Genetic constructs and cell lines were assembled by standard methods (Table S1). All cell lines used in the main text (Table S2) were derived from T-REx-CHO-K1 (Invitrogen). Cell lines were constructed by sequential rounds of Lipofectamine 2000 (Invitrogen) transfection and selection. Stably transfected clones were isolated by limiting dilution or fluorescence-activated cell sorting (FACS). Time-lapse microscopy was performed with cells plated on 24-well glass-bottom plates (Mattek). For plate-bound Delta experiments, IgG-Deltaext was adsorbed to the plate together with 5 μg/ml hamster fibronectin (Innovative Research) prior to cell plating. Before imaging, cells were switched to a low-fluorescence medium, consisting of 5% FBS in αMEM lacking riboflavin, folic acid, phenol red, and vitamin B12. Movies were acquired using an Olympus IX-81 ZDC microscope, equipped with a 37°C environmental chamber supplying 5% CO2, a 20X 0.7 NA objective, and automated acquisition software (MetaMorph). Western blots for Gal4 were obtained using standard protocols. Blots were probed with rabbit anti-Gal4 DBD primary antibody (sc-577, Santa Cruz Biotechnology, 1:200) followed by incubation with horseradish peroxidase-labeled anti-rabbit IgG secondary antibody (Amersham, 1:2000). Bands were quantified using a VersaDoc gel imaging system. qRT-PCR was performed using standard protocols based on the RNeasy kit (Qiagen) and iScript cDNA synthesis kit (Bio-Rad). Co-culture experiments were analyzed for YFP fluorescence using a FACScalibur flow cytometer (Becton Dickinson) and standard protocols. Movies were analyzed in several stages. First, individual cell nuclei were identified on CFP images using a custom Matlab-based algorithm based on edge detection and thresholding of constitutively expressed H2B-Cerulean fluorescence. Then, for analysis of single-cell expression trajectories, individual nuclei were tracked across frames using custom software (Matlab, C) based on the SoftAssign algorithm (supplementary). All single-cell trajectories were validated manually. For further details see supplementary.