1. Volume 155, Issue 4, Pages 909-921 (November 2013)
A Zebrafish Embryo Culture System Defines Factors that Promote Vertebrate Myogenesis across Species 
Cong Xu, Mohammadsharif Tabebordbar, Salvatore Iovino, Christie Ciarlo, Jingxia Liu, Alessandra Castiglioni, Emily Price, Min Liu, Elisabeth R. Barton, C. Ronald Kahn, Amy J. Wagers, Leonard I. Zon 
Cell 
Volume 155, Issue 4, Pages (November 2013)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1
A Chemical Genetic Screen to Identify Modifiers of Skeletal Muscle Development
(A) myf5-GFP;mylz2-mCherry double-transgenic expression recapitulates expression of the endogenous genes. myf5-GFP is first detected at the 11-somite stage. mylz2-mCherry expression is not observed until 32 hpf. Scale bars represent 200 μm.
(B) myf5-GFP;mylz2-mCherry embryos were dissociated at the oblong stage and cultured in zESC medium. Images were taken 48 hr after plating. Scale bars represent 250 μm.
(C) Expression of myogenic genes measured by qRT-PCR. Blastomere cells were cultured and harvested at 24 hr. Error bars represent SD from the triplicate reactions. All values are normalized to β-actin and expression data of basic medium (gray bars, lacking bFGF) are set to 1.
(D) Expression of myogenic genes by different populations of blastomere cells isolated by FACS after 24 hr culture with bFGF. Error bars represent SD from triplicate reactions. Expression data are normalized to β-actin, and expression values of double-negative population (blue bars) are set to 1.
See also Figure S1.
Cell  , DOI: ( /j.cell )
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4. Figure 2
Chemical Genetic Screens to Identify Modifiers of Skeletal Muscle Development
(A) Schematic of a high-throughput image-based chemical screening assay. Approximately 800 myf5-GFP;mylz2-mCherry double-transgenic embryos were collected and dissociated at the oblong stage. Resulting blastomere cells were aliquotted into four 384-well plates with preadded chemicals. After 2 days, the 384-well plates were imaged and analyzed using a Celigo cytometer.
(B) Sample images from the modifier screen. Hits could be grouped into three categories. Category I has only myf5-GFP expression. Category II has a decreased amount of both fluorescent colors. Category III has increased expression of both. Scale bars represent 250 μm.
(C) Hits from the enhancer screen. Six chemicals that increase the GFP and mCherry signals were identified. Scale bars represent 250 μM.
See also Figure S2 and Tables S1 and S2.
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5. Figure 3
Forskolin Treatment Elevates cAMP Level and Increases Proliferation of Satellite Cells from Both Healthy and Dystrophic Mice
(A) Satellite cells from C57BL/6J mice were cultured and treated with DMSO or forskolin in the absence or presence of bFGF, as indicated. Total cell number was determined after 5 days (mean ± SEM, n = 4). Data are presented as fold change, normalized to “DMSO no bFGF” controls.
(B) Fold change in cell number from cultures of mdx satellite cells after 5 days in vitro with bFGF alone, DMSO + bFGF, or forskolin + bFGF (mean ± SEM, n = 4). Data are normalized to “DMSO +bFGF” controls.
(C) Concentration of cAMP in cultured satellite cells is increased after treatment with 25 μM, 50 μM, or 100 μM of forskolin, as compared to the DMSO-treated cells (mean ± SD, n = 5). Data are normalized “DMSO” controls.
(D) Experimental scheme for myogenic colony-forming assay. Satellite cells were isolated from C57BL/6J mice, and a single cell was plated into each well of 96-well plates. Cells were cultured for 6 days and treated with DMSO or forskolin. The number of wells containing a myogenic colony and the number of cells in each colony were counted after 6 days.
(E) Clonal plating efficiency of satellite cells is not altered by forskolin treatment, as compared to DMSO-treated cells (mean ± SD, n = 4).
(F) Forskolin treatment increases the number of myogenic cells arising from a single satellite cell in each well, showing an increase in cell proliferation. Data are plotted as the % of colonies containing the indicated number of cells (mean ± SD, n = 4). H, p < 0.05; HH, p < 0.01.
