Functional analysis of mSix1-8 in chick.

<p>(A) Schematic diagram of mutation analysis of mSix1-8 enhancer in the DRG. Various mSix1-8-EGFP reporters were co-electroporated into the left side of the neural tube with the control wild-type reporter (mSix1-8wt-mRFP1) at HH14, and fluorescence intensities of EGFP and mRFP1 in the DRG were examined at 48 h.p.e. (B) Results of mutation analysis of mSix1-8 in the DRG. mSix1-8wt-mRFP1 (red) was co-electroporated with various EGFP constructs (green): mSix1-8wt (Ba, Ba'), mSix1-8NR1-4m (Bb, Bb'), mSix1-8TCF/LEFm2 (Bc, Bc'), mSix1-8SMADm (Bd, Bd'), mSix1-8bHLH12m (Be, Be') and mSix1-8POU12m (Bf, Bf'). Numbers in brackets correspond to the bar numbers (1–9) in C. The wild-type reporter (Ba-Bf, Ba') marked the DRG (drg) and weakly the neural tube (nt), while all mutations reduced EGFP levels (Bb'-Bf'). mSix1-8NR1-4m almost completely abolished mSix1-8 enhancer activity (Bb, Bb'). The image is a dorsal view of the trunk region, and anterior is to the left. (C) Quantification of the effect of various mutations on Six1-8 enhancer activity in the DRG. The relative EGFP/mRFP1 levels were calculated for each embryo by measuring five DRG, and are shown relative to the value obtained from the wild-type reporter (100%). Data are mean±SD. The relative EGFP level detected in the DRG that received reporters with various mutations was significantly lower (*<i>p</i><0.001) than that of embryos received wild-type reporter. 1: wild-type (n = 7), 2: NR1m (n = 5), 3: NR2m (n = 6), 4: NR1-4m (n = 8), 5: TCF/LEFm2 (n = 6), 6: SMADm (n = 8), 7: bHLH12m (n = 6), 8: POU12m (n = 6), 9: YYm (n = 7). (D) Schematic diagram of mutation analysis of mSix1-8 enhancer in cranial ganglia. Three mSix1-8-EGFP reporters were co-electroporated into the entire epiblast with the control wild-type reporter (mSix1-8wt-mRFP1) at HH4-5, and the fluorescence intensities of EGFP and mRFP1 in the head region were examined at 48 h.p.e. (E) Mutation analysis of mSix1-8 in cranial ganglia. mSix1-8wt-mRFP1 (red) was co-electroporated with various EGFP constructs (green): mSix1-8wt (Ea, Ea', Ea''), mSix1-8NR1-4m (Eb, Eb', Eb'') and mSix1-8TCF/LEFm2 (Ec, Ec', Ec''). Numbers in brackets correspond to the bar numbers in F. The wild-type reporter (Ea-Ec, Ea') marked the trigeminal ganglion (V), geniculate (VII)/vestibuloacoustic (VIII) ganglion complex (VII/VIII) and weakly the otic vesicle (ov), while the two mutations reduced EGFP levels (Eb'-Ec'). mSix1-8NR1-4m almost completely abolished mSix1-8 enhancer activity (Eb, Eb'). mSix1-8wt shows weak enhancer activity in the posterior optic cup (opc, Eb and Ec). Each image is a lateral view of the left side of the head/neck. Anterior is to the left and dorsal is to the top. (F) Quantification of the effect of the two mutations on mSix1-8 enhancer activity in the trigeminal ganglion. The relative EGFP/mRFP1 levels were calculated for each embryo by measuring trigeminal ganglia of both sides, and are shown relative to the value obtained from the wild-type reporter (100%). Data are mean±SD. The relative EGFP level detected in trigeminal ganglia that received reporters with the two mutations was significantly lower (*<i>p</i><0.001) than that of embryos received wild-type reporter. 1: wild-type (n = 5), 2: NR1-4m (n = 8), 3: TCF/LEFm2 (n = 7). drg: dorsal root ganglia, nt: neural tube, opc: optic cup, ov: otic vesicle, V: trigeminal ganglion, VII/VIII: VII/VIII ganglion complex. Scale bars: 0.5 mm.</p

Pattern and specificity of mSix1-8-NLSCre-mediated recombination in embryos.

