Regulation of Transcriptional Bursting and Spatial Patterning in EarlyDrosophilaEmbryo Development

Nascent RNA synthesis often occurs in periods of high transcriptional activity, interspersed with basal or no activity periods. This phenomenon, known as transcriptional bursting, drives high intercellular variability in gene expression levels. How do key patterning genes in earlyDrosophila melanogasterembryos overcome this variability to establish precise spatial patterns for tissue development? To address this question, we study single cell transcriptional activity from MS2-based live imaging data of four transgenic constructs (rho, Kr, snashadow,snaproximal enhancers) and the endogenousevegene. We developed an algorithm to infer promoter states in hundreds of cells within the embryo using transcriptional activity data. Results show that while mean transcription levels exhibit spatial gradients, the burst duration and interburst timing remain surprisingly invariant across the embryo and different constructs. The time between consecutive bursts was consistent with a memoryless exponential distribution, whereas the burst duration exhibited tighter control with lesser stochastic variations. Our analysis identified two regulatory mechanisms for gene expression gradients: (1) similar burst-timing statistics across genes, with the activity time (the time from the first to the last burst) being modulated to regulate distinct expression levels; (2) for the same gene with different enhancers (snashadow andsnaproximal), we observed changes in the mean burst duration and variability of the inter-burst timing. This study provides a comprehensive approach to analyzing transcriptional bursting kinetics, revealing activity time as a major regulator of spatiotemporal expression patterning in early embryonic development.