See also Figure S3.
Cell  , DOI: ( /j.cell )
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6. Figure 4
Forskolin-Treated Satellite Cells Exhibit Effective Differentiation In Vitro
(A) Experimental scheme. Satellite cells from C57BL/6J mice were cultured in the presence of bFGF for 5 days. Cells were harvested on day 5, and equal numbers of cells were induced to differentiate in the presence of forskolin or DMSO.
(B) Images of satellite cells differentiated in the presence of DMSO (left) or forskolin (right) and stained for myosin heavy chain (MHC, red) and nuclei (blue). Scale bars represent 200 μm.
(C) Quantification of percentage of nuclei in myotubes after satellite cell differentiation in the presence of forskolin or DMSO (mean ± SEM, n = 4). Differentiation potential of satellite cells is unaffected by forskolin (p = nonsignificant [NS]).
(D) Satellite cells from C57BL/6J mice were cultured with bFGF and forskolin/DMSO for 5 days. Cells were harvested on day 5, and equal numbers of cells were induced to differentiate after removal of the compound.
(E) Images of DMSO- (left) or forskolin (right)-treated satellite cells differentiated after removal of compound and were stained for MHC (red) and nuclei (blue). Scale bars represent 200 μm.
(F) Quantification of percentage of nuclei in myotubes after differentiation of forskolin- or DMSO-treated cells (mean ± SEM, n = 5). Forskolin-treated satellite cells show no defect in myotube formation.
See also Figure S4.
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7. Figure 5
Forskolin-Treated Cultured Satellite Cells Retain Immunophenotypic Characteristics of Freshly Isolated Satellite Cells and Engraft Skeletal Muscle In Vivo
(A) Representative FACS plots depict CD45−SCA-1−MAC1− cells gated for the CXCR4+ and β1 Integrin+ subset of freshly isolated (left) and cultured satellite cells (initially sorted as 100% CXCR4+ and β-1 Integrin+) treated with DMSO (middle) or forskolin (right).
(B) Average frequency (mean ± SEM, n = 6) of CXCR4+β1-Integrin+ cells among cultured satellite cells treated with DMSO or forskolin, quantified by FACS. Most cultured satellite cells were treated with either DMSO or forskolin retain expression of CXCR4 and β1 Integrin.
(C) Experimental scheme. GFP+ satellite cells were harvested from β-actin-GFP mice and were transplanted into the TA muscle of recipient mdx mice, injured 1 day previously by injection of cardiotoxin. Cells were transplanted either immediately after isolation or following 5 days in culture with DMSO or forskolin treatment.
(D) Total number of cultured GFP+ satellite cells that were obtained from 6,000 freshly isolated cells and used for transplantation into mdx muscle. Cell number was, on average, 2.5 times greater in forskolin-treated as compared to DMSO-treated cultures (mean ± SD, n = 6).
(E) Transverse frozen section of TA muscle from mdx mice transplanted with 6,000 freshly isolated satellite cells (left), cultured DMSO-treated satellite cells expanded from 6,000 freshly isolated cells (middle), or cultured forskolin-treated satellite cells expanded from 6,000 freshly isolated cells (right). Laminin staining is shown in red. Scale bars represent 200 μm.
(F) Transverse frozen sections of TA muscle from mdx mice transplanted with 200,000 cultured DMSO-treated (left) or 200,000 cultured forskolin-treated (right) GFP+ satellite cells. Laminin staining is shown in red. Scale bars represent 200 μm.
(G) Quantification of donor-derived (GFP+) myofibers in mdx muscles transplanted with freshly isolated, DMSO-cultured, or forskolin-cultured satellite cells (mean ± SD, n = 6).
(H) Quantification of donor-derived myofibers in mdx muscle transplanted with 200,000 cultured DMSO-treated or 200,000 cultured forskolin-treated satellite cells (mean ± SD, n = 4).
