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

果蝇的转录因子和形态发生蛋白Dorsal，假定核沿着 背腹（DV）轴的早期胚胎与高水平的腹侧定位的核和进行性 侧核和背核的浓度较低。激活一些基因并抑制其他基因，Dorsal 决定合子靶基因沿着DV轴在核浓度中的表达模式， 依赖的方式。这种核梯度维持在几个核有丝分裂期间发生 胚胎发育的合胞体阶段，但尚不清楚是否需要持续的背功能， 这一时期或只是暂时的，或者是否背功能的时间要求不同，其目标 基因.此外，直到最近才发现Dorsal的浓度呈现动态变化， 在单个核周期内的单个核中的浓度， 在细胞核内，沿着DV轴沿着的不同位置的细胞核内的蛋白质在合胞体发育过程中发生变化。这些 Dorsal的动态行为可能有助于对运动的鲁棒性和可再现性的空间和时间控制， Dorsal的许多靶基因通过调控机制仍然没有特征。提供洞察力 暴露于浓度依赖性形态因子的水平和时间如何影响模式 活动，将追求以下两个实验目标：具体目标1：开发光遗传学 方法来控制活动和/或水平的背。将使用几种方法来激活或 通过蓝光照明，具有精细尺度时间和空间精度的背侧。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 细胞核与细胞质定位。具体目标2：阐明背侧和背侧之间的关系 蛋白质动态行为及其合子靶基因沿着胚背- 腹轴将使用NanoString技术检测约70个靶基因的表达， 固定的胚胎已经经历了不同持续时间的光遗传学调节，和MS 2-MCP RNA 基于茎环的成像方法将用于进一步表征与 这些基因在活胚胎中的子集。当背部活动为以下任一种情况时，靶基因的反应方式 在胚胎发育早期的特定时间被激活或扰动， 背动力学对发育的影响除了提供新的见解Dorsal的作用，在控制这一点， 范式基因调控网络，这些调查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， 胚胎以及各种各样的其他情况下，特别是那些动态行为是一个 功能的重要组成部分。

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.