Additional file 2: Figure S2. of Identification in GRMD dog muscle of critical miRNAs involved in pathophysiology and effects associated with MuStem cell transplantation

Clinical follow-up. Clinical scores of mock GRMD dogs (---) and MuStem cell-injected dogs (—) are represented as mean ± SD. The clinical score of each GRMD dog was assessed weekly and expressed as a percentage of a theoretical healthy dog score. Limits of the MuStem cell delivery window are indicated (dashed lines). (PDF 32 kb

Sites of β-galactosidase activity in transgenic mouse embryos.

<p>All sites showed only selective cells positive for enhancer activation. DRGs = dorsal root ganglia; E = embryonic day of gestation; MN = motoneurons; OFT = cardiac outflow tract; PA = pharyngeal arch; PSM = pre-somitic mesoderm.</p

<i>In vitro</i> reporter assays support an additive combinatorial effect of transcription factors upon the FGF10 intronic enhancer.

<p>LUC-<i>FGF10</i>-Int1, which construct placed the luciferase gene under the control of the FGF10-Int1 element, was transfected alone or together with <i>ISL1</i>, <i>GATA4</i> and <i>TBX20</i> expression vectors into 10T1/2 cells. Each factor alone potentiated luciferase expression and these effects were additive in combination.</p

Bioinformatics analyses of the human <i>FGF10</i> locus surrounding the first exon.

<p><b>A</b>: Alignment of genomic regions around and within the human [hg18] <i>FGF10</i> locus to those of frog [xenTro2], chicken [galGal3], opossum [monDom4], mouse [mm9], dog [canFam2] and rhesus macaque [rheMac2] with colored regions >90% identical and the vertical scale ranging from 50% (bottom) to 100% (top). Color code for genomic features at <a href="http://ecrbrowser.dcode.org/ecrInstructions/ecrInstructions.html" target="_blank">http://ecrbrowser.dcode.org/ecrInstructions/ecrInstructions.html</a>. The <i>FGF10</i>-Pr1, <i>FGF10</i>-Pr2 and FGF10-Int1 regions examined in this study are boxed. <b>B</b>: A non-canonical predicted site for GATA-type transcription factors is 52 nucleotides 5′ to the ISL1 cognate sequence in <i>FGF10</i>-Int1 in the direction of transcription on the – strand in humans, mice and (not shown) macaque and opossum. <b>C</b>: Nucleotide sequence of the <i>FGF10</i>-Int1 enhancer module and position of conserved putative transcription factor binding sites as predicted by rVista (<a href="http://rvista.dcode.org" target="_blank">http://rvista.dcode.org</a>). All indicated human sites are identical to those of the macaque and mouse except for the SMAD prediction, only found in mouse; the ISL1, GATA and HOXA7 sites are also identical to the opossum, and the ISL1, NKX2-5 and TBX sites are also identical to the dog.</p

Expression of <i>ISL1</i> and <i>GATA4</i> transcripts in the human heart between 26 and 38 days of gestation.

<p><b>A–H</b>: <i>ISL1 in situ</i> at Carnegie stages (CS)12 (26–28 days post fertilization [dpf]), CS13 (28–31 dpf), CS14 (32–33 dpf) and CS15 (34–36 dpf) respectively. <b>E–H</b> are magnifications of <b>A–D</b> respectively. <b>I–K</b> show <i>GATA4</i> expression in adjacent sections to <b>B–D</b>. <b>A</b>: <i>ISL1</i> is expressed at CS12 in foregut endoderm, splanchnic mesoderm, and early motoneurons. <b>B, F</b>: At CS13, <i>ISL1</i> is transcribed by mesenchyme around the cardiac OFT and pharyngeal arches. <i>ISL1</i> expression continues in the splanchnic mesoderm between the trachea and OFT, and is visible in dorsal root ganglia, at CS14 (<b>C, G</b>) and CS15 (<b>D, H</b>). <b>I–K</b>: <i>GATA4</i> is expressed in the endocardium and myocardium of the arterial pole at CS13, CS14 and CS15 (<b>I, J, K</b> respectively). <b>Inset</b>: RT-PCR of <i>ISL1</i>, <i>GATA4</i>, <i>GATA5</i>, <i>GATA6</i>, <i>FGF10</i> and positive control <i>ACTB</i> mRNAs in embryonic human hearts at stages CS13-16 (to 40 dpf). Abbreviations: drg, dorsal root ganglia; es, esophagus; fb, forebrain; fg, foregut; ph, pharynx; nt, neural tube; oft, OFT; ra, right atrium; t, trachea. Arrows, motoneurons. Bar: 110 µm (A–D, I) and 55 µm (E–H, J, K).</p

Flow chart of the statistical filtration of microarray data.

<p>Procedure leading to classification for each of the three pairwise comparisons of healthy, GRMD and GRMD^MuStem dog muscle samples.</p

Functional characterization of GRMD dog model.

<p>A selection of Gene Ontology (GO) terms significantly enriched in the groups of genes up- and down-regulated between GRMD dog model and healthy dog. Associated with the GO term, the total numbers of genes assigned with this annotation are indicated, followed in the rectangle by the number of genes observed (on left) and expected by chance (on right). Numbers in bold indicate a significant over-representation of the corresponding GO term. Enrichments are colour-coded according to the associated p-value from red (enriched) to blue (depleted).</p

Clinical follow-up.

<p>(A) Mean±SD clinical scores of mock GRMD dogs and MuStem cell-injected dogs (GRMD^MuStem). The clinical score of each GRMD dog was assessed weekly and expressed as a percentage of a theoretical healthy dog score. Limits of the MuStem cell delivery window are indicated (dashed lines). (B) Right lateral view of a GRMD^MuStem dog, #7G^Mu. (C) Right lateral view of mock GRMD dog, #4G. Note the anterior weight transfer and plantigrady.</p

Histological analysis of the <i>Biceps femoris</i> muscle of 9-month old dogs.

<p>Healthy (#3H), GRMD (#6G), and GRMD^MuStem (#7G^Mu) dog muscles are presented respectively in left, mid and right panel. (A, B, C) Muscle tissue presentation after hemalun eosin safran staining. (B) Mock GRMD dogs display a typical dystrophinopathic pattern with diffuse anisocytosis, hypertrophic hyaline fibres (open arrowhead), centronucleated fibres (arrow), necrotic fibres (*), regenerative foci (black arrowhead) and multifocal thickening of the endomysial space by fibrosis (highlighted by the saffron yellow staining). (C) The dystrophinopathic pattern is remodelled in GRMD^MuStem dogs: anisocytosis is milder, hypertrophic hyaline fibres and necrotic fibres are less numerous, whereas centronucleated fibres are more abundant (arrow) compared to GRMD dogs. Inset, another picture taken on the same tissue sample with a higher magnification that displays centronucleated fibre. (D, E, F) Regenerative activity of muscle fibres in healthy, GRMD and GRMD^MuStem dogs, as indicated after immunolabelling specific to the developmental isoform of the myosin heavy chain (MyHCd). (G, H, I) Dystrophin expression in healthy, GRMD and GRMD^MuStem dogs. (A-B-C; G-H-I) Scale bar = 200 μm (in set Scale bar = 100 μm). (D, E, F) Scale bar = 500 μm.</p

Description of the ten dogs used in the <i>in vivo</i> study.

<p>Description of the ten dogs used in the <i>in vivo</i> study.</p