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to examine temporal miRNA expression patterns during the first 5 d post-fertilization (dpf) of zebrafish development (unpublished data).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,...

A number of genes have been implicated in regeneration, but the regulation of these genes, particularly pertaining to regeneration in higher vertebrates, remains an interesting and mostly open question. We have studied microRNA (miRNA) regulation of regeneration and found that an intact miRNA pathway is essential for caudal fin regeneration in zebrafish. We also showed that miR-203 directly targets the Wnt signaling transcription factor Lef1 during this process. Repression of Lef1 by miR-203 blocks regeneration, whereas loss of miR-203 results in excess Lef1 levels and fin overgrowth. Expression of Lef1 from mRNAs lacking 3 UTR recognition elements can rescue the effects of excess miR-203, demonstrating that these effects are due to specific regulation of lef1 by miR-203. Our data support a model in which regulation of Lef1 protein levels by miR-203 is a key limiting step during regeneration.M ost vertebrates, including humans, are unable to regenerate the majority of lost or damaged tissues. In contrast, zebrafish are able to regenerate various damaged tissues, including fins, hearts, retinas, and spinal cords (1). For fins, regeneration relies on the formation of blastema cells, stem cell-like cells that either are recruited to the damaged area or originate from the de-differentiation of cells in the area (2, 3). Zebrafish caudal fins undergo isometric growth (i.e., fin grows in proportion to body size) throughout life, and understanding the regulatory mechanisms for controlling such growth remains a key question. The fin is composed of multiple bony rays that grow autonomously and are made up of bony segments, termed lepidotrichia. Each ray is composed of two hemirays, which create a protective shell around nerves, blood vessels, and mesenchymal cells. Fins grow through the addition of bone to the distal tip of the fin. Regeneration proceeds through at least five steps: wound healing, mesenchymal disorganization or reorganization, blastema formation, outgrowth, and termination (1, 4). miRNAs are a recently discovered class of genes that regulate gene expression at the posttranscriptional level and are required for development, stem cell maintenance, and renewal (5-18). Recently, Yin et al. (19) showed that fibroblast growth factor (Fgf) signaling alters the expression of multiple miRNAs during regeneration. One of the miRNA targets of Fgf signaling, miR-133, targets mps1, which encodes a kinase that regulates blastemal proliferation. Interestingly, these authors also found that various other markers of regeneration were indirectly activated on the reduction of miR-133 levels, suggesting that overall regulation of regeneration by miRNAs might be quite complex. Here we show that an intact miRNA pathway indeed is essential for regeneration. Furthermore, we show that in addition to regulation of Fgf signaling during regeneration, Wnt signaling also subject is to miRNA regulation through miR-203 control of Lef1.To examine global miRNA expression patterns in regenerating fins, we first conducted microarrays. Cauda...

MicroRNAs (miRNAs) are highly conserved small RNAs that act as translational regulators of gene expression, exerting their influence by selectively targeting mRNAs bearing complementary sequence elements. These RNAs function in diverse aspects of animal development and physiology. Because of an ability to act as rapid responders at the level of translation, miRNAs may also influence stress response. In this study, we show that the miR-8 family of miRNAs regulates osmoregulation in zebrafish embryos. Ionocytes, which are a specialized cell type scattered throughout the epidermis, are responsible for pH and ion homeostasis during early development before gill formation. The highly conserved miR-8 family is expressed in ionocytes and enables precise control of ion transport by modulating the expression of Nherf1, which is a regulator of apical trafficking of transmembrane ion transporters. Ultimately, disruption of miR-8 family member function leads to an inability to respond to osmotic stress and blocks the ability to properly traffic and/or cluster transmembrane glycoproteins at the apical surface of ionocytes.

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Background: microRNAs (miRNAs) are small (~22 nt) non-coding RNAs that regulate cell movement, specification, and development. Expression of miRNAs is highly regulated, both spatially and temporally. Based on direct cloning, sequence conservation, and predicted secondary structures, a large number of miRNAs have been identified in higher eukaryotic genomes but whether these RNAs are simply a subset of a much larger number of noncoding RNA families is unknown. This is especially true in zebrafish where genome sequencing and annotation is not yet complete.