Cell fate specification occurs through the tightly regulated expression of key transcription factors at precise levels, times, and places during development. The expression of these determinants is controlled by enhancers and promoters. This proposal focuses on two cases where the level and timing of gene expression are especially important for cell fate specification in the Drosophila visual system. A number of mutations have been shown to cause abnormal gene regulation and cell fate, making a better understanding of these processes highly relevant to understanding the genetic basis of disease. Live imaging of stochastic gene activation in the visual system: In Aim 1, I will examine the stochastic specification of cell fate in the Drosophila retina, where otherwise equivalent R7 photoreceptor precursors have a particular probability of taking one of two fates important for color vision. This decision is controlled by the stochastic, cell-intrinsic expression of the transcription factor Spineless. I will examine two possible sources of spineless stochasticity: variability in transcriptional activation at the level of the promoter, and variation in chromatin accessibility. In order to quantify the underlying transcriptional dynamics, I will image gene expression in real time using the MS2 and PP7-based live transcriptional imaging systems. MS2 live imaging has recently revolutionized the study of gene regulation in the Drosophila embryo by providing a new level of quantitative measurement. This approach combined with direct tagging by CRISPR/Cas9 of the spineless locus will allow me to determine the origin of stochasticity in this cell fate decision. Factors that influence stochastic fate: In Aim 2, I will directly test two models that predict how different factors influence the probability of spineless expression. To directly test the role of transcriptional initiation vs. chromatin state, I will modify the spineless basal promoter using CRISPR/Cas9 by replacing it with characterized promoters that have been shown to initiate expression more or less robustly in the Drosophila embryo, for instance through recruitment of paused RNA Polymerase II. This will allow me to test the role of the promoter in stochastic fate decisions. If stochastic outcomes are unaffected by such changes, I will test the role of local chromatin state via local, targeted changes in chromatin state to evaluate the effect on the stochastic ratio of fates produced. This approach will be complemented by live imaging of transcription, which will provide an additional means of assessing the quantitative effects of specific modifications. Studying the role of transcriptional timing and levels in temporal transitions in neuroblast fate: Aim 3 will examine a series of temporal transitions between five different transcription factors in medulla neuroblasts that generate neural diversity in the fly brain. Although we know that there is cross regulation among these temporal transcripti...