Figures and data
Characterizing NMJ integrity in muscles dissected from wild-type controls and end-stage G93A mice with or without NaBu feeding.
(A) Representative compacted z-stack scan images of whole-mount EDL, soleus diaphragm extraocular muscles stained with antibodies against neurofilament medium chain (NF, labeling axons), synaptophysin (SYP, labeling axon terminals) and Alexa Fluor 488 conjugated α-Bungarotoxin (BTX, labeling AChRs on muscle membrane). scale bars, 50 μm. (B) Mean ratios of well innervated NMJs (SYP signals are present in >60% of BTX positive area), partially innervated NMJs (SYP signals are present in 30-60% of BTX positive area) and poorly innervated NMJs (SYP signals are present in <30% of BTX positive area) and in different types of muscles dissected from WT controls and end-stage G93A mice with or without NaBu feeding (tens to hundreds of NMJs were measured per muscle type per mice, also see Figure 1-figure supplement 1 and Figure 1-table supplement 1). * P < 0.05; ** P < 0.01; **** P < 0.0001; ns, not significant.
Quantification of peri-NMJ SC abundance in muscles dissected from wild-type controls and end-stage G93A mice with or without NaBu feeding.
(A) Representative compacted z-stack scan images of whole-mount EDL, soleus diaphragm extraocular muscles stained with antibodies against Pax7 (labeling SCs), SYP and BTX-Alexa Fluor 488. scale bars, 50 μm. (B) To measure the abundance of SCs around NMJs, circles of 75 μm diameter were drawn around the NMJs and the number of nuclear Pax7+ cells (co-localized with DAPI) were counted. Scale bars, 10 μm. (C) Mean number of SCs around NMJs in different types of muscles dissected from WT controls and end-stage G93A mice with or without NaBu feeding (tens to hundreds of NMJs were measured per muscle type per mice, see Figure 2-table supplement 1). * P < 0.05; ** P < 0.01; **** P < 0.0001; ns, not significant.
Isolation of SCs from wildtype and G93A muscles using fluorescence activated cell sorting.
(A) Representative FACS profiles during the isolation of Vcam1+CD31-CD45-Sca1- cells (gate P5-4). The corresponding population hierarchies are shown right to the plots. For single antibody staining controls, unstained controls and viability test, see Figure 3-figure supplement 1 and Figure 3-figure supplement 2. (B) Comparing the percentage of events in the P5-4 gate between cells isolated from WT and G93A mice. * P < 0.05; *** P < 0.001; ns: not significant.
Proliferation and differentiation properties of FACS-isolated SCs in culture.
(A) Representative images of FACS-isolated SCs cultured for 4 days in growth medium stained with antibodies against Pax7, Ki67 (proliferating cell marker), MyoD (myogenic lineage marker). Measurement results for the ratios of Pax7+Ki67+, Pax7+Ki67- and Pax7-Ki67- cells are shown in the right two panels. Scale bars, 100 μm. * P < 0.05; ** P < 0.01; **** P < 0.0001; ns, not significant. (B) Representative images of FACS-isolated SCs cultured for 4 days in growth medium and 2 days in differentiation medium stained with antibodies against Pax7, Ki67, myosin heavy chain (MHC, differentiated myotube marker). Measurement results for the ratios of Pax7+Ki67+ and Pax7+Ki67- cells, as well as the fusion indices are shown in the right two panels.
Proliferation and differentiation properties of FACS-isolated SCs with different NaBu treatment conditions.
(A) qRT-PCR based relative quantification of Hmga2, Ccnd1 and P21 expression in FACS-isolated G93A HL SCs cultured with different doses of NaBu in growth medium for 1 day (n = 6). *** P < 0.001; **** P < 0.0001. (B) Left two panels show the representative images of FACS-isolated G93A HL and diaphragm SCs cultured in growth medium for 4 days and differentiation medium for 2 days and experienced different NaBu treatment conditions and stained with Pax7, Ki67 and MHC antibodies. Cells in top row were not treated with NaBu. Cells in the middle row were treated with NaBu for 1 day before the induction of differentiation (Day 4 of culture). Cells in the bottom row were treated with NaBu continuously for 3 days from the day before the induction of differentiation (Day 4-6 of culture). Scale bars, 100 μm. Measurement results for the ratios of Pax7+Ki67+ and Pax7+Ki67- cells, as well as the fusion indices are shown in the right two panels. * P < 0.05; ns, not significant.
Comparing the transcriptomic profile and homeostasis preferences of SCs from different muscle origins cultured in growth and differentiation medium.
