Fig 2: Immunoreactivity of TDP-43, FUS/TLS, MBNL1 and MBNL2 in the cerebellum of control and SCA7 patients. Representative immunohistochemically labelled cerebellar sections (counterstained with hematoxylin) from a SCA7 patient and a control with no neurological disease. a TDP-43 immunoreactivity was increased in the nucleus of PCs in the cerebellum of a SCA7 patient compared to control PCs where it remained diffuse with few TDP-43 dots. b Phosphorylated TDP-43 was strongly labeled in the nucleus of a PC of a SCA7 patient; only rare nuclear granules were observed in the nucleus of control PCs. c FUS/TLS shows increased immunoreactivity with FUS/TLS-positive small dots in the nucleus of a PC in a SCA7 patient compared to a control PC where FUS/TLS was almost not detectable in the nucleus. d Nuclear accumulation of the MBNL1 protein was higher in an atrophic PC in a SCA7 patient compared to PCs from a control. e MBNL2 diffuse immunoreactivity is increased in the nucleus and cytosol of a SCA7 PC, compared to control PCs where MBNL2 immunoreactivity is low in the cytoplasm and almost undetectable in the nucleus. Bars: 20 µm

Fig 3: 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 ?DSM-O48 cells carrying mutatnt Dmpk, Six5 and Mbnl1. b Images of cultured ?DSM-O48-1 haploid ESCs and newborn SC pups from ?DSM-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)

Fig 4: SC mice carrying heterozygous mutations in Dmpk, Six5, Mbnl1 and Dmwd show CDM phenotypes. a Phase-contrast image of cultured ?DSMD-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 ventricular posterior wall attenuation in DSMD-QKO SC mice (2/7, black arrow). Scale bars, 1 mm

Fig 5: Inducible and SMC-specific knockout of Srf in vivo reduces SMC splicing of Vcl, Cald1, and Myocd. To address the in vivo relevance of Myocd-Srf-driven splicing, we generated mice allowing for inducible deletion of Srf in SMCs (Myh11-CreERT2 x Srffl/fl mice injected with tamoxifen, referred to as knockout, KO). Compared to wild type mice (WT), staining for Srf (green), which is a predominately nuclear protein, was reduced in SMCs in the aorta and urinary bladder (A). White lines in the micrographs highlight the SMC layers of the aorta and bladder, respectively. This was confirmed by RT-qPCR, showing reduction of Srf along with three contractile markers in both aorta (B), and bladder (C). We focused on the bladder in view of better Srf depletion and could confirm sizeable reduction of Srf and SMC markers by western blotting (D). Contraction in response to cumulative addition of the muscarinic agonist carbachol (E), depolarization with KCl (60 mM, F), and stimulation with the phosphatase inhibitor Calyculin A (1 µM, G), was reduced, confirming loss of contractility in KO vs. WT bladder. In addition to these expected phenotypes, we also observed a reduction of Rbpms (H), but not of Rbfox2 (I) by RT-qPCR. Splicing of Vcl, Cald1, and Mbnl1 was altered as shown using PCR and agarose gel electrophoresis (J). Myocd splicing (exon 2a) was also examined. Myocd splicing changed from the SMC variant containing exon 2a (+ Ex2a), towards a variant that lacks exon 2a (- Ex2a, panel J, bottom). Silencing of either RBPMS or RBFOX2 similarly favored the heart variant (- Ex2a) of MYOCD in cultured human coronary SMCs (K). Altered splicing of Vcl, and Cald1 in KO bladder was confirmed by western blotting (L)