Fig. S1. Weight of embryos during development. Embryos are collected at different time points (E12.5, E14.5, E16.5 and E18.5) from matings between
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1 Fig. S1. Weight of embryos during development. Embryos are collected at different time points (E12.5, E14.5, E16.5 and E18.5) from matings between Myod +/ or Myod / females and Myod +/ ;Igf2 +/ males and weighed. Embryos obtained from Myod +/ females showed a Mendelian distribution. For weight data, all litters were pooled. wt, Myod +/ and Myod / mutants have similar weight curves, showing no effect of the Myod mutation on the weight of the embryos before birth. By contrast, embryos carrying a deleted Igf2 show a reduction in weight ranging from 20% at E12.5 to 40% just before birth. This reduction is identical in Igf2 +/, Myod +/ ;Igf2 +/ and in Myod / ;Igf2 +/ embryos, showing that Igf2 is epistatic on Myod for the weight phenotype.
2 Fig. S2. Fiber repartition size in intercostal muscle. (A,B) Transverse sections of E18.5 posterior limb from wt (A) and DM (B) embryos stained with Hematoxylin and Eosin. (C) Immunostaining for laminin on E18.5 diaphragm sections from the four genotypes. Fiber cross-sectional area (as measured by MetaMorph software) is presented (n=5 for each genotype); au, arbitrary units. In the absence of Igf2 protein, intercostal fibers exhibit a diminution of their cross-sectional area. (D) E18.5 intercostal muscle sections were immunostained with antibodies to laminin. Muscle fiber number was counted (using MetaMorph software) for the four genotypes.
3 Fig. S3. Ossification of the sternum at E18.5. E18.5 embryos are stained with Alizarin Red (bone) and Alcian Blue (cartilage). Rib cages from Myod +/, Myod +/ ;Igf2 +/ and DM embryos are shown with arrows pointing to ossification segments of the sternum. The white arrow indicates the fifth ossification segment, which is missing in the DM embryo. wt and Myod +/, n=3; Igf2 +/ and Myod +/ ;Igf2 +/, n=2; DM, n=5. Fig. S4. Diaphragm formation during development. (A) Sagittal sections of E13.5 wt, Igf2 +/, Myod / and DM embryos were immunostained for Pax7 (α-pax7, green) and laminin (α-laminin, red). Stars indicate the position of the diaphragm. (B) Sagittal sections of E18.5 wt, Igf2 +/, Myod / and DM embryos were immunostained for Pax7 (green). DM diaphragms are thinner than wt, Myod / and Igf2 +/ diaphragms. wt, Myod +/, Igf2 +/, Myod +/ ;Igf2 +/ and DM, n=5.
4 Fig. S5. Interactions of Myod with the H19-Igf2 locus. (A) Expression level of Igf2 mrna as assessed by RT-qPCR in diaphragm and limb muscle samples from H19 Δ3 /+ ;Myod +/ and H19 Δ3 /+ ;Myod / embryos. Genotyping of H19 Δ3 mice is normally performed by probing for the inserted neo gene by PCR. However, since the Myod mutants also contain a neo insertion, the identification of the H19 Δ3 versus wt embryos required an RT-qPCR step to detect presence or absence of H19 expression. In the absence of the H19 gene, Myod status does not affect Igf2 expression. (B) ChIP-Seq data showing the position of the peak of Myod binding in the H19-Igf2 locus. The genes of the region are indicated by black boxes. Red boxes show the endodermal and mesodermal enhancers described in the literature. ICR, imprinting control region; HUC, H19 upstream conserved region; CS, conserved sequence. (C) 3C experiment showing the interactions between the mesodermal enhancer CS9 (located at +25 kb from the start of the H19 gene) and other regions of the locus. The 4 kb region corresponds to the localization of the ICR upstream of the H19 gene. Interactions occur with the H19 (p-h19) and Igf2 (P0, P1 and P2-P3) promoters. Location of the ICR, H19 and Igf2 genes and CS9 enhancer are shown by rectangles.
5 Movie 1. Contraction of the diaphragm after electric stimulation of the phrenic nerve of Igf2 +/ single-mutant and DM E18.5 embryos.