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8. Figure 6
Treatment of Human iPSCs with Muscle-Promoting Cocktail Induces Skeletal Muscle Differentiation
(A and B) Gene expression analysis of EBs (A) and monolayer cells (B). Cells were harvested at the times indicated for RNA extraction. Ectodermal, endodermal, and mesodermal genes were analyzed by quantitative RT-PCR using GAPDH as a housekeeping gene. Changes in gene expression are indicated relative to undifferentiated iPSCs (day 0). Bars represent the SD of three independent experiments (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001).
(C) Immunostaining of differentiated iPSCs. Under terminal differentiating procedures (day 36), most of the cells express Desmin (red) and Myogenin (green), forming multinucleated myofibers. Cells were also stained with Hoechst (blue). The number on each panel represents the percentage of cells expressing Desmin and Myogenin. Scale bars represent 100 μm.
(D) Representative electron microscopy image of differentiated BJ iPSCs at day 36, magnification ×52,700.
(E) Immunoelectron microscopy staining using skeletal-muscle-specific antimyosin heavy and light chain. Black dots indicate gold cross-linked particles to secondary antibody, magnification ×52,700.
See also Figures S5 and S6.
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9. Figure 7
Engraftment of Human iPSC-Derived Muscle Progenitors into Immune-Compromised Mice
Representative hematoxylin and eosin staining (H&E) images and immunostaining on TA sections of preinjured NSG mice injected with 1 × 105 iPSCs at day 14 of differentiation. Muscles injected with BJ, 00409, or iPSC-derived cells stain positively for human δ-Sarcoglycan protein (red). Fibers were counterstained with Laminin (green). No staining is observed in PBS-injected mice or when fibroblast cells were transplanted. Because the area of human cell engraftment could not be specifically distinguished on H&E stained sections, which must be processed differently from sections for immunostaining, the H&E images shown do not represent the same muscle region as that shown in immunofluorescence images. Scale bars represent 100 μm, n = 3 per sample.
See also Figure S7.
Cell  , DOI: ( /j.cell )
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10. Figure S1
Myogenesis Suppressors Identified In Vitro also Affect Muscle Development In Vivo, Related to Figure 1
(A) Higher magnification of myf5-GFP;mylz2-mCherry blastomere cells cultured in zESC medium with bFGF. Scale bar represents 50 μm.
(B) myf5-GFP;mylz2-mCherry embryos injected with 400pg con-MO or co-injected with 200pg myf5-MO and 200pg myoD-MO lack both myf5-GFP and mylz2-mCherry expression. Images taken at 30 hpf. Scale bars represent 200 μm.
(C) myf5-GFP;mylz2-mCherry embryos injected with 400pg con-MO or co-injected with 200pg myf5-MO and 200pg myoD-MO were disassociated at the oblong stage and cultured in zESC medium with bFGF. No myf5-GFP or mylz2-mCherry expression was detected in the culture of double morphants. Images were taken 26 hr after plating. Scale bars represent 200 μm.
Cell  , DOI: ( /j.cell )
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11. Figure S2
Chemical Genetics Screens to Identify Modifiers of Skeletal Muscle Development, Related to Figure 2
(A) Embryos were treated with chemical “hits” at the sphere stage. Once the control embryos reached the 6-somite stage, they were collected for whole-mount in situ hybridization with myoD riboprobe. Examples of hits verified to perturb muscle development in vivo. Flat mounted embryos stained with the myoD riboprobe at the 6-somite stage are shown with anterior to the left. Embryos were treated with (±)-6-Chloro-PB hydrobromide, LY , 1-ACYL-PAF, or ATRA. All chemicals were from the CHB library and were diluted 300-fold. The level of myoD reduction was determined by comparing the intensity of myoD expression in these embryos with that in control embryos treated with DMSO.
(B) myf5-GFP;mylz2-mCherry embryos were collected at the oblong stage and dissociated. The resulting blastomere cells were aliquoted into a 96-well plate with pre-added chemicals. The culture medium contained 10 ng/ml bFGF to promote muscle development. Cells were treated with 50 μM tyrphostin AG 879, 50 μM tyrphostin AG 808, 50 μM tyrphostin AG 555, 50 μM PD 98059, 10 μM U0126, 10 μM LY , 10 μM wortmannin, or 20 μM rapamycin. Cells were imaged after 2 days using a Celigo cytometer for GFP, mCherry, and bright-field signals. Scale bar represents 250 μm.
