MicroRNA-30a regulates zebrafish myogenesis through targeting the transcription factor Six1

Jenean H. O'Brien 1 Laura Hernandez-Lagunas 0 Kristin Bruk Artinger ( 0 Heide L. Ford ) 1 0 Department of Craniofacial Biology, School of Dental Medicine, University of Colorado Anschutz Medical Campus , Aurora, CO 80045 , USA 1 Department of Pharmacology, School of Medicine, University of Colorado Anschutz Medical Campus , Aurora, CO 80045 , USA Precise spatiotemporal regulation of the SIX1 homeoprotein is required to coordinate vital tissue development, including myogenesis. Whereas SIX1 is downregulated in most tissues following embryogenesis, it is re-expressed in numerous cancers, including tumors derived from muscle progenitors. Despite crucial roles in development and disease, the upstream regulation of SIX1 expression has remained elusive. Here, we identify the first direct mechanism for Six1 regulation in embryogenesis, through microRNA30a (miR30a)mediated repression. In zebrafish somites, we show that miR30a and six1a and six1b (hereafter six1a/b) are expressed in an inverse temporal pattern. Overexpression of miR30a leads to a reduction in six1a/b levels, and results in increased apoptosis and altered somite morphology, which phenocopies six1a/b knockdown. Conversely, miR30a inhibition leads to increased Six1 expression and abnormal somite morphology, revealing a role for endogenous miR30a as a muscle-specific miRNA (myomiR). Importantly, restoration of six1a in miR30a-overexpressing embryos restores proper myogenesis. These data demonstrate a new role for miR30a at a key node in the myogenic regulatory gene network through controlling Six1 expression. - INTRODUCTION Embryonic skeletal trunk muscle in vertebrates arises from progenitor cells in the paraxial mesoderm that are induced to form the primary myotome. These precursor cells differentiate into both fast- and slow-twitch muscle. Early myogenesis is regulated through expression of basic helix-loop-helix (bHLH)-domaincontaining myogenic regulatory factors (MRFs), which are controlled by a complex genetic network that includes Sine oculis homeobox (SIX) and Eyes absent (EYA) transcriptional regulators (Bryson-Richardson and Currie, 2008; Heanue et al., 1999). SIX family members are homeodomain-containing proteins that contribute to the development of muscle, and many other tissues, through promoting the transcription of genes involved in cell proliferation, survival, differentiation, migration and invasion (Christensen et al., 2008). SIX1 and SIX4 directly activate expression of several MRFs in the mouse, including myogenic differentiation 1 (Myod1), myogenic factor 5 (Myf5), myogenin (Myog) and Myf6 (Giordani et al., 2007; Grifone et al., 2005; Hinits et al., 2007; Spitz et al., 1998). In parallel, MRF transcription can be regulated by the homeodomain-containing paired box gene 3 (PAX3) during primary myogenesis (Buckingham and Relaix, 2007), the expression of which is controlled by SIX1 and SIX4, along with their co-transcriptional activators EYA1 and EYA2, in a subset of developing muscles (Grifone et al., 2007; Grifone et al., 2005). Because the SIX family of transcription factors are upstream activators of the myogenic program, their regulation during muscle development is crucial. During early myogenesis, knockdown of mouse and zebrafish Six1 results in severe muscle hypoplasia and a decrease in fast-twitch fibers (Bessarab et al., 2008; Grifone et al., 2005; Laclef et al., 2003a; Nord et al., 2013), demonstrating a role for Six1 in both muscle progenitor activation and the promotion of fast muscle differentiation. However, overexpression of Six1 in zebrafish also prevents fast twitch fiber formation (Nord et al., 2013), indicating that improper Six1 levels, either too high or low, can negatively affect early muscle differentiation. To date, mechanisms controlling Six1 expression during embryogenesis have not been elucidated in any tissue. Because microRNAs (miRs) can tightly regulate protein levels in a developmental context, we examined potential miR-mediated control of Six1. MiRs are small, non-coding RNAs known to exert essential spatiotemporal gene regulation in a diverse array of developmental and disease programs, including myogenesis (Chen et al., 2009; Sayed and Abdellatif, 2011; Yekta et al., 2008). MiRs function by base-pairing to a seed sequence located in target mRNAs, mediating mRNA degradation or translational repression (Bartel, 2009; Filipowicz et al., 2008). In both mice and zebrafish, recent studies aimed at eliminating the function of an enzyme essential for general miR-processing, Dicer, have demonstrated important roles for miRs in embryonic myogenesis, because the resulting phenotype is decreased muscle mass and abnormal muscle fiber morphology (Mishima et al., 2009; ORourke et al., 2007). In addition, members of the miR1 and miR206, and miR133 families, referred to as muscle-specific miRNA (myomiRs) (Goljanek-Whysall et al., 2012; McCarthy, 2008), are known to regulate genes that participate in adult myoblast activation, including Histone Deacetylase 4, DNA Polymerase a and Connexin 43 (Anderson et al., 2006; Chen et al., 2006; Goljanek-Whysall et al., 2012; Kim et al., 2006). However, few miRs have been identified that directly control early myogenic transcriptional regulators. Of the known embryonic MRF transcriptional activators, only Pax3 is reported to be miR-regulated (Gagan et al., 2012). We focused our investigation on miRs conserved across species, and identified miR30a as a potential regulator of zebrafish Six1 and of myogenesis. Previously, miR185 has been shown to regulate SIX1 expression in a kidney cancer xenograft model (Imam et al., 2010); however, this miR is not present in zebrafish, nor is it reported to be expressed embryonically. The miR30 family includes five members (ae) that share the same eight-nucleotide seed sequence and are conserved from zebrafish to humans. During Xenopus embryogenesis, the miR30 family regulates pronephros development through targeting the transcription factor Xlim1/Lhx1, and miR30a specifically is implicated in hepatobiliary duct formation in zebrafish (Agrawal et al., 2009; Hand et al., 2009). In situ analyses in Xenopus embryos also revealed expression of miR30 family members in the somites (Agrawal et al., 2009), which is consistent with a role in myogenic regulation. Here, utilizing molecular and embryological techniques in the zebrafish, we demonstrate that miR30a function is crucial for primary myogenesis. Furthermore, we demonstrate in vivo that the contribution of miR30a to muscle development is through direct modulation of Six1 protein expression. RESULTS miR30a and six1a/b expression inversely correlate during primary myogenesis Because precise modulation of Six1 protein levels is essential for proper myogenesis, we hypothesized that Six1 could be regulated through a miR-mediated mechanism. Owing to a whole-genome duplication event early in the teleost lineage, there are two SIX1 orthologs in zebrafish, six (...truncated)