Transgenic mice expressing tunable levels of DUX4 develop characteristic facioscapulohumeral muscular dystrophy-like pathophysiology ranging in severity

Figures

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  Fig. 1. Transgene map and FSHD-like model generation. a The synthesized FLExDUX4 (FLExD) transgene, flanked by heterologous lox sites (loxP and lox511), was inserted in the antisense orientation to the Rosa26 promoter and maintains the intron/exon structure and cis mRNA regulatory features, including the PAS and DAE, of the human chromosome 4q35 DUX4 gene. When crossed with ACTA1-MCM mice, the bi-transgenic offspring have the capacity for dosage-dependent, TMX-inducible unidirectional recombination of the transgene, resulting in DUX4 expression exclusively in skeletal muscle and transcribed from the Rosa26 promoter, processed, and terminated in exon 3 using the DUX4 PAS. b Genomic PCR indicating percent transgene recombination in different muscles from FLExD/+ mice and bi-transgenic ACTA1-MCM;FLExD mice with no TMX or 3 days after a single IP injection of 5 mg/kg TMX (low), or two IP injections of 10 mg/kg TMX (high). TA, tibialis anterior; Sol, soleus; GA, gastrocnemius; Qua, quadriceps; Diaph, diaphragm

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  Fig. 2. All bi-transgenic ACTA1-MCM;FLExD mouse models show mosaic patterns of nuclear DUX4-FL expression. Tibialis anterior muscles were analyzed by IF for DUX4-FL protein expression over 28 days (moderate model, panels c–j) or 9 days (severe model, panels o–t). Muscles from the mild model, bi-transgenic ACTA1-MCM;FLExD without TMX (panels m and n), showed low steady levels of DUX4-FL immunostaining. Muscles from ACTA1-MCM mice treated with TMX at MD14 (panels a and b) or SD9 (k and l) showed no DUX4-FL signal and served as negative controls. Red, DUX4 IF; Blue, DAPI staining; Green, WGA staining. Scale bar = 50 μm

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  Fig. 3. DUX4-FL target genes, but not DUX4-fl mRNA, show TMX dose-dependent increased expression. Gastrocnemius muscles from 13–14-week-old female mice isolated 9 days after TMX administration were assayed for gene expression levels by qRT-PCR. a Levels of DUX4-fl mRNA are not significantly different between the FLExD mice and the three bi-transgenic models. However, mRNA expression of bWdfc3 and cTrim36 are increased significantly in all bi-transgenic models compared with FLExD. Both Wdfc3 and Trim36 are significantly increased by moderate and severe TMX induction compared with no TMX. Trim36 mRNA levels are significantly increased in the severe compared to moderate models. All expression is normalized to RpL37 expression. MT, equivalent level of cDNA from 17Abic FSHD1 myotubes [18]. Data are mean ± S.D. with significance calculated using Welch’s t test, *p < 0.05, **p < 0.01

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  Fig. 4. The moderate and severe FSHD-like mouse models show significant decline in treadmill performance. Mild, moderate, and severe FSHD-like mice were assessed for treadmill performance, as described in the methods. a The moderately affected female mice (red line) were assayed prior to TMX injection (D0) and 3, 6, 10, 16, 23, and 29 days post-injection (DPI) and compared with age-matched female bi-transgenic mice (green line) and female ACTA1-MCM mice similarly injected with TMX (blue line). b The moderately affected male mice (red line) were assayed prior to TMX injection (D0) and at 2, 6, 10, 14, 17, 21, 24, and 28 DPI and compared with aged-matched male bi-transgenic mice (green line) and male ACTA1-MCM mice similarly injected with TMX (blue line). c Severely affected female mice (red line) were assayed prior to TMX injection (D0) and 2, 5, and 7 DPI (# D8, mice were too affected to be assayed) and compared with female bi-transgenic mice (green line) and ACTA1-MCM mice similarly injected with TMX (blue line). d Severely affected male mice (red line) were assayed prior to TMX injection (D0) and 2, 5, 7, and 8 DPI and compared with age-matched male bi-transgenic mice (green line) and…

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  Fig. 5. Ex vivo muscle function analysis shows DUX4-induced muscle weakness. EDL muscles from female ACTA1-MCM/+ with TMX (MCM, blue, n = 9), mild model (MCM;FLExD Mild, green, n = 9), moderate model (MCM;FLExD Mod, yellow, n = 6), and severe model (MCM;FLExD Severe, red, n = 3) were assayed for maximum twitch, maximum tetanus, and force frequency (Figure S7, not normalize) and then normalized, here, to muscle cross sectional area to provide a specific twitch, b specific tetanus, and c specific force frequency at MD14 and SD10. d Muscle stiffness calculated from eccentric contractions analysis showed all three model mice have stiffer muscle than control. Data are mean ± S.D. with significance *p < 0.05, **p < 0.01, ***p < .001. For panel c, blue asterisks indicate significance compared with MCM +TMX control mice, green asterisks indicate significance compared with mild bi-transgenic mice, and yellow asterisks indicate significance compared with moderate model mice