(C) bFGF treatment activates MEK/ERK and phosphoinositide-3-kinase (PI3K)-AKT signaling pathway in cultured blastomere cells. myf5-GFP;mylz2-mCherry embryos were collected at the oblong stage and dissociated. Resulting blastomere cells were cultured with or without bFGF. Cells were collected after 2 hr for Western blot.
(D) Hits from a screen without supplementation of bFGF in the culture medium. myf5-GFP and mylz2-mCherry signals were quantified by Image J and normalized to the DMSO-treated sample. Some inhibitors identified in this sensitized screen (e.g., SB216763) were missed in the initial screen, possibly due to toxicity or other unanticipated issues associated with the limited dose range that can be assayed in such a chemical genetic screen (see also Figure S6).
(E) 1-cell-stage myf5-GFP (green);mylz2-mCherry (red) embryos were injected with 250 pg fgf8-MO and 250 pg fgf24-MO and rescued by adding 10 μM forskolin at the dome stage. Scale bars represent 200 μm.
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12. Figure S3
Forskolin Treatment Restores Proliferation of mdx Satellite Cells, and Transplantation of Forskolin-Treated Wild-Type Satellite Cells Provides Dystrophin Expression to Dystrophic Muscle, Related to Figure 3
(A) Satellite cells from C57BL/6J (wild-type) or mdx mice were cultured with DMSO or forskolin in the presence of bFGF. Cell numbers were determined at day 5. Mdx satellite cells show defective in vitro expansion under control conditions (compare gray bar and blue bar) and forskolin treatment increases the number of cells recovered after culture of mdx satellite cells (compare blue and red bars). Data are presented as fold change (mean ± SEM, n = 4), normalized to “DMSO + bFGF” controls.
(B) Immunofluorescence images of forskolin-cultured cells stained for MyoD (top), Pax7 (middle) and nuclei (DAPI, bottom). Scale bars represent 100 μm.
(C) Transverse frozen section of mdx muscle transplanted with cultured forskolin-treated GFP+ satellite cells showing GFP (left, green) and Dystrophin (right, red) in the engrafted fibers. Scale bars represent 50 μm.
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13. Figure S4
Forskolin and bFGF Do Not Have a Synergistic Effect on Differentiation of Mouse Satellite Cells, Related to Figure 4
(A) Experimental scheme for data shown in (B, C). Satellite cells from C57BL/6J mice were cultured in the presence of bFGF for 5 days. In order to test potential synergistic effects of bFGF and forskolin on satellite cell differentiation in vitro, cells cultured with bFGF were harvested on day 5 and equal numbers of cells were replated under pro-differentiation conditions with forskolin or DMSO in the presence or absence of bFGF.
(B) Images of satellite cells differentiated with DMSO (top row) or forskolin (bottom row) in the absence (left column) or presence (right column) of bFGF. Cultures were stained for Myosin Heavy Chain (MHC, red) and nuclei (DAPI, blue). Scale bars represent 200 μm.
(C) Quantification of percentage of nuclei in myotubes after differentiation of satellite cells in the presence of forskolin or DMSO with or without bFGF (mean ± SD, n = 3). The differentiation potential of satellite cells appears to be reduced slightly in the presence of bFGF when the cells are also treated with forskolin during differentiation.
(D) Experimental scheme for data shown in (E, F). Satellite cells from C57BL/6J mice were cultured in the presence of bFGF and forskolin/DMSO treatment for 5 days. In order to test the differentiation potential of satellite cells treated with forskolin during both the proliferation and differentiation phases, cells were harvested on day 5 and equal numbers of cells were induced to differentiate in the continued presence of forskolin or DMSO.
(E) Images of differentiated satellite cells treated with DMSO (left) or forskolin (right) during proliferation and differentiation. Myotubes are stained for MHC (red) and nuclei (DAPI, blue). Scale bars represent 200 μm.
