Dosage effect of multiple genes accounts for multisystem disorder of myotonic dystrophy type 1

Figures

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  Fig. 1. SC mice carrying heterozygous mutations in Dmpk, Six5 and Mbnl1 exhibit typical DM1-associated muscle defects. a Diagram of DSM-TKO SC mice generated by ICAHCI of ΔDSM-O48 cells carrying mutatnt Dmpk, Six5 and Mbnl1. b Images of cultured ΔDSM-O48-1 haploid ESCs and newborn SC pups from ΔDSM-O48-1 cells. Scale bar, 100 μm. c Body weight analysis of DSM-TKO and WT SC mice (n > 5 per group, means ± SD). d EMG analysis of TKO SC mice (WT SC mice, n = 3; DSM-TKO SC mice, n = 2, 4-month old). e-g Muscle weakness of DSM-TKO SC mice was determined by treadmill test (e) (n = 5 per group, 4-month old), forelimb grip strength test (f) (WT SC mice, n = 5; DSM-TKO SC mice, n = 8, 4-month old) and rotarod test (g) (n = 5 per group, 4-month old). Unpaired Student’s t-test, *P < 0.05. h H&E staining of TA muscles from DSM-TKO and WT SC mice, DSM-TKO mice showing nuclear clump (green arrow) and atrophic fiber (white arrow) (WT SC mice, n = 11; DSM-TKO SC mice, n = 7, 4-month old). Unpaired Student’s t-test, *P < 0.05. Scale bars, 50 μm. i TA myofiber CSA analysis (n = 3 per group, 4-month old)

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  Fig. 2. Generation of Dmwd+/− SC mice through ICAHCI of haploid cells carrying mutant Dmwd. a Schematic of the sgRNA targeting Dmwd. b Sequences of the Dmwd gene in two cell lines (ΔDmwd-O48-1 and ΔDmwd-O48-2) carrying CRISPR-Cas9-induced gene modifications. c Phase-contrast image of ΔDmwd-O48-1 cell line. Scale bar, 100 μm. d Newborn SC pups generated from ΔDmwd-O48-1 cells. e Average body weight of Dmwd+/− SC mice and WT SC mice (n > 8 per group, means ± SD). f Transcription analysis of Dmwd in TA muscles showed that the transcription level of Dmwd was significantly lower in Dmwd+/− SC mice compared with WT SC mice (n = 4 per group, 4-6-month old). Unpaired Student’s t-test, **P < 0.01. g Representative images of H&E staining of TA muscles from DMWD+/− SC mice and WT SC mice (4-6-month old). Scale bars, 50 μm. h CSA analysis of TA muscle showed muscle wasting in DMWD+/− SC mice (n = 3 per group, 4-6-month old). i Representative images of H&E staining showed normal histological structure of diaphragm and small intestine in DMWD+/− SC mice (4-6-month old). Scale bars, 200 µm for diaphragm; 50 µm for small intestine

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  Fig. 3. SC mice carrying heterozygous mutations in Dmpk, Six5, Mbnl1 and Dmwd show CDM phenotypes. a Phase-contrast image of cultured ΔDSMD-O48-1 haploid cells. Scale bar, 100 μm. b 23% of newborn DSMD-QKO SC pups died during perinatal period (upper), while others survived (lower). c H&E staining of diaphragm sections from DSMD-QKO and WT SC pups (P1). Scale bars, 100 μm. d Diaphragm CSA analysis of DSMD-QKO and WT SC mice (P1) (n = 3 per group). Unpaired Student’s t-test, ****P < 0.0001. Scale bars, 50 μm. e Immunofluorescent staining of bungarotoxin (red) and neurofilament H (green), and complexity analysis of NMJs of DSMD-QKO and WT SC mice (P1) (WT SC pups, n = 2; DSMD-QKO SC pups, n = 5). Unpaired Student’s t-test, **P < 0.01. Scale bars, 50 μm. f H&E staining of TA muscle sections (P1) showing less myofiber in DSMD-QKO SC mice (2/5). Scale bars, 50 μm. g H&E staining of TA muscle of DSMD-QKO and WT SC (P0) mice and the percentage of myofibers containing centrally located nuclei in TA muscle (n = 3 per group). Yellow arrows indicate the myofibers with central nuclei. Unpaired Student’s t-test, *P < 0.05. Scale bars, 50 μm. h H&E staining of heart cryo-sections (P17) showing left…

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  Fig. 4. DM1-related pathogenic phenotypes in adult SC mice carrying quadruple mutations. a EMG analysis indicates typical waxing-waning myotonia in QKO SC mice (WT SC mice, n = 3; DSMD-QKO SC mice, n = 5, 4-6-month old). b Forelimb grip strength test (WT SC mice, n = 5; DSMD-QKO SC mice, n = 7, 4-6-month old). Unpaired Student’s t-test, **P < 0.01. c TA CSA analysis (n = 3 per group, 4-6-month old). d Nuclear clump analysis of TA muscle (WT SC mice, n = 6; DSMD-QKO SC mice, n = 3, 4-6-month old). Unpaired Student’s t-test, **P < 0.01. e The percentage of myofibers containing centrally located nuclei in TA muscle of QKO and WT SC mice (P30) (n = 3 per group). Unpaired Student’s t-test, *P < 0.05. f Representative images of ATPase staining and fiber type analysis (n = 4 per group, 4-6-month old). Unpaired Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars, 100 μm. g Dust-like opacities in eyes of DSMD-QKO SC mice (3/7, 4-6-month old). h The percentage of myofibers containing centrally located nuclei in TA muscles of QKO and WT F1 mice (n = 5 per group, P0). Unpaired Student’s t-test, ***P < 0.001

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  Fig. 5. Decreased differentiation potential of MuSCs from TKO or QKO DM1 mice. a Representative images of PAX7 immunostaining (pink) in TA muscle sections from adult TKO and QKO mice (4-6-month old, n = 3 per group). White arrows indicate PAX7-positive cells. Scale bars, 50 µm. Unpaired Student’s t-test. NS, no significant differences. b In vitro differentiation of MuSCs (n = 3 per group, 2-month old). One-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars, 200 μm. c In vivo differentiation of EGFP-WT MuSCs and RFP-QKO MuSCs (n = 2 per group, 2-month old). R-TA represents TA of right hind leg, while L-TA represents TA of left hind leg. One-way ANOVA, ****P < 0.0001. NS, no significant differences. Scale bar, 100 μm. d Venn diagrams show that the number of common DEGs between mutant and WT samples dramatically decreased following MuSC differentiation (from Day 0 to Day 2). e Heat map of stem cell markers and differentiation markers in DM1 and WT MuSCs. f Venn diagrams show that the number of common mis-splicing genes (skipped exon events) between mutant and WT samples dramatically decreased following MuSC differentiation (from Day 0 to Day 2)

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  Fig. 6. Large-scale screening and potential applications of DM1 mice. a Diagram of large-scale screening for small molecules that can promote differentiation of DM1 MuSCs. b Molecular structure of T5381948. c Images show that T5381948 could specifically improve the in vitro differentiation of DSM-TKO MuSCs. Scale bars, 200 μm. Unpaired Student’s t-test; ***P < 0.001; NS, no significant differences. d Overview of the generation and applications of DM1 mouse model with quadruple heterozygous mutations