**4. Materials and Methods**

#### *4.1. Animals and Embryos Culture*

Adult *Ciona intestinalis* were collected along the coasts of Roscoff (France) by the fishing service of the Station Biologique de Roscoff. Animals were maintained in aquaria filled with artificial sea water (Instant Ocean; salinity ~32%) and provided with a circulation system, as well as mechanical, chemical, and biological filters. Constant light conditions were preferred to promote gamete production [29]. For each experiment, gametes from three adults were obtained surgically from the gonoducts and in vitro cross-fertilization was performed. Embryos were reared in petri dishes in filtered artificial sea water buffered with 1 M HEPES (ASWH; pH 8.0) at 18 ± 1 ◦C up to the stages of interest (gastrula stage; neurula stage; initial, early, mid, and late tailbud stages; larva stage). Then, they were dechorionated in ASWH containing 1% sodium thioglycolate and 0.05% protease; fixed in 4% paraformaldehyde, 0.5 M NaCl, and 0.1 M 3-(*N*-morpholino)propanesulfonic acid (pH 7.5) for 90 min; dehydrated in ethanol series (30%, 50%, and 70%); and stored at −20 ◦C.

#### *4.2. Reagents*

Antagomirs are chemically modified, cholesterol-conjugated single-stranded RNA analogues complementary to the mature miRNA sequences, commonly applied in miRNAs knockdown research [28]. Based on *C. intestinalis* sequence, a specific anti-miR-7 antagomir (AmiR-7: 5 - CSASACAAAAUCACUAGUCUUSCSCSAS-Chol-3 ) was designed and synthetized by Dharmacon (USA).
