Project Summary: Lineage and signaling mechanisms specify cell fates, often yielding stereotypical patterns of cell types. In some contexts, these inputs regulate fate choices in a probabilistic way to produce random patterns. The mechanisms controlling stochastic cell fate specification, in which a cell randomly chooses between fates with a set probability, are poorly understood. Stochastic cell fate specification is critical for the generation of photoreceptors, olfactory receptors, motor neurons, interneurons, immune cells, and stem cells. Breakdowns cause disorders, including vision impairments, anosmia, autism, immunodeficiencies, and lymphoma. The main goal of this project is to determine how cell-intrinsic mechanisms including transcription and chromatin regulation (Aim 1) and extrinsic mechanisms involving cell-cell signaling (Aim 2) control stochastic fate specification. The fly eye is an ideal paradigm to study stochastic fate specification because its development involves a simple, random, binary fate choice. The retina contains a random mosaic of two R7 photoreceptor subtypes, controlled by the stochastic expression of the transcription factor Spineless (Ss), yielding SsON and SsOFF R7s. How transcriptional regulation and chromatin compaction are integrated to turn genes randomly on or off during development is poorly understood (Aim 1). We found that an enhancer drives ss transcription in precursor cells to open the ss locus. Transcription ceases and the ss locus compacts in a subset of cells. In terminal R7s in which chromatin compacts, ss is repressed. In R7s with open chromatin, ss is reactivated. As transitions in ss transcription and chromatin states are critical for this fate choice, we developed strategies to study ss in live, developing eyes. To understand how the dynamics of ss expression influence R7 subtype fate, we will examine ss transcription using a reporter system. To relate the dynamics of transcriptional and chromatin states, we will use DNA visualization systems to mark the ends of the ss locus and simultaneously live image transcription and chromatin compaction. To determine the roles of transcription and chromatin factors, we will examine ss regulation in mutant conditions. As features of this mechanism are observed in bacteria and mouse, our studies will characterize fundamental mechanisms controlling stochastic cell fate specification across species. The stochastic mosaic of R7 subtypes is overlaid on the stereotypical morphology of the fly eye, providing the opportunity to study how development of these patterns intersects (Aim 2). Hedgehog (Hh) and Decapentaplegic (Dpp) signaling, together with the transcription factor Homothorax (Hth), generates the regular morphology of the eye. We found that the Hh-Dpp-Hth network regulates stochastic ss expression. To address how this network regulates ss, we will use mutant and ectopic expression strategies to determine the roles of pathway members. To understand how ...