Optogenetic control of spatio-temporal patterning in early embryogenesis

 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，以指定局部细胞命运并正确绘制身体和器官形成。在过去的十年中，已经做了大量的工作来定量描述这些模式-测量梯度的形状和动力学的建立。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，光遗传学的新兴工具首次使直接控制蛋白质活性的空间模式和动力学成为可能。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，我们的目标是应用光为基础的控制在果蝇的发育信号，MAPK激活。通过这种精细的控制，我们将测量改变信号的幅度，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。 该提案旨在在发育背景下设计MAPK的第一个光遗传学输入。在产生和验证表达我们的光遗传学系统的转基因果蝇后，我们将这些果蝇与缺失MAPK活化配体的果蝇杂交，所述MAPK活化配体通常指定头部和尾部结构。这将使我们能够测试，如果当地的光诱导 MAPK活性可以挽救这些胚胎中的头/尾形成。然后，我们可以系统地扰动光的空间范围和动力学，以确定细胞感知到的这些参数中的哪一个，从而确定其命运。为了更好地理解MAPK信号如何在一个非常不同的发育事件中被重新利用，即神经源性外胚层特化，我们将再次将我们的光遗传学系统引入缺乏控制这种模式化的配体的果蝇中。这将使我们不仅能够比较信号的解释方式，还能够比较两个胚胎发生事件之间的信号处理方式。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，并揭示了两个基本问题：理解信号的哪些参数实际上决定了细胞的命运，以及相同的分子信号如何在不同的背景下决定命运。

 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.