(A) PCA analysis and hierarchical clustering results of different RNA-Seq samples. Samples with suffix “G” were SCs cultured in growth medium for 4 days. Suffix “D” denote SCs cultured in growth medium for 4 days and differentiation medium for 2 days. Suffix “NaBu3” denote SCs treated with 0.5 mM NaBu continuously for 3 days (Day 4-6 of culture). Dashed-line circles in PCA plot highlight samples close to each other in distance. (B) We identify genes differentially expressed in EOM SCs compared to their diaphragm and HL counterparts cultured in growth medium (leftmost panel) and differentiation medium (middle panel) by screening for genes shared by the four DEG lists (gold, magenta, pink and green in color) shown in the Venn diagrams. All DEG lists were generated based on the standard of |log2FC|>= 0.4 and FDR<= 0.001. Yellow box highlights the shared genes. To identify NaBu treatment signature genes, we screened for genes shared by the two DEG lists (green and gold in color) shown in the rightmost Venn diagram. (C) Dot plots in the upper row show the Log2FC of activation signature genes (magenta) and differentiation signature genes (cyan) identified in the 1119, 3318 and 3018 genes highlighted in yellow boxes in (B). Dot plots in the lower row show the Log2 FC of quiescence signature genes identified in the yellow-box highlighted genes in (B). Additionally, genes belonging to the following subgroups of quiescent signature genes: most commonly used SC markers, Notch signaling components and pluripotency signaling components are colored in dark blue, red and gold, respectively.
Functional annotation analysis of EOM SC signature genes and NaBu treatment signature genes and qRT-PCR results.
(A) Top KEGG pathway, GO_P and GO_C annotations of the 478 EOM signature genes (expressed higher in EOM SCs compared to diaphragm and HL counterparts cultured in both growth medium and differentiation medium). Arrows highlight annotations related to axon guidance and cell migration. (B) Top KEGG pathway, GO_P and GO_C annotations of the 3018 NaBu treatment signature genes shown in Figure 6B, rightmost panel. (C) qRT-PCR based relative quantification of Hmga2 (activation signature gene), Actn3 (differentiation signature gene), Notch3 (quiescence signature gene) and Cxcl12 (axon guidance molecule) expression in HL, diaphragm and EOM SCs cultured in growth and differentiation medium, respectively. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant. RNA samples were collected from 3-6 rounds of sorting and sorted cells were split into 3 replicates. (D) qRT-PCR based relative quantification of Hmga2, Actn3, Notch3 and Cxcl12 expression in G93A HL and diaphragm SCs with or without the 3-day NaBu treatment. RNA samples were collected from 6 rounds of sorting and sorted cells were split into 3 replicates.
Coculture of AAV transduced SC derived myotubes with rat spinal motor neurons in compartmentalized microfluidic chambers. (A)
Schematic representation of XonaChip spatial configuration and coculture experiment timeline. NBPM: Neurobasal Plus Medium; RSMNs: rat spinal motor neurons. (B) Representative composite images of RSMNs cocultured with: 1. WT HL SC-derived myotubes transduced with AAV-CMV-eGFP; 2. G93A HL SC-derived myotubes transduced with AAV-CMV-eGFP; 3. G93A HL SC-derived myotubes transduced with AAV-CMV-Cxcl12-IRES-eGFP; 4. G93A EOM SC-derived myotubes transduced with AAV-CMV-eGFP. Arrows highlight RSMN neurites crossed the microgrooves but stayed close to the edges and grew backward. Also see Figure 8-figure supplement 2A. Boxed regions are enlarged below. Scale bars: 50 μm. (C) Zoom-in views of RSMN neurites innervated or missed the AChR clusters (BTX positive patches). Boxed regions are further enlarged below. Scale bars: 50 μm, 10 μm. (D) Measured lengths of the longest neurites derived from RSMN cells (presumably axons) crossing the 450 μm microgrooves. n = 24, 31, 34,31 cells for the 4 groups, respectively (from 3 rounds of coculture experiments).
Coculture of AAV transduced SC derived myotubes with rat spinal motor neurons within the same compartment. (A)
Representative composite images of RSMNs cocultured with: 1. WT HL SC-derived myotubes transduced with AAV-CMV-eGFP; 2. G93A HL SC-derived myotubes transduced with AAV-CMV-eGFP; 3. G93A HL SC-derived myotubes transduced with AAV-CMV-Cxcl12-IRES-eGFP; 4. G93A EOM SC-derived myotubes transduced with AAV-CMV-eGFP. The timeline was the same as that in Figure 8 but RSMNs were seeded on top of myotubes. Boxed regions are enlarged in (B). Scale bars: 50 μm. (B) Zoom-in views of innervated and non-innervated AChR clusters on myotubes having contact with RSMN neurites. Arrows highlight branching phenomena observed in RSMN neurites innervating AChR clusters. Scale bars: 10 μm. (C) Quantification of innervation ratios of AChR clusters (area > 10 μm2) in myotubes in contact with RSMN neurites. n = 37, 48, 40, 52 z-stack images for the 4 groups, respectively (from 3 rounds of coculture experiments). Also see methods for detailed description. *** P < 0.001; **** P < 0.0001; ns, not significant. (D) Graphic summary of the potential mechanisms contributing to EOM NMJ integrity maintenance during ALS progression mimicked by NaBu treatment.
