PDF(Sci-hub)
Abstract：
Metamorphosis takes place within the life cycle of most marine invertebrates. The marine ascidian is a classical model to study complex cellular processes and underlying molecular mechanisms involved in its larval metamorphosis. The detailed molecular signaling pathways remain elusive, though extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinase (JNK) have been revealed to regulate cell migration, differentiation, and apoptosis in ascidian larval organ regression and juvenile organ development. MicroRNAs (miRNAs) are small non-coding RNAs that modulate gene expression at the post-transcriptional level. Large numbers of miRNAs have been demonstrated to be involved in many developmental and metamorphic processes. However, the identification of miRNAs in ascidian larval metamorphosis has not yet been investigated.
Totally, 106 known and 59 novel miRNAs were screened out through RNA-sequencing of three small RNA libraries from 18 to 21-h post-fertilization (hpf) tailbud embryos as well as from 42 hpf larvae (after tail regression) in Ciona savignyi. Expression profiling of miRNAs was confirmed by quantitative real-time PCR, showing that the expression levels of csa-miR-4040, csa-miR-4086, csa-miR-4055, csa-miR-4060, csa-miR-216a, csa-miR-216b, csa-miR-217, csa-miR-183, and csa-miR-92c were significantly higher in 42 hpf larvae, whereas those of csa-miR-4018a, csa-miR-4018b, and csa-miR-4000f were higher in 18 and 21 hpf embryos; then, their expression in 42 hpf larvae became significantly low. For these 12 miRNAs, whose expression levels significantly changed, we predicted their target genes through the combination of miRanda and TargetScan. This prediction analysis revealed 332 miRNA-target gene pairs that were associated with the ERK, JNK, and transforming growth factor beta signaling pathways, suggesting that the identified miRNAs are involved in the regulation of C. savignyi larval metamorphosis via controlling the expression of their target genes. Furthermore, we validated the expression of five selected miRNAs by northern blotting. Among the selected miRNAs, the expression patterns of csa-miR-4018a, csa-miR-4018b, and csa-miR-4000f were further examined by whole-mount in situ hybridization. The results showed that all three miRNAs were specifically expressed in a cell population resembling mesenchymal cells at the head and trunk part in swimming larvae but not in metamorphic larvae. Utilizing the luciferase assay, we also confirmed that miR-4000f targeted Mapk1, suggesting that the csa-miR-4018a/csa-miR-4018b/csa-miR-4000f cluster regulates larval metamorphosis through the Mapk1-mediated signaling pathway.
Totally, 165 miRNAs, including 59 novel ones, were identified from the embryos and larvae of C. savignyi. Twelve of them showed significant changes in expression before and during metamorphosis. In situ hybridization and northern blotting results revealed that three miRNAs are potentially involved in the signaling regulatory network for the migration and differentiation of mesenchymal cells in larval metamorphosis. Furthermore, the luciferase reporter assay revealed that Mapk1 is a target of csa-miR-4000f. Our results not only present a list and profile of miRNAs involved in Ciona metamorphosis but also provide informative cues to further understand their function in ascidian larval metamorphosis.
Totally, 106 known and 59 novel miRNAs were screened out through RNA-sequencing of three small RNA libraries from 18 to 21-h post-fertilization (hpf) tailbud embryos as well as from 42 hpf larvae (after tail regression) in Ciona savignyi. Expression profiling of miRNAs was confirmed by quantitative real-time PCR, showing that the expression levels of csa-miR-4040, csa-miR-4086, csa-miR-4055, csa-miR-4060, csa-miR-216a, csa-miR-216b, csa-miR-217, csa-miR-183, and csa-miR-92c were significantly higher in 42 hpf larvae, whereas those of csa-miR-4018a, csa-miR-4018b, and csa-miR-4000f were higher in 18 and 21 hpf embryos; then, their expression in 42 hpf larvae became significantly low. For these 12 miRNAs, whose expression levels significantly changed, we predicted their target genes through the combination of miRanda and TargetScan. This prediction analysis revealed 332 miRNA-target gene pairs that were associated with the ERK, JNK, and transforming growth factor beta signaling pathways, suggesting that the identified miRNAs are involved in the regulation of C. savignyi larval metamorphosis via controlling the expression of their target genes. Furthermore, we validated the expression of five selected miRNAs by northern blotting. Among the selected miRNAs, the expression patterns of csa-miR-4018a, csa-miR-4018b, and csa-miR-4000f were further examined by whole-mount in situ hybridization. The results showed that all three miRNAs were specifically expressed in a cell population resembling mesenchymal cells at the head and trunk part in swimming larvae but not in metamorphic larvae. Utilizing the luciferase assay, we also confirmed that miR-4000f targeted Mapk1, suggesting that the csa-miR-4018a/csa-miR-4018b/csa-miR-4000f cluster regulates larval metamorphosis through the Mapk1-mediated signaling pathway.
Totally, 165 miRNAs, including 59 novel ones, were identified from the embryos and larvae of C. savignyi. Twelve of them showed significant changes in expression before and during metamorphosis. In situ hybridization and northern blotting results revealed that three miRNAs are potentially involved in the signaling regulatory network for the migration and differentiation of mesenchymal cells in larval metamorphosis. Furthermore, the luciferase reporter assay revealed that Mapk1 is a target of csa-miR-4000f. Our results not only present a list and profile of miRNAs involved in Ciona metamorphosis but also provide informative cues to further understand their function in ascidian larval metamorphosis.
