EDGE: Creation of a Genetically Tractable Cephalopod Model using the Hawaiian Bobtail Squid

鲸类是行为最复杂的无脊椎动物，表现出与高等脊椎动物相当的复杂性。它们有一个高度复杂的伪装系统，使它们能够瞬间融入环境。他们有先进的学习和记忆能力。它们的大脑很大（普通章鱼的神经细胞是老鼠的6倍）。这些创新和其他创新都是沿着一条独立的轨迹从脊椎动物进化而来的，脊椎动物是唯一的另一个同样先进的群体。通过研究头足类，可以了解到很多关于生物复杂性的信息，然而，由于缺乏合适的头足类模型系统，这些研究一直滞后。在过去的30年里，基因上易于处理的模式生物，如果蝇和实验室小鼠，推动了生物学的发现。该项目旨在将夏威夷短尾乌贼发展成为第一个遗传上易于驾驭的头足类动物模型。这个物种是理想的，因为它很小，它可以在整个生命周期中培养，并且在短短两个半月内达到性成熟。该项目涉及在海洋生物实验室建立一个大规模的培养设施，为基因操作提供标本。畜牧方法是完善的，通过多代培养这些生物体。最后，开发了基因组编辑技术。在这个项目下开发的所有资源，包括动物和基因敲除方法，都通过研讨会与头足类动物爱好者的科学界分享。其他努力包括协助将头足类动物生物学纳入本科生的教育课程，并纳入公众宣传活动，如在科学博物馆举办展览。作为一个群体，鞘翅目头足类动物表现出非凡的新颖性，其中许多有望促进跨学科的生物发现。例如，它们拥有迄今为止所有无脊椎动物中最大的大脑，并且表现出与高级脊椎动物相当的行为复杂性。通过融合，它们进化出了相机型的眼睛，它们附属物的生物力学能力激发了机器人的设计。与海洋微生物的显著共生使一些物种成为研究共生的理想模型。最近测序的基因组和转录组已经发现了分子的新奇。例如，在章鱼中，细胞粘附分子的大规模扩张可能有助于解释复杂的神经系统是如何组装的。其他研究表明，头足类动物以前所未有的水平使用RNA编辑来使遗传信息多样化。总之，这些现象强调了头足类动物的巨大潜力，以扩大我们对生物学中基本和未探索领域的理解;然而，该领域一直在努力推进，没有一个遗传上易于处理的模型。头足类动物群落缺乏一个模式，因为这些生物的饲养是困难的。这项工作克服了头足类动物研究的一个关键瓶颈，通过开发Euprymna cooperes，夏威夷短尾乌贼，成为一个遗传上易于处理的模型。这个物种是理想的，因为它很小，它可以通过它的生命周期培养，它很快达到性成熟。为了实现项目目标，建立了一个生命周期培养设施，并开发了使用CRISPR-Cas9进行基因组编辑的方案。该设施生产的动物提供给科学界。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的知识价值和更广泛的影响审查标准进行评估来支持。

Cephalopods are the most behaviorally complex invertebrates, exhibiting sophistication on par with higher vertebrates. They have a highly complex system for camouflage, enabling them to blend in with their environment instantaneously. They have an advanced ability for learning and memory. They have large brains (the common octopus has 6 times as many nerve cells as a rat). These and other innovations have evolved along an independent trajectory from the vertebrates, the only other similarly advanced group. By studying cephalopods, much can be learned regarding biological complexity; however, these studies have lagged due to the lack of a suitable cephalopod model system. Over the past 30 years, genetically tractable model organisms like the fruit fly and lab mouse have driven biological discovery. This project aims to develop the Hawaiian Bobtail Squid into the first genetically tractable cephalopod model. This species is ideal because it is small, it can be cultured through its entire lifecycle, and it reaches sexual maturity in as little as two and a half months. The project involves the establishment of a large-scale culture facility at the Marine Biological Laboratory to provide specimens for genetic manipulation. Husbandry methods are perfected to culture these organisms through multiple generations. Finally, techniques for genome editing are developed. All resources developed under this project, including animals and methods for gene knockouts, are shared with the scientific community of cephalopod enthusiasts through workshops. Additional efforts include assistance with the incorporation of cephalopod biology into educational curricula for undergraduate students, and into public outreach activities, such as exhibits at science museums. As a group, the coleoid cephalopods exhibit extraordinary novelties, many that are poised to promote biological discovery across disciplines. For example, they have by far the largest brains of any invertebrate, and show behavioral sophistication comparable to advanced vertebrates. Through convergence, they have evolved camera-type eyes and the biomechanical capabilities of their appendages inspire robotic design. Conspicuous symbioses with marine microbes make some species ideal models for studying symbioses. Recently sequenced genomes and transcriptomes have uncovered molecular novelties. In octopus, for example, large-scale expansions of cell adhesion molecules may help explain how complex nervous systems are assembled. Other studies indicate that cephalopods use RNA editing at unprecedented levels to diversify genetic information. Taken together, these phenomena underscore the vast potential of cephalopods to broaden our understanding of fundamental and unexplored area in biology; however, the field has struggled to advance without a genetically tractable model. The cephalopod community has lacked a model because the husbandry of these organisms is difficult. This work overcomes a key bottleneck in cephalopod research by developing Euprymna scolopes, the Hawaiian Bobtail Squid, into a genetically tractable model. This species is ideal because it is small, it can be cultured through its lifecycle, and it reaches sexual maturity rapidly. To accomplish the project goals, a lifecycle culture facility is established and protocols for genome editing using CRISPR-Cas9 are developed. Animals produced by this facility are offered to the scientific community. Protocols for husbandry and genome editing are disseminated through workshops.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.