In a second approach to understand the pathogenic mechanisms, we asked whether the pattern of Zic1 expression was consistent with a specific role in coronal suture biogenesis. We found a previously undescribed, transient zone of Zic1 expression in the supraorbital region at E11.5 (Figure 4) that is spatially and temporally overlapping with that of En1 (Figure 4) and is consistent with an early instructive role for Zic1 in the supraorbital regulatory center; combining these observations with the finding of increased en-2 expression driven by mutant ZIC1 constructs in the Xenopus experiments, we propose that the coronal synostosis phenotype associated with the human mutations might be attributable to alteration of EN1 expression in the supraorbital regulatory center, thus disrupting the patterning of the coronal suture at a very early stage in its development. The induction of engrailed expression by ZIC1 orthologs is well characterized in both Drosophila15,46 and Xenopus38 and is thought to act through the Wnt signaling pathway.27 A further component of this signaling network is likely to be Lmx1b, which encodes a LIM-homeodomain protein and is upregulated in early neural crest of Xenopus.47 In the mouse, Lmx1b is prominently expressed in the supraorbital region at E11.5, and homozygous mutants have severely abnormal cranial sutures.48 In humans, heterozygous mutations in LMX1B usually cause nail-patella syndrome (MIM: 161200),49 but a specific missense mutation in the N-terminal arm of the homeodomain has been associated with craniosynostosis.1