Coordinated eye growth in sophisticated arthropod visual systems

任何成像眼睛最重要的特征之一是正确聚焦，这依赖于透镜和视网膜之间的精确间隔。大量的数据显示了这种协调是如何在脊椎动物中建立的。然而，一些最基本的问题，关于眼睛的组成部分是如何协调在无脊椎动物仍然没有答案。该项目主要关注朝阳潜水甲虫，因为它们可以在分子水平上进行操作，而且它们的幼虫有非常复杂的眼睛，其焦点可以量化，因此它们是分析这些关键因素的特别有价值的系统。为了测试所识别的机制的一般性，并在特定情况下进一步剖析它们，在遗传上强大的果蝇模型上进行协同实验。使用宿主实验室最近开发的微型检眼镜评价两只动物的眼睛。初步研究结果表明，甲虫和苍蝇的眼睛都能够在没有视觉反馈的情况下发展出非常精确的焦点。如果得到证实，无脊椎动物的眼睛可能成为一个重要的通用模型，用于精确调整器官发育，易于评估，并可以在分子遗传水平上进行操作。该项目的执行涉及培训许多本科生和研究生，他们也将积极参加外联活动。潜水甲虫幼虫的主眼是复杂的视觉器官，在第二至第三龄的过渡期迅速增长到原来大小的130%。这种眼睛的伸长很可能涉及渗透压的变化。透镜也需要改造，这需要相当长的时间（~8 h），可能涉及PI和Co-PI先前合作鉴定的10种透镜蛋白中的一些。该项目的目标1使用了一种独特的实时成像技术（微型检眼镜），可以直接观察视网膜并测量非常小的无脊椎动物眼睛的屈光状态（聚焦到无限远或近视或远视），包括潜水甲虫幼虫和个体D。黑腹小眼鱼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，以测试视觉输入是否是这些动物眼睛正常生长所必需的。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敲除鉴定的透镜蛋白来研究复杂的Thermonectus透镜如何改变焦距。对D.黑胃探索特定发现的普遍性。预计这种综合方法将提供一些第一批数据，以解决长期以来的问题，眼睛的生长是如何协调的节肢动物。这一奖项反映了NSF的法定使命，并已被认为是值得通过评估使用基金会的智力价值和更广泛的影响审查标准的支持。

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.