CEK8/Ephrin-A4 defines the tendon field boundary through WNT-TGF-β crosstalk in the chicken embryo

The development of tendons in vertebrate limbs occurs through the precise regulation of genes and signaling pathways between the skeletal elements and the ectoderm. Although it is known that SCX is a crucial regulator of tendon development, the establishment and maintenance of the tenogenic regions remain unclear. This study explores the role of CEK8 (Ephrin-A4) in early tendon differentiation in the chicken limb and its interaction with WNT/β-catenin and TGF-β pathways during tendon blastema formation. Our results show that Cek8 expression mirrors Scx in the developing tendon blastema but is localized at the boundary of SCX-positive cells. Inhibiting CEK8 upregulates Scx and other tenogenic genes; however, the effects are not sustained, indicating that CEK8 initially suppresses but later supports tenogenic differentiation. Through functional assays using protein-soaked beads in the tendon blastema, we determined that WNT signaling initially upregulates but later inhibits Cek8 expression in a time-dependent manner. TGF-β signaling induces a wider Cek8 expression domain compared to Scx, while CEK8 is necessary for the effects of TGF-β to maintain Scx induction. Inhibiting CEK8 expands the β-catenin and SMAD2/3 regions, favoring a tenogenic phenotype. These results identify CEK8 as a regulator of tendon progenitor architecture, likely involved in establishing positional information, which functions in association with WNT and TGF-β to control tenogenic differentiation both spatially and temporally. Our study provides new insights into the morphogenetic signals and cell interactions that occur during chicken tendon development.