Alternative methods for miR-7 knockdown in Ciona

MicroRNAs are small, non-coding RNAs of 21-23 nucleotides that regulate gene expression at posttranscriptional level (1). miR-7 mature sequence is evolutionary conserved across Bilateria (2). During embryonic development miR-7 neural expression was reported in mammals and teleosts as well as in animals as distantly related as annelids. The first in vivo studies were performed on fly and demonstrated that miR-7 mutants develop defects in sensory structures only under stressful conditions (4). In humans, the majority of studies concerning this miRNA were focused on its role in cancer, where miR-7 seemds to be contextspecific as evidence exists for both its oncogenic and tumor suppressor roles (3). Moreover, in neural stem cells, miR-7 plays a crucial role in synapse formation as its overexpression induced a significant increase of synapsin gene expression in the derived neurons (5). A miR-7 sequence, identical to human one, is also present in the ascidian Ciona intestinalis genome and its neural expression appears to be conserved. In Ciona tailbuds and larvae, mature transcripts were found in the sensory vesicle. To characterize miR-7 function, we started performing knockdown experiments by injecting the commercial AntagomiR-7 (Dharmacon) and Peptide Nucleic Acids (PNAs). PNAs are synthetic mimics of natural oligonucleotides, in which each ribose phosphate unit is replaced by an aminoethylglycine unit. PNAs bind complementary DNA and RNA strands with excellent affinity and sequence specificity, and exhibit greater mismatch selectivity compared to natural oligonucleotides. The hatching ratio of control and injected samples was not affected by anti miR-7 injections. Injected embryos displayed similar morphology to controls. In situ hybridization experiments revealed that although the nervous system was differentiated properly, the expression of Ci-syn was drastically reduced in injected embryos. Synapsins are neuronal proteins involved in neurotransmitter release, neurite elongation and synapse formation (6). Therefore, our first results are consistent with what reported in human, rising the hypothesis that the neural role of miR-7 is conserved between vertebrates and ascidians.