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

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。指示细胞类型的一种特别常见的机制涉及通过其表面的蛋白质进行交流。泽勒实验室在一种名为海鞘的简单动物模型中研究了这一过程，该模型允许他们观察单个细胞并操纵其蛋白质的功能。该项目将利用一种名为光遗传学的方法，在这种方法中，一种特定颜色的光操纵这些细胞表面蛋白的功能，以改变细胞之间的交流方式。通过在不同的时间照射海鞘胚胎，他们可以精确地操纵细胞交流的过程，以了解这一机制是如何运作的。这将是光遗传学在海鞘动物中的首批应用之一，他们将为更广泛的科学界提供他们开发的一套工具。此外，他们将为本科生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，他们将通过开发一项向儿童演示光遗传学原理的活动与公众分享这项工作，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演讲中。海鞘幼虫周围神经系统(PNS)由一系列纤毛感觉神经元组成，部分是由Notch介导的侧抑制形成的。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Jagge，这表明更复杂的Notch信号，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，结合组织特异性表达的CRISPR试剂，通过精确的时空控制来操纵Notch配体的功能。通过在PNS发育的不同时间单独和同时操纵两个Notch配体的功能，它们将梳理出侧向抑制和诱导对图案化的贡献。这将是光遗传学在海鞘动物中的最早应用之一，他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文稿，解释光遗传学原理，并将纳入我们实验室在SDSU海洋实验室的年度开放演示中。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.