The Interplay Between Notch Ligands is Critical for Epidermal Sensory Neuronal Specification and Spatial Patterning in the Ascidian Embryo

缺口配体之间的相互作用对于海鞘胚胎的表皮感觉神经元规格和空间模式至关重要

• 批准号: 1938531
• 负责人: Robert Zeller
• 金额: $ 65.48万
• 依托单位: San Diego State University Foundation
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-15 至 2025-02-28
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1938531&HistoricalAwards=false
• 关键词: Interplay; Between; Notch; Ligands; Critical

During the process of embryonic development, a fertilized egg develops into an organism composed of many different types of cells such as muscle and nerve. A particularly common mechanism that instructs cells what type to become involves communication via proteins on their surface. The Zeller laboratory studies this process in a simple animal model called an ascidian that allows them to observe individual cells and manipulate the functions of their proteins. This project will utilize an approach called optogenetics, in which a specific color of light manipulates the function of these cell surface proteins to alter how cells communicate with one another. By illuminating the ascidian embryo at different times, they can manipulate the process of cell communication precisely to learn how this mechanism operates. This will be one of the first applications of optogenetics in ascidians and they will provide the broader scientific community with the set of tools that they develop. In addition, they will develop learning modules for both undergraduate and graduate-level courses to provide students with an opportunity to use optogenetics. Lastly, they will share this work with the public by developing an activity that demonstrates the principles of optogenetics to children that will be incorporated into our lab’s annual open house presentation at the San Diego State University Marine Laboratory.The ascidian larval peripheral nervous system (PNS) is composed of a series of ciliated sensory neurons that are patterned, in part, by Notch-mediated lateral inhibition. The sensory neurons express two Notch ligands, Delta and Jagged, at different times and in different spatial domains suggesting that more complex Notch signaling, including lateral induction, may play a role in spatial patterning. To investigate this, the Zeller laboratory will utilize an ontogenetically-regulated transcription factor, coupled with tissue-specifically expressed CRISPR reagents to manipulate Notch ligand function with precise spatiotemporal control. By manipulating the function of both Notch ligands individually and simultaneously at different times during PNS development they will tease apart patterning contributions from lateral inhibition and induction. This will be one of the first uses of optogenetics in ascidians and they will provide reagents to the broader scientific community. Using optogenetics-based approaches in other experimental systems will provide a level of precise spatiotemporal regulation that is currently difficult to achieve. They will develop an optogenetics module that will be introduced into one or more advanced undergraduate laboratory courses at San Diego State University and introduce graduate students and postdoctoral fellows to optogenetics by developing a laboratory module in the ascidian section of the Embryology Course at the Marine Biology Laboratory in Woods Hole. Finally, to expose the general public to optogenetics, they will develop an interactive presentation for children that will explain optogenetics principles that will be incorporated into our lab’s annual open house presentation at the SDSU Marine Laboratory.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

在胚胎发育过程中，受精卵发育成由许多不同类型的细胞组成的有机体，如肌肉和神经。一个特别常见的机制，指示细胞成为什么类型，涉及通过其表面的蛋白质进行通信。Zeller实验室在一个简单的动物模型中研究了这一过程，该模型被称为海鞘，允许他们观察单个细胞并操纵其蛋白质的功能。该项目将利用一种称为光遗传学的方法，其中特定颜色的光操纵这些细胞表面蛋白质的功能，以改变细胞相互通信的方式。通过在不同时间照射海鞘胚胎，他们可以精确地操纵细胞通讯过程，以了解这种机制是如何运作的。这将是光遗传学在海鞘中的首批应用之一，他们将为更广泛的科学界提供他们开发的一套工具。此外，他们将为本科和研究生课程开发学习模块，为学生提供使用光遗传学的机会。最后，他们将与公众分享这项工作，通过开发一项活动，向儿童展示光遗传学的原理，该活动将被纳入我们实验室在圣地亚哥州立大学海洋实验室的年度开放日演示中。海鞘幼虫外周神经系统（PNS）由一系列纤毛感觉神经元组成，部分由Notch介导的侧抑制形成。感觉神经元在不同的时间和不同的空间域表达两种Notch配体Delta和Jagged，这表明更复杂的Notch信号传导，包括侧向诱导，可能在空间模式中发挥作用。为了研究这一点，Zeller实验室将利用个体遗传学调节的转录因子，再加上组织特异性表达的CRISPR试剂，以精确的时空控制来操纵Notch配体功能。通过在PNS发育过程中的不同时间单独和同时操纵两种Notch配体的功能，他们将梳理来自侧抑制和诱导的图案化贡献。这将是光遗传学在海鞘中的首次应用，它们将为更广泛的科学界提供试剂。在其他实验系统中使用基于光遗传学的方法将提供目前难以实现的精确时空调节水平。他们将开发一个光遗传学模块，该模块将被引入圣地亚哥州立大学的一个或多个高级本科实验室课程，并通过在伍兹霍尔海洋生物学实验室的胚胎学课程的海鞘部分开发一个实验室模块，向研究生和博士后研究员介绍光遗传学。最后，为了让公众了解光遗传学，他们将为儿童开发一个互动演示，解释光遗传学原理，并将其纳入我们实验室在SDSU海洋实验室的年度开放日演示中。该奖项反映了NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。