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  Fig. 6. Comparison of gene expression in muscle from FSHD-like models compared with controls. a–d Scatter plots showing differentially expressed genes in RNA-seq data derived from gastrocnemius muscles isolated from aFLExD/+ mice, b bi-transgenic mild model mice, c bi-transgenic moderate model mice, and d bi-transgenic severe model mice (y-axis), each compared with ACTA1-MCM control mice (x-axis). Significantly upregulated genes are indicated in red, and downregulated genes are indicated in blue. e–h Above scatter plots highlighting the DUX4-induced genes similarly induced by DUX4 in C2C12 myoblasts [25]

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  Fig. 7. GO enrichment analysis of differentially expressed genes in FSHD-like severity model mice. GO enrichment analysis for DUX4-induced genes identified in (left to right) C2C12 cells [25], mild FSHD-like mice, moderate FSHD-like model mice, and severe FSHD-like model mice. The size of the beige-colored circles indicates the total number of differentially expressed genes (> 1.4-fold), colored circles indicate the proportion of genes from significantly enriched GO terms, that are in turn offspring of GO superterms (see the “Methods” section and Table S4) relating to mus. Upregulated genes are represented in the top half and downregulated genes are represented in the lower half. The number of genes in each grouping is indicated (n)

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  Fig. 8. Skeletal muscles from the mild and moderately affected FSHD-like mouse models show signs of dystrophic histopathology. Representative cryosections of tibialis anterior, gastrocnemius, soleus, and quadriceps of indicated transgenic animals were analyzed with H&E staining. Female (a–d) ACTA1-MCM mice treated with 1× 5 mg/kg TMX, (e–h) mild model bi-transgenic mice without TMX, and (i–x) moderate model bi-transgenic mice treated 1× with 5 mg/kg TMX. The TMX-injected ACTA1-MCM control mice were assayed at MD28. The TMX-injected bi-transgenic mice were assayed at (i–l) MD3, (m–p) MD6, (q–t) MD14, and (u–x) MD 28. All mice had performed the treadmill exhaustion analysis, as described, 2× per week starting the week prior to TMX injection. Scale bar = 100 μm

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  Fig. 9. Skeletal muscle from the severely affected FSHD-like mice shows signs of a severe myopathy. Representative cryosections of tibialis anterior, gastrocnemius, soleus, and quadriceps from indicated transgenic animals were analyzed with H&E staining. a–dACTA1-MCM mice treated with 2× 10 mg/kg TMX, e–h mild model bi-transgenic mice without TMX, and (i–t) severe model bi-transgenic mice treated 2× with 10 mg/kg TMX. The TMX-injected controls were assayed at SD9. The TMX-injected bi-transgenic mice were assayed at (i–l) SD3, (m–p) SD6, and (q–t) SD9. All mice had performed the treadmill exhaustion analysis, as described, 1 week prior to TMX injection, then 2, 5, and 7 DPI. Scale bar = 100 μm

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  Fig. 10. Skeletal muscles from FSHD-like mouse models develop centralized nuclei. Soleus and TA muscle histological sections from control ACTA1-MCM (blue) and mild (green), moderate (yellow), and severe (red) FSHD-like model mice that had undergone treadmill exhaustion analysis 2× per week (see Figs. 7 and 8), were stained with H&E and counted for myofibers with centralized nuclear. Muscles were assayed at the indicated time points for each model. The ACTA1-MCM +TMX controls were assayed at MD28 and SD9, as appropriate. Data is plotted as percent of myofibers with centralized nuclei. Data are mean ± S.D. with significance calculated by one-way ANOVA using Prism 7. *p < .05, **p < .01, ***p < .001, #p < .001 for ACTA1-MCM compared with all other samples in the group.

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  Fig. 11. Induced DUX4-fl expression causes regeneration of muscle fibers marked with eMyHC expression. Histological cross sections from TA muscles were dissected from mild, moderate, and severe FSHD-like model mice that had undergone treadmill exhaustion analysis 2× per week (see Figs. 7 and 8). Age-matched female control mice were used for the moderate model analysis and male control mice for the severe model analysis. Muscles were assayed from ACTA1-MCM TMX controls at a MD14, b SD3, and c SD9. Representative images of mild model (k, l, s, t), female moderate model (e–i and magnified images f–j), and male severe model (m–q and magnified images n–r) at indicated time point. Quantitative analysis of data is shown in Figure S12. Scale bars = 100 μm

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  Fig. 12. Muscles from the moderate FHSD-like model mice undergo apoptosis in response to induced DUX4-fl expression. TA muscles from female moderate model mice that had undergone treadmill exhaustion analysis 2× per week (see Fig. 8) were assayed at MD6 (a, e), MD14 (b, f), and MD28 (c, g) or at MD28 for matched mild model mice (d, h). Green signal in the TUNEL assay (a–d) indicates nuclei undergoing apoptosis, compared with DAPI staining (e–h) showing all nuclei in the same histological sections. Scale bar = 200 μm. Quantitative analysis of data is shown in Figure S14