Cooperative Roles of Pds5a and Pds5b Constrain Long-Range Chromatin Interactions in Vertebrate Embryos

Three-dimensional (3D) genome organization is dynamically restructured during early vertebrate development, yet how chromatin domains are established remains poorly understood. In particular, the contribution of individual cohesin regulators to this process during embryogenesis is unclear. PDS5 proteins are key modulators of cohesin dynamics, but their depletion has been reported to cause context-dependent and sometimes contrasting architectural effects in cultured cells. Here, we investigated the roles of the cohesin regulators Pds5a and Pds5b during early development using the medaka embryo. Developmental transcriptome analysis revealed distinct but overlapping expression dynamics of pds5a and pds5b around the transition from zygotic genome activation to gastrulation. Morpholino-mediated depletion of either paralog resulted in only mild morphological phenotypes, whereas simultaneous depletion caused more severe developmental defects. In situ Hi-C analysis showed that single depletion of pds5a or pds5b induced only modest changes in 3D genome organization. In contrast, double depletion led to pronounced architectural alterations, including increased long-range chromatin contacts and de novo formation of extended chromatin loops. Transcriptome analysis revealed largely shared, with some condition-specific, gene expression changes in both single- and double-knockdown embryos, indicating that transcriptional effects can occur even in the absence of major architectural disruption. Together, our findings demonstrate that Pds5a and Pds5b act cooperatively to constrain cohesin-mediated long-range interactions during embryogenesis and highlight the importance of analyzing cohesin regulator function within a developmental context to understand how 3D genome organization is established in vivo.