RNA-Seq reveals dynamic changes of gene expression in key stages of intestine regeneration in the sea cucumber Apostichopus japonicus. [corrected]

Abstract

Background: Sea cucumbers (Holothuroidea; Echinodermata) have the capacity to regenerate lost tissues and organs. Although the histological and cytological aspects of intestine regeneration have been extensively studied, little is known of the genetic mechanisms involved. There has, however, been a renewed effort to develop a database of Expressed Sequence Tags (ESTs) in Apostichopus japonicus, an economically-important species that occurs in China. This is important for studies on genetic breeding, molecular markers and special physiological phenomena. We have also constructed a library of ESTs obtained from the regenerative body wall and intestine of A. japonicus. The database has increased to ~30000 ESTs.

Results: We used RNA-Seq to determine gene expression profiles associated with intestinal regeneration in A. japonicus at 3, 7, 14 and 21 days post evisceration (dpe). This was compared to profiles obtained from a normally-functioning intestine. Approximately 5 million (M) reads were sequenced in every library. Over 2400 up-regulated genes (>10%) and over 1000 down-regulated genes (~5%) were observed at 3 and 7dpe (log2Ratio ≥ 1, FDR ≤ 0.001). Specific "Go terms" revealed that the DEGs (Differentially Expressed Genes) performed an important function at every regeneration stage. Besides some expected pathways (for example, Ribosome and Spliceosome pathway term), the "Notch signaling pathway," the "ECM-receptor interaction" and the "Cytokine-cytokine receptor interaction" were significantly enriched. We also investigated the expression profiles of developmental genes, ECM-associated genes and Cytoskeletal genes. Twenty of the most important differentially expressed genes (DEGs) were verified by Real-time PCR, which resulted in a trend concordance of almost 100% between the two techniques.

Conclusion: Our studies demonstrated dynamic changes in global gene expression during intestine regeneration and presented a series of candidate genes and enriched pathways that contribute to intestine regeneration in sea cucumbers. This provides a foundation for future studies on the genetics/molecular mechanisms associated with intestine regeneration.

Figure 1. The change level of global differential expression genes during intestine regeneration in sea cucumber A. japonicus.

The principle "FDR≤0.001 and the absolute value of log2Ratio≥1” was used as a threshold to screen DEGs. The figure showed that not only a large number of genes were differentially expressed, but the change fold of differentially expression was at a high level, especially at the early stage of intestine regeneration.

Figure 2. The expression pattern of top significantly differentially expressed genes at 3dpe, 7dpe, 14dpe and 21dpe.

Expression differences were shown in different colors. Red mean up regulation and green mean down regulation.

Figure 3. Real-time PCR analysis for top 10 up-regulated and 10 down-regulated genes.

(A). LDP: low density lipoprotein-related protein 2-like; GL 2: GL12416-like isoform 2; speA: speedy A; RAP: regeneration associated protein; OTD: orthodenticle SCF: solute carrier family 6 member 9 transcript-like; CB: cyclin B3 TFP: TFP250 HE7: Hu/elav isoform 7; his1: cleavage stage histone H1. (B). Rp2: Rp2 Lipase; CRBP: cellular retinol-binding protein type 1b; FCBP: fatty acid binding protein 2, intestinal; PCSK: proprotein convertase subtilisin/kexin type 9; AMY: alpha-amylase; GAP: FG-GAP repeat family protein; LOC: LOC495367 protein; TAG: triacylglycerol lipase, pancreatic; LYS: lysozyme; CBG: cytosolic beta-glucosidase-like. Different lowercase letters indicate significant differences (P<0.05). Values indicate the mean ± S.E. (N = 5).

Figure 4. Distribution of gene ontology (GO) terms of differentially expressed genes during intestine regeneration.

The percentage of GO-terms in the categories “Molecular function”,“Biological Process” and “Cellular component” was shown.