Comparative transcriptomics in Syllidae (Annelida) indicates that posterior regeneration and regular growth are comparable, while anterior regeneration is a distinct process

Abstract

Background: Annelids exhibit remarkable postembryonic developmental abilities. Most annelids grow during their whole life by adding segments through the action of a segment addition zone (SAZ) located in front of the pygidium. In addition, they show an outstanding ability to regenerate their bodies. Experimental evidence and field observations show that many annelids are able to regenerate their posterior bodies, while anterior regeneration is often limited or absent. Syllidae, for instance, usually show high abilities of posterior regeneration, although anterior regeneration varies across species. Some syllids are able to partially restore the anterior end, while others regenerate all lost anterior body after bisection. Here, we used comparative transcriptomics to detect changes in the gene expression profiles during anterior regeneration, posterior regeneration and regular growth of two syllid species: Sphaerosyllis hystrix and Syllis gracilis; which exhibit limited and complete anterior regeneration, respectively.

Results: We detected a high number of genes with differential expression: 4771 genes in S. hystrix (limited anterior regeneration) and 1997 genes in S. gracilis (complete anterior regeneration). For both species, the comparative transcriptomic analysis showed that gene expression during posterior regeneration and regular growth was very similar, whereas anterior regeneration was characterized by up-regulation of several genes. Among the up-regulated genes, we identified putative homologs of regeneration-related genes associated to cellular proliferation, nervous system development, establishment of body axis, and stem-cellness; such as rup and JNK (in S. hystrix); and glutamine synthetase, elav, slit, Hox genes, β-catenin and PL10 (in S. gracilis).

Conclusions: Posterior regeneration and regular growth show no significant differences in gene expression in the herein investigated syllids. However, anterior regeneration is associated with a clear change in terms of gene expression in both species. Our comparative transcriptomic analysis was able to detect differential expression of some regeneration-related genes, suggesting that syllids share some features of the regenerative mechanisms already known for other annelids and invertebrates.

Keywords: Annelida; Hox genes; JNK; PL10; RNA-seq; Regeneration; Syllidae; Transcriptome; β-Catenin.

Fig. 1

Regeneration timeline of the specimens sequenced for transcriptomic data. Bisection was performed in the midbody site and the amputees were fixed for sequencing in the first stages of regeneration: stage 1 (healing), stage 2 (early blastema development), stage 3 (late blastema development), and stage 4 (patterning/cap regeneration). Anterior regeneration sequencing cover stages 1–3; posterior regeneration covers all the stages. Time-scale of experimentation: 12 days for Sphaerosyllis hystrix and 8 days for Syllis gracilis (see Methods)

Fig. 2

Light microscopy pictures of the regenerating Sphaerosyllis hystrix. a, b, c, g, h, i anterior regeneration. d, e, f, j, k, l posterior regeneration. Amputation was performed in the midbody region and the regenerating animals were observed for 14 days post amputation (dpa). Immediately after body bisection, the wound is closed by invagination through muscle contraction. Anterior regeneration starts by wound healing (1–3 dpa) and the formation of a small blastema (a). The anterior blastema is formed after 4–6 dpa and no differentiated organ is regenerated until 12 dpa (b, c, g). An incomplete prostomium (head) appeared after 13 dpa, bearing eyes (h), and a pair of minute antennae in 14 dpa (i). Posterior regeneration proceeds more quickly: healing occurred in 2 dpa, the blastema developed from 2 to 4 dpa, and a pygidium with a pair of cirri was first seen after 9 dpa (d, e, f). From 10 to 14 dpa, amputees had regrown new pygidia and a maximum of four posterior segments (j–l). All pictures are in dorsal view. Scale bar 0.2 mm. White dashed lines show amputation level. Black dashed lines show the regenerated eyes. Abs: an, antenna; ey, eye

Fig. 3

Light microscopy pictures of the regenerating Syllis gracilis. a, b, c, g, h, i anterior regeneration. d, e, f, j, k, l posterior regeneration. Anterior and posterior regeneration of S. gracilis were observed during 8 dpa. The wound is completely healed after 2 dpa and a blastema develops during the following days in both anterior and posterior regeneration. After 8dpa, the blastema was still elongating during anterior regeneration (a–c, g–i). Regarding posterior regeneration, the blastema differentiated between 4 and 7 dpa; after 8 dpa a pygidium bearing three short cirri was restored (d–f, j–l). All pictures are in dorsal view. Scale bar 0.2 mm. White dashed lines show amputation region

Fig. 4

Heatmaps of differentially expressed genes during regeneration (FDR < 0.001). a Sphaerosyllis hystrix results. b Syllis gracilis results. Note that some of the genes can be up-regulated in more than one condition. Values in centred log₂(fpkm+ 1). AR: anterior regeneration, PR: posterior regeneration, NR: non-regenerating. See Additional file 2: Table S1 and Additional file 3: Table S7 for detailed results

Fig. 5

Results of gene ontology annotation of DE genes. Only the ten most significant enriched GO terms are plotted. a AxP comparison and b AxN comparison for Sphaerosyllis hystrix. c AxP comparison and d AxN comparison for Syllis gracilis. CAT: category; BP: biological process, CC: cellular component, MF: molecular function. Z-score is useful to know if the expression of genes belonging to a certain GO term is more likely to be decreasing (negative) or increasing (positive) and it is calculated as the number of up-regulated genes minus the number of down-regulated genes divided by the square root of the gene count [58]. Up-regulated genes have logFC> 0, and down-regulated genes have logFC< 0. Inner boxes size is based on the p-value and represents the significance of the enrichment of each GO term. Output data of the GOplot analyses is available in Additional file 2: Tables S5 and S6, and Additional file 3: Tables S11 and S12