6 Table S1. Primers for genotyping Myod, Igf2 and H19 mutants; RT- qpcr of H19, Igf2, Myod, Srf, cact and skact; RT- and qpcr of mir p; and the Srf ChIP experiment Gene PCR primers Annealing temperature Igf2 CTAGCTCAAAGCCCTGCGTTTCTTTC TGCGCTGACAGCCGGAACAC 58 C CCCAGGGCATCTATGATTCTGCCGA Myod TGTAGTAGGCGGTGTCGTAGCC 62 C Genotyping PGKR1 MutH19F AGGGGAGGAGTAGAAGGTGGCGCGGAA AATGGGAAACAGAGTCACG H19 Δ13 MutH19R wt H19 F GACAGTGGGAGTGGCACCTT CCATCTTCATGGCCAACTTCT 58 C wt H19 R CTAGAGCTCGCTGATCAGCCT Gapdh ACAGTCCATGCCATCACTGCC GCCTGCTTCACCACCTTCCTTG 58 C Tbp GGTATCTGCTGGCGGTTTGG GCCCTGAGCATAAGGTGGAA Myod GGGCCGCTGTAATCCATCATG CTGCCTTCTACGCACCTGGA Quantitative expression Igf2 H19 CGACGGTTGGCACGGCTTGA GGTGCTTCTCATCTCTTTGG GGAGACTAGGCCAGGTCTC GCCCATGGTGTTCAAGAAGGC Srf CACCTACCAGGTTGTCGGAAT GCTGTCTGGATTGTGGAGGT cact ACTCTCTTCCAGCCCTCCTTTCATT GGAGCCAGTGCAGTGATTTCCTT skact CGTGAAGCCTCACTTCCTACC AGAGCCGTTGTCACACACAA
7 mir-483-5p RT stem-loop primer: 5'-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACCTCCCTT-3' PCR primer F: 5'-CCGGAAGACGGGAGAAGAGA-3' R: 5'-GTATCCAGTGCGTGTCGTGGAGT-3' Srf ChIP primers Myod DRR F: 5'-GCCCGCAGTAGCAAAGTAAG-3' R: 5'-GAAACCGGATCCAACTAGCA-3' Myod CArG F: 5'-GCCTAGCCAGACCAACATTC-3' R: 5'-CTTTGATTTCCCCCTGTCCT-3' Il4 intron F: 5'-AGAATGAAAGGCCCCAAAGT-3' R: 5'-GGGAGGACAGATCTCTGGTG-3'
8 Table S2. Primers for 3C-qPCR analysis of CS9 3C-qPCR BamHI site No Anchor (CS9) PCR primers Annealing temperature ATGACCACCAGATGTCAAGCTCG 62 C CTGCTCCGTGTGAGTTCCTTGG 64 C AGGACCGCAAATCAGACAAGGG 62 C AGCCTGCGTTTCTTTCTCCAGG 62 C GGCCCTCCATCTTGTCTCTTCC 64 C GTGGCAAGGAAAGTGAAGGAGG 62 C CAAGATAAGGACTCATTAGGCCTAGG 63 C ATGGCCCCATTAGAGAGCTACTG 62 C GACACAGGCTGGGCTATGTTTTC 62 C CTGTGACAGTGGTATGCACCAAG 62 C CTGGCCTGAGTACCTCTCCAC 64 C GTCCTCTGCCTTCTGGACTTTGG 64 C TTAGCTCTGGCTCACCCATCTG 62 C GCCTGAATACCCAAGACCTCATAC 63 C ACACGAAGGTTGGGGAGATAGG 62 C CCAGAGCAGGATGTGAGAGGG 64 C TAGGCGGGAGACATAGAAACTGC 62 C GCAGGGTTGCCAGTAAAGACTG 62 C GCCTTGTCGTAGAAGCCGTCTG 64 C TGGAATGTGGGGAGACAAACAGC 62 C CATACCGGGCAGTAGACCTGAC 64 C CCTCCCAGGTCCTGAAGAATAC 62 C CTTTAGGTAGCCCAAGGCTCAG 62 C CCGTCCTTTGGGCATAGCTTCC 64 C
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