MiR-92 Family Members Form a Cluster Required for Notochord Tubulogenesis in Urochordate Ciona savignyi

Abstract

MicroRNAs are frequently clustered in the genome and polycistronically transcribed, regulating targeted genes in diverse signaling pathways. The miR-17-92 cluster is a typical miRNA cluster, playing crucial roles in the organogenesis and homeostasis of physiological processes in vertebrates. Here, we identified three miRNAs (csa-miR-92a, csa-miR-92b, and csa-miR-92c) that belonged to the miR-92 family and formed a miRNA cluster in the genome of a urochordate marine ascidian Ciona savignyi. Except for miR-92a and miR-92b, other homologs of the vertebrate miR-17-92 cluster members could not be identified in the Ciona genome. We further found that the mature sequences of urochordate miR-92 family members were highly conserved compared with the vertebrate species. The expression pattern revealed that three miR-92 family members had consistent expression levels in adult tissues and were predominantly expressed in heart and muscle tissue. We further showed that, at the embryonic and larval stages, csa-miR-92c was expressed in the notochord of embryos during 18–31 h post fertilization (hpf) by in situ hybridization. Knockout of csa-miR-92c resulted in the disorganization of notochord cells and the block of lumen coalescence in the notochord. Fibroblast growth factor (FGF), mitogen-activated protein kinase (MAPK), and wingless/integrated (Wnt)/planar cell polarity (PCP) signaling pathways might be involved in the regulatory processes, since a large number of core genes of these pathways were the predicted target genes of the miR-92 family. Taken together, we identified a miR-92 cluster in urochordate Ciona and revealed the expression patterns and the regulatory roles of its members in organogenesis. Our results provide expression and phylogenetic data on the understanding of the miR-92 miRNA cluster’s function during evolution.

ELMOD3-Rab1A-Flotillin2 cascade regulates lumen formation via vesicle trafficking in Ciona notochord

“…Based on the target sequences of CRISPR/Cas9, all sgRNAs were synthesized and cloned into the Cr-U6 > sgRNA(F + E) vector (Addgene number: 59 986) for expressing the sgRNA. The electroporation mixes were as described previously with some modifications [56]. The CRISPR/Cas9 system contained the PCR product of sgRNA (control group) or the mix PCR products of the targeted gene sgRNA (knockout group) (50 µl), plasmid of Cs-brachyury (3k) > NLS: Cas9:: NLS:: P2A:: mCherry (40 µg), embryos (300 µl) and electroporation buffer (420 µl).…”