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Results: 5

1.
Fig. 5.

Fig. 5. From: MicroRNA regulation of the paired-box transcription factor Pax3 confers robustness to developmental timing of myogenesis.

Model illustrating that the complete silencing of Pax3 in committed myoblasts confers robustness to developmental timing of differentiation; see Discussion for details.

Katarzyna Goljanek-Whysall, et al. Proc Natl Acad Sci U S A. 2011 July 19;108(29):11936-11941.
2.
Fig. 1.

Fig. 1. From: MicroRNA regulation of the paired-box transcription factor Pax3 confers robustness to developmental timing of myogenesis.

Expression of miR-1 and miR-206 is inversely correlated with Pax3 in developing muscle. (A) Somite sections and schematics illustrating the distribution of miR-1, miR-206, and Pax3 in dermomyotome lips (dml, vll) and myotome (m); probes are indicated above each panel. (B) Whole-mount views of forelimbs (fl) and hindlimbs (hl) at HH stages 24 and 28, hybridized with Pax3, miR-1, or miR-206 probes as indicated. Pax3 transcripts detected at HH24 were significantly decreased by HH28, concomitant with increased miR-206 expression at that stage. miR-1 was detected faintly in the HH28 hindlimb but strongly in the heart (ht) and somites. Stippled lines indicate outline of limbs, and colored asterisks indicate equivalent regions. (C) Double in situ hybridization showing ectopic miR-206 expression (red, detecting GFP from miR-206 expression vector) leading to a loss of Pax3 transcripts (purple); a whole mount and a section are shown.

Katarzyna Goljanek-Whysall, et al. Proc Natl Acad Sci U S A. 2011 July 19;108(29):11936-11941.
3.
Fig. 2.

Fig. 2. From: MicroRNA regulation of the paired-box transcription factor Pax3 confers robustness to developmental timing of myogenesis.

The 3′-UTR of Pax3 is regulated by miR-1 and miR-206. (A) Alignment of target sites TS1 and TS2 with miR-1 and miR-206; Materials and Methods provides the genomic positions. (B) Normalized luciferase activity shown for Pax3–3′-UTR sensor constructs cotransfected with miR-1, miR-206, or miR-140 as indicated by (+). Activity was plotted relative to control (no miRNA). Experiments were repeated four times with triplicate samples and two independent DNA plasmid preparations. Error bars represent SE (n = 12). The constructs used are indicated above the graphs; TSm represents constructs with mutant target sites. (C) qPCR of Pax3 expression in RuGli cells. Pax3 transcript levels in mock-transfected control cells (lane 1) were reduced after transfection of miR-206 (lane 3) and miR-1 (lane 4). Cotransfection of antimiR-206 (AM) with miR-206 (lane 2) restored Pax3 expression. (D) Western blot analysis showing reduced Pax3 protein in RuGli cells transfected with miR-206 (lane 3) and miR-1 (lane 4). Protein levels were unaffected after cotransfection of antimiR-206 (AM) with miR-206 (lane 2).

Katarzyna Goljanek-Whysall, et al. Proc Natl Acad Sci U S A. 2011 July 19;108(29):11936-11941.
4.
Fig. 4.

Fig. 4. From: MicroRNA regulation of the paired-box transcription factor Pax3 confers robustness to developmental timing of myogenesis.

miRNA affects somite myogenesis predominantly via Pax3. (A) FITC-labeled TP MO, designed to prevent miR-1/miR-206 interactions with both target sites in the Pax3 3′-UTR, were electroporated. After overnight incubation, somites were microdissected. (B–D) Western blot (B) and qPCR (C) demonstrated increases in Pax3 protein and transcripts after electroporation with TP1 plus TP2 compared with contralateral noninjected somites or somites electroporated with control morpholinos (MO). qPCR (C) and in situ hybridization (D) demonstrated a corresponding loss of myogenin expression (Upper); myogenin expression in control morpholino-injected somites was comparable to that in noninjected somites from the opposite side (Lower). (E–H) Electroporation of Pax3/GFP expression vectors (red) led to localized loss of myogenin (blue), consistent with the concept that elevated Pax3 levels are incompatible with differentiation (Upper). (E and G) Electroporation of Pax3 splice morpholinos (red) had no effect on myogenin expression (blue; Lower). (H) Western blot analysis confirmed reduced Pax3 expression in somites after Pax3 MO electroporation. (F) Cotransfection of Pax3 splice-MO (red) rescued antagomiR-206–induced loss of myogenin transcripts and restored normal expression of myogenin (blue; Upper), whereas control morpholino (red) did not rescue the antagomir-206 mediated inhibition of myogenin expression (blue; Lower).

Katarzyna Goljanek-Whysall, et al. Proc Natl Acad Sci U S A. 2011 July 19;108(29):11936-11941.
5.
Fig. 3.

Fig. 3. From: MicroRNA regulation of the paired-box transcription factor Pax3 confers robustness to developmental timing of myogenesis.

Delayed myogenic differentiation after antagomir-mediated inhibition demonstrates a requirement for miR-1 and miR-206 activity. Antagomirs injected into presomitic mesoderm of HH12–14 embryos are indicated above each panel. Myogenic differentiation was assessed after 24 h by in situ hybridization with a myogenin probe. (A–D) Injection of scrambled antagomir had no effect (A); however, injection of antagomir-206 (B) or a 1:1 mixture of antagomir-206 and antagomir-1 (D) led to a loss of myogenin expression. Injection of antagomir-1 also affected myogenic differentiation (C). (E) Quantification of phenotypes observed. (F, F′, and G) Sections immunostained for Pax3 in the dermomyotome (red) and MF20 in the myotome (green) confirmed the inhibition of myogenic differentiation after antagomir-206 injection (F) compared with control embryos (G). F′ is a higher-magnification view of F illustrating the increased number of Pax3-positive cells in the myotome (white arrowheads). Fewer myosin heavy chain (MF20)-positive cells were detected on the injected side, indicated by an asterisk, see also Fig. S4. (H and I) qPCR demonstrating the opposite effects on Pax3 and myogenin transcript levels in antagomiR-206 injected somites. (J and K) Western blots of dissected somites treated as indicated revealing increased Pax3 protein levels in antagomiR-206 injected somites with no change apparent on Pax7 protein levels. (L) Northern blot showing that antagomiR-1 specifically affects miR-1 and antagomiR-206 affects miR-206.

Katarzyna Goljanek-Whysall, et al. Proc Natl Acad Sci U S A. 2011 July 19;108(29):11936-11941.

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