Developmental regulation of cranial tendon fibroblast diversity and ECM interactions

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Project Summary/Abstract Tendons and ligaments are fundamental components of a functional musculoskeletal system. Tendons attach muscles to bone and interact with these tissues at distinct attachment sites called entheses (bone) and myotendinous junctions (MTJs, muscle). These interactions cause unique changes in gene expression and extracellular matrix (ECM) production that allow tendons to bear the forces exerted by muscle contraction. Transcription factors such as Scleraxis (Scx) specify early tendon progenitor cells (TPCs) and regulate ECM production. We previously discovered a critical ECM scaffolding protein called Thrombospondin-4b (Tsp4b) in zebrafish that is regulated by Scx, required for tendon maintenance and conserved in human tendons. How different types of tendon fibroblasts (tenocytes) are specified and influence ECM assembly at entheses or MTJs remains unclear. The current proposal addresses these issues using the advantages of the zebrafish for single cell RNA sequencing, in vivo imaging and genetic manipulation. The long-term goal of the proposed research is to understand the spatial dynamics of gene regulation and ECM assembly in tendons/ligaments and their regulation by mechanical force. Three primary hypotheses guide the research: 1) distinct subtypes of TPCs develop at entheses and MTJs in response to force, 2) ECM secreted by tenocytes regulates force-dependent signals that alter these distinct modes of gene expression in tenocytes and 3) retinoic acid is a novel force-dependent signal controlling tendon development. Aim 1 will perform scRNA-seq in tenocytes and see how force alters gene expression profiles. Aim 2 will study roles for Tsp4b, ECM, and TGF-beta signaling in force-dependent gene expression in tendons. Aim 3 will study the roles of RA signaling in tendons, and its responses to mechanical load. Each aim combines novel single cell approaches, genetic manipulation, live imaging and quantitative methods for physiological stimulation of muscles to get at mechanisms of tendon cell specification and ECM assembly in response to force.