Reduction in length of the yolk cell extension, which together with the somite defects resulted in an overall ventral Title Loaded From File curvature of the embryonic axis. As a negative control for the knockdown studies an unrelated microRNA was selected to ensure the phenotypes observed were specific to knockdown of the Title Loaded From File miR-30 family and was not a generic consequence of morpholino introduction. MicroRNA-140 was chosen as it has no reported similarity to any members of the miR30 family and previous expression analysis in zebrafish has shown that miR-140 is expressed in the palatal skeleton and head cartilage [42,43] No phenotype was observed in these embryos (Fig. S1C).miR-30 Misregulation Affects Hh Pathway ActivityZebrafish mutants for well characterised molecular pathways have been reported and multiple studies point to the developmental consequences of perturbing Hedgehog (Hh) signalling, which shows acute dosage sensitivity [44?6]. We noticed that the phenotype we generated by alterations in the level of the miR-30 family mimics misregulation of the Hh pathway, displaying downwards curvature of the embryos and characteristic U-shaped somites associated with Hh pathway misregulation (Fig. 2B) [14,47]. To determine whether the miR-30 knock down phenotype was due to a mis-regulation of Hh signalling we analysed ptc1 expression as a read out of Hh activity (Fig. 2E ) [48,49]. Ptc1 encodes an Hh ligand receptor, transcription of which is activated by Hh signalling [48]. In situ hybridisation of 24 hpf embryos injected with the miR-30 morpholino exhibited increased ptcmiR-30 Targets smoothened in Zebrafish MuscleFigure 2. The miR-30 family is required during early embryonic development to regulate Hh pathway activity. Embryo somite structure at 24 hpf is shown (A ). Ptc1 expression analysis was used as a read out of Hh pathway activity, showing elevated levels in miR-30 morpholino and dnPKA treated embryos when compared to wild type embryos (E ). Slow muscle fibre number was quantified by immunohistochemistry using the S58 antibody (yellow/green) and MF20 staining (red). Embryo sections are orientated dorsal side upwards (I ). Images are shown of wild type embryos (A, E, I), miR-30 morpholino treated embryos (B, F, J), dnPKA treated embryos (C, G, K) and miR-30 overexpression embryos (D, H, L). 23148522 doi:10.1371/journal.pone.0065170.gexpression (Fig. 2F) suggesting upregulation of the pathway. As a positive control for Hh pathway activation dnPKA mRNA (dominant negative Protein Kinase A) was injected into zebrafish embryos (Fig. 2C,G,K). Protein kinase A is a negative regulator of Hedgehog signaling and injection of dnPKA leads to overactivation of the pathway [47]. There is significant similarity between the embryos treated with dnPKA and the miR-30 knockdown embryos, with primary defects in the early patterning and establishment of the somites resulting in U shaped somites and overall curvature of the embryo. To further verify that miR-30 levels are linked to Hh pathway activity a miR-30 RNA sequence duplex was overexpressed in zebrafish embryos (Fig. 2D,H,L) and showed reduced ptc1 expression (Fig. 2H), suggesting that the microRNA family is involved in regulating Hh pathway activity. These experiments indicate that the miR-30 family has a negative regulatory role on the level of Hedgehog signaling during zebrafish embryonic development.uninjected controls (Fig. 2I ). Sixty somite sections were analysed for each treatment from 24 hpf embryos. Analysis of the m.Reduction in length of the yolk cell extension, which together with the somite defects resulted in an overall ventral curvature of the embryonic axis. As a negative control for the knockdown studies an unrelated microRNA was selected to ensure the phenotypes observed were specific to knockdown of the miR-30 family and was not a generic consequence of morpholino introduction. MicroRNA-140 was chosen as it has no reported similarity to any members of the miR30 family and previous expression analysis in zebrafish has shown that miR-140 is expressed in the palatal skeleton and head cartilage [42,43] No phenotype was observed in these embryos (Fig. S1C).miR-30 Misregulation Affects Hh Pathway ActivityZebrafish mutants for well characterised molecular pathways have been reported and multiple studies point to the developmental consequences of perturbing Hedgehog (Hh) signalling, which shows acute dosage sensitivity [44?6]. We noticed that the phenotype we generated by alterations in the level of the miR-30 family mimics misregulation of the Hh pathway, displaying downwards curvature of the embryos and characteristic U-shaped somites associated with Hh pathway misregulation (Fig. 2B) [14,47]. To determine whether the miR-30 knock down phenotype was due to a mis-regulation of Hh signalling we analysed ptc1 expression as a read out of Hh activity (Fig. 2E ) [48,49]. Ptc1 encodes an Hh ligand receptor, transcription of which is activated by Hh signalling [48]. In situ hybridisation of 24 hpf embryos injected with the miR-30 morpholino exhibited increased ptcmiR-30 Targets smoothened in Zebrafish MuscleFigure 2. The miR-30 family is required during early embryonic development to regulate Hh pathway activity. Embryo somite structure at 24 hpf is shown (A ). Ptc1 expression analysis was used as a read out of Hh pathway activity, showing elevated levels in miR-30 morpholino and dnPKA treated embryos when compared to wild type embryos (E ). Slow muscle fibre number was quantified by immunohistochemistry using the S58 antibody (yellow/green) and MF20 staining (red). Embryo sections are orientated dorsal side upwards (I ). Images are shown of wild type embryos (A, E, I), miR-30 morpholino treated embryos (B, F, J), dnPKA treated embryos (C, G, K) and miR-30 overexpression embryos (D, H, L). 23148522 doi:10.1371/journal.pone.0065170.gexpression (Fig. 2F) suggesting upregulation of the pathway. As a positive control for Hh pathway activation dnPKA mRNA (dominant negative Protein Kinase A) was injected into zebrafish embryos (Fig. 2C,G,K). Protein kinase A is a negative regulator of Hedgehog signaling and injection of dnPKA leads to overactivation of the pathway [47]. There is significant similarity between the embryos treated with dnPKA and the miR-30 knockdown embryos, with primary defects in the early patterning and establishment of the somites resulting in U shaped somites and overall curvature of the embryo. To further verify that miR-30 levels are linked to Hh pathway activity a miR-30 RNA sequence duplex was overexpressed in zebrafish embryos (Fig. 2D,H,L) and showed reduced ptc1 expression (Fig. 2H), suggesting that the microRNA family is involved in regulating Hh pathway activity. These experiments indicate that the miR-30 family has a negative regulatory role on the level of Hedgehog signaling during zebrafish embryonic development.uninjected controls (Fig. 2I ). Sixty somite sections were analysed for each treatment from 24 hpf embryos. Analysis of the m.