Journal: bioRxiv

Article Title: c-MAF transduces motor neuron firing to sustain fast-glycolytic myofibers and neuromuscular junctions

doi: 10.64898/2026.02.05.703983

Figure Lengend Snippet: a , weighted nearest neighbor (WNN) UMAP integrating snATAC-seq and snRNA-seq from tibialis anterior muscle 7 days post-denervation. Left panel colored by cluster identity shows major populations. Right panel colored by experimental group shows control (CTL, red) versus denervated (D, blue) b , Dot plot of marker gene expression across cell clusters. Y-axis lists 15 clusters; X-axis displays marker genes. Dot size indicates percent expression; color intensity (purple gradient) shows average expression level. Validates cluster identities through fiber type-specific Myh expression and cell type-specific markers. c , Coverage plots showing chromatin accessibility at f Myh locus for Myh4 /IIb, Myh2 /IIa, and Myh1 /IIx in control (CTL) and denervated (D) TA, with at the right violin plots showing the expression level of the corresponding gene. Below, in black each gene is represented. d , UMAP subset focusing on type IIb myonuclei. Left (CTL) shows control IIb nuclei right (D) shows resilient denervated IIb nuclei that retain spatial clustering with control population (D_IIb_res), suggesting maintenance of transcriptional/chromatin accessibility identity despite denervation. e , Coverage plots comparing Myh1 and Myh4 loci in resilient denervated population. Cyan tracks showing denervated nuclei; red tracks showing control nuclei. Violin plots (right) indicate gene expression. Myh1 and Myh4 genes are presented below corresponding the coverage plots. f , Venn diagram comparing differentially expressed genes between IIb denervated nuclei which cluster with the control (resilient myonuclei, 305 genes, orange) and those (non-resilient, 725 genes, green) that do not cluster with the control. Overlap shows 268 shared dysregulated genes; 37 unique to resilient, 457 unique to non-resilient.

Article Snippet: For cryosections, antibodies included MYH2 (1:100, SC-71 clone, DSHB), MYH4 (1:200, BF-F3 clone, DSHB), MYH1 (1:100, 6H1 clone, DSHB), MYH7 (1:40, BA-F8, DSHB), Laminin (1:750, L9393, Sigma), FOXO3A (1:400, 75D8, Cell Signaling), and c-MAF (1:100, HPA028289, Sigma-Aldrich).

Techniques: Control, Marker, Gene Expression, Expressing

Journal: bioRxiv

Article Title: c-MAF transduces motor neuron firing to sustain fast-glycolytic myofibers and neuromuscular junctions

doi: 10.64898/2026.02.05.703983

Figure Lengend Snippet: a , UMAP visualization of single-nucleus RNA-seq from plantaris muscle 7 days post-tenotomy (mechanical unloading) and sham-operated controls. Cells colored by cluster identity reveal 11 distinct populations: IIb (orange, type IIb/ Myh4 myonuclei), IIx (red, type IIx/ Myh1 myonuclei), FAPs (yellow, fibro-adipogenic progenitors), IIa (green, type IIa/ Myh2 myonuclei), Macrophages (teal), FAPS- Dpp4 + (cyan), Neuromuscular cells (blue), Tenocytes (light blue), Endothelial (purple), MuSC (magenta, muscle stem cells), Schwann cells (pink), MTJ (pink, myotendinous junction). Myonuclei clusters segregate by Myh isoform expression demonstrating fiber type-specific transcriptional states. b , Same UMAP colored by experimental condition using group.by function. Cyan indicates Ctrl-Pltr (control plantaris); pink indicates T-Pltr (tenotomized plantaris). Both conditions distribute across all clusters, enabling paired comparison of tenotomy effects within each cell type. c , Venn diagram analysis of Ingenuity Pathway Analysis (IPA) upstream regulators (URs). Left: Overlap between Tenotomy (57 unique URs) and Denervation (212 unique URs) showing 31 shared regulators, indicating partially overlapping stress responses. Right: Triple Venn diagram among the 212 unique denervation URs showing fiber type-specific ones: type IIa (82 unique), IIb (45 unique), and IIx (36 unique) myonuclei. Central overlaps show shared regulators: 5 (IIa/IIb), 12 (all three), 12 (IIb/IIx), and 20 (IIa/IIx). d , Table of IIb-specific upstream regulators showing z-scores and p-values across fiber types (IIa, IIb, IIx). Columns display regulator name, activation z-score, and statistical significance for each subtype. Red cells indicate activated pathways; blue indicates inhibited pathways. Red rectangles highlight two key regulators: KAT2B (Lysine acetyltransferase) showing its specific inhibition in IIb fibers, and HDAC5 (class IIa histone deacetylase) showing its specific activation. This inverse regulation suggests coordinated chromatin remodeling where HDAC5 activation and KAT2B inhibition converge to alter acetylation landscapes during denervation of fast glycolytic IIb myofibers.

