Distinct patterns of gene expression during regeneration and asexual reproduction in the annelid Pristina leidyi

Abstract

Regeneration, the ability to replace lost body parts, is a widespread phenomenon in the animal kingdom often connected to asexual reproduction or fission, since the only difference between the two appears to be the stimulus that triggers them. Both developmental processes have largely been characterized; however, the molecular toolkit and genetic mechanisms underlying these events remain poorly unexplored. Annelids, in particular the oligochaete Pristina leidyi, provide a good model system to investigate these processes as they show diverse ways to regenerate, and can reproduce asexually through fission under laboratory conditions. Here, we used a comparative transcriptomics approach based on RNA-sequencing and differential gene expression analyses to understand the molecular mechanisms involved in anterior regeneration and asexual reproduction. We found 291 genes upregulated during anterior regeneration, including several regeneration-related genes previously reported in other annelids such as frizzled, paics, and vdra. On the other hand, during asexual reproduction, 130 genes were found upregulated, and unexpectedly, many of them were related to germline development during sexual reproduction. We also found important differences between anterior regeneration and asexual reproduction, with the latter showing a gene expression profile more similar to that of control individuals. Nevertheless, we identified 35 genes that were upregulated in both conditions, many of them related to cell pluripotency, stem cells, and cell proliferation. Overall, our results shed light on the molecular mechanisms that control anterior regeneration and asexual reproduction in annelids and reveal similarities with other animals, suggesting that the genetic machinery controlling these processes is conserved across metazoans.

Keywords: Annelida; Pristina leidyi; differential gene expression; paratomic fission; regeneration; reproduction.

Figure 1

Anterior regeneration and asexual reproduction in Pristina leidyi. Brief description of major events, schematic representation and bright‐field microscopy images of the different stages defined for (a) anterior regeneration and (b) asexual reproduction through paratomic fission in the annelid P. leidyi. Scale bars = 100 μm (based on Zattara & Bely, 2011).

Figure 2

Gene expression patterns during anterior regeneration and asexual reproduction. (a) Hierarchically clustered heatmap of the 40 most upregulated, annotated, and nonredundant genes from pairwise comparisons of anteriorly regenerating, asexually reproducing and control individuals. Upregulated genes in both anteriorly regenerating and fissioning comparisons are shown in bold. (b) Scaled Venn diagram showing the overlapped annotated genes upregulated in both regeneration and asexual reproduction conditions. (c) Enriched Gene Ontology terms are associated with the 35 upregulated genes shared between anterior regeneration and asexual reproduction (molecular function category).

Figure 3

Gene expression patterns during anterior regeneration. (a) Volcano plot displaying the –log₁₀ p value (false discovery rate [FDR]) as a function of fold change in the regenerating and control individuals. Labeled genes are discussed in the text. (b) Hierarchically clustered heatmap of the most important upregulated annotated and nonredundant genes in this comparison, and their categories according to the main function discussed. (c) Gene Ontology enrichment analysis of the upregulated annotated genes. (d) Gene ontology enrichment analysis of the downregulated annotated genes.

Figure 4

Gene expression patterns during asexual reproduction by paratomic fission. (a) Volcano plot displaying the –log₁₀ p value (false discovery rate [FDR]) as a function of fold change in fissioning and control individuals. Labeled genes are discussed in the text. (b) Hierarchically clustered heatmap of the most important upregulated annotated and nonredundant genes in this comparison, and their categories according to the main function discussed. (c) Gene Ontology enrichment analysis of upregulated annotated genes. (d) Gene Ontology enrichment analysis of downregulated annotated genes.

Figure 5

Experimental design and sampling for RNA‐sequencing. After a 1‐week starvation period, worms were selected for the three experimental groups: (1) control group, with nonregenerative and nonreproducing individuals, (2) anterior regenerating group, with worms amputated at segment 7 and fixed 72 h post amputation, and (3) asexually reproducing group, with individuals showing a fission zone at the second stage. Three replicates per condition were prepared, consisting of a pool of 20 individuals each, at the same experimental stage. Following RNA extraction and complementary DNA library construction, the samples were sequenced using Illumina technology.