Transcriptomic analysis of early stages of intestinal regeneration in Holothuria glaberrima

Abstract

Echinoderms comprise a group of animals with impressive regenerative capabilities. They can replace complex internal organs following injury or autotomy. In holothurians or sea cucumbers, cellular processes of intestinal regeneration have been extensively studied. The molecular machinery behind this faculty, however, remains to be understood. Here we assembled and annotated a de novo transcriptome using RNA-seq data consisting of regenerating and non-regenerating intestinal tissues from the sea cucumber Holothuria glaberrima. Comparisons of differential expression were made using the mesentery as a reference against 24 h and 3 days regenerating intestine, revealing a large number of differentially expressed transcripts. Gene ontology and pathway enrichment analysis showed evidence of increasing transcriptional activity. Further analysis of transcripts associated with transcription factors revealed diverse expression patterns with mechanisms involving developmental and cancer-related activity that could be related to the regenerative process. Our study demonstrates the broad and diversified gene expression profile during the early stages of the process using the mesentery as the focal point of intestinal regeneration. It also establishes the genes that are the most important candidates in the cellular processes that underlie regenerative responses.

Figure 1

Diagram illustrating the intestine composition of normal and regenerating stages in H. glaberrima. (A) Normal intestine and attached mesentery. The other end of the mesentery (not shown) is attached to the body wall. (B) Mesentery one day after evisceration of the intestine. (C) Mesentery three days following evisceration Distribution of tissues in normal intestine and mesentery and in regenerating mesentery/intestine at 1 and 3 days post-evisceration (dpe).

Figure 2

Gene expression patterns of RNA-seq and correlation with RT-qPCR. (A) Volcano plot of differentially expressed transcripts (DETs) at day 1 of regeneration. (B) Volcano plot of DETs at day 3 of regeneration. (C) Bar plots show upregulated and downregulated DETs in one or both days of regeneration. (D) Plot showing correlation line, squared Pearson correlation coefficient and p-value between 12 expression values (Log2FC) of seven genes in regeneration stages compared to the normal stage. RNA-seq values are located in the x axis and RT-qPCR values are located on the y axis. Green colored points are from day 1 values, while red colored are from day 3.

Figure 3

Gene ontology enrichment analysis of differentially expressed genes (DEGs) at day 1 and day 3 of regeneration using DAVID database. Ontology domains comprise biological process (BP), cellular component (CC) and molecular function (MF). The GeneRation is a measure of the gene count divided by the number of genes in the total group of genes assayed that belong to the specific gene category.

Figure 4

Pathway enrichment analysis of differentially expressed genes (DEGs) at day 1 and day 3 of regeneration using KEGG database. The GeneRation is a measure of the number of genes in the differentially expressed set divided by the total number of genes for that specific pathway found in KEGG.

Figure 5

Heatmap of differentially expressed transcription factors (DETFs) in non-regenerating stage mesentery (Normal), and regenerating (days 1 3) intestines. DETFs showing a similar expression profiles are grouped in the dendrogram and labeled by colors in the right part of the heatmap. Logarithm count measures for expression values encompass non-negative integer values from the expression analysis.