Article Snippet: For cryosections, antibodies included MYH2 (1:100, SC-71 clone, DSHB), MYH4 (1:200, BF-F3 clone, DSHB), MYH1 (1:100, 6H1 clone, DSHB), MYH7 (1:40, BA-F8, DSHB), Laminin (1:750, L9393, Sigma), FOXO3A (1:400, 75D8, Cell Signaling), and c-MAF (1:100, HPA028289, Sigma-Aldrich).

Techniques: RNA Sequencing, Expressing, Control, Comparison, Activation Assay, Inhibition, Histone Deacetylase Assay

Journal: bioRxiv

Article Title: c-MAF transduces motor neuron firing to sustain fast-glycolytic myofibers and neuromuscular junctions

doi: 10.64898/2026.02.05.703983

Figure Lengend Snippet: a , Immunofluorescence of distal hindlimb cryosections showing MYH isoform distribution and Laminin (white, fiber boundaries). Top rows: MYH1 (cyan, type IIx), MYH2 (green, type IIa), MYH7 (red, type I). Bottom rows: MYH4 (cyan, type IIb), MYH2 (green), MYH7 (red). Columns show: Control HSA-Cre + (leftmost), myofiber constitutive HSA-Cre;c-Maf flox/flox , myofiber inducible HSA-CreERT2;c-Maf flox/flox at 7 days, 15 days, and 30 days post-tamoxifen, Region A deleted (f Myh super enhancer deletion, EnhA -/- ), and ALS 90 days old. Major muscles labeled in control: TA (tibialis anterior), Tibia, EDL (extensor digitorum longus), FHL-TP (flexor hallucis longus-tibialis posterior), Sol (soleus), Plant (plantaris), Gas (gastrocnemius). c-Maf loss shows fiber type transition in peroneal group muscles (FHL-TP, central TA) with preserved EDL. ALS models displayed similar regional fiber type switch. b , RT-qPCR of fast-glycolytic genes in inducible c-Maf mutant 7 and 15 dpi of Tamoxifen. Bottom: Myh4 and Myh1 showing temporal changes. c-Maf loss reduces Myh4 expression while delayed Myh1 increase. Top shows Pvalb (parvalbumin), Atp2a1 (SERCA1), and Mybpc2 (fast MyBP-C) downregulation. White bars: control (CTL); pink bars: 7 dpi; red bars: 15 dpi. *p < 0.05 by one-way ANOVA with individual data points. c , RT-qPCR at 4 weeks post-tamoxifen comparing circadian timepoints ZT03 and ZT15. Left: Myh4 expression is reduced only at ZT03 (red bars) versus controls (white bars). Right: Myh1 upregulation in KO at ZT03. *p < 0.05, **p < 0.01 showing sustained fiber type shift. d , RT-qPCR on 90 days old ALS G93A mice comparing circadian timepoints ZT03 and ZT15. Top c-Maf expression sharply down regulated (grey bars) versus control (white bars). Middle left: Myh4 expression is constitutively reduced versus controls. Middle right: Myh1 is upregulated specifically at ZT03. Bottom left Myh4 expression only in 90 days old ALS mice is upregulated at ZT15. Bottom right same with Myh1. e, RNA-FISH on isolated TA fibers at ZT03 and ZT15, 4 weeks post-tamoxifen, targeting pre-mRNA of Myh4 (red), Myh1 (yellow), Myh2 (green). DAPI (blue) marks myonuclei. Yellow box shows high-magnification view of transcription foci shown in panel f . g, c-Maf mutant myofibers show increased myonuclei expressing non-dominant Myh isoforms, more pronounced at ZT03 than ZT15, indicating spatio-temporal discoordination of the fMyh locus. Violin plots, white color control, red c-Maf mutant. The % of heterogeneity is the % of f Myh allele that differs from the dominant f Myh allele per myofiber as estimated by Rnascope experiments. *p < 0.05, ***p < 0.001 ****p < 0.0001 by one-way ANOVA.

