Analysis of cranial tenocyte heterogeneity reveals a role for Wnt signaling in tendon attachments.

Tenocytes secrete the extracellular matrix (ECM) of tendons and ligaments in response to mechanical forces from the muscles and bones to which they attach. Although these tissues are often injured and weaken with age, we know little about the genetic mechanisms controlling their development or maintenance. Through single-cell RNA sequencing (scRNA-seq) of connective tissues in the embryonic zebrafish head, we identify distinct subpopulations of developing tenocytes and ligamentocytes. Spatially distinct transcriptional cell signatures, particularly for ECM genes, correlate with the type of tendon/ligament (i.e. longer load-bearing skeletal attachments versus soft tissue attachments) as well as tenocyte locations within tendons (i.e. skeletal entheses versus myotendinous junctions). Combinatorial in situ analyses confirm spatial co-expression of genes defining many of these subsets of tendon or ligament cells. From pathway analysis, the scRNA-seq data also suggest a role for canonical Wnt signaling in tenocyte development. Genetic and pharmacological Wnt manipulations alter tenocyte aggregation and cause ectopic cranial muscle attachments. These findings reveal previously unappreciated spatial and functional heterogeneity in tenocytes during embryogenesis and define a role for Wnt signaling in attachment patterning and morphogenesis.