During early development gene expression patterns progressively emerge as cell fates are determined and the embryo takes form. The regulation of patterning occurs across a broad range of spatial and temporal scales. These scales range from the molecular scale dynamics of regulatory proteins binding to genomic loci to activate or repress expression, to the organization of chromatin and distribution of proteins within nuclei at sub-micron to micron scales, to the emergence of domains of gene expression at the scales of hundreds of microns to millimeters. Here I will discuss the application of high-resolution light-sheet microscopy, single molecule tracking, and new analysis approaches to functionally bridge data acquired across this wide range of spatial and temporal scales. I will present data on how transient assemblies of transcriptional regulators shape the nucleoplasm and regulate gene expression during embryogenesis in Drosophila melanogaster. I will discuss how combining imaging data with systematic mutagenesis of key transcription factors allows us to dissect how protein-protein interactions influences the ability of transcription factors to efficiently find and bind their genomic targets and regulate transcriptional activity.