Cloning and characterization of zebrafish smad2, smad3 and smad4

smad genes encode transcription factors involved in the signal transduction of members of the TGFbeta superfamily. We report here the cloning, characterization and genomic mapping of smad2, smad3 and smad4 from the zebrafish, Danio rerio. In Xenopus, smad2 overexpression has been shown to interfere with gastrulation and dorsal cell fate specification. However, full-length zebrafish smad2, although functionally active in Xenopus explants, has no effect when overexpressed in zebrafish embryos. In contrast, an N-terminally truncated, constitutively active version of Smad2 protein causes severe dorsalization or partial secondary axis formation, pointing to a role of Smad2 during mesoderm and axis formation. The temporal and spatial expression patterns of zebrafish smad2, 3 and 4 were investigated by developmental RT-PCR and whole mount in-situ hybridization. All three genes show strong and ubiquitous maternal expression. Zygotic expression is weak and ubiquitous in the case of smad2, and strong and ubiquitious in the case of smad4, while smad3 shows a spatially restricted zygotic expression pattern. It is expressed in migrating neural crest cells of the trunk and a subset of cells in the diencephalon in close proximity to the expression domain of the Nodal-related protein Cyclops/Ndr2/Znr1, a potential signal upstream of Smad2/3 required for eye-field separation and floor plate specification. Overexpression of truncated smad2 in cyclops mutant embryos leads to a rescue of the eye and floorplate defects. These data suggest that Smad2 acts as a mediator of Nodal signals during zebrafish midline signaling, while Smad3 might be involved in later steps of eye field separation.