Volume 24, Issue 5, Pages 554-561 (March 2013) mir-125a-5p-Mediated Regulation of Lfng Is Essential for the Avian Segmentation Clock  Maurisa F. Riley, Matthew S. Bochter, Kanu Wahi, Gerard J. Nuovo, Susan E. Cole  Developmental Cell  Volume 24, Issue 5, Pages 554-561 (March 2013) DOI: 10.1016/j.devcel.2013.01.024 Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 The Lfng 3′ UTR Is an Evolutionarily Conserved Target of mir-125a-5p (A) By qRT-PCR mir-125a-5p 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 = SD). (B) Lfng 3′ UTR schematic showing high conservation among mouse, human, and chicken Lfng 3′ UTRs (mVista Bray et al., 2003). Conserved regions shown as colored boxes. Positions of TargetScan predicted miRNA binding sites define the first nucleotide of the 3′ UTR as 1. (C) RNA in situ analysis of mir-125a-5p in whole-mount mouse embryos at E9.5 (a) and E10.5 (b) and in HH10 chick embryos (c). Section in situs of E10.5 mouse embryos (d, sagittal; e, transverse) demonstrate mir-125a-5p expression in the ectoderm and mesoderm, but not in the neural tube, notochord, or tail gut. Arrows indicate most recent somite boundary; nt, neural tube; not, notochord; tg, tailgut. In situs with negative control probe on adjacent sections did not exhibit staining (b′, c′, d′, e′). (D) Transfection of pre-mir-125a-5p (125a) significantly reduces luciferase expression from pmir-mLfng3′ UTR (pMIR-REPORT + mouse Lfng 3′ UTR) compared to transfection of a negative control pre-mir (NC#1). Mutations of the mir-125a-5p binding sites at either end of the 3′ UTR (MUT1-3) abrogate this effect. (E) Transfection of pre-mir-125a-5p significantly reduces luciferase expression from pmir-cLfng3′ UTR (pMIR-REPORT + chicken Lfng 3′ UTR). Mutations in the mir-125a-5p (MUT) binding site abrogate this effect. Two-way ANOVA, Bonferroni post hoc; ∗p < 0.05, ∗∗p < 0.01, error bar = SD. See Figure S1 for related analyses. Developmental Cell 2013 24, 554-561DOI: (10.1016/j.devcel.2013.01.024) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 Inhibition of mir-125a-5p Perturbs Somitogenesis in Chicken Embryos and Stabilizes Lfng Transcripts in the PSM (A) Electroporation of anti-NC#1MO has no effect on somite morphology (a), while electroporation of anti-mir-125aMO results in abnormal somite morphology with absent (dashed line) or disorganized (arrows) intersomitic boundaries in electroporated regions (b). (B) Uncx4.1 staining is reduced and diffuse in embryos electroporated with anti-mir-125aMO (dashed line, b) compared to anti-NC#1MO (a). Note relatively normal somites in the older region of the anti-mir-125aMO embryo in the region that is less positive for the morpholino (square bracket). The same embryo is pictured in (Aa) and (Ba). (C) MyoD expression is disorganized in somites electroporated with anti-mir-125aMO (dashed line, b) compared to embryos electroporated with anti-NC#1MO (a). Note normal myotomes in the unelectroporated regions of the anti-mir-125aMO embryo (square brackets). (D) Two hours postelectroporation with anti-NC#1MO, endogenous Lfng is observed in the three described phases (Pourquié and Tam, 2001). In contrast, in anti-mir-125aMO-positive embryos, robust, noncyclic Lfng expression is observed in the caudal PSM of all embryos (d, n = 15/15). (E) In situ analysis with a probe specific for the Lfng intron demonstrates that both control embryos (a and b) and embryos electroporated with anti-mir-125aMO (c and d) exhibit dynamic patterns of newly transcribed Lfng RNA. (F) c-hairy-1 expression is cyclic in control embryos (a–c) or in embryos electroporated with anti-mir-125aMO. (d–f) Right-hand panels reflect the fluorescein signal demarcating the electroporated regions of each embryo. Fl, fluorescein. Degree of electroporation efficiency designated as ++, strong; +, moderate; (+), weak; −, negative. Arrowheads