Mashanov VS et al. (2014), Transcriptomic changes during regeneration of t...

ECB-ART-43437

BMC Genomics 2014 May 12;15:357. doi: 10.1186/1471-2164-15-357.

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Transcriptomic changes during regeneration of the central nervous system in an echinoderm.

Mashanov VS , Zueva OR , García-Arrarás JE .

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BACKGROUND: Echinoderms are emerging as important models in regenerative biology. Significant amount of data are available on cellular mechanisms of post-traumatic repair in these animals, whereas studies of gene expression are rare. In this study, we employ high-throughput sequencing to analyze the transcriptome of the normal and regenerating radial nerve cord (a homolog of the chordate neural tube), in the sea cucumber Holothuria glaberrima. RESULTS: Our de novo assembly yielded 70,173 contigs, of which 24,324 showed significant similarity to known protein-coding sequences. Expression profiling revealed large-scale changes in gene expression (4,023 and 3,257 up-regulated and down-regulated transcripts, respectively) associated with regeneration. Functional analysis of sets of differentially expressed genes suggested that among the most extensively over-represented pathways were those involved in the extracellular matrix (ECM) remodeling and ECM-cell interactions, indicating a key role of the ECM in regeneration. We also searched the sea cucumber transcriptome for homologs of factors known to be involved in acquisition and/or control of pluripotency. We identified eleven genes that were expressed both in the normal and regenerating tissues. Of these, only Myc was present at significantly higher levels in regeneration, whereas the expression of Bmi-1 was significantly reduced. We also sought to get insight into which transcription factors may operate at the top of the regulatory hierarchy to control gene expression in regeneration. Our analysis yielded eleven putative transcription factors, which constitute good candidates for further functional studies. The identified candidate transcription factors included not only known regeneration-related genes, but also factors not previously implicated as regulators of post-traumatic tissue regrowth. Functional annotation also suggested that one of the possible adaptations contributing to fast and efficient neural regeneration in echinoderms may be related to suppression of excitotoxicity. CONCLUSIONS: Our transcriptomic analysis corroborates existing data on cellular mechanisms implicated in regeneration in sea cucumbers. More importantly, however, it also illuminates new aspects of echinoderm regeneration, which have been scarcely studied or overlooked altogether. The most significant outcome of the present work is that it lays out a roadmap for future studies of regulatory mechanisms by providing a list of key candidate genes for functional analysis.

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Species referenced: Echinodermata
Genes referenced: abat irak1bp1 LOC100887844 LOC115919910 LOC115925415 LOC373385 LOC587837 LOC588471 pou2f1

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	Figure 1. The model organism used in this study: Holothuria glaberrima Selenka, 1867 (Echinodermata: Holothuroidea).
	Figure 2. Histological organization of the radial organ complex in non-injured (A, B) and regenerating animals on day 2 (C, D), 12 (E, F), and 20 (G, H) post injury. All micrographs are longitudinal paraffin sections, except for A insert, which is a cross section. All sections were stained with Heindenheinâ€™s azan. The right column (B, D, F, H) shows high magnification views of the boxed areas in the corresponding images of the left column (A, C, E, G, respectively). e, epidermis; hc, hyponeural canal; lm, longitudinal muscle; rnc, radial nerve cord; wvc, water-vascular canal. Arrows show the location of the plane of injury. The arrowhead in F shows the growing tip of the regenerating radial nerve cord.
	Figure 3. Summary of major steps involved in library preparation, sequencing, assembly, and annotation workflow.

	Figure 5. Comparison of mRNA expression levels as determined by RNA-seq and qRT-PCR. Comparisons were performed for 21 genes at three time points of regeneration relative to uninjured animals (for the list of genes and numerical values, see Additional file 5). The red line is the linear regression line.
	Figure 6. Diagram showing clustering of functional annotation terms associated with differentially expressed genes during the radial organ complex regeneration. Differentially expressed genes that are up-regulated (right column) or down-regulated (left column) at a given time point of regeneration were analyzed for significantly enriched functional annotation terms, which were clustered using DAVID. Functional annotation clusters with enrichment scores (in negative log scale) > 1.3 are shown. The corresponding detailed data resulting from DAVID output are listed in Additional file 6).
	Figure 7. Clustering of co-expressed genes. The left row shows a heatmap produced by AutoSOME with clusters of co-expressed genes. Only eight clusters containing more than 100 genes are shown. The middle column shows median log2 fold-change in expression (vs uninjured animals) of the genes contained in each cluster. The clustered transcripts were analyzed with DAVID for significantly enriched functional annotation terms. The right column shows representative functional categories and their enrichment score.
	Figure 8. Putative regulation of co-expressed transcripts by transcription factors (TFs). Co-expressed genes contained in clusters identified with AutoSOME (FigureÂ 7) were analyzed for over-representation of putative TF binding sites using oPOSSUM.
	Figure 9. Expression of pluripotency factors in regeneration of the radial organ complex in the sea cucumber H. glaberrima. Summary of the digital expression assay and homology search against the UniProt database. Red and blue colors indicate significant (adjusted p < 0.001, more than two fold change in expression level) up-regulation or down-regulation, respectively, in the regenerating tissues as opposed to uninjured organs. NS, no significant changes in expression was observed. *Note that the gene designated as Oct1 is included in this list because it is the only Oct homolog found in the H. glaberrima transcriptome. Likewise, Oct1/2 is the only Oct in the sea urchin genome [44]. In mammals, there are a number of other Oct genes that perform various essential roles. One of them, Oct4 was used to induce pluripotency in mammalian somatic cells [41,45].

	Figure 11. Suppression of excitotoxicity as a possible adaptation contributing to efficient neural regeneration in echinoderms. (A) The relevant genes in the transcriptome of the sea cucumber H. glaberrima. Their expression levels (relative to the uninjured tissues) and results of homology search against the UniProt database. Red and blue colors indicate significant (adjusted p < 0.001, more than two fold change in expression level) up-regulation or down-regulation, respectively. (B) A diagram illustrating a hypothetical mechanism of excitotoxicity suppression in the injured radial nerve cord of H. glaberrima. Down-regulation of genes coding for two enzymes, GCPII and Abat, results in decreased glutamate production. Decreased activity of GCPII also leads to accumulation of its substrate, NAAG, which further inhibits release of glutamate from presynaptic terminals, but stimulates production of TGF-beta, which promotes neuronal survival. Down-regulation of ionotropic glutamate receptors (Glur1, Glur4) reduces the overstimulation of the post-synaptic membrane by glutamate and thus prevents downstream neurotoxic cascades from being triggered.

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