Structure-function analysis of the EGF-CFC family member Cripto identifies residues essential for nodal signalling

Cripto is the founding member of the family of EGF-CFC genes, a class of extracellular factors essential for early vertebrate development. In this study we show that injection of Cripto recombinant protein in mid to late zebrafish Maternal-Zygotic one-eyed pinhead (MZoep) blastulae was able to fully rescue the mutant phenotype, thus providing the first direct evidence that Cripto activity can be added extracellularly to recover oep-encoded function in zebrafish early embryos. Moreover, 15 point mutations and two deletion mutants were generated to assess in vivo their functional relevance by comparing the ability of cripto wild-type and mutant RNAs to rescue the zebrafish MZoep mutant. From this study we concluded that the EGF-CFC domain is sufficient for Cripto biological activity and identified ten point mutations with a functional defective phenotype, two of which, located in the EGF-like domain, correspond to loss-of-function mutations. Finally, we have developed a three-dimensional structural model of Cripto protein and used it as a guide to predict amino acid residues potentially implicated in protein-protein interaction.