Zic3 represses anterior digit development in tetrapods

Pentadactyl limbs represent a conserved morphological feature among tetrapods, with anterior digits considered more important than posterior digits for refined movement. While posterior digit formation is governed by graded expression of the Shh and 5’Hox genes, the regulatory mechanisms underlying anterior digit development, especially digit I (DI), remain poorly defined. This study identified an anterior expression pattern of Zic3 in the limb buds of representative tetrapods, including humans, which exerted an inhibitory effect on skeletal development. Zic3 was highly expressed in the anterior region of limb buds at early developmental stages, with species-specific divergence emerging during later development. Overexpression of Zic3 significantly delayed chondrogenesis and ossification, leading to bone shortening but not loss. Furthermore, RNA sequencing demonstrated that Zic3 down-regulated key genes associated with skeletal development, including Cytl1, Sox9, Ihh, Ptch1, Runx2, and Wnt16. These findings demonstrate that Zic3 acts as a conserved inhibitor of anterior skeletal maturation and contributes to the molecular asymmetry of tetrapod limb development.