Visualizing DNA folding and RNA in embryos at single-cell resolution

Establishment of cell types during development requires precise interactions between genes and distal regulatory sequences. Our understanding of how these interactions look in three dimensions, vary across cell types in complex tissue, and relate to transcription remains limited. Here we describe optical reconstruction of chromatin architecture (ORCA), a method to trace the DNA path in single cells with nanoscale accuracy and genomic resolution reaching 2 kilobases. We applied ORCA to a Hox gene cluster in cryosectioned Drosophila embryos and labelled ~30 RNA species in parallel. We identified cell-type-specific physical borders between active and Polycomb-repressed DNA, and unexpected Polycomb-independent borders. Deletion of Polycomb-independent borders led to ectopic enhancer-promoter contacts, aberrant gene expression, and developmental defects. Together, these results illustrate an approach for high-resolution, single-cell DNA domain analysis in vivo, reveal domain structures that change with cell identity, and show that border elements contribute to formation of physical domains in Drosophila.