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.面肩肱型肌营养不良症（Facioscapulohumeral Muscular Dystrophy, FSHD）与转录激活因子DUX4（transcription activator DUX4）的异常去抑制密切相关。该效应仅在少数细胞中发生，因此需开展单细胞分析。然而，单细胞/单细胞核RNA测序（single cell/nucleus RNA-seq）无法完全捕获多核大型肌管（multinucleated large myotubes）的转录组。为规避上述局限，本研究采用了多重误差稳健荧光原位杂交（Multiplexed Error Robust Fluorescent In Situ Hybridization, MERFISH）空间转录组学（spatial transcriptomics）技术，可实现亚细胞分辨率（subcellular resolution）下的RNA转录本表达谱分析。我们同时对体外分化的对照样本、同基因D4Z4收缩突变体（isogenic D4Z4 contraction mutant）及1型FSHD患者的肌管与未融合单核细胞（unfused mononuclear cells, MNCs），以及其中的单个细胞核，开展了140个基因的空间分布分析，其中包含24个DUX4直接靶基因。研究发现，肌细胞核可根据DUX4靶基因（DUX4 target genes）的表达情况分为两个转录簇，该特征仅存在于患者/突变体来源的细胞核中；而未融合单核细胞则依据发育状态（developmental state）进行聚类。患者/突变体肌管可分为"FSHD高表达（FSHD-hi）"与"FSHD低表达（FSHD-lo）"两种状态，前者以DUX4靶基因高表达及肌肉基因表达下调为特征。拟时间分析（Pseudotime analyses）显示，成肌细胞分化（myoblast differentiation）存在明显的分支：一条为对照肌管分支，另一条为FSHD高表达肌管分支；多核的FSHD高表达肌管中，表达DUX4靶基因的细胞核数量不等。与对照组相比，患者/突变体肌管中与细胞外基质（extracellular matrix）及应激基因本体论（stress gene ontologies）相关的基因共表达模块（gene co-expression modules）发生了显著改变。我们还鉴定出DUX4基因网络内的不同亚通路，它们对疾病表型存在差异化贡献。综上，本研究基于MERFISH的分析实现了多核细胞的高效基因网络表达谱分析，并首次在成肌细胞分化过程中特异性鉴定出FSHD诱导的转录组改变。