GSE160090
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. Overall design: 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.