Xiaoni Li

The sea cucumber genome provides insights into morphological evolution and visceral regeneration

Apart from sharing common ancestry with chordates, sea cucumbers exhibit a unique morphology and exceptional regenerative capacity. Here we present the complete genome sequence of an economically important sea cucumber, A. japonicus, generated using Illumina and PacBio platforms, to achieve an assembly of approximately 805 Mb (contig N50 of 190 Kb and scaffold N50 of 486 Kb), with 30,350 protein-coding genes and high continuity. We used this resource to explore key genetic mechanisms behind the unique biological characters of sea cucumbers. Phylogenetic and comparative genomic analyses revealed the presence of marker genes associated with notochord and gill slits, suggesting that these chordate features were present in ancestral echinoderms. The unique shape and weak mineralization of the sea cucumber adult body were also preliminarily explained by the contraction of biomineralization genes. Genome, transcriptome, and proteome analyses of organ regrowth after induced evisceration provided insight into the molecular underpinnings of visceral regeneration, including a specific tandem-duplicated prostatic secretory protein of 94 amino acids (PSP94)-like gene family and a significantly expanded fibrinogen-related protein (FREP) gene family. This high-quality genome resource will provide a useful framework for future research into biological processes and evolution in deuterostomes, including remarkable regenerative abilities that could have medical applications. Moreover, the multiomics data will be of prime value for commercial sea cucumber breeding programs.

Differential Expression of miRNAs in the Respiratory Tree of the Sea Cucumber Apostichopus japonicus Under Hypoxia Stress

The sea cucumber, an important economic species, has encountered high mortality since 2013 in northern China because of seasonal environmental stress such as hypoxia, high temperature, and low salinity. MicroRNAs (miRNAs) are important in regulating gene expression in marine organisms in response to environmental change. In this study, high-throughput sequencing was used to investigate alterations in miRNA expression in the sea cucumber under different levels of dissolved oxygen (DO). Nine small RNA libraries were constructed from the sea cucumber respiratory trees. A total of 26 differentially expressed miRNAs, including 12 upregulated and 14 downregulated miRNAs, were observed in severe hypoxia (DO 2 mg/L) compared with mild hypoxia (DO 4 mg/L) and normoxic conditions (DO 8 mg/L). Twelve differentially expressed miRNAs were clustered in severe hypoxia. In addition, real-time PCR revealed that 14 randomly selected differentially expressed miRNAs showed significantly increased expressions in severe hypoxia and the expressions of nine miRNAs, including key miRNAs such as Aja-miR-1, Aja-miR-2008, and Aja-miR-184, were consistent with the sequencing results. Moreover, gene ontology and pathway analyses of putative target genes suggest that these miRNAs are important in redox, transport, transcription, and hydrolysis under hypoxia stress. Notably, novel-miR-1, novel-miR-2, and novel-miR-3 were specifically clustered and upregulated in severe hypoxia, which may provide new insights into novel “hypoxamiR” identification. These results will provide a basis for future studies of miRNA regulation and molecular adaptive mechanisms in sea cucumbers under hypoxia stress.

Understanding regulation of microRNAs on intestine regeneration in the sea cucumber Apostichopus japonicus using high-throughput sequencing

The sea cucumber, as a member of the Echinodermata, has the capacity to restore damaged organs and body parts, which has always been a key scientific issue. MicroRNAs (miRNAs), a class of short noncoding RNAs, play important roles in regulating gene expression. In the present study, we applied high-throughput sequencing to investigate alterations of miRNA expression in regenerative intestine compared to normal intestine. A total of 73 differentially expressed miRNAs were obtained, including 59 up-regulated miRNAs and 14 down-regulated miRNAs. Among these molecules, Aja-miR-1715-5p, Aja-miR-153, Aja-miR-252a, Aja-miR-153-5p, Aja-miR-252b, Aja-miR-2001, Aja-miR-64d-3p, and Aja-miR-252-5p were differentially expressed over 10-fold at 3days post-evisceration (dpe). Notably, real-time PCR revealed that Aja-miR-1715-5p was up-regulated 1390-fold at 3dpe. Moreover, putative target gene co-expression analyses, gene ontology, and pathway analyses suggest that these miRNAs play important roles in specific cellular events (cell proliferation, migration, and apoptosis), metabolic regulation, and energy redistribution. These results will provide a basis for future studies of miRNA regulation in sea cucumber regeneration.

