Molecular and Functional Divergence of Zebrafish Sox Paralogs Controlling Endoderm Formation and Left–Right Patterning

Endoderm, one of three primary germ layers of vertebrate embryos, makes major contributions to the respiratory and gastrointestinal tracts and associated organs, including the liver and pancreas. In mammals, transcription factor (TF) SOX17 is vital for endoderm organ formation and can induce endoderm progenitor identity. Duplication of ancestral sox17 before or during the early evolution of ray-finned fishes produced paralogs sox32 and sox17 in zebrafish. Sox32 is required for specification of endoderm and progenitors of the left-right (LR) organizer (Kupffer's Vesicle, KV), with Sox17 a downstream target of Sox32 implicated in further KV development. Phenotypic evidence, therefore, suggests functional similarities between zebrafish Sox32 and Sox17 and mammalian SOX17. Here, we directly compare these orthologs and paralogs, 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 High Mobility Group domain from its paralog 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 Sox17 C-terminal domain are essential for its role in establishing correct organ asymmetry. Overall, our results illuminate the molecular basis for functional divergence of Sox32 and Sox17 in vertebrate endoderm development and LR patterning, and reveal that alterations in specific domains of both TFs at different points during the evolution of fish are critical to their distinct and essential functions.