forelimbs (0 dpa vs. 6 dpa), in triplicate. Each replicate contained at least 4 regenerating tissues. Average values +/- SEM are plotted. (*, **, *** = p-value < 0.01, 0.005 and 0.001 respectively). (b) STRING interactions among 11 blastema-associated genes (larger nodes) and 60 additional common differentially expressed genes (smaller nodes) with interaction scores greater than 98% of all 3,262 possible interactions. Nodes are colored according to fold-change observed in zebrafish and labeled according to zebrafish gene nomenclature provided by Ensembl.
Fig 6
Fig 6. miR-21 regulation of blastema-associated genes.
(a) Network of blastema-associated genes, miR-21 and predicted miR-21 targets differentially expressed in three model systems. STRING interactions with 11 common blastema-associated genes, miR-21, miR-31, miR-181, and 50 additional common differentially expressed genes with common predicted miRNAs binding sites. STRING interactions are scaled by the thickness and color of edges score. Nodes representing the miRNAs, predicted target mRNAs and blastema-associated genes are colored according to fold change observed in zebrafish and labeled according to zebrafish gene nomenclature provided by Ensembl. (b) qRT-PCR validation of five common predicted miR-21 target genes downregulated in regenerating zebrafish caudal fins (0 dpa vs. 4 dpa), bichir pectoral fins (0 dpa vs. 7 dpa) and axolotl forelimbs (0 dpa vs. 6 dpa). Each data point represents average values +/- SEM from triplicate reactions, each containing at least 4 biological samples. (*, **, *** = p-value < 0.01, 0.005 and 0.001 respectively).
Fig 7
Fig 7. Conserved gene regulatory circuit for appendage regeneration.
Following injury, three miRNAs (miR-21, miR-31 and miR-181c) are commonly upregulated and target a set of five commonly downregulated genes shown with blue lines. These five target genes have functional interactions (green lines) with a set of 11 common blastema-associated genes from STRING [58]. Higher interaction scores are shown by thicker edges with darker green colors. These blastema-associated genes have diverse functions including growth factor signaling, extracellular matrix remodeling, signal transduction, transcription and protein homeostasis. Nodes representing the miRNAs and mRNAs are colored according to fold change observed in zebrafish and labeled according to zebrafish gene nomenclature provided by Ensembl.

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