Graphical Abstract Highlights d Hi-M simultaneously reveals 3D chromatin organization and transcriptional activity d Hi-M and Hi-C maps agree across several orders of magnitude d Chromatin is spatially compacted into TADs after the midblastula transition d TAD internal organization dramatically changes upon transcriptional activation

Acquisition of cell fate is thought to rely on the specific interaction of remote cis-regulatory modules (CRMs), e.g. enhancers, and target promoters. However, the precise interplay between chromatin structure and gene expression is still unclear, particularly within multicellular developing organisms. Here we employ Hi-M, a single-cell spatial genomics approach, to detect CRM-promoter looping interactions within topologically associating domains (TADs) during early Drosophila development. By comparing cis-regulatory loops in alternate cell types, we show that physical proximity does not necessarily instruct transcriptional states. Moreover, multi-way analyses reveal multiple CRMs spatially coalesce to form hubs. Loops and CRM hubs are established early during development, prior to the emergence of TADs. Moreover, CRM hubs are formed, in part, via the action of the pioneer transcription factor Zelda and precede transcriptional activation. Our approach provides insight into the role of CRM-promoter interactions in defining transcriptional states, as well as distinct cell types.