Wnt Signaling Pathway Linked to Intestinal Regeneration via Evolutionary Patterns and Gene Expression in the Sea Cucumber Apostichopus japonicus

Abstract

Many echinoderms are regenerative species that exhibit exceptional regenerative capacity, and sea cucumber is a representative organism that could regenerate the whole intestine after evisceration. There are many signaling pathways participate in the regeneration process, but it is not clear which is essential for the intestinal regeneration. In this study, we performed genome-wide comprehensive analyses on these regeneration-related signaling pathways, and found the Wnt signaling pathway was one of the most conservative pathways among regenerative species. Additionally, among these signaling pathways, we found that the Wnt signaling pathway was the only one under positive selection in regenerative echinoderms, and the only one enriched by differentially expressed genes during the intestinal regeneration. Thus, it suggests both coding sequence and gene expression of the Wnt signaling pathway have been shaped by natural selection to provide the genetic architecture for intestinal regeneration. Wnt7, Fz7, and Dvl are the three positively selected genes and also happen to be three upstream genes in the Wnt signaling pathway. They are all significantly upregulated at the early stages of regeneration, which may contribute significantly to the early activation of Wnt signaling and the initiation of intestinal regeneration. Expression knockdown of Wnt7 and Dvl by RNA interference significantly inhibit intestinal extension, implying that they are essential for intestinal regeneration. As an important regeneration-related gene, the downstream gene c-Myc is also conserved and highly expressed during the whole regeneration stages, which may make the Wnt/c-Myc signaling to be an important way to promote intestinal regeneration. Therefore, it is reasonable to conclude that the Wnt signaling pathway is the chosen one to play an important role in intestinal regeneration of sea cucumbers, or even in the regeneration of other echinoderms.

Keywords: Wnt signaling pathway; intestinal regeneration; natural selection; positive selection; sea cucumber.

FIGURE 1

Methods of identifying key pathways and genes in regeneration. The methods combined the results of gene gain/gene loss analysis, positive selection analysis, and DGE analysis.

FIGURE 2

Comparative analysis of genes in regeneration-related signaling pathways of regenerative animals. The comparative analysis are performed on seven regeneration-related signaling pathways of six species. The genes present in genomes are markered in green background, and the genes absent in genomes are markered in other colors according to the legends.

FIGURE 3

Wnt gene family in echinoderms. (A) Phylogenetic tree of the Wnt gene family of three echinoderms and Saccoglossus kowalevskii. The phylogenetic tree is constructed using the maximum likelihood (ML) method with 1000 bootstraps and Bayesian inference (BI). Yellow circles indicate the support values of ML analysis larger than 80%. The support values of BI analysis are displayed beside each node. (B) Comparison of Wnt gene family members across echinoderms and S. kowalevskii. Empty indicate the loss of particular Wnt genes, and overlapping boxes represent duplicated Wnt genes.

FIGURE 4

Differential expression analysis of genes during intestinal regeneration. (A) Expression profiles of the DEGs at different intestinal regeneration time points: 0 (C), 3 day (3d), 5 day (5d), 7 day (7d), 14 day (14d), 21 day (21d). DEGs are divided into five groups that significantly expressed at 3d (3d versus C), 5d (5d versus 3d), 7d (7d versus 5d), 14d (14d versus 7d), and 21d (21d versus 14d). (B) Significantly enriched pathways of DEGs that significantly upregulated at 3d. RichFactor is the ratio of the number of DEGs in this pathway term to the number of all genes in this pathway term.

FIGURE 5

Differential expression analysis of Wnt signaling pathway genes. (A) Expression profiles of the DEGs at different intestinal regeneration time points: 0 (C), 3 day (3d), 5 day (5d), 7 day (7d), 14 day (14d), 21 day (21d). Genes with different colors are correspondent to the color of genes in the plot B. (B) Genes involved in Wnt signaling pathway. Genes from upstream to downstream of the Wnt signaling pathway are in different color.

FIGURE 6

Real-time PCR analysis of four genes during intestinal regeneration. Normal tissues of the intestine were treated as the control group. The values were normalized against NADH. Data are the mean ± standard deviation of the triplicate experiments. Different lowercase letters indicate significant differences (p < 0.05).

FIGURE 7

The intestinal regeneration after injection of dsRNA and inhibitor. (A) Photos of the intestinal regeneration after injection of inhibitor (10 and 2 mg/kg), dsWnt7, dsDvl, and dsEGFP (Control). As the performance of the negative controls of dsRNA and inhibitor were similar, only the control of dsRNA was displayed. (B) Distributions of the intestinal lengths of different treatment groups. Independent samples t-test was used to test the significant differences. ^∗∗p < 0.01, ^∗p < 0.05, and N.S., significant differences.