**2. Results**

#### *2.1. miR-7 in Ciona intestinalis*

Comparing miR-7 mature sequences in different animal models, we observed that miR-7 is highly conserved also in basal chordates: *C. intestinalis* miR-7 differs from that of *Homo sapiens*, only by the deletion of the terminal uracil. This feature is shared with another tunicate species, *Oikopleura dioica*, while in the available transcriptomes of two other ascidians, miR-7 mature sequences are completely conserved (Figure 1B).

In *C. intestinalis* genome, the miR-7 gene resides within the last intron of the heterogeneous nuclear ribonucleoprotein K (hnRNP K) gene, oriented in the same direction as the Ci-hnRNP K transcription unit (Figure 1D).

### *2.2. Genes Expression Profile*

To determine the expression pattern of miR-7 mature transcripts during *C. intestinalis* development, standard in situ hybridization protocol [29] was ineffective. When hybridization with DIG-labeled Locked Nucleic Acid (LNA; Exiqon, Vedbaek, Denmark) probes were carried out overnight, unspecific stains were always detected in mesenchymal cells of late tailbud and larva trunk (Figure 2A). Extending the hybridization step to five days and increasing the hybridization temperature (5 ◦C more than the recommended temperature) was found to be optimal for miRNA detection with LNA probes, as confirmed by miR-124 results (Figure 2B). Performing this modified protocol, we found that miR-124 mature transcripts were abundantly present in all of the nervous system of *C. intestinalis* larva, as previously reported by Zeller and co-workers [30].

**Figure 2.** Whole mount in situ hybridization of *C. intestinalis* embryos. (**A**) Unspecific stain in mesenchymal cells of larva trunk, obtained when hybridization with LNA probes was performed overnight; (**B**) miR-124 expression in larval central and peripheral nervous system; (**C**) miR-7 expression at late tailbud stage: signal is clearly visible in the posterior ventral part of the sensory vesicle; (**D**,**E**) miR-7 expression at larva stage: the signal persists in the posterior ventral region and faintly extends in the neural ganglion; (**F**) hnRNP K expression at mid tailbud stage: signal appears more intense in the epidermal sensory neurons; (**G**,**H**) hnRNP K expression at larva stage: signal persists in epidermal sensory neurons and extends all over the trunk. (**A**,**E**–**H**) Scale bar = 60 μm; (**B**–**D**) Scale bar = 100 μm.

Using this protocol, we found that miR-7 expression started in the central nervous system at the late tailbud stage, but only in the ventral posterior part of the sensory vesicle (Figure 2C). At larval stage, the signal persisted in this region and faintly extended in the neural ganglion (Figure 2D,E).

hnRNP K, the gene hosting miR-7, was ubiquitously expressed at early developmental stages; but from mid tailbud stage, the signal was more intense in the epidermal sensory neurons, i.e., ascidian peripheral nervous system, of both trunk and tail (Figure 2F). This expression persisted at the late tailbud and larval stages, and a strong signal was also detectable all over the trunk (Figure 2G,H).

#### *2.3. miR-7 Downregulation by PNAs*

To evaluate PNAs effectiveness in miRNA knockdown, we designed a 22-mer PNA complementary to *C. intestinalis* miR-7 (PNA-a7, Figure 1C) and a PNA scrambled sequence with the same base composition of PNA-a7 (PNA-sc7, Figure 1C). Then, PNA-a7 and PNA-sc7 were microinjected in *C. intestinalis* eggs before in vitro fertilization. Moreover, we performed microinjections employing the commercial AntagomiR (AmiR-7), commonly used in miRNAs knockdown studies [28], and we compared the effects with those obtained with PNAs.

Preliminary trials revealed that the highest non-lethal concentrations were 0.7 mM for PNAs (PNA-a7 and PNA-sc7) and 0.3 mM for AmiR-7. Based on these results, PNAs solution seemed less toxic than AmiR-7, as embryos injected with concentrations higher than 0.3 mM AmiR-7 died before completing embryogenesis. All the following analyses were performed on embryos injected with 0.7 mM PNAs or 0.3 mM AmiR-7.

The developing rates of controls (injected with only the vital dye, Fast Green) and embryos injected with PNA-sc7, PNA-a7 or AmiR-7 were comparable, ranging from 69% (PNA-a7) to 72% (PNA-sc7) and no difference in sample morphology was recorded.

The specificity of PNA-a7 as well as AmiR-7 for miR-7 in *C. intestinalis* was previously checked by blast search in its genome by using cin-miR-7 (MIMAT0006091) as query. No identity with other microRNAs were found. Few mRNAs (for example: NLRC5-like or FAM192A-like mRNAs) having some sequence identity with miR-7 were obtained but the query coverage was lower, as there was some similarity but not for the entire sequence of miR-7.

To verify miRNA downregulation by PNAs, we first evaluated miR-7 expression by in situ hybridization. Results revealed that miR-7 expression was drastically reduced in embryos injected with PNA-a7 (Figure 3A), while miR-7 was normally expressed in embryos injected with PNA-sc7 (Figure 3B).

**Figure 3.** *C. intestinalis* miR-7 expression at the late tailbud stage in embryos injected with (**A**) PNA-a7 and (**B**) PNA-sc7. Scale bar = 40 μm.

Then, we checked the expression of some pan-neural genes: Ci-ETR [31] and Ci-Syn [32] in all the injected embryos. Ci-ETR expression was normal as its signal was observed throughout the central nervous system and in the epidermal sensory neurons of all injected samples (Figure 4A,C,E,G). Nevertheless, the expression of Ci-Syn was reduced in embryos injected with PNA-a7 (87%, *n* = 28) and AmiR-7 (91%, *n* = 32), compared to PNA-sc7 (*n* = 41). In control embryos and in embryos injected

with PNA-sc7, the hybridization signal occurred in most of the sensory vesicle, in the motor ganglion, and extended into the posterior neural tube (Figure 4B,D). In embryos injected with PNA-a7, Ci-Syn transcripts were detected only in a subpopulation of neurons in the sensory vesicle and in the motor ganglion, and not detected in the posterior neural tube (Figure 4F). The same expression pattern was present in embryos injected with AmiR-7 (Figure 4H).

**Figure 4.** Whole mount in situ hybridization of *C. intestinalis* control embryos and embryos injected with PNA-sc7, PNA-a7, and AntagomiR (AmiR-7). (**A**,**C**,**E**,**G**) Ci-ETR expression in central and peripheral nervous system of late tailbud embryos; (**B**,**D**,**F**,**H**) Ci-Syn expression at late tailbud stage: in control and PNA-sc7 injected embryos, signal is detectable in most of the sensory vesicle, in the motor ganglion and along the posterior neural tube. In embryos injected with PNA-a7 and AmiR-7, transcripts are present only in a subpopulation of neurons in the sensory vesicle and in the motor ganglion, while no signal is recorded in posterior neural tube. Arrows indicate the posterior limit of the signal. (**A**,**C**,**E**,**G**) Scale bar = 15 μm. (**B**,**D**,**F**,**H**) Scale bar = 10 μm.
