Optogenetic control of spatio-temporal patterning in early embryogenesis

 描述(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，以确定局部细胞的命运，并适当地绘制出身体和器官的形成。在过去的十年里，已经做了大量的工作来定量地描述这些模式--测量梯度的形状和它们建立的动力学。然而，可用于研究这些模式的遗传工具非常不适合干扰它们。基因扰动通常会完全破坏模式，或者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动的空间模式和动态成为可能。与扩散的化学刺激不同，光图案可以被清晰地定义并随意移除。在这里，我们的目标是应用基于光的控制来控制果蝇的发育信号，即MAPK的激活。有了这种精细的控制，我们将测量信号的幅度、持续时间和空间范围变化的后果，并定量比较信号在不同发育阶段的解释方式。这项提议的目的是在发展的背景下设计第一个光基因对MAPK的输入。在产生并验证了表达我们的光遗传系统的转基因果蝇后，我们将把这些果蝇与那些缺乏MAPK激活配体的果蝇杂交，MAPK激活配体通常指定头部和尾部结构。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的头/尾的形成。然后，我们可以系统地扰乱光的空间范围和动力学，以确定细胞感知到这些参数中的哪些参数，从而确定其命运。为了更好地了解MAPK信号在一个非常不同的发育事件-神经性外胚层规格-中如何被重新利用，我们将再次将我们的光遗传系统引入缺乏控制这种模式的配体的果蝇中。这将使我们不仅能够比较信号被解释的方式，而且还可以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活体发育系统中的首次应用，并阐明了两个基本问题：理解信号的哪些参数实际上决定了细胞命运，以及相同的分子信号如何在不同的环境中指定命运。

 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.