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Fig. S1. Localization of β-catenin in control and HSA-β-catflox(ex3)/+ muscles. Muscle cross-section of P0 mice of indicated genotypes were stained with antibodies against β-catenin and visualized by Alexa Fluor 488-conjugated secondary antibodies. AChR clusters were labeled with α-BTX and myonuclei were labeled with DAPI. The genotyping primers were 5′-GCCTGCATTACCGGTCGATGCAACGA-3′ and 5′-GTGGCAGATGGCGCGGCAACACCATT-3′ for HSA- and HB9-Cre with a 750-bp PCR product and 5′-CTAAGCTTGGCTGGACGTAAACTC-3′ (exon3), 5′-ACGTGTGGCAAGTTCCGCGTCATCC-3′ (AS5 exon4) and 5′-GGTAGTGGTCCCTGCCCTTGACAC-3′ (F1intron) with 1025-bp PCR product for β-catenin(ex3) and 1.5 kb for wild-type β-catenin. The primers for TOP-EGFP reporter were: 5′-TCACGAACTCCAGCAGGACCATG-3′ and 5′-CCTGGTCGAGCTGGACGGCGAC-3′. Generation and genotyping of HSA-β-cat−/− mutant and β-catloxP/loxP control mice have been described previously (Li et al., 2008).
Fig. S2. Normal muscle structure in HSA-β-catflox(ex3)/+ mice. (A) Hematoxylin and Eosin staining of diaphragm sections of P0 control and HSA-β-catflox(ex3)/+ mice. (B,C) No difference in muscle thickness (B) (P>0.05, n=10, t-test) or cross-sectional area of muscle fibers (C) (P>0.05, n=52, t-test) between the two genotypes. (D) Normal contractile units in HSA-β-catflox(ex3)/+ muscles. Shown are representative electron microscopy images of P0 muscles of indicated genotypes. No change in the distance between Z-lines and the alignment of M-line in HSA-β-catflox(ex3)/+ muscles.
Fig. S3. Aberrant innervation of motor axons in HSA-β-catflox(ex3)/flox(ex3) homozygous mice. (A) Whole-mount staining of left hemi-diaphragms of P0 mice of indicated genotypes. Phrenic nerves and terminals were stained with anti-NF/synaptophysin antibodies which were visualized with Alexa Fluor 488-conjugated goat anti-rabbit antibodies. Arrowheads, secondary nerve branches; arrow, ectopic axon; V, ventral; L, lateral; M, medial. (B) Decreased number of secondary nerve branches in HSA-β-catflox(ex3)/flox(ex3) muscles. (**P<0.01, n=7 in control, and n=4 in HSA-β-catflox(ex3)/flox(ex3), t-test). (C) Increased length of secondary, tertiary, quaternary and 5th branches in HSA-β-catflox(ex3)/flox(ex3) muscles. (**P<0.01, n=7, one-way ANOVA).
Fig. S4. Augmented motor axon arborization in intercostal muscles and tibialis anterior muscles in HSA-β-catflox(ex3)/+ mice. (A,D) Intercostal muscles (A) and tibialis anterior muscles (D) of indicated genotypes were stained whole-mount for nerve (green) and AChR (red). White dashed lines indicate the width of AChR endplate zone. Arrowheads indicate arborized axons. (B,C,E,F) Quantitative data are shown in B (**P<0.01, n=13, t-test); C (**P<0.01, n=9, t-test); E and F, (**P<0.01, n=11, t-test).
Fig. S5. Similar motoneuron numbers in HSA-β-catflox(ex3)/+ and control mice. (A) Cross sections of P0 spinal cords (C3-C5) were immunostained with anti-HB9 antibody (green) and counterstained with DAPI (blue). Dashed yellow lines outline the spinal cords. Insets showed enlarged areas with two groups of motoneurons circled. (B-D) Quantitative analysis of motoneuron numbers of hemi-ventral column (B), lateral motor column (LMC) (C) or medial motor column (MMC) (D). (P>0.05, n=6, t-test).
Fig. S6. Normal SV2 staining at HSA-β-catflox(ex3)/+ NMJs. (A) Left hemi-diaphragm was whole-mount stained for SV2 (green) and AChR clusters (red). (B) Quantitative analysis of data shown in A. (P>0.05, n=6, t-test).
Fig. S7. No apparent deficits in HB9-β-catflox(ex3)/+ NMJs. (A) Specific expression of truncated β-catenin (arrow) in the spinal cord, but not other tissues, in HB9-β-catflox(ex3)/+ and no expression in the spinal cord of control mice. (B) Whole-mount staining of left hemi-diaphragms of P0 mice. Phrenic nerves were stained with rabbit antibodies to NF/synaptophysin (green) and α-BTX for AChR clusters (red). Arrowheads indicate secondary branches. V, ventral; L, lateral; M, medial. (C-H) Quantitative analysis of data shown in B. No difference was observed in the primary branch location (C), number of secondary branches (D), length of secondary branches (E), number of AChR clusters (F), AChR cluster size (G) or endplate band width (H). (P>0.05, n=4, t-test).
