Optogenetic control of spatio-temporal patterning in early embryogenesis

早期胚胎发生中时空模式的光遗传学控制

• 批准号: 9121646
• 负责人: Heath E Johnson
• 金额: $ 5.43万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9121646
• 关键词: Automobile Driving; Cells; Chemicals; Complex; Defect; Development; Diffusion; Drosophila genus; Drosophila melanogaster; Ectoderm; Embryo; Embryonic Development; Engineering; Event; Future; Gene Expression; Genetic; Head; Human; Immersion Investigative Technique; Kinetics; Lead; Ligands; Light; Location; MAP Kinase Gene; Maps; Measures; Methods; Microinjections; Molecular; Nature; Nerve; Neurons; Organ; Organism; Outcome; Pathway interactions; Pattern; Physiologic pulse; Polychlorinated Biphenyls; Process; Protein Dynamics; Reading; Research; Series; Shapes; Signal Transduction; Son of Sevenless Proteins; Specific qualifier value; Staging; Stimulus; Structure; System; Tail; Testing; Time; Tissues; To specify; Transgenic Organisms; Work; base; cofactor; developmental disease; fly; improved; in vivo; loss of function; migration; mutant; neuroblast; novel; optogenetics; prevent; programs; public health relevance; spatiotemporal; tool; transcription factor; transmission process

 DESCRIPTION (provided by applicant): Development is a complex process that requires spatial and temporal coordination of signals to specify local cell fates and properly map out body and organ formation. In the last decade, much work has been done to quantitatively characterize these patterns - measuring shape of gradients and the kinetics of their establishment. Yet the genetic tools available to study these patterns are very poorly suited to perturb them. Genetic perturbations typically destroy patterns completely, or alter multiple features of their spatiotemporal distribution simultaneously. In contrast, the emerging tools of optogenetics make it possible for the first time to directly control the spatial patterns and dynamics of protein activity. Unlike chemical stimuli which undergo diffusion, light patterns can be sharply defined and removed at will. Here we will aim to apply light based control over a developmental signal, MAPK activation, in Drosophila melanogaster. With this fine control we will measure the consequences of varying the amplitude, duration and spatial range of the signal, and quantitatively compare how the signal is interpreted at different developmental stages. This proposal aims to engineer the first optogenetic inputs to MAPK in a developmental context. After generating and validating transgenic flies which express our optogenetic system, we will cross these flies with ones which are depleted of the MAPK activating ligand which normally specifies the head and tail structures. This will enable us to test if local light-induced MAPK activity can rescue head/tail formation in these embryos. We can then systematically perturb the spatial range and dynamics of light to determine which of these parameters is sensed by the cell to specify its fate. To better understand how the MAPK signal is repurposed in a very different developmental event, that of the neurogenic ectoderm specification, we will once again introduce our optogenetic system into flies depleted of the ligand which controls this patterning. This will enable us to compare not only the way the signal is interpreted but also how it processed between the two embryogenesis events. Successful completion of these aims will mark the first use of optogenetics in an in vivo developmental system and shed light on two fundamental questions: understanding what parameters of a signal actually specify cell fate as well as how the same molecular signal specifies fates in different contexts.

 描述（由申请人提供）：发育是一个复杂的过程，需要信号的空间和时间协调，以指定局部细胞的命运并正确规划身体和器官的形成。在过去的十年中，人们做了很多工作来定量描述这些模式——测量梯度的形状及其建立的动力学。然而，可用于研究这些模式的遗传工具非常不适合扰乱它们。遗传扰动通常会完全破坏模式，或同时改变其时空分布的多个特征。相比之下，新兴的光遗传学工具首次使得直接控制蛋白质活性的空间模式和动态成为可能。与经历扩散的化学刺激不同，光图案可以被清晰地定义并随意去除。在这里，我们的目标是对果蝇中的发育信号（MAPK 激活）应用基于光的控制。通过这种精细控制，我们将测量改变信号幅度、持续时间和空间范围的后果，并定量比较信号在不同发育阶段的解释方式。 该提案旨在在发育背景下设计 MAPK 的第一个光遗传学输入。在生成并验证表达我们的光遗传学系统的转基因果蝇后，我们将这些果蝇与那些通常指定头部和尾部结构的 MAPK 激活配体耗尽的果蝇杂交。这将使我们能够测试局部光诱导 MAPK 活性可以挽救这些胚胎中头/尾的形成。然后，我们可以系统地扰动光的空间范围和动态，以确定细胞感知到哪些参数来指定其命运。为了更好地理解 MAPK 信号如何在一个非常不同的发育事件（神经源性外胚层规范）中被重新利用，我们将再次将我们的光遗传学系统引入到控制这种模式的配体耗尽的果蝇中。这将使我们不仅能够比较信号的解释方式，还能比较信号在两个胚胎发生事件之间的处理方式。这些目标的成功完成将标志着光遗传学在体内发育系统中的首次应用，并阐明两个基本问题：了解信号的哪些参数实际上指定细胞命运，以及相同的分子信号如何指定不同背景下的命运。