Bi-fated tendon-to-bone attachment cells are regulated by shared enhancers and KLF transcription factors

The connection between different tissues is vital for the development and function of all organs and systems. In the musculoskeletal system, the attachment of elastic tendons to stiff bones poses a mechanical challenge that is solved by the formation of a transitional tissue, which allows the transfer of muscle forces to the skeleton without tearing. Here, we show that tendon-to-bone attachment cells are bi-fated, activating a mixture of chondrocyte and tenocyte transcriptomes, which is regulated by sharing regulatory elements with these cells and by Krüppel-like factors transcription factors (KLF). To uncover the molecular identity of attachment cells, we first applied high-throughput bulk and single-cell RNA sequencing to murine humeral attachment cells. The results, which were validated by in situ and single-molecule in situ hybridization, reveal that together with the expression of unique sets of genes, the attachment cells express hundreds of chondrogenic and tenogenic genes. In search for the underlying mechanism, we performed ATAC sequencing and found that attachment cells share a significant fraction of accessible intergenic chromatin areas with either tenocytes or chondrocytes. Epigenomic analysis further revealed transcriptional enhancer signatures for the majority of these regions. We then examined a subset of these regions using transgenic mouse enhancer reporter assay. Results verified the shared activity of some of these enhancers, supporting the possibility that the transcriptome of attachment cells is regulated by enhancers with shared activities in tenocytes or chondrocytes. Finally, integrative chromatin and motif analyses, as well as the transcriptome data, indicated that KLFs are regulators of attachment cells. Indeed, blocking the expression of Klf2 and Klf4 in the developing limb mesenchyme led to abnormal differentiation of attachment cells, establishing these factors as key regulators of the fate of these cells. In summary, our findings show how the molecular identity of bi-fated attachment cells enables the formation of the unique transitional tissue that connects tendon to bone. More broadly, we show how mixing the transcriptomes of two cell types through shared enhancers and a dedicated set of transcription factors can lead to the formation of a new cell fate that connects them. Sox9+/Scx+ FACS-sorted attachment progenitors from E13.5 dissected forelimbs (proximal side). The cells were genetically co-labeled by Sox9-CreERT2;Rosa26-tdTomato+;ScxGFP+ and were prepared for 10x Chromium single-cell RNA-Seq.

不同组织间的连接对于所有器官和系统的发育与功能均至关重要。在肌肉骨骼系统中，弹性肌腱与坚硬骨骼的附着面临力学挑战，而过渡组织（transitional tissue）的形成可解决这一难题，使肌肉力量能够在不发生撕裂的情况下传递至骨骼。本研究发现，肌腱-骨附着细胞具有双分化命运，可同时激活软骨细胞（chondrocyte）与腱细胞（tenocyte）的混合转录组表达模式，这一过程通过与这两类细胞共享调控元件（regulatory elements），并受Krüppel样因子转录因子（Krüppel-like factors transcription factors, KLF）调控实现。为揭示附着细胞的分子特征，我们首先对小鼠肱骨附着细胞开展了高通量批量RNA测序（high-throughput bulk RNA sequencing）与单细胞RNA测序（single-cell RNA sequencing）。经原位杂交（in situ hybridization）与单分子原位杂交（single-molecule in situ hybridization）验证的研究结果显示：附着细胞除表达特异性基因集之外，还可表达数百个成软骨相关与腱系相关基因。为探究背后的调控机制，我们进行了ATAC测序（ATAC sequencing），结果发现附着细胞与腱细胞或软骨细胞共享大量可及的基因间染色质区域。表观基因组分析（epigenomic analysis）进一步揭示，上述区域中的绝大多数均带有转录增强子（transcriptional enhancer）特征。随后，我们通过转基因小鼠增强子报告基因实验（transgenic mouse enhancer reporter assay）对其中部分区域进行了验证，结果证实部分增强子存在共享活性，这支持了“附着细胞的转录组由在腱细胞或软骨细胞中具有活性的共享增强子调控”这一观点。最后，整合染色质与基序分析（motif analyses）结果，并结合转录组数据，我们发现KLF家族是附着细胞的调控因子。实验证实，在发育中的肢间充质（limb mesenchyme）中阻断Klf2与Klf4的表达会导致附着细胞分化异常，由此确定这两类因子是该细胞命运的关键调控者。综上，本研究揭示了具有双分化命运的附着细胞如何通过其分子特征，形成连接肌腱与骨骼的独特过渡组织。从更广泛的视角来看，本研究证实，通过共享增强子与一组特异性转录因子混合两类细胞的转录组，可催生连接二者的全新细胞命运。本研究使用的样本为从E13.5解剖分离的前肢（近端侧）中通过荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）获得的Sox9+/Scx+附着祖细胞。这些细胞经Sox9-CreERT2;Rosa26-tdTomato+;ScxGFP+进行遗传共标记，并制备用于10x Chromium单细胞RNA测序。