Article Snippet: For cryosections, antibodies included MYH2 (1:100, SC-71 clone, DSHB), MYH4 (1:200, BF-F3 clone, DSHB), MYH1 (1:100, 6H1 clone, DSHB), MYH7 (1:40, BA-F8, DSHB), Laminin (1:750, L9393, Sigma), FOXO3A (1:400, 75D8, Cell Signaling), and c-MAF (1:100, HPA028289, Sigma-Aldrich).

Techniques: Immunofluorescence, Control, Muscles, Labeling, Quantitative RT-PCR, Mutagenesis, Expressing, Isolation, RNAscope

Journal: bioRxiv

Article Title: c-MAF transduces motor neuron firing to sustain fast-glycolytic myofibers and neuromuscular junctions

doi: 10.64898/2026.02.05.703983

Figure Lengend Snippet: a, Quantification of fiber type composition in tibialis anterior (TA) muscle from c-Maf mutant mice generated using constitutive HSA-Cre (Top) or inducible HSA-CreERT2 (Bottom), assessed by Myosin heavy chain (MYH) isoform immunostaining. Wild-type (black bars) and c- Maf knockout (white/gray bars) muscles show significant reduction in type IIb (MYH4) fiber content with compensatory increases in type IIx (MYH1) and type IIa (MYH2) fibers following c- Maf deletion. b, Cross-sectional area (CSA µm 2 ) measurements of individual fiber types in TA (top) and EDL (bottom) muscles from HSA-Cre (left) and HSA-CreERT2 (right) mutant mice. Selective atrophy of MYH4-expressing type IIb fibers is observed exclusively in TA muscle, with no CSA changes detected in EDL, revealing region-specific vulnerability to c-Maf loss despite comparable fiber type composition. Note that the CSA of MYH4 myofibers is around 3000 µm 2 in the TA, while of 1200 µm 2 in EDL.

Article Snippet: For cryosections, antibodies included MYH2 (1:100, SC-71 clone, DSHB), MYH4 (1:200, BF-F3 clone, DSHB), MYH1 (1:100, 6H1 clone, DSHB), MYH7 (1:40, BA-F8, DSHB), Laminin (1:750, L9393, Sigma), FOXO3A (1:400, 75D8, Cell Signaling), and c-MAF (1:100, HPA028289, Sigma-Aldrich).

Techniques: Mutagenesis, Generated, Immunostaining, Knock-Out, Muscles, Expressing

Journal: bioRxiv

Article Title: c-MAF transduces motor neuron firing to sustain fast-glycolytic myofibers and neuromuscular junctions

doi: 10.64898/2026.02.05.703983

Figure Lengend Snippet: a , Immunofluorescence of distal hindlimb cryosections comparing muscle-specific large Maf family mutant models ( HSA-Cre;c-Maf flox/flox , HSA-Cre;Mafb flox/flox and Mafa null mice). Four panels showing transverse sections from: wild-type (WT), c-Maf single mutant, Mafa single mutant, and Mafb single mutant mice. Myosin heavy chain (MYH) isoforms visualized by immunostaining: MYH7 (blue, slow oxidative), MYH2 (red, fast oxidative), MYH1 (white, fast oxidative-glycolytic), MYH4 (green, fast glycolytic), and Laminin (magenta, basal lamina marking fiber boundaries). White arrows indicating flexor hallucis longus (FHL) and tibialis posterior (TP) muscles showing region-specific fiber type transition in c-Maf mutant. b , RT-qPCR quantification of genes specifically dysregulated in c-Maf mutant compared to Mafb and Mafa mutants. Seven bar graphs show relative mRNA expression for: Klk1b26 , Prkaa2 , Fbxo32 , Foxo3 , Fkbp5 , Fbxo31 , and Eif4e . X-axis categories: CTL (control), total Mafa mutant, muscle specific Mafb mutant, muscle specific c-Maf mutant . Statistical comparisons by one-way ANOVA with post-hoc testing. Asterisks indicate significance: *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: For cryosections, antibodies included MYH2 (1:100, SC-71 clone, DSHB), MYH4 (1:200, BF-F3 clone, DSHB), MYH1 (1:100, 6H1 clone, DSHB), MYH7 (1:40, BA-F8, DSHB), Laminin (1:750, L9393, Sigma), FOXO3A (1:400, 75D8, Cell Signaling), and c-MAF (1:100, HPA028289, Sigma-Aldrich).

