IDBR: A workstation for optogenetics in embryogenesis and regeneration

细胞行为与多细胞模式形成的协调是发育、再生、癌症、进化和合成生物学的核心问题。分子和细胞生物学取得了显著的进步，因为开发了实验控制生化信号的工具。然而，生物电信号——模式控制的一个迷人而重要的物理层——仍然知之甚少。在多种脊椎动物和无脊椎动物模型系统中，利用靶向的、定量的、分子水平的离子通量变化进行功能实验，涉及发育（左右不对称、颅面图案、眼睛诱导）、再生（脊椎动物的尾巴和肢体再生，涡虫的前后极性）中的特定生物电事件，现在正被应用于原位癌症的检测和抑制。通过开发1)允许体内生理特性分子水平变化的工具，2)建立合成生理和分子数据的定量计算机模型，3)充实详细显示生物物理信号如何产生、传播和转导成下游典型生化/转录反应的途径，以及4)向相关领域的许多实验室传播方案和试剂，生物电已经进入了一个新时代。至关重要的是，该领域由于缺乏工具而受到阻碍：变革性影响需要许多实验室能够对体内离子通量和跨膜电压施加严格的时空控制。光遗传学（表达光敏离子通道）是一项令人兴奋的进步，但从未应用于可兴奋细胞（神经和肌肉）之外，因为现有的设备不允许灵活控制大范围内足够明亮的光，而这在研究发育或再生系统（器官、整个动物或生物工程结构）时是必要的。该项目将开发一个自动化的、高度通用的光遗传学研究站，扩展过去现有的技术，使模型系统中的电压梯度实验控制成为可能。该平台围绕计算机控制的显微镜构建，适用于从小动物到单个细胞的任何事物，该平台将允许任何能够访问分子生物学和显微镜的实验室在任何情况下对生理学和生物电的作用进行筛选和有针对性的实验。这将通过改变体内功能生理学实验的计划、执行和分析的技术状态，显著影响几个基本领域。Aim 1利用商业伙伴关系以及工程和光学领域的当地合作者，建立一个基于光的平台，以控制任何所需细胞群的静息电位。目的2通过在非洲爪蟾系统中控制干细胞衍生物和调节器官模式的原理证明应用来验证该系统。关键是，蛋白质/mRNA谱不能完全决定细胞行为；这个IDBR平台将改变功能电生理学领域，并有助于破解生物电密码，因为它首次允许许多实验室轻松收集功能生理数据。在任何感兴趣的细胞/组织中直接控制电压将使定量理解生物物理（翻译后）参数如何与基因调控网络相互作用，以决定模式形成和组织/器官功能。为几个社区带来一个全新的监管方面，将使该领域真正了解生物物理梯度的作用和信息内容。拟议的活动将涉及培养一名少数民族博士生和一名有才华的年轻博士后，研究发育生物学前沿的高度跨学科技术，以及基础科学和工程工业之间的合作优势。天然生物电场形成了一种微妙的“支架”，决定着生物结构的生长和形态，引导着基因网络的活动。任何实验室控制活体组织内部自然生物电场的能力将改变我们理解和控制组织和器官形状的能力。这将对理解复杂身体部位的进化具有重要意义，并最终推动新的合成生物学和生物工程应用。这项技术的直接受益者不仅是本科生和其他学生，他们将在最先进的学习模块中使用这些设备，而且整个社会将最终受益于这项技术在生物医学上的应用（体外器官的生物工程，再生袖子，以及由活组织制成的强大计算设备的开发）。

The orchestration of cell behavior into multicellular pattern formation is a central issue for developmental, regenerative, cancer, evolutionary, and synthetic biology. Molecular and cell biology has made remarkable strides because tools were developed for the experimental control of biochemical signals. However, Bioelectric signals - a fascinating and important physical layer of pattern control - remain poorly understood. Functional experiments using targeted, quantitative, molecular-level changes in ion fluxes in a variety of vertebrate and invertebrate model systems implicated specific bioelectric events in development (left-right asymmetry, craniofacial patterning, eye induction), regeneration (tail and limb regeneration in vertebrates, and anterior-posterior polarity in planaria), and is now being applied to detection and suppression of cancer in situ. By developing 1) tools that allow molecular-level changes in physiological properties in vivo, 2) building quantitative computer models synthesizing physiological and molecular data, 3) fleshing out pathways that show in detail how biophysical signals are produced, propagated, and transduced into downstream canonical biochemical/transcriptional responses, and 4) disseminating protocols and reagents to many labs in related fields, bioelectricity has been brought into a new age. Crucially, the field is held back by a lack of tools: transformative impact requires the ability for many labs to be able to exert tight spatio-temporal control over ion flux and transmembrane voltage in vivo. Optogenetics (expression of light-sensitive ion channels) is an exciting advance, but has never been applied outside of excitable cells (nerve and muscle) because available devices do not allow flexible control of sufficiently-bright light delivered over large areas - a necessity when working with developing or regenerating systems (organs, whole animals, or bioengineered constructs). This project will develop an automated, highly versatile, optogenetics research station that extends past existing technology to enable experimental control of voltage gradients in model systems. Built around a computer controlled microscope that is suitable for working with anything from small animals to individual cells, this platform will allow any lab that has access to molecular biology and microscopy to perform screens and targeted experiments on the role of physiology and bioelectricity in any context. This will significantly impact several basic fields by transforming the state of the art in how functional in vivo physiology experiments are planned, executed, and analyzed. Aim 1 capitalizes on commercial partnerships and local collaborators in engineering and optics to build a platform for light-based control of resting potential in any desired cell groups. Aim 2 validates the system by proof-of-principle applications in the control of stem cell derivatives in vivo and regulation of organ patterning in the Xenopus laevis system.Crucially, protein/mRNA profile does not fully determine cell behavior; this IDBR platform will transform the field of functional electrophysiology, and help to crack the bioelectrical code, by, for the first time, allowing many labs to easily collect functional physiomic data. Direct control of voltage in any cell/tissue of interest will enable quantitative understanding of how biophysical (post-translational) parameters interact with gene regulatory networks in determining pattern formation and tissue/organ function. Bringing a whole new aspect of regulation to several communities will allow the field to truly understand the role and information content of biophysical gradients.The proposed activity will involve the training of a minority PhD student and talented young post-doctoral fellow in highly interdisciplinary techniques at the forefront of developmental biology, at the collaborative edge between basic science and engineering industry. Natural bioelectric fields form a kind of subtle "scaffold" that determines the growth and form of biological structures, and guides the activity of gene networks. The ability for any lab to control the natural bioelectric fields inside living tissues will transform our ability to understand and control the shape of tissues and organs. This will have important implications for understanding evolution of complex body parts, as well as ultimately driving novel the synthetic biology and bioengineering applications. The direct beneficiaries of this technology will be not only the undergraduates and other students who will use such devices in state-of-the-art learning modules, but society as a whole, which will ultimately benefit from applications of this technology to biomedicine (bioengineering of organs in vitro, regenerative sleeves, and development of robust computational devices made of living tissues).