Sp8 and Sp6 regulatory functions in the limb bud ectoderm

Sp8 and Sp6 are two closely related Sp genes expressed in the limb ectoderm, where they regulate proximo-distal and dorso-ventral patterning. Genetic studies in mice have shown that they act in a dose-dependent manner, with Sp8 exerting a substantially stronger effect than Sp6. Here, we integrate ChIPmentation-seq and RNA-seq analyses to elucidate their genome-wide regulatory networks and mechanisms of action. Our results show that Sp8 has a dual mode of action either directly binding GC-rich motifs or indirectly binding AT-rich motifs through Dlx. In contrast, Sp6 primarily acts through AT-rich motifs, via interaction with cofactors such as Dlx5. Both factors regulate key components of the Wnt, FGF, and BMP signaling pathways, central to limb patterning, and display both cooperative and independent roles. We further show that Sp8 and Sp6 can form homo- and heterodimers, as well as interact with Dlx5, revealing a complex regulatory network. Our findings provide molecular insight into limb morphogenesis and have potential implications for understanding congenital limb malformations.