Comparison of pigment composition and melanin content among white, light-green, dark-green, and purple morphs of sea cucumber, Apostichopus japonicus

Sea cucumber, Apostichopus japonicus (Selenka), is a commercially important marine species in China. Among the differently colored varieties sold in China, white and purple sea cucumbers have the greatest appeal to consumers. Identification of the pigments that may contribute to the formation of different color morphs of sea cucumbers will provide a scientific basis for improving the cultivability of desirable color morphs. In this study, sea cucumbers were divided into four categories according to their body color: white, light green, dark green, and purple. The pigment composition and contents in the four groups were analyzed by high performance liquid chromatography (HPLC). The results show that the pigment contents differed significantly among the white, lightgreen, dark-green, and purple sea cucumbers, and there were fewer types of pigments in white sea cucumber than in the other color morphs. The only pigments detected in white sea cucumbers were guanine and pteroic acid. Guanine and pteroic acid are structural colors, and they were also detected in light-green, dark-green, and purple sea cucumbers. Every pigment detected, except for pteroic acid, was present at a higher concentration in purple morphs than in the other color morphs. The biological color pigments melanin, astaxanthin, β-carotene, and lutein were detected in light-green, dark-green, and purple sea cucumbers. While progesterone and lycopene, which are also biological color pigments, were not detected in any of the color morphs. Melanin was the major pigment contributing to body color, and its concentration increased with deepening color of the sea cucumber body. Transmission electron microscopy analyses revealed that white sea cucumbers had the fewest epidermal melanocytes in the body wall, and their melanocytes contained fewer melanosomes as well as non-pigmented pre-melanosomes. Sea cucumbers with deeper body colors contained more melanin granules. In the body wall of dark-green and purple sea cucumbers, melanin granules were secreted out of the cell. The results of this study provide evidence for the main factors responsible for differences in coloration among white, light-green, darkgreen, and purple sea cucumbers, and also provide the foundation for further research on the formation of body color in sea cucumber, A. japonicus.

Metabolic responses to intestine regeneration in sea cucumbers Apostichopus japonicus

Sea cucumbers are excellent models for studying organ regeneration due to their striking capacity to regenerate most of their viscera after evisceration. In this study, we applied NMR-based metabolomics to determine the metabolite changes that occur during the process of intestine regeneration in sea cucumbers. Partial least-squares discriminant analysis showed that there was significant differences in metabolism between regenerative intestines at 3, 7, and 14days post evisceration (dpe) and normal intestines. Changes in the concentration of 13 metabolites related to regeneration were observed and analyzed. These metabolites included leucine, isoleucine, valine, arginine, glutamate, hypotaurine, dimethylamine, N,N-dimethylglycine, betaine, taurine, inosine, homarine, and histidine. Three important genes (betaine-aldehyde dehydrogenase, betaine-homocysteine S-methyltransferase 1, and dimethylglycine dehydrogenase) were differentially expressed to regulate the levels of betaine and N,N-dimethylglycine during intestine regeneration. These results provide an important basis for studying regenerative mechanisms and developing regenerative matrixes.