<p>(A) The structure of a transgene used to generate the mSix1-8-NLSCre transgenic mouse line. mSix1-8 (538 bp) is placed upstream of the tkintron (the HSV thymidine kinase gene promoter and chimeric intron) to drive the expression of NLSCre. The polyA signal is from SV40. The entire expression unit is flanked by two tandem copies of the core region of the HS4 insulator (ins). The positions of the genotyping PCR primers (arrowheads) and the size of the PCR product (406 bp) are shown. Selected restriction sites are also indicated. (B-H) Localization of ß-Gal-positive cells in mSix1-8-NLSCre/R26R-LacZ double transgenic embryos. At E9.0 (Ba), the earliest sign of the appearance of ß-Gal-positive cells was detected in the otic pit region (white square bracket). A close-up view (Bb) shows signals in three scattered cells (black arrowheads) in the otic pit. A section through the posterior part of the otic pit confirmed the presence of ß-Gal-positive cells in the pits of both sides (Bc). At E9.5 (Ca), ß-Gal-positive cells were noted in the developing trigeminal ganglion (black square bracket) and olfactory placode (yellow square bracket) in addition to the otic pit (white square bracket). A close-up view (Cb) shows signals in scattered cells in and around the olfactory placode (white arrowheads). At E9.75 (Da), signals were detected in the developing geniculate ganglion. In a close-up view (Db) showed signals in the ventral portion of the otic vesicle (black arrow). At E10.5 (Ea), clear signals were detected in all the cranial sensory ganglia (V, VII, VIII, IX and X), cells in and around the olfactory epithelium (Eb, a close-up view) and in the DRG. At E11.0 (F), the intensity of the signals in the sensory organs became stronger. Signals were also found in the mesenchyme of forelimb bud (Ea, F-H), hindlimb bud (F-H), branchial arches (Ea, Eb, F-H), and the maxillary process (F-H). In all panels of whole mount embryos, anterior is to the left, dorsal is to the top, and all panels are lateral views. In the transverse section shown in Bc, dorsal is to the top. ba: branchial arches, drg: dorsal root ganglia, fb: forebrain, fl: forelimb bud, hb: hindbrain, hl: hindlimb bud, mp: maxillary process, oe: olfactory epithelium, op: olfactory placode, otp: otic pit, ov: otic vesicle, V: trigeminal ganglia, VII: geniculate ganglia, VII/VIII: VII/VIII ganglion complex, IX: petrosal ganglion, X: nodose ganglion. Scale bars: 2 mm (H), 1 mm (Ba, Ca, Da, Ea, F, G), 0.2 mm (Bb), 0.5 mm (Cb, Db, Eb), 0.1 mm (Bc).</p

PCR primers used to introduce mutations in mSix1-8.

<p>Positions corresponding to the predicted transcription factor binding sites are underlined. Mutated nucleotides are in lower case.</p><p>PCR primers used to introduce mutations in mSix1-8.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

Functional analysis of mSix1-8 in mouse.

