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

显微注射和成像套件，用于评估鱼类对环境变化的生理反应的遗传和分子基础

• 批准号: RTI-2023-00082
• 负责人: Zimmer, Alex
• 金额: $ 6.38万
• 依托单位: University of New Brunswick
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744881
• 关键词: Microinjection; imaging; suite; assessing; genetic

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.

沿海生态系统的盐度、温度和溶解氧在季节性和日周期中波动很大，给常驻鱼类带来了极大的挑战。气候变化预测预测这些周期的极端情况会增加，但目前还不清楚这将如何影响鱼类种群。预测气候变化的影响至关重要的是了解对这些压力源的生理反应，因为这些反应影响动物的生存和表现。该申请用于购买荧光立体显微镜和显微注射系统，这些系统构成了Alex Zimmer和Ben Speers-Roesch博士研究所必需的综合套件。他们的计划旨在了解鱼类在不断变化的环境中生存所使用的反应，使用加拿大大西洋地区具有生态重要性的鱼类，mummichog（Fundulus heteroclitus），并将解决三个广泛的研究主题：离子调节的发育机制。Mummichog是广盐性离子调节和离子转运细胞（ionocytes）功能的模型，但对其敏感的早期生命阶段的离子调节生理学知之甚少。体视显微镜将被用来表征离子细胞群体的表达在发展中，他们的反应，不断变化的盐度，使用几种荧光标记技术。显微注射系统将用于使用靶向转录因子（例如，foxi3）了解这些离子细胞如何在生活史中对广盐性能力做出贡献。生理可塑性的机制基础。生理可塑性，即适应环境变化的能力，是生活在动态环境中的生物体的重要特征。广盐性，像其他塑料性状，是依赖于潜在的机制，可以微调，以满足环境的要求。环境调节机制的重要性，如鳃中Na +/K +-ATP酶亚型的盐度依赖性表达，将通过使用显微注射系统通过基因编辑消除特定基因亚型的表达来评估广盐性和其他可塑性性状。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传感器是瞬时受体电位离子通道（TRPC）。TRPC对冷（TRPM8）或热（TRPV1）有反应，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开发缺乏特异性TRPC表达的mummichog突变株系。这些线将用于辨别TRPC在控制对自然和人为热应力的反应中的作用，包括耐热性的变化和活动水平的变化，以节省能源。