Volume 41, Issue 4, Pages 382-391.e5 (May 2017) A Molecular Switch Regulating Cell Fate Choice between Muscle Progenitor Cells and Brown Adipocytes  Yitai An, Gang Wang, Yarui Diao, Yanyang Long, Xinrong Fu, Mingxi Weng, Liang Zhou, Kun Sun, Tom H. Cheung, Nancy Y. Ip, Hao Sun, Huating Wang, Zhenguo Wu  Developmental Cell  Volume 41, Issue 4, Pages 382-391.e5 (May 2017) DOI: 10.1016/j.devcel.2017.04.012 Copyright © 2017 Elsevier Inc. Terms and Conditions

Developmental Cell 2017 41, 382-391. e5DOI: (10. 1016/j. devcel. 2017 Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Inducible Deletion of Pax7 in MPCs Resulted in Upregulation of Multiple Brown Adipocyte-Related Genes and a Cell Fate Change to Brown Adipocytes in Culture (A) Experimental scheme for (B) and (C). (B) MPCs were isolated from P12 Pax7CreER/+ (control) and Pax7CreER/flox (KO) mice and cultured for 2 days before harvest. Total RNAs were isolated and subjected to RNA-seq. Selected genes with differential expression levels in MPCs from the control and Pax7-KO mice are shown. (C) MPCs from the control and Pax7-KO mice were isolated by FACS and cultured in growth media for 36 hr. The relative expression of selected genes was measured by qRT-PCR. Data are presented as means ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (D) Experimental scheme for (E) and (F). MPCs were isolated by FACS from P21 Pax7CreER/+:Rosa-stop-YFP (control) and Pax7CreER/flox:Rosa-stop-YFP (KO) mice. (E and F) Cells were cultured in the pro-adipogenic media for 5 days followed by oil red O staining (E) or UCP1 immunostaining (F). In (F), the right panels are enlarged images of the areas in the white boxes in the left panels. Scale bars, 50 μm. Developmental Cell 2017 41, 382-391.e5DOI: (10.1016/j.devcel.2017.04.012) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 MyoD and Myf5 Suppressed the BA Fate by Transcriptionally Repressing Prdm16 (A) FACS-isolated MPCs from 2-month-old wild-type mice were first transfected with different siRNAs as indicated. Cells were then cultured in the pro-adipogenic media for 5 days followed by oil red O staining (top panels; scale bar, 50 μm) or UCP1 immunostaining (red) (bottom panels; scale bar, 20 μm). The ratio of the UCP1+ BAs was quantified by counting more than 100 cells from three randomly chosen fields. The nuclei were counterstained by DAPI (blue). (B) FACS-isolated MPCs were infected with adenoviruses expressing shLacZ or shMyoD (n = 3 mice). The mRNA expression of selected genes was measured by qRT-PCR 36 hr after infection. (C) Quadruplicate C2C12 cells were first transfected with siRNAs as indicated and then cultured in the pro-adipogenic media for 4 days. Left: 3/4 of cells were subjected to oil red O staining. Scale bar, 50 μm. Middle: Quantification of the intracellular oil red O by measuring the absorbance at 500 nm. Right: 1/4 of the cells were subjected to western blotting. Data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Developmental Cell 2017 41, 382-391.e5DOI: (10.1016/j.devcel.2017.04.012) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 E2f4 Is a Direct Transcription Target of MyoD/Myf5 MPCs used below Were Isolated by FACS from 2-Month-Old Wild-Type Mice (A) Sorted cells were fixed after either 1 hr of plating (i.e., quiescent satellite cells or QSC) or 1 day of culturing (i.e., activated satellite cells or ASC) followed by immunostaining for E2F4 (green). (B) MPCs from three mice were separately infected with adenoviruses expressing indicated shRNAs; 36 h later, the E2f4 mRNA levels were measured by qRT-PCR. (C) MPCs were transfected with siRNAs as indicated. E2F4 protein levels were measured by western blotting. (D) Top: schematic of the E2f4 promoter. TSS, transcription start site; blue boxes, exons; black box, the E box. Bottom: ChIP assays (n = 3) were performed using lysates from proliferating C2C12 myoblasts with a MyoD antibody or an immunoglobulin G control. The fold enrichment of MyoD on the specific E box in the E2f4 promoter is shown. NS, non-specific site. (E and F) MPCs (E) and C2C12 myoblasts (F) were transfected with siRNAs as indicated; 24 hr post transfection, cells were cultured in the pro-adipogenic media for 4 days followed by oil red O staining. Data are presented as mean ± SD. ∗∗p < 0.01, ∗∗∗p < 0.001. Scale bars, 30 μm. Developmental Cell 2017 41, 382-391.e5DOI: (10.1016/j.devcel.2017.04.012) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Prdm16 Is a Direct Transcription Target of the E2F4/p107/p130 Repressive Complex (A) FACS-isolated MPCs in triplicate were transfected with siRNAs as indicated. Relative gene expression was measured by qRT-PCR 24 hr after transfection. (B) Top: schematic of the Prdm16 promoter. #1 and #2 indicate the predicted E2F4 binding sites (black boxes). Blue box, exons. Bottom: ChIP assays (n = 3) were performed using lysates from proliferating C2C12 myoblasts with specific antibodies as indicated. The fold enrichment of E2F4, p107, or p130 on two potential E2F4 binding sites in the Prdm16 promoter and a known E2F4 binding site in the PPARγ promoter (control) is shown. (C–F) C2C12 cells in triplicate were co-transfected with various luciferase reporters, siRNAs, and/or cDNA expression vectors as indicated. Cells were harvested 24 hr after transfection and luciferase activities were measured. In (C) and (D), a luciferase reporter construct carrying a 1.5-kb mouse Prdm16 proximal promoter was used. In (E), two luciferase reporter constructs were used, each carrying a short fragment of the Prdm16 promoter with one of the two (i.e., #1, #2) potential E2F4 binding sites. In (F), two luciferase reporter constructs were used, one carrying three copies of the wild-type E2F4 binding site (CGCAGC) found in site #2 and the other three copies of the mutated E2F4 binding site (CTCCTC). In (A)–(F), Fold changes were calculated. Data are presented as means ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Developmental Cell 2017 41, 382-391.e5DOI: (10.1016/j.devcel.2017.04.012) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 Knockdown of Myod Postnatally Promoted MPC to BA Conversion In Vivo at Low Efficiency (A) Experimental scheme for (B). CTX, cardiotoxin. (B) Representative TA muscle sections from Pax7CreER(Gaka):Rosa-stop-YFP mice infected with shLacZ- or shMyoD-expressing adenoviruses following the scheme in (A) were subjected to immunostaining for UCP1 (red) and YFP (green). Nuclei were counterstained by DAPI (blue). The UCP1+/YFP+ cells are indicated by white arrowheads. Scale bar, 20 μm. (C) Quantification of UCP1+/YFP+ cells on TA sections from (B). Sixteen sections from four pairs of TA muscles were counted. Data are presented as means ± SD. ∗∗∗p < 0.001. Developmental Cell 2017 41, 382-391.e5DOI: (10.1016/j.devcel.2017.04.012) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 Inducible Ablation of Pax7 in Embryos Promoted BAT Development Tamoxifen was injected into pregnant mice (Pax7CreER/+ × Pax7flox/+:Rosa-stop-YFP) to induce Pax7 deletion in E10.5 embryos. Embryos or YFP+ cells were isolated at various time points as indicated. (A and B) YFP+ progenitor cells were isolated by FACS from the control (i.e., Pax7CreER/+:Rosa-stop-YFP) and KO (i.e., Pax7CreER/flox:Rosa-stop-YFP) embryos at E12.5 (A) or E13.5 (B). In (A), the relative mRNA levels of selected genes were determined by qRT-PCR (n = 3 pairs of embryos). In (B), YFP+ cells were allowed to attach to the culture plates overnight and then subjected to immunostaining for MyoD (green). Nuclei were counterstained with DAPI (blue). Representative images (left) and the ratio of MyoD+ cells (right) are shown (cells in ten random fields from two pairs of embryos were counted). Scale bar, 20 μm. (C and D) The control and Pax7-KO embryos were isolated at E16.5 and the corresponding transverse sections of the interscapular regions were subjected to immunostaining (C) for UCP1 (red) and YFP (green) or H&E staining (D). The interscapular BAT (iBAT) is marked by black dotted lines. Number (#) denotes different embryos. Scale bars, 50 μm (C) and 200 μm (D). (E) Quantification of the size of the iBAT from (D) by SPOT5.0. (F) The control and Pax7-KO P1 pups were examined. The iBAT was dissected from individual pups and weighed. Representative images (top) and the weight of the iBAT from individual pups (bottom) are shown (n = 9 pairs). In all quantifications, data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Developmental Cell 2017 41, 382-391.e5DOI: (10.1016/j.devcel.2017.04.012) Copyright © 2017 Elsevier Inc. Terms and Conditions