Techniques: Immunofluorescence, Mutagenesis, Immunostaining, Muscles, Quantitative RT-PCR, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Multiple Defects in Muscle Regeneration in the HSALR Mouse Model of RNA Toxicity

doi: 10.3390/ijms262210985

Figure Lengend Snippet: TA muscle in HSALR at 28 days post injury. ( A ) Representative image of H&E-stained TA muscle sections 28 days post injury (dpi). Scale bars =Scale bars = 50 μm. ( B ) The percentage of regenerated fibers with central nuclei (CN = 1 ( p = 4.5507 × 10 −5 ), CN = 2 ( p = 0.000563), and CN ≥ 3 ( p = 0.0012845)). n = 5 mice per group; n = 1100 fibers for WT and 1206 fibers for HSALR. ( C ) Quantification shows a similar level of MuSCs per 10× field in TA of HSALR compared to WT mice at 28 days post injury. n = 5 mice/group. At least five fields are counted per mouse. ( D , E ) Quantitative RT-PCR also shows no significant change in the expression of Col1a1 and Col3a1 mRNA in TA of HSALR mice compared to wildtype mice at 28 days post injury. ns-not significant. n = 5 mice per group; Student’s t -test; error bars are mean ± SEM. ( F ) MYH2 IF (Red) and MYH4 IF (green) in TA muscle sections 28 days post injury (BaCl 2 ) show an increased number of MYH2 fibers in HSALR mice compared to WT mice. There was no significant difference in the expression of MYH2 and MYH4 in non-injured muscle (saline) from the same HSALR and WT mice. Nuclei are stained with DAPI (blue). Scale bars = 100 μm. ( G ) Quantitative RT-PCR shows a significant increase in Myh2 mRNA expression in TA of HSALR mice during regeneration (0–28 days post injury) compared to wildtype mice. ( H ) Quantitative RT-PCR shows a significant decrease in Myh4 mRNA expression in skeletal muscle (TA) of HSALR mice during regeneration (0–28 days post injury) compared to wildtype mice. ns-not significant. n = 5 mice per group; * p < 0.05, ** p < 0.01; *** p < 0.001, Student’s t -test; error bars are mean ± SEM.

Article Snippet: Primary antibodies were anti-PAX7 (1:20, PAX7-c, DSHB), anti-MYH3 (1:200, clone F1.652, DSHB), anti-MYH2(1:20, SC-71-c, DSHB), anti-MYH4 (1:10, BF-F3-c, DSHB), anti-MYH7 (1:20, BA-D5 (DSHB), anti-laminin (1:1000, catalog L9393, Sigma-Aldrich), and anti-MyoD rabbit polyclonal (1:200, NBP1-54153, Novus, Chesterfield, MO, USA).

Techniques: Staining, Quantitative RT-PCR, Expressing, Saline

Journal: International Journal of Molecular Sciences

Article Title: Multiple Defects in Muscle Regeneration in the HSALR Mouse Model of RNA Toxicity

doi: 10.3390/ijms262210985

Figure Lengend Snippet: HSALR muscle shows fiber type switching at 28 days post injury in 2-month-old mice. ( A ) Representative image of H&E-stained TA muscle sections at 28 days post injury (dpi). Scale bars are shown. ( B , C ) MYH2 IF (Red), and MYH4 IF (green) in TA sections from uninjured TA (saline) ( B ) and 28 days post injury (28 dpi). ( C ) show an increased proportion of MYH2 fibers in the regenerated TA muscles of HSALR mice as compared to WT mice. Nuclei are stained with DAPI (blue). Scale bars are shown.