indicate the most recent somite boundary. See Figure S2 for fluorescein images as well as analysis of mir-125a-5p by in situ in electroporated embryos. Developmental Cell 2013 24, 554-561DOI: (10.1016/j.devcel.2013.01.024) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 Blocking Interactions between mir-125a-5p and Lfng Perturbs Somitogenesis and Segmentation Clock Function (A) Schematic of the Lfng 3′ UTR and the TPs used, with the single mir-125a-5p binding site in chicken. Approximate positions of Lfng-TPCtrl (blue) and Lfng-TPmir-125a (red) are shown (not to scale). (B) Electroporation of Lfng-TPctr has no effect on somite morphology (a and a′), while electroporation of Lfng-TPmir-125a results in abnormal somite morphology with absent (dashed lines) or disorganized (∗) intersomitic boundaries (b and b′). (C) Uncx4.1 expression is disorganized and sometimes reduced in Lfng-TPmir-125a-positive embryos (dashed line, b and b′) compared to embryos electroporated with Lfng-TPctr (a and a′). Note relatively normal somites in the less positive region of (b) (square bracket). (D) MyoD expression in the somites of Lfng-TPmir-125a-positive embryos (b and b′) indicates that myotomes are formed, but somite compartments are of irregular sizes (∗) compared to embryos electroporated with Lfng-TPctr (a and a′). In some regions of the embryo, MyoD expression is strongly downregulated or delayed (dashed line, b). (E) Two hours postelectroporation, cyclic expression of endogenous Lfng is observed in Lfng-TPCtrl embryos (a–c), while robust, noncyclic Lfng expression is observed in the caudal PSM of Lfng-TPmir-125a-positive embryos (d, n = 14/14). Fl: fluorescein as described in Figure 2. Arrowheads indicate the most recent somite boundary. See Figure S3 for analysis of target protector activity in cell lines, and Figure S4 for fluorescein images. Developmental Cell 2013 24, 554-561DOI: (10.1016/j.devcel.2013.01.024) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 Long-Term Inhibition of Interactions between mir-125a-5p and Lfng Perturbs the Oscillatory Expression of Genes Linked to the Segmentation Clock via Feedback (A) Eight hours postelectroporation, cyclic expression of endogenous Lfng is observed in Lfng-TPCtrl embryos (a–c) while endogenous Lfng expression is noncyclic, with no expression detected in the caudal PSM of Lfng-TPmir-125a-positive embryos (d, n = 15/15). (B) Eight hours postelectroporation, cyclic expression of endogenous c-hairy-1 is observed in Lfng-TPCtrl embryos (a–c), but noncyclic c-hairy-1 expression is observed in the caudal PSM of Lfng-TPmir-125a-positive embryos (d, n = 15/15). Fl, fluorescein; + and ++ indicate relative levels of fluorescein signal, images available in Figure S4. (C) Model for effects of mir-125a-5p in the clock. Stable clock oscillations are governed in part by interlocking feedback loops where Notch activates Lfng and c-hairy-1 (green arrows), while LFNG protein inhibits Notch signaling, and c-hairy-1 protein inhibits its own transcription and that of Lfng (red lines). The lengths of delays imposed by transcription rate and translational efficiency (dashed lines), as well as the half-lives of transcripts and proteins are critical for maintenance of stable oscillations. mir-125a-5p is proposed to increase the rate of Lfng transcript turnover and/or decrease the efficiency of translation. In the absence of mir-125a-5p, (middle) levels of Lfng transcript and LFNG protein increase. In the long term (right), the effect of increased Lfng transcript stability is loss of robust oscillations, with stable, increased levels of c-hairy-1 transcript and stable decreased transcription of Lfng. See also Figure S4 for fluorescein images. Developmental Cell 2013 24, 554-561DOI: (10.1016/j.devcel.2013.01.024) Copyright © 2013 Elsevier Inc. Terms and Conditions