EMT and mesendoderm markers upregulated in neurospheres after 48 h induction with serum and Lif.

<p>(<b>A</b>): Immunostaining for Slug, E-cadherins and N-cadherins (<b>B</b>): Immunostaining for mesendoderm markers Sox17 and Brachyury (T). (<b>C</b>): Expression profile of Slug, N-cadherins, E-cadherins, Sox17 and Brachyury of 48 h serum and Lif induced neurospheres measured by QPCR relative to neurospheres cultured in standard neural stem cell media. Abbreviations: Ncad, N-cadherins; Ecad, E-cadherins. Scale bars: 50 µm.</p

Schematic diagram depicting our interpretation of the results.

<p>Neural stem cells derived from later stages of development have been shown to reprogram to iPS cells via overexpression of <i>Oct4</i> transcription factor (Kim et al., 2009). Signalling alone (BMPs and bFGF), however, can induce dedifferentiation of neural stem cells into a neural crest phenotype (Sailer et al., 2005). TGFβ and Jak/Stat pathways can induce a further dedifferentiation to mesendoderm-like phenotype providing evidence for extracellular signaling regulated cell plasticity of neural stem cells (this work, red arrow).</p

Effect of signalling pathway inhibitors on upregulation of mesendoderm markers during 48 h induction of neurospheres.

<p>Expression levels: ++++ very strong; +++ strong; ++good; + weak; − no expression.</p

Expression of pluripotency associated proteins in serum-free cultured neurospheres and 48 h serum and Lif induced neurospheres.

<p>(<b>A</b>): Morphology in serum-free (upper panel) and 48 h serum/Lif (lower panel) conditions. (<b>B</b>): Transcriptional profile of pluripotency associated factors Oct4, Sox2, c-Myc, Klf4 and Nanog of serum-free cultured neurospheres and 48 h treated neurospheres. Data is expressed as ΔΔCT and normalized to ES cells. (<b>C</b>): Immunostaining for Oct4, Nanog, Sox2, SSEA1 and alkaline phosphatase. Abbreviations: AP, alkaline phosphatase; SSEA1, stage specific embryonic antigen 1. Scale bars: 50 µm.</p

<i>In vitro</i> differentiation of neurospheres in serum and Lif conditions.

<p>(<b>A</b>): Immunostaining for Brachyury (T), Sox17, Nanog and Oct4 at 5 days after induction in serum and Lif conditions (<b>B</b>): At 10 days post induction with serum and Lif, cells exhibit very heterogeneous morphologies, indicating the presence of different cell types. (<b>C</b>): Immunostaining for GFAP and αSMA in 10 day induced cultures. Abbreviations: GFAP, glial fibrillary acidic protein; αSMA, alpha smooth muscle actin. Scale bars: 50 µm.</p

<i>In vivo</i> differentiation of 48 h induced neural stem cells.

<p>Staining of chick embryo paraffin sections for mesenchymal marker N-cadherin, tight junction marker ZO-1, endoderm marker Sox17, and epithelial marker E-cadherin. Although some of the labelled cells express E-cadherin, the staining pattern suggest the cells do not achieve a complete integration to ectoderm lineage. However, high integration towards mesoderm and endoderm lineages is confirmed by N-cadherin, ZO-1 and Sox17 stainings. Although induced (green) cells show higher efficiency of integration, both induced (green) and non-induced (red) cells once incorporated into these tissues express respective lineage markers and acquire similar morphological characteristics to their neighbouring host cells.</p

Mest but Not MiR-335 Affects Skeletal Muscle Growth and Regeneration

<div><p>When skeletal muscle fibers are injured, they regenerate and grow until their sizes are adjusted to surrounding muscle fibers and other relevant organs. In this study, we examined whether <i>Mest</i>, one of paternally expressed imprinted genes that regulates body size during development, and miR-335 located in the second intron of the <i>Mest</i> gene play roles in muscle regeneration. We generated miR-335-deficient mice, and found that miR-335 is a paternally expressed imprinted microRNA. Although both <i>Mest</i> and miR-335 are highly expressed during muscle development and regeneration, only <i>Mest^+/-</i> (maternal/paternal) mice show retardation of body growth. In addition to reduced body weight in <i>Mest^+/-; DMD-null</i> mice, decreased muscle growth was observed in <i>Mest^+/-</i> mice during cardiotoxin-induced regeneration, suggesting roles of Mest in muscle regeneration. Moreover, expressions of <i>H19</i> and <i>Igf2r</i>, maternally expressed imprinted genes were affected in tibialis anterior muscle of <i>Mest^+/-; DMD-null</i> mice compared to <i>DMD-null</i> mice. Thus, Mest likely mediates muscle regeneration through regulation of imprinted gene networks in skeletal muscle.</p></div

Generation of miR-335 deficient mice.

