<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

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

Mest is required for skeletal muscle growth during regeneration.

<p>(A) H&E staining of TA muscles under normal condition (top panel) and 14 days after CTX-induced injury (middle and bottom panels). Bottom panel shows the extended images of a part of middle panel. (B) Average cross section areas of TA muscles in WT (n = 3), <i>Mest</i>^<i>+/-</i> (n = 4) and <i>miR-335</i>^<i>+/Neo</i> mice (n = 6) under normal condition and WT (n = 7), <i>Mest</i>^<i>+/-</i> (n = 4) and <i>miR-335</i>^<i>+/Neo</i> mice (n = 6) 14 days after CTX injury. (C) H&E staining of TA muscles of <i>DMD-null</i>, <i>Mest</i>^<i>+/-</i><i>; DMD-null</i> and <i>miR-335</i>^<i>+/Neo</i><i>; DMD-null</i> mice at 11–13 weeks old. (D) Average cross section areas of TA muscles in <i>DMD-null</i> (n = 8), <i>Mest</i>^<i>+/-</i><i>; DMD-null</i> (n = 4) and <i>miR-335</i>^<i>+/Neo</i><i>; DMD-null</i> mice (n = 4) at 11–13 weeks old. Error bars indicate the s.e.m. ^<i>#</i><i>P</i> = 0.0549 compared with WT mice. Scale bar: 100 μm.</p

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

ErbB signaling is implicated in generation of neurons and mitoses in the sub-ventricular zone.

<p><b>A</b>. Treatment with an ErbB inhibitor AG1478 prevents generation of GFP-expressing neurons in the OT of Tg(<i>pou4f1-hsp70l</i>:<i>GFP</i>) embryos, but not with the control AG43 and DMSO, at 52 hpf. Dotted circle, OT. Scale bar, 100 μm. <b>B</b>. Quantification of <i>pou4f1-hsp70l</i>:GFP intensity in the OT for the experiment shown in A (mean ± s.e.m.; **<i>P</i> < 0.01, ***<i>P</i> < 0.001; n = 8, 10, 16 for AG1478, AG43, DMSO, respectively). <b>C</b>. (left) A timeline of AG1478 treatment and irradiation of UV light on Tg(<i>elavl3</i>:<i>Kaede</i>). (right) Representative embryos showing impaired generation of neurons in the OT (dotted circle) at 52 hpf following AG1478 treatment. Scale bar, 50 μm. <b>D</b>. WISH of embryos at 44 hpf. Expression of <i>neurog1</i> and <i>neurod</i> is increased and diminished, respectively, while expression of <i>her6</i> is obviously unaltered in the OT following AG1478 treatment. Scale bar, 100 μm. <b>E</b>. A coronal section of WISH-stained embryos for <i>neurog1</i> mRNA at 36 hpf. <b>F</b>. Impaired neurogenesis and a decrease in pH3-positive mitotic cells in the SVZ of AG1478-treated Tg(<i>pou4f1-hsp70l</i>:<i>GFP;-8</i>.<i>4neurog1</i>:<i>nRFP</i>) embryos (AG1478) compared to the control embryos (DMSO) at 40 hpf. Scale bar, 10 μm. <b>G</b>. Quantification of the number of pH3-positive cells in the OT for the experiment shown in F, in the VZ (blue) and the SVZ (red) of AG1478-treated embryos and the control (white) embryos (mean ± s.e.m.; *<i>P</i> < 0.05; n = 11, 7 for AG1478, DMSO, respectively).</p

NRG1-II stimulates neurogenesis through ErbB4 as a cell-extrinsic signal.

<p><b>A</b>. Impaired neurogenesis in MO^{<i>erbb4atg</i>}-injected Tg(<i>pou4f1-hsp70l</i>:<i>GFP</i>) embryos at 50 hpf. Dotted circle, OT. Scale bar, 100 μm. <b>B</b>. Quantification of <i>pou4f1-hsp70l</i>:GFP intensity in the OT for the experiment shown in A (mean ± s.e.m.; ****<i>P</i> < 0.0001). <b>C</b>. WISH of MO^{<i>erbb4atg</i>}-injected and the control MO^<i>ctrl</i>-injected embryos for <i>neurog1</i> and <i>neurod</i> mRNAs at 48 hpf. Scale bar, 100 μm. <b>D</b>. A coronal section of WISH-stained embryos for <i>erbb4</i> mRNA at 36 hpf. The approximate site of the section is shown in a lateral view in the inset. <b>E</b>. A decrease in Tyr¹¹⁶²-phosphorylated ErbB4 (pErbB4) in the OT of MO^{<i>nrg1IIE1</i>}-injected embryos (<i>nrg1IIE1</i>) compared to the control MO^{<i>nrg1IIE15m</i>}-injected embryos (<i>nrg1IIE15m</i>) at 40 hpf. Scale bar, 10 μm. <b>F</b>. Quantification of pErbB4-positive area in the OT for the experiment shown in E (mean ± s.e.m; **<i>P</i> < 0.01; n = 8, 9 for MO^{<i>nrg1IIE1</i>}, MO^{<i>nrg1IIE15m</i>}, respectively). <b>G</b>. A timeline of the injection of hNRG1 proteins into the hindbrain ventricle. MO^{<i>nrg1IIE1</i>}-injected embryos with neurogenic phenotypes were segregated at 47 hpf, and they were subjected to intra-ventricle injection of hNRG1 or the control BSA. These embryos were analyzed at 54 hpf. <b>H</b>. Partial rescue of the defective neurogenesis in MO^{<i>nrg1IIE1</i>}-injected Tg(<i>pou4f1-hsp70l</i>:<i>GFP</i>) embryos at 54 hpf following the injection of hNRG1 proteins, compared to the control BSA injection. Scale bar, 100 μm. <b>I</b>. Quantification of <i>pou4f1-hsp70l</i>:GFP intensity in the OT for the experiment shown in H (mean ± s.e.m.; ****<i>P</i> < 0.0001; n = 17 for MO^{<i>nrg1IIE1</i>}_pre, n = 21 for MO^{<i>nrg1IIE15m</i>}_pre, n = 11 for MO^{<i>nrg1IIE1</i>}_BSA, n = 16 for MO^{<i>nrg1IIE1</i>}_hNRG1, n = 18 for MO^{<i>nrg1IIE15m</i>}_post.).</p

