Facioscapulohumeral muscular dystrophy (FSHD) is linked to abnormal derepression 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 multiplexed error-robust fluorescent in situ hybridization (MERFISH) 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 myotubes and unfused mononuclear cells (MNCs) of control, isogenic D4Z4 contraction mutant and FSHD patient samples, as well as the individual nuclei within them. We find myocyte nuclei segregate into two clusters defined by the expression of DUX4 target genes, which is exclusively found in patient/mutant nuclei, whereas MNCs cluster based on developmental states. 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 multinucleated FSHD-hi myotubes. Gene coexpression modules related to extracellular matrix and stress gene ontologies are significantly altered in patient/mutant myotubes compared with the control. We also identify distinct subpathways within the DUX4 gene network that may differentially contribute to the disease transcriptomic phenotype. Taken together, our MERFISH-based study provides effective gene network profiling of multinucleated cells and identifies FSHD-induced transcriptomic alterations during myoblast differentiation.

中文翻译：

---

单细胞空间转录组学揭示面肩肱型肌营养不良症中成肌细胞命运发育分叉后的营养不良轨迹


面肩肱型肌营养不良症 (FSHD) 与转录激活因子 DUX4 的异常去抑制有关。这种效应仅限于一小部分细胞，需要进行单细胞分析。然而，单细胞/核RNA-seq无法完全捕获多核大肌管的转录组。为了规避这些问题，我们使用多重抗错荧光原位杂交 (MERFISH) 空间转录组学，可以以亚细胞分辨率对 RNA 转录本进行分析。我们同时检查了 140 个基因的空间分布，包括 24 个直接 DUX4 靶标、体外分化的肌管和对照的未融合单核细胞 (MNC)、同基因 D4Z4 收缩突变体和 FSHD 患者样本，以及其中的单个细胞核。我们发现肌细胞核分为由 DUX4 靶基因表达定义的两个簇，这仅在患者/突变体细胞核中发现，而 MNC 则根据发育状态进行聚类。患者/突变体肌管处于“FSHD-hi”和“FSHD-lo”状态，前者表现为 DUX4 靶标高表达和肌肉基因表达降低。伪时间分析揭示了成肌细胞分化为对照分支和 FSHD-hi 肌管分支的明显分叉，在多核 FSHD-hi 肌管中发现了不同数量的 DUX4 靶标表达细胞核。与对照相比，患者/突变肌管中与细胞外基质和应激基因本体相关的基因共表达模块显着改变。我们还确定了 DUX4 基因网络中可能对疾病转录组表型有不同贡献的不同子通路。 总而言之，我们基于 MERFISH 的研究提供了多核细胞的有效基因网络分析，并鉴定了成肌细胞分化过程中 FSHD 诱导的转录组改变。