MiR-9 regulates PNS differentiation during development of ascidians (Tunicata)

MicroRNAs are non-coding transcripts of approximately 21 nucleotides highly conserved in different metazoan and acting as post- transcriptional regulators of gene expression. Anti-miR Peptide Nucleic Acids (PNA) are pseudopeptide chains bearing the four nucleobases mimicking the antisense oligonucleotide base-matching properties. PNAs exhibit stronger and more selective binding affinity for complementary nucleic acid strands than natural nucleic acids. Ascidians are marine sessile, filter-feeding animals, which develop through a swimming larva that shows the basic chordate features, comprising a notochord, which runs the length of the tail, and a dorsal tubular central nervous system (CNS). Moreover, ascidian larvae possess a peripheral nervous system (PNS), mainly composed of multiple series of epidermal sensory neurons. We identified and experimental validated the spatio-temporal expression of some miRNAs, known to be expressed preferentially in the nervous system of vertebrates, in two species of ascidians, Ciona intestinalis and Phallusia mammillata. Our results show that in both species, miR-124 is expressed in all the nervous system; miR-7 is expressed in peripheral sensory cells of the larva and the juveniles; miR- 184 expression is limited to the developing nervous system of the larva and extends to the endostyle (an endoderm derivative) of the adult. Mir-9 is expressed, only in C. intestinalis, in few cells of the CNS directly connected to the otolith, a gravity sensory organ. Inhibition of miR-9 by PNA caused a negative regulation of the differentiation of peripheral epidermal sensory neurons. These results give insights into the evolutionary history of miRNA in relation with the emergence of chordate nervous system novelties.