Microinjection and imaging suite for assessing the genetic and molecular basis of physiological responses to changing environments in fishes

沿海生态系统在季节和每日循环中容易受到盐度、温度和溶解氧的大幅波动，对常驻鱼类构成极端挑战。气候变化预报预测，这些周期的极端情况将会增加，但尚不清楚这将如何影响鱼类数量。要预测气候变化的影响，至关重要的是了解对这些应激源的生理反应，因为这些反应会影响动物的生存和性能。这项申请是为了购买荧光立体显微镜和显微注射系统，这构成了对Alex Zimmer博士和Ben Speers-Roesch博士的研究必不可少的综合套件。他们的项目试图了解鱼类在不断变化的环境中使用一种在大西洋加拿大具有重要生态意义的鱼种--粘鼠(Fundulus Heterocltes)--生存时所使用的反应，并将解决三个广泛的研究主题：离子调节的发育机制。Mummichog是研究真盐离子调节和离子转运细胞(离子细胞)功能的模型，但对其敏感早期生命阶段的离子调节生理学知之甚少。立体显微镜将使用几种荧光标记技术来表征发育过程中离子细胞群体的表达，以及它们对盐度变化的反应。显微注射系统将被用来通过针对转录因子(例如foxI3)的遗传操作来消除特定的离子细胞亚型，以了解这些离子细胞如何在生命历史上对泛盐能力做出贡献。生理可塑性的力学基础。生理可塑性，即适应不断变化的环境的能力，是生活在动态环境中的生物体的一个重要特征。像其他塑料特性一样，全盐度依赖于潜在的机制，这些机制可以微调以满足环境需求。环境调节机制的重要性，如钠/钾-三磷酸腺苷酶亚型在鳃中的盐度依赖表达，将通过使用显微注射系统通过基因编辑消除特定基因异构体的表达来评估其对常盐性和其他可塑性性状的重要性。温度感应和热应对反应之间的联系。环境温度的变化会触发生存所需的行为和生理应对反应。脊椎动物的主要温度感受器是瞬时受体电位离子通道(TRPC)。TRPC对冷(TRPM8)或热(TRPV1)做出反应，但TRPC在触发重要应对反应中的作用尚不清楚。显微注射系统将使我们能够培育出缺乏特定TRPC表达的木乃伊突变系。这些品系将被用来辨别TRPC在控制对自然和人为热应激的反应中的作用，包括耐热性的变化和活动水平的变化，以节省能源。

Coastal ecosystems are subject to large fluctuations in salinity, temperature, and dissolved oxygen on seasonal and daily cycles, posing extreme challenges for resident fishes. Climate change forecasts predict increases in the extremes of these cycles, but it is unclear how this will impact fish populations. Critically important to predicting the effects of climate change is an understanding of physiological responses to these stressors, as these responses influence animal survival and performance. This application is for the purchase of a fluorescence stereomicroscope and a microinjection system that form a comprehensive suite that is essential to the research of Drs. Alex Zimmer and Ben Speers-Roesch. Their programs seek to understand responses used by fishes to survive in changing environments using an ecologically important fish species in Atlantic Canada, the mummichog (Fundulus heteroclitus), and will address three broad research themes: Developmental mechanisms of ion regulation. Mummichog is a model for euryhaline ion regulation and the function of ion-transporting cells (ionocytes), but little is known regarding ionoregulatory physiology of its sensitive early life stages. The stereomicroscope will be used to characterize the expression of ionocyte populations over development, and their response to changing salinity, using several fluorescent labeling techniques. The microinjection system will be used to eliminate specific ionocyte subtypes using genetic manipulations targeting transcription factors (e.g., foxi3) to understand how these ionocytes contribute to euryhaline capacity over life history. Mechanistic basis of physiological plasticity. Physiological plasticity, the ability to adjust to changing environments, is an important feature of organisms that live in dynamic environments. Euryhalinity, like other plastic traits, is dependent upon underlying mechanisms that can be fine-tuned to meet environmental demands. The importance of environmentally-regulated mechanisms, such as the salinity-dependent expression of Na+/K+-ATPase isoforms in the gills, to euryhalinity and other plastic traits will be assessed by eliminating the expression of specific gene isoforms via gene editing using the microinjection system. Linkages between temperature sensing and thermal coping responses. Changes in environmental temperature trigger behavioral and physiological coping responses needed to survive. The primary temperature sensors in vertebrates are the transient receptor potential ion channels (TRPCs). TRPCs respond to cold (TRPM8) or hot (TRPV1), but the role of TRPCs in triggering vital coping responses is unclear. The microinjection system will allow us to develop mutant lines of mummichog lacking the expression of specific TRPCs. These lines will be used to discern the roles of TRPCs in controlling responses to natural and anthropogenic thermal stress, including shifts in heat tolerance and changes in activity levels to save energy.