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

Abstract

The mechanical challenge of attaching elastic tendons to stiff bones is solved by the formation of a unique transitional tissue. Here, we show that murine tendon-to-bone attachment cells are bi-fated, activating a mixture of chondrocyte and tenocyte transcriptomes, under regulation of shared regulatory elements and Krüppel-like factors (KLFs) transcription factors. High-throughput bulk and single-cell RNA sequencing of humeral attachment cells revealed expression of hundreds of chondrogenic and tenogenic genes, which was validated by in situ hybridization and single-molecule ISH. ATAC sequencing showed that attachment cells share accessible intergenic chromatin areas with either tenocytes or chondrocytes. Epigenomic analysis revealed enhancer signatures for most of these regions. Transgenic mouse enhancer reporter assays verified the shared activity of some of these enhancers. Finally, integrative chromatin and motif analyses and transcriptomic data implicated KLFs as regulators of attachment cells. Indeed, blocking expression of both Klf2 and Klf4 in developing limb mesenchyme impaired their differentiation.

Single‐Cell Integration Analysis of Heterotopic Ossification and Fibrocartilage Developmental Lineage: Endoplasmic Reticulum Stress Effector Xbp1 Transcriptionally Regulates the Notch Signaling Pathway to Mediate Fibrocartilage Differentiation

“…Based on the proposed temporal analysis characteristics of the gene expression in Figure 2(g) , combined with the results of previous studies, the gene expression characteristics of ectopic ossification demonstrated strong similarities to those of the fibrocartilaginous region of the tendon-bone union [ 8 , 66 – 68 ]. Therefore, we hypothesized that ectopically ossified cells have similar transcriptional characteristics to cells in the fibrocartilaginous zone of the tendon-bone junction region.…”

“…The cell differentiation trajectory of GSE168153 is shown in Supplemental Figure 5B . Moreover, these cells were further annotated based on the results of previous studies ( Figure 4(b) ) [ 66 ]. Accordingly, the proposed temporal expression profile of genes associated with the phenotypic cartilaginous region of tendon fibers is shown in Figure 4(c) .…”

Tendon and multiomics: advantages, advances, and opportunities

“…In addition, the junction showed characteristics of both muscle and tendon, but some proteins, for example, Col22A1 were restricted to the junction 15 . Similarly, single-cell analysis of tendon-to-bone attachment or enthesis, showed clear demarcation between tendon and cartilage to cells at the attachment itself 16 . These cells express a mix of both tenocyte and cartilage transcriptomic profiles.…”

“…In addition, this transitional region expresses transcription factors, such as the Krüppel-like factors (KLFs), Lmo1 , and Gli1 , that may be the master regulators for this cell cluster. Furthermore, it was demonstrated that this region is composed of bi-fated cells that accordingly share a mix of accessible chromatin regions 16 .…”

Development and maintenance of tendons and ligaments

“…Loss of Scx or Sox9 disrupts tendon-bone attachment development throughout the skeleton (Blitz et al, 2013;Killian and Thomopoulos, 2016;Roberts et al, 2019;Sugimoto et al, 2013). Genome-wide transcriptomic and chromatin accessibility assays of the limb attachment cells early in their differentiation have shown that APs, as a population, are marked by a hybrid fibroblast-chondrocyte transcriptional profile with active enhancers shared between tenocytes or ligamentocytes and chondrocytes (Kult et al, 2021). This suggests that the graded structure of the enthesis is derived from attachment cells with a hybrid tendon-cartilage or ligament-cartilage cell fate (Fig.…”