Zebrafish miR-214 modulates Hedgehog signaling to specify muscle cell fate

Abstract

Numerous microRNAs (miRNAs) have been discovered in the genomes of higher eukaryotes, and functional studies indicate that they are important during development. However, little is known concerning the function of individual miRNAs. We approached this problem in zebrafish by combining identification of miRNA expression, functional analyses and experimental validation of potential targets. We show that miR-214 is expressed during early segmentation stages in somites and that varying its expression alters the expression of genes regulated by Hedgehog signaling. Inhibition of miR-214 results in a reduction or loss of slow-muscle cell types. We show that su(fu) mRNA, encoding a negative regulator of Hedgehog signaling, is targeted by miR-214. Through regulation of su(fu), miR-214 enables precise specification of muscle cell types by sharpening cellular responses to Hedgehog.Multicellular organisms such as zebrafish use miRNAs to regulate gene expression in a tissue-or time-specific manner, guiding developmental decisions 1,2 . To identify target genes regulated by miRNAs, we first developed a microarray to examine temporal miRNA expression patterns during the first 5 d post-fertilization (dpf) of zebrafish development (unpublished data). To understand the function of a subset of these miRNAs, we performed loss-of-function experiments using antisense morpholino oligonucleotides complementary to mature miRNAs. Morpholinos have been used extensively in zebrafish as antisense inhibitors of mRNA translation and splicing 3 but are also capable of interfering with miRNA function ( Supplementary Fig. 1 online). Injection of morpholinos designed to block the function of miR-214 (214 MO ) yielded embryos with U-shaped somites at 1dpf (1 dpf) ( Fig. 1a-d). Expression of miR-214 begins during early somitogenesis and continues throughout embryogenesis (Fig. 1e). In situ hybridization showed that miR-214 is expressed in somites at 1 dpf ( Fig. 1f,g; see also ref. 2).Somites are transient embryonic structures derived from paraxial mesoderm that give rise to muscle and skeleton 4 . Presomitic mesodermal cells immediately adjacent to the notochord (adaxial cells) are highly influenced by Hedgehog and give rise to the slow-twitch muscle lineage 5,6 . Lateral presomitic cells give rise to fast-twitch muscle fibers and experience little stimulation by Hedgehog initially, whereas later-developing fast-muscle fates are dependent on Hedgehog signaling 7 . There are two slow muscle cell types that require precise COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. Hedgehog signals for proper development: superficial slow fibers (SSFs), which migrate from the midline to populate the surface of the myotome, and slow muscle pioneers that remain close to the midline 6,8 . Muscle pioneers require higher levels of and longer exposure to Hedgehog for proper specification than SSFs and can be distinguished from slow muscle fibers by the expression of the transcription factor Engrailed (Eng) 4,...

Chemical modification and design of anti-miRNA oligonucleotides

“…45 Not surprisingly, PMOs have also proven to be effective anti-miRNA compounds in Zebrafish. Flynt et al 46 reported knockdown of miR-214 in Zebrafish, demonstrating a role for this miRNA in muscle cell development. Like PNAs, neutral PMOs are difficult to use with mammalian cells.…”

Regulation of zebrafish fin regeneration by microRNAs

“…2B). We also co-injected antisense morpholinos complementary to miR-203 (i.e., miR-203 MO ) that function to block the activity of endogenous miR-203 and thus should lead to increased fluorescence (31,33). As a control, GFP lacking the lef1 3Ј UTR also was injected into single-cell zebrafish embryos with and without exogenous miR-203 and miR-203 MO (Fig.…”

“…Western Blot Analyses. At 1 dpf, embryos were dechorionated, deyolked, and sonicated in lysis buffer as described previously (33). Then Ϸ30 embryos were pooled, and one-sixth of the resultant volume was loaded into each lane (Fig.…”

MicroRNA‐214 is aberrantly expressed in cervical cancers and inhibits the growth of HeLa cells

“…Recent studies have shown that miRNAs are aberrantly expressed in different cell types and at different developmental stages, suggesting that miRNAs play important roles in cell growth (8)(9)(10)(11)(12), differentiation (13,14), metabolism (15), and programmed cell death (16). Moreover, previous studies have demonstrated that a number of miRNAs are also involved in tumor pathogenesis and the development of different types of human cancers, including cervical (17), ovarian (8,18), lung (19), breast (9), colon (10), and prostate cancers (12).…”

“…miR-214 was first explored for its role in apoptosis (20). Later, several studies documented that miR-214 is involved in the murine aging process (21), modulates Hedgehog signaling to specify muscle cell fate (14), and induces cell survival and cisplatin resistance by targeting PTEN in human ovarian cancer (8). However, the role of miR-214 in cervical cancer cells remains unknown.…”