TRIM21 of Micropterus salmoides exerts antiviral roles against largemouth bass ulcer syndrome virus.

Tripartite motif 21 (TRIM21), a member of the TRIM family, plays an important role in apoptosis, autophagy and ubiquitination in human, and has been proven to play antiviral roles in different organisms. In this study, the TRIM21 gene of Micropterus salmoides (MsTRIM21) was cloned, and it encoded 376 amino acids, which showed 89.3% similarity with Micropterus dolomieu and 38.3% with homo sapiens. Bioinformatics analysis revealed MsTRIM21 contained four domains: C4HC3-type RING-variant (RINGv), coiled coil, PRY and SPRY. The high expression level of MsTRIM21 could be detected in liver, stomach and muscle of healthy Micropterus salmoides, and it was significantly upregulated in head kidney, muscle, gill and brain and significantly down-regulated in the stomach of Micropterus salmoides infected with largemouth bass ulcer syndrome virus (LBUSV). The overexpression of MsTRIM21 could significantly inhibit the viral replication in vitro, evidenced by the reduction of CPE severity and the downregulation of the viral gene transcription. In addition, the overexpression of MsTRIM21 could significantly increase the expression level of interferon regulatory factor (IRF) 3, IRF7, myxovirus resistance 1 (Mx1), interferon stimulated gene 15 (ISG15), double-stranded RNA-activated protein kinase (PKR) and tumor necrosis factor α (TNF-α) in vitro, indicating the enhancement of innate immune response and inflammatory response, which may directly affect the replication of LBUSV. Thus, these results provide new lights on the roles of fish TRIM21 in innate immune response against iridovirus.

Gene Regulation during Carapacial Ridge Development of Mauremys reevesii: The Development of Carapacial Ridge, Ribs and Scutes.

The unique topological structure of a turtle shell, including the special ribs-scapula relationship, is an evolutionarily novelty of amniotes. The carapacial ridge is a key embryonic tissue for inducing turtle carapace morphologenesis. However, the gene expression profiles and molecular regulatory mechanisms that occur during carapacial ridge development, including the regulation mechanism of rib axis arrest, the development mechanism of the carapacial ridge, and the differentiation between soft-shell turtles and hard-shell turtles, are not fully understood. In this study, we obtained genome-wide gene expression profiles during the carapacial ridge development of Mauremys reevesii using RNA-sequencing by using carapacial ridge tissues from stage 14, 15 and 16 turtle embryos. In addition, a differentially expressed genes (DEGs) analysis and a gene set enrichment analysis (GSEA) of three comparison groups were performed. Furthermore, a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was used to analyze the pathway enrichment of the differentially expressed genes of the three comparative groups. The result displayed that the Wnt signaling pathway was substantially enriched in the CrTK14 vs. the CrTK15 comparison group, while the Hedgehog signaling pathway was significantly enriched in the CrTK15 vs. the CrTK16 group. Moreover, the regulatory network of the Wnt signaling pathway showed that Wnt signaling pathways might interact with Fgfs, Bmps, and Shh to form a regulatory network to regulate the carapacial ridge development. Next, WGCNA was used to cluster and analyze the expression genes during the carapacial ridge development of M. reevesii and P. sinensis. Further, a KEGG functional enrichment analysis of the carapacial ridge correlation gene modules was performed. Interesting, these results indicated that the Wnt signaling pathway and the MAPK signaling pathway were significantly enriched in the gene modules that were highly correlated with the stage 14 and stage 15 carapacial ridge samples of the two species. The Hedgehog signaling pathway was significantly enriched in the modules that were strongly correlated with the stage 16 carapacial ridge samples of M. reevesii, however, the PI3K-Akt signaling and the TGF-ß signaling pathways were significantly enriched in the modules that were strongly correlated with the stage 16 carapacial ridge samples of P. sinensis. Furthermore, we found that those modules that were strongly correlated with the stage 14 carapacial ridge samples of M. reevesii and P. sinensis contained Wnts and Lef1. While the navajo white 3 module which was strongly correlated with the stage 16 carapacial ridge samples of M. reevesii contained Shh and Ptchs. The dark green module strongly correlated with the stage 16 carapacial ridge samples of P. sinensis which contained Col1a1, Col1a2, and Itga8. Consequently, this study systematically revealed the signaling pathways and genes that regulate the carapacial ridge development of M. reevesii and P. sinensis, which provides new insights for revealing the molecular mechanism that is underlying the turtle's body structure.

Global Analysis of Transcriptome and Translatome Revealed That Coordinated WNT and FGF Regulate the Carapacial Ridge Development of Chinese Soft-Shell Turtle.

The turtle carapace is composed of severely deformed fused dorsal vertebrae, ribs, and bone plates. In particular, the lateral growth in the superficial layer of turtle ribs in the dorsal trunk causes an encapsulation of the scapula and pelvis. The recent study suggested that the carapacial ridge (CR) is a new model of epithelial-mesenchymal transition which is essential for the arrangement of the ribs. Therefore, it is necessary to explore the regulatory mechanism of carapacial ridge development to analyze the formation of the turtle shell. However, the current understanding of the regulatory network underlying turtle carapacial ridge development is poor due to the lack of both systematic gene screening at different carapacial ridge development stages and gene function verification studies. In this study, we obtained genome-wide gene transcription and gene translation profiles using RNA sequencing and ribosome nascent-chain complex mRNA sequencing from carapacial ridge tissues of Chinese soft-shell turtle at different development stages. A correlation analysis of the transcriptome and translatome revealed that there were 129, 670, and 135 codifferentially expressed genes, including homodirection and opposite-direction differentially expressed genes, among three comparison groups, respectively. The pathway enrichment analysis of codifferentially expressed genes from the Kyoto Encyclopedia of Genes and Genomes showed dynamic changes in signaling pathways involved in carapacial ridge development. Especially, the results revealed that the Wnt signaling pathway and MAPK signaling pathway may play important roles in turtle carapacial ridge development. In addition, Wnt and Fgf were expressed during the carapacial ridge development. Furthermore, we discovered that Wnt5a regulated carapacial ridge development through the Wnt5a/JNK pathway. Therefore, our studies uncover that the morphogenesis of the turtle carapace might function through the co-operation between conserved WNT and FGF signaling pathways. Consequently, our findings revealed the dynamic signaling pathways acting on the carapacial ridge development of Chinese soft-shell turtle and provided new insights into uncover the molecular mechanism underlying turtle shell morphogenesis.