Many eukaryotic transcription factors (TFs) contain intrinsically disordered low-complexity sequence domains (LCDs), but how these LCDs drive transactivation remains unclear. We used live-cell single-molecule imaging to reveal that TF LCDs form local high-concentration interaction hubs at synthetic and endogenous genomic loci. TF LCD hubs stabilize DNA binding, recruit RNA polymerase II (RNA Pol II), and activate transcription. LCD-LCD interactions within hubs are highly dynamic, display selectivity with binding partners, and are differentially sensitive to disruption by hexanediols. Under physiological conditions, rapid and reversible LCD-LCD interactions occur between TFs and the RNA Pol II machinery without detectable phase separation. Our findings reveal fundamental mechanisms underpinning transcriptional control and suggest a framework for developing single-molecule imaging screens for drugs targeting gene regulatory interactions implicated in disease.

Small-molecule fluorophores are important tools for advanced imaging experiments. We previously reported a general method to improve small, cell-permeable fluorophores which resulted in the azetidine-containing 'Janelia Fluor' (JF) dyes. Here, we refine and extend the utility of these dyes by synthesizing photoactivatable derivatives that are compatible with live-cell labeling strategies. Once activated, these derived compounds retain the superior brightness and photostability of the JF dyes, enabling improved single-particle tracking and facile localization microscopy experiments.

Recent analysis of a novel strain of transgenic zebrafish (gutGFP) has provided a detailed description of the early morphological events that occur during the development of the liver and pancreas. In this paper, we aim to complement these studies by providing an analysis of the morphological events that shape the zebrafish intestinal epithelium. One of our goals is to provide a framework for the future characterization of zebrafish mutant phenotypes in which intestinal epithelial morphogenesis has been disrupted. Our analysis encompasses the period between 26 and 126 h post-fertilization (hpf) and follows the growth, lumen formation and differentiation of a continuous layer of endoderm into a functional intestinal epithelium with three morphologically distinct segments: the intestinal bulb, mid-intestine and posterior intestine. Between 26 hpf and 76 hpf, the entire intestinal endoderm is a highly proliferative organ. To make a lumen, the zebrafish endoderm cells undergo apical membrane biogenesis, adopt a bilayer configuration and form small cavities that coalesce without cell death. Thereafter, the endoderm cells polarize and differentiate into distinct cell lineages. Enteroendocrine cells are distinguished first at 52 hpf in the caudal region of the intestine in a new stable transgenic line, Tg[nkx2.2a:mEGFP]. The differentiation of mucin-containing goblet cells is first evident at 100 hpf and is tightly restricted to a middle segment of the intestine, designated the mid-intestine, that is also demarcated by the presence of enterocytes with large supranuclear vacuoles. Meanwhile, striking expansion of the lumen in the rostral intestine forms the intestinal bulb. Here the epithelium elaborates folds and proliferating cells become progressively restricted to a basal compartment analogous to the crypts of Lieberkühn in mammals. At 126 hpf, the posterior intestine remains an unfolded monolayer of simple columnar epithelium.