Ype was 6.25 mM, which achieved rescue of the somite structure in 70 of embryos. To evaluate the phenotypic rescue, embryos were monitored up to 24 hpf and the resulting phenotype was assessed for improved overall morphology and somite structure. Cyclopamine rescue yielded Title Loaded From File miR-30 morpholino Title Loaded From File treated embryos with more obvious chevron-shaped somites (Fig. 5K). Ventral curvature of the embryos was improved leading to an overall extended morphology similar to that in wild-type embryos (Fig. 5E). Detailed analysis of the somite structure was carried out on the four somites immediately posterior to the yolk cell extension at 24 hpf following cyclopamine rescue. Analysis of the somite boundaries showed that miR-30 morpholino embryos treated with cyclopamine had an improved angular somite structure (Fig. 5K) that more closely resembled that of the wild type embryo somite (Fig. 5E). In parallel both uninjected and miR-30 morpholinoinjected embryos were treated with identical amounts of DMSO to act as a negative control which produced no effect on the phenotypes of the resulting embryos (Fig S4C+D). Furthermore, a reduction in ptc1 expression was observed following cyclopamine rescue of miR-30 morpholino embryos indicating that Hh pathway activity had been reduced (Fig. 5L). Immunohistochemical analysis revealed that following cyclopamine treatment the number of slow muscle fibres in miR-30 morpholino treated embryos (38.0369.90) reduced to the wild type range (23.0163.13) with an average of 24.163.58 slow muscle fibres per somite (p = 0.0784) (Fig. 5G, 5J, 5M and Table S1). Together our results indicate that cyclopamine inhibition of Smoothened suppresses the phenotype associated with loss of miR-30 function, supporting the hypothesis that miR-30 modulates Hh signalling by regulation of smoothened.DiscussionIn the current study we have demonstrated that inhibition of the miR-30 microRNA family causes elevated ptc1 expression and increased numbers of superficial slow muscle fibres during zebrafish muscle development, consistent with an increase in Hh pathway activity. These features are a result of direct targeting of the Hh transmembrane receptor smoothened by the microRNA family, representing a novel role for miR-30 in muscle fibre specification and distribution. This is supported 23148522 by the observation that miR-30 overexpression, and hence Hh pathway activity reduction, can be rescued by coinjection with Shh mRNA but not with dnPKA mRNA. The inhibition of Smoothened is critical to controlled levels of Hh activity within a cell, a function that is attributed to the interaction of the Smoothened protein with Ptc [62]. It has been shown that Ptc acts sub-stoichiometrically to suppress Smoothened, demonstrating a catalytic mode of action rather than a direct interaction between the two pathway components [63]. However,miR-30 Targets smoothened in Zebrafish MuscleFigure 3. miR-30 directly targets the 39UTR of the Hedgehog transmembrane receptor smoothened. (A ) Embryos injected with 3 different GFP reporter mRNAs; (A,B) the GFP ORF plus tandem perfect target sites (GFP-PTS), (C,D) GFP ORF plus the smo 39UTR sequence (GFP-SMO) and (E,F) the GFP ORF without UTR sequence (GFP- no UTR). Constructs were injected either alone (A,C,E) or with the miR-30 duplex RNA (B,D,F). (G) Western blot validation on lysates of GFP injected embryos with and without the miR-30 duplex (H) Densitometric analysis of GFP protein levels normalised against a-tubulin loading contr.Ype was 6.25 mM, which achieved rescue of the somite structure in 70 of embryos. To evaluate the phenotypic rescue, embryos were monitored up to 24 hpf and the resulting phenotype was assessed for improved overall morphology and somite structure. Cyclopamine rescue yielded miR-30 morpholino treated embryos with more obvious chevron-shaped somites (Fig. 5K). Ventral curvature of the embryos was improved leading to an overall extended morphology similar to that in wild-type embryos (Fig. 5E). Detailed analysis of the somite structure was carried out on the four somites immediately posterior to the yolk cell extension at 24 hpf following cyclopamine rescue. Analysis of the somite boundaries showed that miR-30 morpholino embryos treated with cyclopamine had an improved angular somite structure (Fig. 5K) that more closely resembled that of the wild type embryo somite (Fig. 5E). In parallel both uninjected and miR-30 morpholinoinjected embryos were treated with identical amounts of DMSO to act as a negative control which produced no effect on the phenotypes of the resulting embryos (Fig S4C+D). Furthermore, a reduction in ptc1 expression was observed following cyclopamine rescue of miR-30 morpholino embryos indicating that Hh pathway activity had been reduced (Fig. 5L). Immunohistochemical analysis revealed that following cyclopamine treatment the number of slow muscle fibres in miR-30 morpholino treated embryos (38.0369.90) reduced to the wild type range (23.0163.13) with an average of 24.163.58 slow muscle fibres per somite (p = 0.0784) (Fig. 5G, 5J, 5M and Table S1). Together our results indicate that cyclopamine inhibition of Smoothened suppresses the phenotype associated with loss of miR-30 function, supporting the hypothesis that miR-30 modulates Hh signalling by regulation of smoothened.DiscussionIn the current study we have demonstrated that inhibition of the miR-30 microRNA family causes elevated ptc1 expression and increased numbers of superficial slow muscle fibres during zebrafish muscle development, consistent with an increase in Hh pathway activity. These features are a result of direct targeting of the Hh transmembrane receptor smoothened by the microRNA family, representing a novel role for miR-30 in muscle fibre specification and distribution. This is supported 23148522 by the observation that miR-30 overexpression, and hence Hh pathway activity reduction, can be rescued by coinjection with Shh mRNA but not with dnPKA mRNA. The inhibition of Smoothened is critical to controlled levels of Hh activity within a cell, a function that is attributed to the interaction of the Smoothened protein with Ptc [62]. It has been shown that Ptc acts sub-stoichiometrically to suppress Smoothened, demonstrating a catalytic mode of action rather than a direct interaction between the two pathway components [63]. However,miR-30 Targets smoothened in Zebrafish MuscleFigure 3. miR-30 directly targets the 39UTR of the Hedgehog transmembrane receptor smoothened. (A ) Embryos injected with 3 different GFP reporter mRNAs; (A,B) the GFP ORF plus tandem perfect target sites (GFP-PTS), (C,D) GFP ORF plus the smo 39UTR sequence (GFP-SMO) and (E,F) the GFP ORF without UTR sequence (GFP- no UTR). Constructs were injected either alone (A,C,E) or with the miR-30 duplex RNA (B,D,F). (G) Western blot validation on lysates of GFP injected embryos with and without the miR-30 duplex (H) Densitometric analysis of GFP protein levels normalised against a-tubulin loading contr.