<p>(A) Mutation analysis of mSix1-8 in mouse. Wild-type (mSix1-8wt-LacZ, Aa) and mutated [(mSix1-8NR1-4m-LacZ, Ab) and (mSix1-8TCF/LEFm2-LacZ, Ac)] transgenes were used for transgenesis and ß-Gal localization was examined at E10.5. Embryos injected with the wild-type transgene showed ß-Gal activity specifically in the trigeminal ganglion, the VII/VIII ganglion complex and epibranchial placode/ganglia (Aa)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136666#pone.0136666.ref017" target="_blank">17</a>]. However, ß-Gal activity was almost completely lost in an embryo carrying NR1-4m mutation, with the exception of a small number of ectopic ß-Gal-positive cells in the cervical area (Ab). In an embryo carrying TCF/LEFm2 mutation, ß-Gal activity was lost in the sensory organs and ganglia, with the exception of ectopic activity in the somites (Ac). drg: dorsal root ganglia, so: somites, V: trigeminal ganglion, VII/VIII: VII/VIII ganglion complex, IX: petrosal ganglion, X: nodose ganglion. Scale bars: 1 mm. (B) Summary of the phenotypes of transgenic mouse embryos. transgenic embryos: the total number of transgenic embryos obtained using each transgene, pattern A: number of embryos with ß-Gal staining pattern in which the signal in the trigeminal ganglion stands out as the major ß-Gal-positive domain (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136666#pone.0136666.g003" target="_blank">Fig 3Aa</a> represents a typical pattern A staining), pattern B: number of embryos with ß-Gal staining in which signals in the cranial ganglia was reduced while those in the DRG were relatively unaffected, embryos with ectopic expression: number of embryos with an ectopic LacZ staining, embryos without sensory ganglia-expression: number of embryos without a LacZ staining in the sensory ganglia. Parts of the results obtained using the wild-type transgene were reported previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136666#pone.0136666.ref017" target="_blank">17</a>].</p

mSix1-8-NLSCre induces sensory neuron-specific recombination in mouse embryos.

<p>(A-I) Immunofluorescence analysis of Cre-mediated recombination in mSix1-8-NLSCre/R26R-LacZ double transgenic embryos. Distribution of ß-Gal was examined at E10.5 (A-D, H), E11.5 (E) and E12.5 (F, G). At E10.5, ß-Gal (Aa) co-localized with a neuron marker ISL1/2 (Aa') in the trigeminal ganglion (yellow signals in Aa''), geniculate-vestibuloacoustic ganglion complex (Ba, Ba', Ba''), petrosal ganglion (Ca, Ca', Ca'') and nodose ganglion (Da, Da', Da'') in transverse sections through the cervical area. In contrast, the same sections stained for a glial marker SOX10 showed no overlap of ß-Gal (green)-positive cells with SOX10 (red)-positive cells in all cranial sensory ganglia (Ab, Bb, Cb, Db). At E11.5, ß-Gal (Ea) also co-localized with ISL1/2 (Ea') in the DRG (yellow signals in Ea'') in a transverse section through the trunk region but not with SOX10 on the same section (Eb). In the frontal sections through the OE at E12.5 (F, G), ß-Gal (green)-positive cells were detected not only in the OE (F, G) but also in the vomeronasal organ (G) and in the surrounding mesenchyme along the TUBB3 (red)-positive axons of the olfactory and vomeronasal sensory neurons (white arrowheads). Many ß-Gal-positive cells were also found in the "migratory mass (white square bracket)" comprising placode-derived migratory cells and axons of olfactory sensory neurons located ventral to the forebrain (F). (H) At E10.5, ß-Gal (green)-positive cells were found in the OE (demarcated by white dotted line) and in an aggregate of TUBB3-positive cells (white arrowheads) located next to the OE. (I) Immunofluorescence analysis of Six1-8 enhancer activity in chick. Frontal section through the olfactory pit of a representative chick embryo at 48 h.p.e. EGFP (green) derived from pT2A-BB-mSix1-8-EGFP was detected in the OE and in an aggregate of TUBB3 (red)-positive cells (yellow arrow) located subjacent to the OE, a likely avian homolog of the rodent migratory mass. DAPI was used for nuclear staining (blue, Aa'', Ab, Ba'', Bb, Ca'', Cb, Da'', Db, Ea'', Eb, F-I). In all panels, dorsal is to the top and midline is to the left (A-E, H) or right (F, G, P). drg: dorsal root ganglia, fb: forebrain, mm: migratory mass, oe: olfactory epithelium, ov: otic vesicle, vno: vomeronasal organ, V: trigeminal ganglia, VII: geniculate ganglia, VIII: vestibuloacoustic ganglion, IX: petrosal ganglion, X: nodose ganglion. Scale bars: 0.1 mm (A-I).</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

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