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

果蝇转录因子和形态原，背部，呈梯度分布的核沿 早期胚胎的背腹轴，腹侧核内高水平且渐进性 在外侧核和背侧核中的浓度较低。激活一些基因，抑制其他基因，背部 确定合子靶基因在核浓度下沿DV轴的表达模式- 依赖的态度。这种核梯度在几次有丝分裂过程中保持不变。 胚胎发育的合体阶段，但尚不清楚是否需要连续的背部功能 这一时期或仅仅是暂时的，或者背部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，背部的浓度呈现动态变化 在单个核周期内的单个核中的浓度以及背部的浓度 在合胞体发育过程中，沿DV轴不同位置的核内的变化。这些 背部的动态行为可能有助于健壮和可重复的空间和时间控制 背部的许多靶基因通过调节机制实现，但这些机制仍未确定。提供洞察力 浓度依赖的形态生物质的暴露水平和时间如何影响图案化 活动，将追求以下两个实验目标：具体目标1：发展光遗传 控制背部活动和/或水平的方法。将使用几种方法来激活或 通过蓝光照明，以精细的时间和空间精度灭活背部。这些 方法将使用不同的策略来调节背部蛋白的稳定性或完整性，或者其 核质定位与胞质定位。特定目标2：阐明背侧和背侧的关系 蛋白质在胚胎背部的动态行为及其合子靶基因的表达 腹轴。将使用纳米串技术来检测~70个目标基因的表达 固定的胚胎已经经历了不同时间的光遗传调制，以及MS2-MCP RNA 基于茎环的成像方法将被用来进一步表征与 活胚胎中这些基因的子集。靶基因在背部活动时的反应方式 在早期胚胎发育的特定时间被激活或被扰乱将提供对该角色的重要洞察 背部动力对发育的影响。除了提供了对背部在控制这一过程中的作用的新的见解 聚合的基因调控网络，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/调控因子的光遗传学方法 胚胎以及各种各样的其他上下文，特别是那些动态行为是 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.