Investigating microRNA-target interactions during skeletal muscle development in chicken embryos

MicroRNAs (miRNAs), short non-coding RNAs, which act post-transcriptionally to regulate gene expression, are of widespread significance and have been implicated in many biological processes during development and disease, including muscle disease. In addition to the myomiRs, which are miRNAs highly enriched in striated muscles, recent advances in sequencing technology and bioinformatics led to the identification of a large number of miRNAs in vertebrates and other species. However, for many of these miRNAs specific roles, in particular during myogenesis, have not yet been determined. Here, I investigated the potential functions of miR-128, confirmed an interaction with one of its candidate targets, Eya4, and looked at the impact of its knock-down on skeletal myogenesis in the chicken embryo. The expression pattern of miR-128, as well as 22 other somitic miRNAs, were characterised by LNA in situ hybridisation (LNA ISH). Eya4 was identified as a candidate ‘muscle’ target of miR-128 by computational analysis. Its expression pattern was characterised; miR-128 and Gga-Eya4 displayed similar profiles in developing somites. Using the miRanda algorithm potential miRNA binding sites were identified in the 3’ untranslated region (UTR) of other transcription factors, which along with Eya4 are members of the PAX-SIX-EYA-DACH (PSED) network (Six1/4, Eya1/2/3, and Dach1). These miRNA/target interactions were examined in vitro and in vivo. Gga-Eya4 was confirmed as a target of miR-128 as well as miR-206 by luciferase reporter assays. MiR-128/Gga-Eya4 interaction was validated by RNA ISH and RT-qPCR after antagomiR (AM)-128 injection in chicken embryos. Knock-down of miR-128 resulted in a significant de-repression of Gga-Eya4 expression; an increase in Gga-Six4 and Gga-Pax3 expression was also observed, whereas Gga-MyoD1 expression was decreased. With this project, using a combination of cell-based experiments and animal studies, I showed that miR-128 could play an important role in the regulation of skeletal myogenesis in the chicken embryo by targeting Gga-Eya4, a member of the PSED network

Investigating microRNA-target interactions during skeletal muscle development in chicken embryos

MicroRNAs (miRNAs), short non-coding RNAs, which act post-transcriptionally to regulate gene expression, are of widespread significance and have been implicated in many biological processes during development and disease, including muscle disease. In addition to the myomiRs, which are miRNAs highly enriched in striated muscles, recent advances in sequencing technology and bioinformatics led to the identification of a large number of miRNAs in vertebrates and other species. However, for many of these miRNAs specific roles, in particular during myogenesis, have not yet been determined. Here, I investigated the potential functions of miR-128, confirmed an interaction with one of its candidate targets, Eya4, and looked at the impact of its knock-down on skeletal myogenesis in the chicken embryo. The expression pattern of miR-128, as well as 22 other somitic miRNAs, were characterised by LNA in situ hybridisation (LNA ISH). Eya4 was identified as a candidate ‘muscle’ target of miR-128 by computational analysis. Its expression pattern was characterised; miR-128 and Gga-Eya4 displayed similar profiles in developing somites. Using the miRanda algorithm potential miRNA binding sites were identified in the 3’ untranslated region (UTR) of other transcription factors, which along with Eya4 are members of the PAX-SIX-EYA-DACH (PSED) network (Six1/4, Eya1/2/3, and Dach1). These miRNA/target interactions were examined in vitro and in vivo. Gga-Eya4 was confirmed as a target of miR-128 as well as miR-206 by luciferase reporter assays. MiR-128/Gga-Eya4 interaction was validated by RNA ISH and RT-qPCR after antagomiR (AM)-128 injection in chicken embryos. Knock-down of miR-128 resulted in a significant de-repression of Gga-Eya4 expression; an increase in Gga-Six4 and Gga-Pax3 expression was also observed, whereas Gga-MyoD1 expression was decreased. With this project, using a combination of cell-based experiments and animal studies, I showed that miR-128 could play an important role in the regulation of skeletal myogenesis in the chicken embryo by targeting Gga-Eya4, a member of the PSED network

Fine-tuning of the PAX-SIX-EYA-DACH network by multiple microRNAs controls embryo myogenesis

MicroRNAs (miRNAs), short non-coding RNAs, which act post-transcriptionally to regulate gene expression, are of widespread significance during development and disease, including muscle disease. Advances in sequencing technology and bioinformatics led to the identification of a large number of miRNAs in vertebrates and other species, however, for many of these miRNAs specific roles have not yet been determined. LNA in situ hybridisation has revealed expression patterns of somite-enriched miRNAs, here we focus on characterising the functions of miR-128. We show that antagomiR-mediated knockdown (KD) of miR-128 in developing chick somites has a negative impact on skeletal myogenesis. Computational analysis identified the transcription factor EYA4 as a candidate target consistent with the observation that miR-128 and EYA4 display similar expression profiles. Luciferase assays confirmed that miR-128 interacts with the EYA4 3′UTR. In vivo experiments also suggest that EYA4 is regulated by miR-128. EYA4 is a member of the PAX-SIX-EYA-DACH (PSED) network of transcription factors. Therefore, we identified additional candidate miRNA binding sites in the 3′UTR of SIX1/4, EYA1/2/3 and DACH1. Using the miRanda algorithm, we found sites for miR-128, as well as for other myogenic miRNAs, miR-1a, miR-206 and miR-133a, some of these were experimentally confirmed as functional miRNA target sites. Our results reveal that miR-128 is involved in regulating skeletal myogenesis by directly targeting EYA4 with indirect effects on other PSED members, including SIX4 and PAX3. Hence, the inhibitory effect on myogenesis observed after miR-128 knockdown was rescued by concomitant knockdown of PAX3. Moreover, we show that the PSED network of transcription factors is co-regulated by multiple muscle-enriched microRNAs

miR-133-mediated regulation of the Hedgehog pathway orchestrates embryo myogenesis

