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

Shiri Kult,Axel Visel,Marco Osterwalder,Ronnie Blecher-Gonen,Lydia Farack,Elazar Zelzer,Sharon Krief,Tsviya Olender,Tomer-Meir Salame,Dena Leshkowitz,Hadas Keren-Shaul,Svetalana Markman,Shani Ben-Moshe,Ido Amit,Shalev Itzkovitz,Terence D Capellini

doi:10.7554/elife.55361

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.

Highlights

The function of the musculoskeletal system relies on the proper assemblage of its components, namely skeletal tissues, muscles, and tendons
GO analysis of these genes yielded terms relating to regulation of cytokine and IL-12 production, as well as response to laminar fluid shear stress (Figure 1—figure supplement 3C). These results clearly show that the transcriptome of the attachment cells includes a mixture of tenocyte and chondrocyte genes, many of which are involved in ECM organization and developmental processes, in addition to a unique subgroup of genes that are upregulated in these cells
We studied the activity of eight elements, which were selected because they were associated with bona fide marker genes for tenocytes or chondrocytes, and were found to be expressed in the attachment cells

Summary

Introduction

The function of the musculoskeletal system relies on the proper assemblage of its components, namely skeletal tissues (bone, cartilage, and joints), muscles, and tendons. The attachment of tissues composed of materials with large differences in their mechanical properties is highly challenging. Elastic tendons, which have a Young’s modulus (a measure of stiffness) in the order of 200 megapascal, are attached to the much harder bone, with a modulus in the order of 20 gigapascal. This disparity makes the connection between these two tissues a mechanical weak point, which is subject to higher incidence of tearing by both external and internal forces acting on the musculoskeleton during movement.

Objectives

Methods

Results

Conclusion