Single-Cell Spatial Transcriptomics Reveals a Dystrophic Trajectory Following a Developmental Bifurcation of FSHD Myoblast Cell Fates
Facioscapulohumeral Muscular Dystrophy (FSHD) is linked to abnormal de-repression of the transcription activator DUX4. This effect is localized to a low percentage of cells, requiring single cell analysis. However, single cell/nucleus RNA-seq cannot fully capture the transcriptome of multinucleated large myotubes. To circumvent these issues, we use MERFISH (Multiplexed Error Robust Fluorescent In Situ Hybridization) spatial transcriptomics that allows profiling of RNA transcripts at a subcellular resolution. We simultaneously examined spatial distributions of 140 genes, including 24 direct DUX4 targets, in in vitro differentiated control, isogenic D4Z4 contraction mutant and FSHD1 patient myotubes and unfused mononuclear cells (MNCs), as well as the individual nuclei within them. We find myocyte nuclei segregate into 2 clusters defined by expression of DUX4 target genes, which is exclusively found in patient/mutant nuclei, while MNCs cluster based on developmental state. Patient/mutant myotubes are found in “FSHD-hi” and “FSHD-lo” states with the former signified by high DUX4 target expression and decreased muscle gene expression. Pseudotime analyses reveal a clear bifurcation of myoblast differentiation into control and FSHD-hi myotube branches, with variable numbers of DUX4 target expressing nuclei found in multi-nucleated FSHD-hi myotubes. Gene co-expression modules related to extracellular matrix and stress gene ontologies are significantly altered in patient/mutant myotubes compared to control. We also identify distinct sub-pathways within the DUX4 gene network that differentially contribute to the disease phenotype. Taken together, our MERFISH-based study provides effective gene network profiling of multinucleated cells and uniquely identifies FSHD-induced transcriptomic alterations within myoblast differentiation.