Skeletal myogenesis serves as a paradigm to investigate the molecular mechanisms underlying exquisitely regulated cell fate decisions in developing embryos. The evolutionary conserved miR-133 family of microRNAs is expressed in the myogenic lineage, but how it acts remains incompletely understood. Here we performed genome-wide differential transcriptomics of miR-133 knock-down (KD) embryonic somites, the source of vertebrate skeletal muscle. This revealed extensive downregulation of Sonic hedgehog (Shh) pathway components: patched receptors, Hedgehog interacting protein, and the transcriptional activator, Gli1. By contrast Gli3, a transcriptional repressor, was de-repressed and confirmed as a direct miR-133 target. Phenotypically, miR-133 KD impaired myotome formation and growth by disrupting proliferation, extracellular matrix deposition and epithelialization. Together this suggests that miR-133 mediated Gli3 silencing is critical for embryonic myogenesis. Consistent with this idea we found that activation of Shh signalling by either purmorphamine, or KD of Gli3 by antisense morpholino (MO) rescued the miR-133 KD phenotype. We identify a novel Shh/MRF/miR-133/Gli3 axis that connects epithelial morphogenesis with myogenic fate specification

A database of microRNA expression patterns in Xenopus laevis

MicroRNAs (miRNAs) are short, non-coding RNAs around 22 nucleotides long. They inhibit gene expression either by translational repression or by causing the degradation of the mRNAs they bind to. Many are highly conserved amongst diverse organisms and have restricted spatio-temporal expression patterns during embryonic development where they are thought to be involved in generating accuracy of developmental timing and in supporting cell fate decisions and tissue identity. We determined the expression patterns of 180 miRNAs in Xenopus laevis embryos using LNA oligonucleotides. In addition we carried out small RNA-seq on different stages of early Xenopus development, identified 44 miRNAs belonging to 29 new families and characterized the expression of 5 of these. Our analyses identified miRNA expression in many organs of the developing embryo. In particular a large number were expressed in neural tissue and in the somites. Surprisingly none of the miRNAs we have looked at show expression in the heart. Our results have been made freely available as a resource in both XenMARK and Xenbase

Expression of <i>Xenopus</i> MicroRNAs in the developing somites.

<p>Expression patterns of <i>Xenopus</i> laevis miRNA are shown at stage 33/34 both in wholemounts and transverse sections from the trunk. All embryos are lateral views with anterior to the left. (A) miRs-14, 17-5p, 499, 18b, 363-3p, 30a-5p, 302 and 220c show expression in the head and trunk. Sections show expression in the somites, neural tube and notochord. miR-499 and 302 also shows expression in the surface ectoderm. (B) miRs 1a, 1b and 206 show expression only in the trunk which is confined to the somites in sections. (C) miR-184 shows expression in the head and trunk. sections show expression in the nural tube and somites but bot the notochord. (D) mir-15b shows expression in the head and trunk. Sections show expression in the somites and dorsal neural tube. (E) mir133 family shows differential expression. Mir-133a and d show expression in the head and trunk and in the somites, neural tube and notochord. miR-133b and c show expression in the trunk and sections show only expression in the somites.</p

Identification and expression of novel <i>Xenopus</i> MicroRNAs.

<p>(A) Predicted pre-miRNA structures of <i>Xenopus</i> laevis mir-GNW8, 9, 11, 12 and 13. The 5p and 3p miRNAs are highlighted on the predicted hairpin precursor with the most abundant sequence highlighted in green and the lower abundance sequence in pink (B) Normalised coverage plots showing the relative expression by number of reads of the 3p and 5p miRNAs from the libraries made from unfertilised egg, stage 7–8, stage 12, stages 18–20, stages 34–36 and stage 56 <i>Xenopus</i> laevis embryos. (C) Expression patterns of the novel miRNAs at stage 12/13 and stage 28/29.</p

<i>Xenopus</i> MicroRNAs show expression in a diverse number of developing organs and cell types.

<p>Expression patterns of <i>Xenopus</i> laevis miRNAs are shown at varying stages. Arrowheads point to relevant expression. Views are lateral with anterior to left except (C, D and K) which is a ventral view with anterior to the left and (E) and (F) which are dorsal views with anterior to the bottom. MiR-34b, pancreas; miR-128, brain; miR-107, gut; miR-122, liver; miR-126, blood vessels; miR-200a, olfactory placodes; miR-455, liver; miR-30d, brain; miR-100, brain; miR-96, brain and olfactory placodes, miR-125, brain and gut, miR-200b, olfactory placode.</p