Somites are embryonic precursors of the axial skeleton and skeletal muscles and ALK inhibitor 1 establish the segmental vertebrate body plan. situations that have critical requirements for tight post-transcriptional regulation. Introduction Somites are cohorts of cells that bud from the anterior end of the presomitic mesoderm (PSM) and give rise to the axial skeleton and other structures (reviewed in Hirsinger et al. 2000 During somitogenesis the expression levels of numerous genes oscillate in the PSM as part of a segmentation clock that controls the timing of somite formation. The Notch target was the first gene found expressed in this pattern (Palmeirim et al. 1997 In mouse and chick a key oscillatory gene is Lunatic fringe (transcript levels and LFNG protein levels oscillate with a period that matches the rate of somite formation (2 hours in the mouse 90 minutes in the chick) (Dale et al. 2003 Pourquie 2001 Either loss of expression or sustained non-oscillatory activity perturbs somite formation and patterning presumably by altering its oscillatory expression (Dale et al. 2003 Evrard ALK inhibitor 1 et al. 1998 Serth et al. 2003 Zhang and Gridley 1998 It is known that cyclic expression is regulated at the transcriptional level (Cole et al. 2002 but little is known about the post-transcriptional mechanisms that contribute to the rapid oscillations. ALK inhibitor 1 Stable oscillatory expression patterns have been proposed to be regulated by feedback inhibition mechanisms coupled with transcriptional time delays (Lewis 2003 Monk 2003 Some mathematical models of the segmentation clock invoke delayed feedback loops involving regulation of Notch1 and (or in chick). In these models mRNA and protein half-lives of oscillatory genes must be tightly regulated to ensure proper clock function (Feng and Navaratna 2007 Gonzalez and Kageyama 2009 The 3′UTR is evolutionarily conserved and has been proposed to regulate RNA half-life (Chen et al. 2005 Hilgers et al. 2005 One possible source of such regulation would be miRNAs non-coding RNA molecules that direct post-transcriptional repression of protein-coding genes by promoting RNA turnover and/or by decreasing translational efficiency of their target transcripts (reviewed in Bartel ALK inhibitor 1 2004 and one model of oscillatory gene expression has proposed miRNA functions in the clock (Xie et al. 2007 We hypothesized that the oscillatory expression of in the segmentation clock Rabbit Polyclonal to SPHK2 (phospho-Thr614). could require post-transcriptional regulation by miRNAs. Here we identify an miRNA (3′UTR. Inhibiting function or preventing interactions between and endogenous transcripts perturbs somitogenesis and disrupts clock function in the PSM of developing chick embryos. These findings support the hypothesis that regulation of oscillatory genes by miRNAs may provide a mechanism for post-transcriptional control of the segmentation clock. Results mir-125a-5p is expressed in the PSM and targets the 3′UTR To examine the possibility that oscillations might be regulated by miRNAs we assessed the expression of candidate miRNAs in the PSM where the clock is active. By QRT-PCR (Fig 1A) and miRNA microarray (data not shown) we found that mir-125a-5p levels are higher in the mouse PSM than in the mature somites. Thus its expression is enriched in the PSM where is predicted to require a short RNA half life. is proposed to target three sites in the mouse 3′UTR and one of the sites is ALK inhibitor 1 conserved in chicken (Fig. 1B). Whole mount hybridization confirmed specific expression of ALK inhibitor 1 in the PSM of mouse and chicken embryos (Fig 1C panels a – c). Futher expression was observed in mouse embryos in the ectoderm and mesoderm but was largely excluded from the neural tube notochord and tailgut (Fig 1C panels d and e). Figure 1 The 3′UTR is an evolutionarily conserved target of is significantly enriched in the PSM compared to the mature somites of E9.5 mouse embryos (*= p<0.05 Student’s T-Test. Error bars = ... The 3′UTR can be directly targeted by is a direct target of 3′UTR sequence exhibit lower luciferase expression than control vectors in these cells due to the effects of endogenous miRNAs (Fig. S1A). However expression of exogenous causes a further significant reduction in luciferase expression only from vectors containing the mouse or chicken 3′UTR (Fig. 1D-E). In contrast mir-125a-5p binding sites were not identified in the 3′UTRs of other oscillatory genes and expression had no effect on expression of transcripts containing the 3′UTR (Fig. S1B). Mutation of predicted binding sites at either end of the mouse 3′UTR.