A microRNA Cluster-Lefty Pathway is Required for Cellulose Synthesis During Ascidian Larval Metamorphosis.

Synthesis of cellulose and formation of tunic structure are unique traits in the tunicate animal group. However, the regulatory mechanism of tunic formation remains obscure. Here, we identified a novel microRNA cluster of three microRNAs, including , , and in . hybridization and promoter assays showed that cluster was expressed in the mesenchymal cells in the larval trunk, and the expression levels were downregulated during the later tailbud stage and larval metamorphosis. Importantly, overexpression of cluster in mesenchymal cells abolished the cellulose synthesis in larvae and caused the loss of tunic cells in metamorphic larvae, indicating the regulatory roles of cluster in cellulose synthesis and mesenchymal cell differentiation into tunic cells. To elucidate the molecular mechanism, we further identified the target genes of cluster using the combination approaches of TargetScan prediction and RNA-seq data. () was confirmed as one of the target genes after narrow-down screening and an experimental luciferase assay. Furthermore, we showed that was expressed in the mesenchymal and tunic cells, indicating its potentially regulatory roles in mesenchymal cell differentiation and tunic formation. Notably, the defects in tunic formation and loss of tunic cells caused by overexpression of cluster could be restored when was overexpressed in larvae, suggesting that regulated the differentiation of mesenchymal cells into tunic cells through the signaling pathway during ascidian metamorphosis. Our findings, thus, reveal a novel microRNA- molecular pathway that regulates mesenchymal cells differentiating into tunic cells required for the tunic formation in tunicate species.