Deciphering when the pivotal transcription factor Dorsal exerts patterning effects using optogenetics

利用光遗传学破译关键转录因子 Dorsal 何时发挥模式效应

• 批准号: 9612309
• 负责人: Angelike Stathopoulos
• 金额: $ 25.13万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-10 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9612309
• 关键词: Acute; Anterior; Behavior; Biological Assay; Biological Process; Cell Cycle; Cell Nucleus; Circadian Rhythms; DNA Damage; Dependence; Development; Developmental Gene; Dorsal; Drosophila genus; Embryo; Embryonic Development; Enhancers; Exhibits; Exposure to; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic Transcription; Health; Hour; Human; Image; Individual; Inflammation; Investigation; Lateral Dorsal Nucleus; Light; Lighting; Methodology; Methods; Mitotic; Nuclear; Nuclear Grade; Output; Pattern; Positioning Attribute; Process; RNA; Regulation; Regulator Genes; Reproducibility; Role; Specific qualifier value; System; TP53 gene; Technology; Testing; Time; Transcriptional Regulation; biological adaptation to stress; dorsal proteins; experimental study; gastrulation; imaging modality; insight; morphogens; nano-string; nuclear division; optogenetics; promoter; somitogenesis; stem; tool; transcription factor; virtual

The Drosophila transcription factor and morphogen, Dorsal, assumes a graded nuclear distribution along the dorsal-ventral (DV) axis of the early embryo with high levels in ventrally-positioned nuclei and progressively lower concentrations in lateral and dorsal nuclei. Activating some genes and repressing others, Dorsal determines the pattern of expression of zygotic target genes along the DV axis in a nuclear concentration- dependent manner. This nuclear gradient is maintained over several nuclear mitotic divisions occurring during the syncytial stage of embryogenesis, but it is unclear whether Dorsal function is required continuously during this period or only transiently, or whether the temporal requirements for Dorsal function differ among its target genes. Moreover, only recently has it come to light that the concentration of Dorsal exhibits dynamic changes in concentration in individual nuclei within single nuclear cycles and that the concentration of Dorsal present within nuclei at different positions along the DV axis changes over the course of syncytial development. These dynamic behaviors of Dorsal likely contribute to robust and reproducible spatial and temporal control of Dorsal's many target genes through regulatory mechanisms that remain uncharacterized. To provide insight into how levels as well as time of exposure to concentration-dependent morphogens may influence patterning activity, the following two experimental aims will be pursued: Specific Aim 1: To develop optogenetic approaches to control activity and/or levels of Dorsal. Several approaches will be used to either activate or inactivate Dorsal with fine-scale temporal and spatial precision by illumination with blue light. These approaches will use different strategies to modulate either the stability or integrity of Dorsal protein, or its nuclear versus cytoplasmic localization. Specific Aim 2: To elucidate the relationship between Dorsal protein dynamic behavior and the expression of its zygotic target genes along the embryonic dorsal- ventral axis. The expression of ~70 target genes will be examined using NanoString technology applied to fixed embryos that have been subjected to optogenetic modulation for varying durations, and MS2-MCP RNA stem-loop based imaging methods will be used to further characterize dynamic behaviors associated with a subset of these genes in live embryos. The ways in which target genes respond when Dorsal activity is either activated or perturbed at specific times during early embryogenesis will provide important insight into the role of Dorsal dynamics on development. In addition to providing new insight into Dorsal's role in controlling this paradigmatic gene regulatory network, these investigations will optimize and validate powerful new and widely applicable optogenetic approaches for the study of other dynamic transcriptional/regulatory factors operating in the embryo as well as a wide variety of other contexts, particularly those for which dynamic behavior is an important component of function.

果蝇转录因子和形态发生素 Dorsal 沿核呈分级分布 早期胚胎的背腹（DV）轴，腹侧核水平较高，并且逐渐 侧核和背核浓度较低。激活一些基因并抑制其他基因，Dorsal 确定核浓度中合子靶基因沿 DV 轴的表达模式- 依赖方式。这种核梯度在发生期间发生的几次核有丝分裂中得以维持 胚胎发生的合胞体阶段，但尚不清楚在胚胎发生的合胞体阶段是否持续需要Dorsal功能 这段时期或只是短暂的，或者背侧功能的时间要求是否在其目标之间有所不同 基因。此外，直到最近才发现 Dorsal 的浓度呈现动态变化 单核周期内单个核的浓度以及 Dorsal 存在的浓度 在合胞体发育过程中，沿 DV 轴不同位置的细胞核内的变化。这些 Dorsal 的动态行为可能有助于稳健且可重复的空间和时间控制 Dorsal 的许多靶基因通过调节机制仍然未知。提供见解 研究浓度依赖性形态发生素的水平和暴露时间如何影响图案形成 活动，将追求以下两个实验目标： 具体目标 1：开发光遗传学 控制背侧活动和/或水平的方法。将使用多种方法来激活或 通过蓝光照射，以精细的时间和空间精度使 Dorsal 失活。这些 方法将使用不同的策略来调节 Dorsal 蛋白的稳定性或完整性，或其 核定位与细胞质定位。具体目标 2：阐明 Dorsal 之间的关系 蛋白质动态行为及其合子靶基因沿胚胎背侧的表达 腹轴。将使用 NanoString 技术检查约 70 个目标基因的表达 经过不同时间光遗传学调制的固定胚胎，以及 MS2-MCP RNA 基于茎环的成像方法将用于进一步表征与 活胚胎中这些基因的子集。当背侧活动处于以下任一状态时，靶基因的反应方式 在早期胚胎发生过程中的特定时间激活或扰动将为了解该作用提供重要的见解 背侧动力学对发育的影响。除了提供对 Dorsal 在控制这一过程中的作用的新见解之外 范式基因调控网络，这些研究将优化和验证强大的新的和广泛的 适用于研究其他动态转录/调节因子的光遗传学方法 胚胎以及各种其他环境，特别是那些动态行为是重要的环境 功能的重要组成部分。