The role of chromatin dynamics in stochastic cell fate specification

项目摘要：随机细胞命运规范，即细胞在细胞命运之间随机选择，是至关重要的 在开发过程中。随机机制使视觉和嗅觉感受器、运动神经元、免疫多样化 细胞和干细胞。这些机制的故障会导致视力障碍、嗅觉障碍、免疫缺陷， 自闭症和淋巴瘤。我研究了发育中的果蝇视觉系统，作为一个范例来理解 控制随机细胞命运规范的机制。在苍蝇眼内，R7光感受器随机地 在两个亚型之间的二元命运选择，特征是光检测视紫红蛋白(Rh3或 RH4)。这一决定由转录因子无刺(SS)的随机开/关表达控制。 SON R7表达Rh4，SsOFF R7表达Rh3。每个R7独立地使用一个 接受SSON R7命运的可能性为67%，接受SsOFF R7命运的可能性为33%。因此，视网膜有一致的 亚型比例，但独特的随机模式。 我们已经确定了在发育过程中控制ss表达的两步机制。在第一步中，一个 早期的表达脉冲打开了前体R7S中的ss基因位点。在第二步中，轨迹被压缩为变化 度，确定在指定终端R7亚型时重新启动表达的能力。这场决赛 然后，在动物的整个生命周期内保持表情决定。我在调查动态染色质 SS轨迹的紧实度影响随机的R7亚型规格。我假设R7的前体 在表达的早期脉冲之后快速重组ss基因座上的染色质以确定开/关 SS在R7S末端的表达状态。 为了验证这一假设，我将使用固定和实时成像技术来跟踪r7期间的ss压实。 子类型规范。为了在固定的图像中可视化压缩，我开发了一个三色DNA-FISH策略 这标记了ss轨迹、上游区域和下游区域。测量它们之间的3D距离 Regions在R7的开发中提供了特定细胞类型的致密化量化。作为这种方法的补充， 我利用LACO/LACI报告系统开发了一种实时成像方法。将LACO重复插入到 SS的上游和下游区域，我将测量两个Laci报告点之间的3D距离 在区分R7时，将重点放在固定成像无法识别的过渡点。为了方便 为了定量，我开发了一个自动图像分析流水线来评估固定和 活组织(目标1)。我将通过进行atac-seq来鉴定染色质来补充这些方法。 在正常和突变条件下SS基因座的可及性。我将特别关注 晚期增强子，其活性最终决定sSON或ssOFF R7的命运(目标2)。这个项目的成果 我将阐明随机过程中染色质动力学和表达状态之间的调控关系 细胞命运规范。

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.