Zic3 represses the development of anterior digits in tetrapods

Pentadactyl limbs are the typical limb pattern found in tetrapods, with the anterior digits usually more important than the posterior digits for refined movement. Gradients in the levels of the expression of genes such as Shh and the 5’Hox genes provide the foundation for development of posterior digits. However, the regulatory mechanisms for anterior digit development, especially digit I (DI), remain unclear. Here we show the expression pattern of Zic3 in limb buds of representative tetrapods and demonstrate that Zic3 has an inhibitory function on skeletal development. We found that Zic3 is highly expressed in the anterior region of tetrapod limb buds, including human, at early developmental stages and exhibits differentiated species-specific expression patterns at later stages. Overexpression of Zic3 results in both chondrogenesis and ossification being significantly delayed, resulting in bone shortening but not loss. Furthermore, RNA-seq showed that Zic3 downregulates the expression of genes associated with skeletal development, including Cytl1, Sox9, Ihh, Ptch1, Runx2 and Wnt16. Our results demonstrate diverse expression patterns and the crucial functions of Zic3 in tetrapod limb development. These findings shed light on the development of anterior limbs and complement studies on genes involved in posterior limb development.