Development • Supplementary information


Fig. S1. Expression of miR-133 in developing somites. Whole-mount in situ hybridization using double digoxigenin-labelled LNA probes (Exiqon) shows restricted expression of miR-133 in the neural tube (NT), anterior notochord (NC) and medial somites, but no expression in the sclerotome (SC). (A) HH15 stage embryo. 1-3 indicates levels of cryosections shown in panels 1-3, with a schematic representing somite expression. (B) RT-qPCR for miR-133 shows a relative increase of expression in more mature, anterior somites compared with posterior epithelial somites. Eight biological replicates. ***P<0.001; **P=0.001-0.01; *P=0.01-0.1. (C) Comparison of miR-133 and Gli3 expression in developing somites. Higher magnification of somites show miR-133 expressed in nascent myoblasts (MY) and Gli3 in the dermomyotome (DM). MD, mesonephric duct. 

(A) Gene Ontology (GO) terms of genes expressed at higher levels in AM133 injected somites were associated with angiogenesis and tube morphogenesis. (B) However, the false discovery rates (FDRs) for these were significantly higher compared to GO terms associated with the downregulated DE genes. 
cGli1 RT F cGli1 RT R TTGGCTGTCAGAGGGCTACT cGli2 RT F CACCATCAGCTCTGCCTACA cGli2 RT R TGAGCTGGTGTGAGGTTCAG cGli3 RT F CTCACCTCTTTCCAGCGTTC cGli3 RT R AATGTACGGGTGAGGAGTGC cPtch1 RT F GTGGAAGTTGGTGGACGAGT cPtch1 RT R CATGTACTCTGCTGGCCTGA cPtch2 RT F GAGTGAGGAGAAGGCAGGTG cPtch2 RT R CATGGTGACACAGGCATAGG cSmo RT F GACAACCCCAAGAGCTGGTA cSmo RT R CACAAAGAAGCAGGCATTGA cHHIP RT F CCCTCGACGATATGGAAGAA cHHIP RT R GTGGTGATCCACAGCACATC cPax3 RT F AGCAGAGCAACTGGAAGAGC cPax3 RT R GGTGGTTGAAAGCCATCAGT cPax7 RT F GCATCAAATTCGGGAAGAAA cPax7 RT R CTCTTCAAAGGCAGGTCTGG cMyf5 RT F CAACCCCAACCAGAGACTCC cMyf5 RT R GAGTCCGCCATCACATCGGA cMgn RT F GGCTTTGGAGGAGAAGGACT cMgn RT R CAGAGTGCTGCGTTTCAGAG cCDC20 RT F CCCTCAGCTGGAACAGCTAC cCDC20 RT R TGGTGCTGAGTGAAGGTCTG cCDK1 RT F TATAAAGGGCGCCACAAAAC cCDK1 RT R TCTTGAGGTCCATGGAAAGG cFGF8 RT F AGCAGAGCCTGGTGACAGAT cFGF8 RT R TTTCCCCTTCTTGTTCATGC cSNAI1 RT F CGATGCTCAGACCAGGAAAT cSNAI1 RT R AAGGGCTTTTCACCAGTGTG cGREM1 RT F AGGCACTGCACATCACTGAG cGREM1 RT R TCAGGGCAGTTGAGGGTAAC cGAPDH RT F TCTCTGGCAAAGTCCAAGTG cGAPDH RT R TCACAAGTTTCCCGTTCTCAG cGLI3-UTR F GCAGATCT TTTCACTTTCCACAAATCTGG cGLI3-UTR R ATGCTAGC TCAATCTGCC CAGTGAAATT A cGLI3-UTR-MUT F GCCTTATTTTGTGTTATCCTTTAACAGAGAGTTGACAAGATACGTTGACAAAT TTTCACAATGAGG cGLI3-UTR-MUT-R

Fig. S2 .

 S2 Fig. S2. Inhibition of miR-133 leads to a myogenic phenotype in early somites, but not in differentiating somites. Embryos were injected with FITC-labelled antagomir-133 (AM133) at HH14/15 or HH20. Whole mount in situ hybridisation was performed for Myf5, MyoD, Mgn or Pax3 as indicated. (A) 9 hour incubation, (B) 24 hour incubation post-injection. (C) AM133-injected into interlimb somites at HH20 and incubated for 24 hours. Antisense probes (purple), FITC-labelled antagomir (red). Number of embryos indicated on each panel. White arrowheads indicating loss of expression on injected side; black arrowheads indicating reduced expression or no change in expression on injected side. (D) Immunostaining for Cas3, an apoptosis marker, in embryos injected with AM133 at HH14/15 and incubated for 9 hours show no difference in AM133 injected side compared to contralateral non-injected side. Scale bar 50 μm. 




  Development 145: doi:10.1242/dev.159657: Supplementary information Development • Supplementary information 


Fig. S3 .

 S3 Fig. S3. Dermomyotome growth, epithelial organization and basement membrane deposition are not affected injection of a scrambled antagomir. (A) Schematic representation of the experimental procedure. Somites of HH14/15 embryos were injected with FITC-labelled scrambled antagomir (AMscr) and processed for immunostaining after 9 hours. (B) Somites were cryosectioned and immunostained for Pax3, Pax7, FITC and DAPI as indicated. The areas positive for Pax3 or Pax7 staining within the somite were quantified using Fiji/ImageJ. There was no significant difference between injected and non-injected contralateral control side. (C) Immunostaining for pH3, FITC and DAPI as indicated. The number of pH3-positive cells was similar in AMscr injected somites compared to the contralateral side. Scale bar 50 μm; ns, not significant. (D) Immunostaining for F-actin, laminin, FITC and DAPI as indicated. Epithelial organization and basement membrane deposition were not affected and were similar on both sides. Scale bar 50 μm; ns, not significant. (E) Western blot detecting Gli3 protein in somites injected with AMscr or AM133 shows increased level of Gli3Rep 


Fig. S4 :

 S4 Fig. S4: Differential transcriptomics of somites after miR-133 KD.(A) Gene Ontology (GO) terms of genes expressed at higher levels in AM133 injected somites were associated with angiogenesis and tube morphogenesis. (B) However, the false discovery rates (FDRs) for these were significantly higher compared to GO terms associated with the downregulated DE genes. 


Fig. S5 .

 S5 Fig. S5. Purmorphamine mediated activation of the Shh pathway rescues myogenic differentiation in absence of miR-133 function. Co-injection of FITC-labelled antagomir-133 (AM133) and purmorphamine (purm) into HH14/15 embryos was examined by in situ hybridisation for myogenin (Mgn) transcripts, which indicates normal expression after 9 hours incubation (n=6/6). Mgn antisense probe (purple), FITC-labelled antagomir (red). 




  Development 145: doi:10.1242/dev.159657: Supplementary information Development • Supplementary information 




  AGCUGGUAAAAUGGAACCAAA U-3' Sigma control siRNA MISSION siRNA Universal negative control (#1) Sigma; product number SIC001 Gli3 MO 5'-ACGTATCTTGGTCCCTCTCTGTTAA-3' Control MO Standard control from GeneTools Development 145: doi:10.1242/dev.159657: Supplementary information Development • Supplementary information 




  


Table S1 -PCR and mutagenesis primers, mimics and siRNA

 S1
Development • Supplementary information