Generation of post-mitotic neurons requires NRG1-II.

<p><b>A</b>. MOs against <i>nrg1</i> used in this study. Domain structures of NRG1 type I–III isoforms are shown together with the target site (red bar) of each <i>nrg1</i> MO. <b>B</b>. Decreased <i>neurod</i>:EGFP-positive neurons in MO^{<i>nrg1IIE1</i>}-injected embryos (<i>nrg1IIE1</i>) compared to the control 5 nucleotides-mismatched MO^{<i>nrg1IIE15m</i>}-injected embryos (<i>nrg1IIE15m</i>) of Tg(BAC(<i>neurod</i>:<i>GFP</i>);<i>-8</i>.<i>4neurog1</i>:<i>nRFP</i>) at 50 hpf. Scale bar, 10 μm. <b>C</b>. Quantification of <i>neurod</i>:EGFP intensity in the OT for the experiment shown in A (mean ± s.e.m.; **<i>P</i> < 0.01; n = 4 per group). <b>D</b>. WISH of MO^{<i>nrg1IIatg</i>}-injected and the control MO^{<i>nrg1IIatg5m</i>}-injected embryos for <i>neurog1</i> and <i>neurod</i> mRNAs at 48 hpf. Scale bar, 100 μm. <b>E</b>. A coronal section of WISH-stained embryos for <i>nrg1</i> mRNA at 36 hpf. The approximate site of the section is shown in a lateral view in the inset. <b>F</b>. A schematic of a stacked image of confocal sections from a dorsal view and the higher magnification of the OT from a dorsal view (bottom). <b>G</b>. Impaired neurogenesis and a decrease in pH3-positive mitotic cells in the SVZ of MO^{<i>nrg1IIatg</i>}-injected TgBAC(<i>neurod</i>:<i>EGFP</i>) embryos (<i>nrg1IIatg</i>) compared to the control embryos (<i>nrg1IIatg5m</i>) at 40 hpf. Scale bar, 20 μm. <b>H</b>. Quantification of the number of pH3-positive cells in the VZ (blue) and the SVZ (red) of MO^{<i>nrg1II</i>}-injected embryos and the control (white) embryos (mean ± s.e.m.; *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001; n = 6, 6, 4, for <i>nrg1IIE1</i>, n = 6, 5, 5 for <i>nrg1IIE15m</i> at 26, 33, 36 hpf, n = 3, 4 for <i>nrg1IIatg</i>, <i>nrg1IIatg5m</i> at 40 hpf, respectively).</p

FMPs express MyoD in developing limbs.

<p>(<b>A–F</b>) Immunohistochemistry of FMP cells on longitudinal sections of limbs at E16.5 for GFP (A,D), MyoD (B,E) and merged with DAPI (C,F). MyoD-positive (A–C) and -negative (D–F) FMPs are shown. Scale bar = 10 µm. (<b>G</b>) Percentage of MyoD-positive and MyoD-negative cells in (Pax3)GFP+ cells in limbs at E16.5 with data reported as the mean and s.d. of three fetuses. (<b>H–M</b>) Immunocytochemistry of freshly isolated FMPs (H–J) and SCs (K–M) for GFP (H,K), MyoD (I,L), and merged with DAPI (J,M). Arrows in H–J indicate MyoD-positive FMPs. Scale bar = 20 µm. (<b>N</b>) Percentage of MyoD-positive and MyoD-negative cells of freshly isolated FMPs and SCs (<i>n = </i>100 cells par condition). FMPs, fetal skeletal muscle progenitors; SCs, satellite cells.</p

Engrafted FMPs restore dystrophin expression in <i>DMD-null</i> mice.

<p>(<b>A–C</b>) Immunostaining for dystrophin in TA muscles of <i>DMD-null</i> mice injected with medium (A), FMPs (B), and SCs (C) 2 weeks after intramuscular engraftment. Scale bar = 100 µm. (<b>D</b>) Quantification of dystrophin+ fibers in TA engrafted with FMPs (<i>n</i> = 7 recipient mice) and SCs (<i>n</i> = 7 recipient mice). Data is reported as the mean and s.e.m. from all engrafted mice. P-value indicated on the figure is <0.05 (*). (<b>E,F</b>) Immunostaining for dystrophin in TA muscles of <i>DMD-null;nude</i> mice injected with medium (E) and FMPs (F), 24 weeks after engraftment. Scale bar = 100 µm. FMPs, fetal skeletal muscle progenitors; SCs, satellite cells; TA, tibialis anterior.</p