Sp6 and Sp8 transcription factors are necessary mediators of Wnt/ ß Catenin function in the limb ectoderm

The apical ectodermal ridge (AER) is a specialized epithelium located at the distal dorso-ventral (DV) rim of the developing limb that is crucial for limb bud development. The induction of the AER is a complex process that relies on intricate interactions among the FGF, WNT, and BMP signaling pathways operating within the ectoderm and between the ectoderm and mesoderm of the early limb bud. Furthermore, induction of the AER is linked to the establishment of DV patterning. Sp6 and Sp8 are two members of the Specificity Protein family of transcription factors that are expressed in the limb bud ectoderm and function downstream of WNT/ β Catenin signaling and upstream of Fgf8. Their individual genetic inactivations result in a mild syndactyly phenotype for Sp6 and limb truncation, due to the premature regression of the AER, for Sp8. To investigate a possible functional redundancy between Sp6 and Sp8, we generated double Sp6;Sp8 null mutants. We also generated Sp6-null; Sp8-conditional mutants using a Sp8 floxed allele with the Ap2-Cre and with the Msx2-Cre deleter lines. Our results show that double Sp6;Sp8 mutants are tetra-amelic. Initial budding occurs, but Fgf8 and Bmp4 are not activated in the limb ectoderm and the dorsal marker Wnt7a persists throughout the limb bud ectoderm. The phenotype of mutants bearing a single functional copy of Sp6 (Sp6 +/-;Sp8-/-) is indistinguishable from that of the double mutants, whereas the presence of a single functional allele of Sp8 (Sp6-/-;Sp8+/-) results in a Split Hand Foot Malformation phenotype. We conclude that Sp6 and Sp8 work in a redundant manner as indispensable mediators of WNT/ β Catenin signaling in the limb ectoderm and that their function links the Proximo-distal and DV axes.