En1 and Wnt7a interact with Dkk1 during limb development in the mouse

Wnt signaling plays an essential role in induction and development of the limb. Missing digits are one consequence of the reduced Writ signaling in Wnt7a null mice, while extra digits result from excess Wnt signaling in mice null for the Writ antagonist Dkk1. The extra digits and expanded apical ectodermal ridge (AER) of Dkk1-deficient mice closely resemble En1 null mice. To evaluate the in vivo interaction between En] and the canonical Writ signaling pathway, we generated double and triple mutants combining the hypomorphic doubleridge allele of Dkk1 with null alleles of En I and Wnt7a. Reducing Dkk1 expression in Dkk1(d/+) Wnt7a(-/-) double mutants prevented digit loss, indicating that Wnt7a acts through the canonical pathway during limb development. Reducing Dkk1 levels in Dkk](d/d)En1(-/-) double mutants resulted in severe phenotypes not seen in either single mutant, including fused bones in the autopod, extensive defects of the zeugopod, and loss of the ischial bone. The subsequent elimination of Wnt7a in Dkk1(d/d) En1(-/-) Wnt7a(-/-) triple mutants resulted in correction of most, but not all, of these defects. The failure of Wnt7a inactivation to completely correct the limb defects of Dkk1(d/d) En1(-/-) double mutants indicates that Wnt7a is not the only gene regulated by En1 during development of the mouse limb. (C) 2004 Elsevier Inc. All rights reserved.