摘要：
变态发生在大多数海洋无脊椎动物的生命周期内。海洋海鞘是研究其幼虫变态的复杂细胞过程和潜在分子机制的经典模型。详细的分子信号通路仍然难以捉摸，尽管细胞外信号调节激酶（ERKs）和c-JunN末端激酶（JNK）已被发现可以调节细胞迁移，分化,海鞘幼虫器官退化和幼年器官发育的凋亡。微RNA(miRNA)是在转录后水平上调节基因表达的小的非编码RNA。大量miRNA已被证明参与许多发育和变质过程。然而,在海鞘幼虫变态中miRNAs的鉴定尚未被研究。
完全正确,通过对来自Cionasavignyi的18至21小时受精后（hpf）尾芽胚胎以及42hpf幼虫（尾部回归后）的三个小RNA文库进行RNA测序，筛选出106个已知的miRNA和59个新的miRNA。通过定量实时PCR确认miRNA的表达谱,表明csa-miR-4040、csa-miR-4086、csa-miR-4055、csa-miR-4060、csa-miR-216a的表达水平,csa-miR-216b,csa-miR-217、csa-miR-183和csa-miR-92c在42hpf幼虫中显著升高，而那些csa-miR-4018a，csa-miR-4018b,csa-miR-4000f在18和21hpf胚胎中更高；然后，它们在42hpf幼虫中的表达显着降低。对于这12种miRNA，其表达水平显著改变，我们通过miRanda和TargetScan的组合预测了它们的靶基因。该预测分析揭示了332个与ERK相关的miRNA-靶基因对，JNK,和转化生长因子β信号通路，表明鉴定的miRNA通过控制其靶基因的表达参与C.savignyi幼虫变态的调节。此外，我们通过northern印迹验证了5种选择的miRNAs的表达。在选定的miRNA中，csa-miR-4018a的表达模式，csa-miR-4018b,通过全装原位杂交进一步检查csa-miR-4000f。结果表明，所有三种miRNA都在类似于游泳幼虫头部和躯干部分的间充质细胞的细胞群中特异性表达，但在变态幼虫中却不表达。利用荧光素酶测定，我们还证实miR-4000f靶向Mapk1,提示csa-miR-4018a/csa-miR-4018b/csa-miR-4000f簇通过Mapk1介导的信号通路调节幼虫变态.
完全正确,165miRNA,包括59个小说，从C.savignyi的胚胎和幼虫中鉴定出。其中12个在变态前和变态过程中表现出明显的表达变化。原位杂交和Northern印迹结果表明，三种miRNA可能参与了幼虫变态中间充质细胞迁移和分化的信号调节网络。此外，荧光素酶报告基因分析显示Mapk1是csa-miR-4000f的靶标。我们的结果不仅提供了与Ciona变态有关的miRNA的列表和概况，而且还提供了信息线索，以进一步了解它们在腹水幼虫变态中的功能。
完全正确,通过对来自Cionasavignyi的18至21小时受精后（hpf）尾芽胚胎以及42hpf幼虫（尾部回归后）的三个小RNA文库进行RNA测序，筛选出106个已知的miRNA和59个新的miRNA。通过定量实时PCR确认miRNA的表达谱,表明csa-miR-4040、csa-miR-4086、csa-miR-4055、csa-miR-4060、csa-miR-216a的表达水平,csa-miR-216b,csa-miR-217、csa-miR-183和csa-miR-92c在42hpf幼虫中显著升高，而那些csa-miR-4018a，csa-miR-4018b,csa-miR-4000f在18和21hpf胚胎中更高；然后，它们在42hpf幼虫中的表达显着降低。对于这12种miRNA，其表达水平显著改变，我们通过miRanda和TargetScan的组合预测了它们的靶基因。该预测分析揭示了332个与ERK相关的miRNA-靶基因对，JNK,和转化生长因子β信号通路，表明鉴定的miRNA通过控制其靶基因的表达参与C.savignyi幼虫变态的调节。此外，我们通过northern印迹验证了5种选择的miRNAs的表达。在选定的miRNA中，csa-miR-4018a的表达模式，csa-miR-4018b,通过全装原位杂交进一步检查csa-miR-4000f。结果表明，所有三种miRNA都在类似于游泳幼虫头部和躯干部分的间充质细胞的细胞群中特异性表达，但在变态幼虫中却不表达。利用荧光素酶测定，我们还证实miR-4000f靶向Mapk1,提示csa-miR-4018a/csa-miR-4018b/csa-miR-4000f簇通过Mapk1介导的信号通路调节幼虫变态.
完全正确,165miRNA,包括59个小说，从C.savignyi的胚胎和幼虫中鉴定出。其中12个在变态前和变态过程中表现出明显的表达变化。原位杂交和Northern印迹结果表明，三种miRNA可能参与了幼虫变态中间充质细胞迁移和分化的信号调节网络。此外，荧光素酶报告基因分析显示Mapk1是csa-miR-4000f的靶标。我们的结果不仅提供了与Ciona变态有关的miRNA的列表和概况，而且还提供了信息线索，以进一步了解它们在腹水幼虫变态中的功能。