(F) Quantification of percentage of nuclei in myotubes after differentiation of forskolin or DMSO treated cells in the continued presence of the compound (mean ± SD, n = 3). Forskolin-treated and DMSO-treated satellite cells differentiate normally into myotubes in the continued presence of the compound.
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14. Figure S5
Characterization of Pluripotency of and iPSCs and Toxicity Assay of GSK3β Inhibitors on iPSC and EB, Related to Figure 6
(A) Expression of NANOG, OCT4, SSEA3, SSEA4, and TRA1-60 in undifferentiated and iPSCs. Merged images show Hoechst staining (blue, nuclei) and staining for the indicated protein (red).
(B) Bright-field images of BJ, 00409, iPSC colonies. Scale bars represent 100 μm.
(C) Gene expression analysis of undifferentiated and iPSCs. Indicated pluripotency genes were analyzed by quantitative RT-PCR using GAPDH as a housekeeping gene. Changes in gene expression levels are expressed relative to undifferentiated BJ iPSCs. Bars represent the standard deviation of three independent experiments and iPSCs show equivalent expression of pluripotency genes as the well-established BJ iPSC line (Maherali et al., 2008).
(D) Bright-field images showing EB formation of BJ, and iPSCs after 7 days of differentiation. Scale bars represent 100 μm.
(E) BJ monolayer iPSCs were incubated with 0.5 μM of indicated chemicals for 24 hr. Indicated percentages represent the percent live cells (PI-negative and calcein-positive) detected after culture with the indicated compound.
(F) Bright-field images of BJ EBs after 7 days of chemical treatment (0.5 μM; chemicals added at days 1, 3 and 5). Scale bars represent 100 μm.
(G and H) BJ iPSC-derived EB were stimulated with bFGF+BIO (G) or bFGF+forskolin (H). Cells were harvested at the times indicated for RNA extraction. Ectodermal, endodermal and mesodermal genes were analyzed by quantitative RT-PCR using GAPDH as a housekeeping gene. Changes in gene expression are presented relative to undifferentiated BJ iPSCs (Day 0).
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15. Figure S6
Myogenic Differentiation of and iPSCs and Nuclear Expression of PAX7, MYF5, and MYOD1 in BJ EB, Related to Figure 6
(A and B) Gene expression analysis of EB (A) and monolayer cells (B). Cells were harvested at the times indicated for RNA extraction. Ectodermal, endodermal and mesodermal genes were analyzed by quantitative RT-PCR using GAPDH as housekeeping gene. Changes in gene expression levels are expressed relative to undifferentiated iPSCs (Day 0). Bars represent the standard deviation of three independent experiments, (∗p < 0.05, ∗∗ < 0.01, ∗∗∗ < 0.001).
(C) Immunostaining of differentiated iPSCs. Under terminal differentiating procedures (day 36) most of the cells express Desmin (red) and Myogenin (green), forming multinucleated myofibers. Cells were also stained with Hoechst (blue). Scale bars represent 100 μm.
(D) Representative electron microscopy images of differentiated and cells at day 36, magnification x52700.
(E) Immuno-electron microscopy staining using skeletal muscle specific anti-Myosin heavy and light chain. Black dots indicate gold cross-linked particles to secondary antibody, magnification x52700.
(F) Immunostaining of PAX7, MYF5 and MYOD1 in BJ iPSCs (day0) and EBs (day7). Merged images show Hoechst staining (blue, nuclei) and staining for the indicated protein (red). Scale bars represent 100 μm.
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16. Figure S7
Engraftment of Human iPSC-Derived Muscle Progenitors into the Satellite Cell Compartment of Immune-Compromised Mice, Related to Figure 7
Immunostaining of TA sections from pre-injured-NSG mice injected with 1x105 iPSCs at day 14 of differentiation. BJ (A, B), (B), (B) lines showed positivity for human histone H2A (green) and Pax7 (red) proteins. No staining for human histone H2A was observed in PBS injected mice (A). Scale bars represent 50 μm (A) and 25 μm (B). n = 3 per sample. The Pax7 antibody cross-reacts with mouse and human protein, while the H2A antibody is human-specific.
Cell  , DOI: ( /j.cell )
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