<p>(A) Design of constructs used for generation of miR-335 deficient mice. The miR-335 genomic locus was replaced by a floxed neomycin-resistance cassette (loxP-Neo-loxP) to obtain <i>miR-335</i>^<i>+/Neo</i> mice. <i>miR-335</i>^<i>+/-</i> mice (+/-) were generated by crossing male <i>miR-335</i>^<i>+/Neo</i> mice with female CAG-<i>cre</i> transgenic mice. (B) Southern blot analysis of WT and G418 resistant ES clones with 5’ and 3’ probes. †: Non-specific band. (C and D) PCR analysis for targeted allele with genomic DNA in tails of WT (+/+), <i>miR-335</i>^<i>+/Neo</i> (+/Neo), and <i>miR-335</i>^<i>+/-</i> mice (+/-). In (C), an insertion and a deletion of a floxed neomycin-resistance cassette in genomic DNA of <i>miR-335</i>^<i>+/Neo</i> (+/Neo) and <i>miR-335</i>^<i>+/-</i> mice (+/-), respectively, were detected with PCRs amplified with primers shown in Fig 2A (Orange and Green arrows). In (D), a PCR analysis to distinguish alleles for WT (+/+), <i>miR-335</i>^<i>+/-</i> (+/-), and <i>miR-335</i>^<i>-/-</i> mice (-/-) was shown. The WT allele-specific (280 bp) and the mutant allele-specific (306 bp) bands were amplified with the primers shown in Fig 2A (Green arrow). (E, F and G) qRT-PCR for miR-335 was performed in TA muscles isolated from WT, <i>miR-335</i>^<i>+/Neo</i>, and <i>miR-335</i>^<i>+/-</i> or <i>miR-335</i>^<i>-/+</i> mice (n = 3 per genotype). (H and I) qRT-PCR for <i>Mest</i> mRNA was performed in TA muscles of WT, <i>miR-335</i>^<i>+/-</i>, and <i>miR-335</i>^<i>+/Neo</i> mice (n = 3 per genotype). Expression of <i>Mest</i> mRNA and that of miR-335 are normalized to <i>Gapdh</i> and snoRNA-202, respectively. Error bars indicate the s.e.m. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001.</p

Mest is required for body and skeletal muscle growth.

<p>(A) Representative images of 4 weeks old mice in individual genotypes. (B and C) Body weights of male littermate WT (n = 3–22), <i>Mest</i>^<i>+/-</i> (n = 6–15), <i>DMD-null</i> (n = 4–25), and <i>Mest</i>^<i>+/-</i><i>; DMD-null</i> mice (n = 4–22) from 1 to 12 (11–13) weeks old. (D) Body weights of WT (n = 15) and <i>miR-335</i>^<i>+/Neo</i> (n = 12) mice at 6 weeks. (E) Body weights of WT (n = 8–15) and <i>miR-335</i>^<i>+/-</i> mice (n = 15–18) from 1 to 6 weeks old. (F) TA muscle weights of male littermate WT (n = 13), <i>Mest</i>^<i>+/-</i> (n = 6), <i>DMD-null</i> (n = 18), and <i>Mest</i>^<i>+/-</i><i>; DMD-null</i> mice (n = 11) at 6 weeks old. (G) TA/Body weights of male littermate WT, <i>Mest</i>^<i>+/-</i>, <i>DMD-null</i>, and <i>Mest</i>^<i>+/-</i><i>; DMD-null</i> mice at 6 weeks old. (H) TA muscle weights of male littermate WT (n = 3), <i>Mest</i>^<i>+/-</i> (n = 6), <i>DMD-null</i> (n = 4), and <i>Mest</i>^<i>+/-</i><i>; DMD-null</i> mice (n = 4) at 11–13 weeks old. (I) TA/Body weights of male littermate WT, <i>Mest</i>^<i>+/-</i>, <i>DMD-null</i>, and <i>Mest</i>^<i>+/-</i><i>; DMD-null</i> mice at 11–13 weeks old. (J and K) The numbers and average cross section areas of TA muscle fibers of male littermate WT (n = 7) and <i>Mest</i>^<i>+/-</i> mice (n = 4) at 6 weeks. Error bars indicate the s.e.m. *<i>P</i> < 0.05, ***<i>P</i> < 0.001. NS = Not significant.</p

<i>Mest</i> and miR-335 are coordinately expressed in skeletal muscle during postnatal development and regeneration.

<p>(A) qRT-PCRs for <i>Mest</i> mRNA and miR-335 were performed with TA muscles of P0, 6 weeks, and 12 weeks old WT mice (n = 3 per time point). (B) qRT-PCRs for <i>Mest</i> mRNA and miR-335 were performed with TA muscles of 3 months old WT (n = 4) and <i>DMD–null</i> mice (n = 7). (C) qRT-PCRs for <i>Mest</i> mRNA and miR-335 were performed with TA muscles from day 0 to day 10 after CTX injection (n = 3 per time point). (D) A schematic diagram of the Mest and miR-335 genomic region on chromosome 6 in mouse. (E) qRT-PCR for miR-335 was performed in TA muscles of WT and <i>Mest</i>^<i>+/-</i> mice (n = 3 per genotype). Expression of <i>Mest</i> and that of miR-335 are normalized to <i>Gapdh</i> and snoRNA-202, respectively. Error bars indicate the s.e.m. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001 compared with P0 (A), WT (B and E), and day 0 (C).</p