Mechanisms controlling stochastic gene expression during eye development

项目摘要 细胞命运的规定由血统、信号和随机调节输入驱动。这个 控制随机命运规范的机制，其中细胞随机选择 两个或更多的命运，人们对此知之甚少。随机命运规范是多样化的关键 视网膜神经元、嗅觉神经元、运动神经元、免疫细胞和干细胞。 这些机制的故障会导致包括视力在内的人类疾病 功能障碍、嗅觉障碍、自闭症、免疫缺陷和淋巴瘤。这个项目的主要目标是 就是确定染色质状态和转录变异如何控制随机命运 规范，使用苍蝇视网膜中光感受器亚型的随机图案作为 范例。 苍蝇眼包含两个颜色检测R7光感受器亚型的随机马赛克， 由视紫红质4(Rh4)或视紫红质3(Rh3)的表达定义。这个命运的决定是受控制的 通过转录因子无刺(SS)，它在成熟的R7的随机子集中表达。 SSON R7表达Rh4，而SsOFF R7表达Rh3。我们的数据支持两步机制 调控成熟R7中SON/OFF的表达。在步骤1中，早期增强器驱动早期脉冲 在R7前体中打开ss基因座染色质的ss转录。在步骤2中， 转录脉冲停止，染色质可变关闭，定义了晚期的可及性 增强剂。根据染色质压缩的程度，晚期增强剂要么打开 (开放染色质)或保持关闭(关闭染色质)在成熟的R7的生命周期内。多么 转录和染色质紧凑的调节是整合在一起的，以随机启动基因或 在开发过程中，人们对此知之甚少。我们将使用DNA FISH、基因组学、CRISPR和 基于LACO/LACI的活体成像方法评估染色质动力学和 两步机制中监管投入的暂时性(目标1)。 后生动物中驱动随机命运规范的可变性来源的识别 还没有实现。我们的数据表明，转录的可变性(起始、伸长、 频率和持续时间)影响终端R7命运规范。为了测试这一点 假设，我们将使用新生多色RNA FISH和基于MS2/MCP的实时成像来 评估转录参数并将它们与R7亚型命运联系起来(目标2)。成功 这些实验的完成将确定驱动细胞命运决定的可变性来源 并告知在后生动物的发育过程中，分子噪声如何被用来使细胞类型多样化。

Project Summary Cell fate specification is driven by lineage, signaling, and stochastic regulatory inputs. The mechanisms controlling stochastic fate specification, in which a cell randomly chooses between two or more fates, are poorly understood. Stochastic fate specification is critical for diversifying retinal neurons, olfactory sensory neurons, motor neurons, immune cells, and stem cells. Breakdowns in these mechanisms cause debilitating human disorders, including vision impairments, anosmia, autism, immunodeficiencies, and lymphoma. The main goal of this project is to determine how chromatin state and transcriptional variability control stochastic fate specification, using the random patterning of photoreceptor subtypes in the fly retina as a paradigm. The fly eye contains a random mosaic of two color-detecting R7 photoreceptor subtypes, defined by expression of Rhodopsin 4 (Rh4) or Rhodopsin 3 (Rh3). This fate decision is controlled by the transcription factor Spineless (Ss), which is expressed in a random subset of mature R7s. SsON R7s express Rh4, while SsOFF R7s express Rh3. Our data support a two-step mechanism regulating ssON/OFF expression in mature R7s. In step 1, the early enhancer drives an early pulse of ss transcription in R7 precursors that opens the chromatin at the ss locus. In step 2, the transcriptional pulse ceases and chromatin variably closes, defining the accessibility of the late enhancer. Depending on the degree of chromatin compaction, the late enhancer either turns on (open chromatin) or remains off (closed chromatin) for the lifetime of the mature R7. How regulation of transcription and chromatin compaction is integrated to turn genes randomly on or off during development is poorly understood. We will use DNA FISH, genomics, CRISPR, and lacO/LacI-based live imaging approaches to assess the role of chromatin dynamics and the temporality of regulatory inputs in the two-step mechanism (Aim 1). Identification of the source of variability driving stochastic fate specification in metazoans has not been achieved. Our data suggest that variability in transcription (initiation, elongation, frequency, and duration) in individual cells influences terminal R7 fate specification. To test this hypothesis, we will use nascent multi-color RNA FISH and MS2/MCP-based live imaging to assess transcriptional parameters and relate them to R7 subtype fates (Aim 2). Successful completion of these experiments will identify a source of variability that drives a cell fate decision and inform how molecular noise is utilized to diversify cell types during metazoan development.