The role of chromatin dynamics in stochastic cell fate specification

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Project Summary: Stochastic cell fate specification, in which cells randomly choose between cell fates, is critical during development. Stochastic mechanisms diversify visual and olfactory receptors, motor neurons, immune cells, and stem cells. Breakdowns in these mechanisms lead to vision disorders, anosmia, immunodeficiency, autism, and lymphomas. I study the developing Drosophila visual system as a paradigm to understand the mechanisms controlling stochastic cell fate specification. Within the fly eye, R7 photoreceptors make a stochastic binary fate choice between two subtypes, characterized by expression of light-detecting rhodopsins (Rh3 or Rh4). This decision is controlled by the stochastic ON/OFF expression of the transcription factor Spineless (Ss). SsON R7s express Rh4 and SsOFF R7s express Rh3. Each R7 independently makes this random decision with a 67% chance of taking the SsON R7 fate and 33% chance of taking the SsOFF R7 fate. Thus, retinas have consistent subtype ratios, but unique, random patterns. We have identified a two-step mechanism controlling ss expression during development. In step one, an early pulse of expression opens the ss gene locus in precursor R7s. In step two, the locus compacts to varying degrees, determining the ability to reinitiate expression upon specification of terminal R7 subtypes. This final expression decision is then maintained for the lifetime of the animal. I am investigating how dynamic chromatin compaction at the ss locus influences stochastic R7 subtype specification. I hypothesize that R7 precursors rapidly reorganize chromatin at the ss locus following the early pulse of expression to determine the ON/OFF state of ss expression in terminal R7s. To test this hypothesis, I will use fixed and live imaging techniques to track ss compaction during R7 subtype specification. To visualize compaction in fixed images, I developed a three-color DNA-FISH strategy that labels the ss locus, upstream region, and downstream region. Measuring the 3D distance between these regions provides cell-type-specific quantification of compaction in developing R7s. Complementing this method, I developed a live imaging approach utilizing the LacO/LacI reporter system. Inserting LacO repeats into the regions upstream and downstream of ss, I will measure the 3D distance between the two LacI reporter punctae in differentiating R7s, focusing on transition points that cannot be identified with fixed imaging. To facilitate quantification, I developed an automated image analysis pipeline to evaluate chromatin compaction in fixed and live tissue (Aim 1). I will complement these approaches by conducting ATAC-seq to identify chromatin accessibility at the ss locus in normal and mutant conditions. I will focus specifically on the accessibility of the late enhancer whose activity ultimately determines the ssON or ssOFF R7 fate (Aim 2). The results of this project will elucidate the regulatory relationship between chromatin dynamics and expression state during stochastic cell fate specification.