Article Snippet: Primary antibodies were anti-PAX7 (1:20, PAX7-c, DSHB), anti-MYH3 (1:200, clone F1.652, DSHB), anti-MYH2(1:20, SC-71-c, DSHB), anti-MYH4 (1:10, BF-F3-c, DSHB), anti-MYH7 (1:20, BA-D5 (DSHB), anti-laminin (1:1000, catalog L9393, Sigma-Aldrich), and anti-MyoD rabbit polyclonal (1:200, NBP1-54153, Novus, Chesterfield, MO, USA).

Techniques: Staining, Saline, Muscles

Journal: International Journal of Molecular Sciences

Article Title: Multiple Defects in Muscle Regeneration in the HSALR Mouse Model of RNA Toxicity

doi: 10.3390/ijms262210985

Figure Lengend Snippet: Representative images of immunofluorescence staining for MHC type 1 (red), type 2A (green), and type IIX (no staining) from ( A ) DM1 and ( B ) DM2 skeletal muscles. The Type 1 (MYH7) fibers are stained with antibody BA-D5 (DSHB), and the Type 2A (MYH2) fibers are stained with antibody SC-71 (DSHB). Nuclei are stained with DAPI (blue). Scale bars are shown.

Article Snippet: Primary antibodies were anti-PAX7 (1:20, PAX7-c, DSHB), anti-MYH3 (1:200, clone F1.652, DSHB), anti-MYH2(1:20, SC-71-c, DSHB), anti-MYH4 (1:10, BF-F3-c, DSHB), anti-MYH7 (1:20, BA-D5 (DSHB), anti-laminin (1:1000, catalog L9393, Sigma-Aldrich), and anti-MyoD rabbit polyclonal (1:200, NBP1-54153, Novus, Chesterfield, MO, USA).

Techniques: Immunofluorescence, Staining, Muscles

Journal: International Journal of Molecular Sciences

Article Title: Multiple Defects in Muscle Regeneration in the HSALR Mouse Model of RNA Toxicity

doi: 10.3390/ijms262210985

Figure Lengend Snippet: Immunofluorescence staining of muscle fiber types using an anti-MYH2 antibody (red) for type IIA and an anti-MYH4 antibody for type IIB (green) in the tibialis anterior (TA) muscle sections of different mouse models under non-damaged conditions (saline). Nuclei are stained with DAPI (blue). Scale bars = 200 μm.

Article Snippet: Primary antibodies were anti-PAX7 (1:20, PAX7-c, DSHB), anti-MYH3 (1:200, clone F1.652, DSHB), anti-MYH2(1:20, SC-71-c, DSHB), anti-MYH4 (1:10, BF-F3-c, DSHB), anti-MYH7 (1:20, BA-D5 (DSHB), anti-laminin (1:1000, catalog L9393, Sigma-Aldrich), and anti-MyoD rabbit polyclonal (1:200, NBP1-54153, Novus, Chesterfield, MO, USA).

Techniques: Immunofluorescence, Staining, Saline

Journal: International Journal of Molecular Sciences

Article Title: Multiple Defects in Muscle Regeneration in the HSALR Mouse Model of RNA Toxicity

doi: 10.3390/ijms262210985

Figure Lengend Snippet: Immunofluorescence staining of muscle fiber types using an anti-MYH2 antibody (red) for type IIA and an anti-MYH4 antibody for type IIB (green) in tibialis anterior (TA) muscle sections 28 days after barium chloride (BaCl 2 ) injury of different mouse models. Nuclei are stained with DAPI (blue). Scale bars = 200 μm.

Article Snippet: Primary antibodies were anti-PAX7 (1:20, PAX7-c, DSHB), anti-MYH3 (1:200, clone F1.652, DSHB), anti-MYH2(1:20, SC-71-c, DSHB), anti-MYH4 (1:10, BF-F3-c, DSHB), anti-MYH7 (1:20, BA-D5 (DSHB), anti-laminin (1:1000, catalog L9393, Sigma-Aldrich), and anti-MyoD rabbit polyclonal (1:200, NBP1-54153, Novus, Chesterfield, MO, USA).

Techniques: Immunofluorescence, Staining