Extracellular matrix remodeling and matrix metalloproteinases (ajMMP-2 like and ajMMP-16 like) characterization during intestine regeneration of sea cucumber Apostichopus japonicus

Remodeling of extracellular matrix (ECM) regulated by matrix metalloproteinases (MMPs) is essential for tissue regeneration. In the present study, we used immunohistochemistry (IHC) techniques against ECM components to reveal changes of ECM during intestine regeneration of Apostichopus japonicus. The expression of collagen I and laminin reduced apparently from the eviscerated intestine, while fibronectin exhibited continuous expression in all regeneration stages observed. Meanwhile, we cloned two MMP genes from A. japonicus by RACE PCR. The full-length cDNA of ajMMP-2 like is 2733bp and contains a predicted open reading frame (ORF) of 1716bp encoding 572 amino acids. The full-length cDNA of ajMMP-16 like is 2705bp and contains an ORF of 1452bp encoding 484 amino acids. The predicted protein sequences of each MMP contain two conserved domains, ZnMc_MMP and HX. Homology and phylogenetic analysis revealed that ajMMP-2 like and ajMMP-16 like share high sequence similarity with MMP-2 and MMP-16 from Strongylocentrotus purpuratus, respectively. Then we investigated spatio-temporal expression of ajMMP-2 like and ajMMP-16 like during different regeneration stages by qRT-PCR and IHC. The expression pattern of them showed a roughly opposite trend from that of ECM components. According to our results, a fibronectin-dominate temporary matrix is created in intestine regeneration, and it might provide structural integrity for matrix and promote cell movement. We also hypothesize that ajMMP-2 like and ajMMP-16 like could accelerate cell migration and regulate interaction between ECM components and growth factors. This work provides new evidence of ECM and MMPs involvement in sea cucumber regeneration.

Comparative Phospho- and Acetyl Proteomics Analysis of Posttranslational Modifications Regulating Intestine Regeneration in Sea Cucumbers

Sea cucumbers exposed to stressful circumstances eviscerate most internal organs, and then regenerate them rapidly under favorable environments. Reversible protein phosphorylation and acetylation are major modifications regulating protein function. Herein, for the first time, we perform quantitative phospho- and acetyl proteomics analyses of intestine regeneration in a sea cucumber species Apostichopus japonicus. We identified 1,862 phosphorylation sites in 1,169 proteins, and 712 acetylation sites in 470 proteins. Of the 147 and 251 proteins differentially modified by phosphorylation and acetylation, respectively, most were related to cytoskeleton biogenesis, protein synthesis and modification, signal recognition and transduction, energy production and conversion, or substance transport and metabolism. Phosphorylation appears to play a more important role in signal recognition and transduction than acetylation, while acetylation is of greater importance in posttranslational modification, protein turnover, chaperones; energy production and conversion; amino acid and lipid transport and metabolism. These results expanded our understanding of the regulatory mechanisms of posttranslational modifications in intestine regeneration of sea cucumbers after evisceration.

Identification and expression characterization of WntA during intestinal regeneration in the sea cucumber Apostichopus japonicus

Wnt genes encode secreted glycoproteins that act as signaling molecules; these molecules direct cell proliferation, migration, differentiation and survival during animal development, maintenance of homeostasis and regeneration. At present, although the regeneration mechanism in Apostichopus japonicus has been studied, there is a little research on the Wnt signaling pathway in A. japonicus. To understand the potential role of the Wnt signaling pathway in A. japonicus, we cloned and sequenced the WntA gene in A. japonicus. Protein localization analysis showed that WntA protein was ubiquitously expressed in epidermal cells, the muscle and submucosa of the intestinal tissue. After stimulation and evisceration, the dynamic changes in expression of the WntA gene and protein showed that WntA was constitutively expressed during different stages of intestine regeneration in A. japonicus, with higher levels during the early wound healing stage and late lumen formation in the residual and nascent intestinal tissues, indicating its response to intestinal regeneration. Simultaneously, cell proliferation and apoptosis analysis showed that the patterns of cell proliferation were similar to the patterns of WntA protein expression during different intestinal regeneration stages in this organism. Taken together, these results suggested that WntA might participate in intestinal regeneration and may be connected with cell proliferation, apoptosis in different intestinal layers. This research could establish a basis for further examination of WntA functions in A. japonicus and Wnt genes in other echinoderms.