The molecular basis for functional divergence of duplicated SOX factors controlling endoderm formation and left-right...

Endoderm, one of three primary germ layers of vertebrate embryos, makes major contributions to the respiratory and gastrointestinal tracts and associated organs, including liver and pancreas. In mammals, the transcription factor SOX17 is vital for endoderm organ formation and can induce endoderm progenitor identity. Duplication of ancestral sox17 in the teleost lineage produced the paralogues sox32 and sox17 in zebrafish. Sox32 is required for specification of endoderm and progenitors of the left-right organiser (Kupffer's Vesicle, KV), with Sox17 a downstream target of Sox32 that is implicated in further KV development. Phenotypic evidence therefore suggests functional similarities between zebrafish Sox32 and Sox17 and mammalian SOX17. Here, we directly compare these orthologues and paralogues, using the early zebrafish embryo as a biological platform for functional testing. Our results indicate that, unlike Sox32, human SOX17 cannot induce endoderm specification in zebrafish. Furthermore, using hybrid protein functional analyses, we show that Sox32 specificity for the endoderm gene regulatory network is linked to evolutionary divergence in its DNA-binding HMG domain from its paralogue Sox17. Additionally, changes in the C-terminal regions of Sox32 and Sox17 underpin their differing target specificities. Finally, we establish that specific conserved peptides in the C-terminal domain are essential for the role of Sox17 in establishing correct organ asymmetry. Overall, our results illuminate the molecular basis for functional divergence of Sox32 and Sox17 in vertebrate endoderm development and left-right patterning, and the evolution of SoxF transcription factor function.