Coordinated eye growth in sophisticated arthropod visual systems

任何形成图像的眼睛最重要的特征之一是正确聚焦，这依赖于晶状体和视网膜之间的精确间距。大量数据显示了这种协调是如何在脊椎动物中建立起来的。然而，关于无脊椎动物眼睛的组成部分是如何协调的一些最基本的问题仍然没有答案。该项目主要关注于日射潜水甲虫，因为它们可以在分子水平上进行操作，并且它们的幼虫具有极其复杂的眼睛，其焦点可以量化，因此它是分析这些关键因素的一个特别有价值的系统。为了测试已确定机制的普遍性，并在特定情况下进一步剖析它们，在遗传强大的果蝇模型上进行了协同实验。这两只动物的眼睛使用最近在宿主实验室开发的微型检眼镜进行评估。初步研究结果表明，甲虫和苍蝇的眼睛都能在没有视觉反馈的情况下形成惊人的精确聚焦。如果得到证实，无脊椎动物的眼睛可能成为精确调节器官发育的重要通用模型，这种模型很容易评估，并且可以在分子遗传水平上进行操作。该项目的执行涉及到对许多本科生和研究生的培训，他们也将积极参与外展活动。此外，该项目还涉及到协同教学模块的开发。潜水甲虫幼虫的主要眼睛是复杂的视觉器官，在第二到第三龄的过渡阶段，眼睛的大小迅速增长到原来的130%。这种眼睛伸长很可能与渗透压的变化有关。晶状体也需要重组，这需要相当长的时间（约8小时），可能涉及到PI和Co-PI之前合作鉴定的10种晶状体蛋白质中的一些。该项目的第一个目标是使用一种独特的实时成像技术（微型检眼镜），它可以直接观察视网膜，并测量非常小的无脊椎动物的眼睛的屈光状态（聚焦到无限远或近视眼或远视眼），包括潜水甲虫幼虫和个体黑腹瓢虫的眼睛。这项技术与传统的光学方法和潜水甲虫的行为测试相结合，以测试视觉输入对这些动物的正常眼睛发育是否必要。目的2使用物理操作和药理学来评估渗透压对眼管生长的贡献，目的3使用原位杂交、免疫染色和RNAi基因抑制鉴定的晶状体蛋白来研究复杂的Thermonectus晶状体如何改变焦距。有针对性的平行实验对黑腹龙的具体发现的普遍性进行了探讨。预计这种综合方法将为解决节肢动物眼睛生长如何协调这一期待已久的问题提供一些第一手数据。该奖项反映了美国国家科学基金会的法定使命，并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，被认为值得支持。

One of the most important features of any image-forming eye is correct focusing, which relies on the precise spacing between the lens and retina. Substantial data show how this coordination is established in vertebrates. However, some of the most basic questions concerning how eye components are coordinated in invertebrates remain unanswered. This project primarily focuses on Sunburst Diving Beetles as a particularly valuable system for analyzing these key factors because they can be manipulated on the molecular level and their larvae have extremely sophisticated eyes, the focus of which can be quantified. To test for the generality of the identified mechanisms, and in specific instances dissect them further, synergistic experiments are performed on the genetically powerful fly model Drosophila melanogaster. The eyes of both animals are evaluated using a micro-ophthalmoscope that has recently been developed in the host laboratory. Preliminary findings suggest that both beetle and fly eyes are able to develop astonishingly precise focus in the absence of visual feedback. If confirmed, invertebrate eyes could become an important general model for precisely tuned organ development that is easily assessable and can be manipulated at the molecular-genetic level. The execution of this project involves training of many undergraduate and graduate students, who will also actively participate in outreach activities. In addition, the project involves the development of synergistic teaching modules.The principal eyes of diving beetle larvae are sophisticated visual organs that rapidly grow to ~130% of their original size at the second to third instar transition. It is likely that such eye elongation involves changes in osmotic pressure. The lens also needs to be reformed, which takes considerably longer (~8 h) and likely involves some of the 10 lens proteins that have been identified through a previous collaboration of the PI and Co-PI. Aim 1 of the project uses a unique live imaging technique (the micro-ophthalmoscope) that allows direct visualization of the retina and measurement of the refractive states (focusing into infinity or being near- or far-sighted) of very small invertebrate eyes, including those of the diving beetle larvae and individual D. melanogaster ommatidia. This technique is used in combination with traditional optical methods and behavioral tests in diving beetles to test if visual input is necessary for proper eye growth in these animals. Aim 2 uses physical manipulations and pharmacology to evaluate the contribution of osmotic pressure to eye-tube growth and aim 3 uses in situ hybridization, immunostaining, and RNAi knockdowns of the identified lens proteins to investigate how the complex Thermonectus lenses change focal lengths. Targeted parallel experiments on D. melanogaster explore the generality of specific findings. It is expected that this integrative approach will provide some of the first data to address the long overdue question of how eye growth is coordinated in arthropods.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.