Danionella cerebrum as a transparent vertebrate adult model for studying immune-related biological processes and diseases

需要一个光学可及的成年脊椎动物模型来研究体内的动态细胞过程。我们的目标是发展 并描述大脑丹尼奥氏菌的特征，这是一种与斑马鱼有亲缘关系的硬骨鱼，在成年后仍保持透明，用于研究 免疫相关的生物学过程及其对多器官疾病进展的影响。透明的成人模特 对于免疫相关过程的实时成像研究尤其重要，因为它具有动态和复杂的性质 免疫细胞的异质性和可塑性，以及疾病进展过程中渗入的免疫细胞的参与 作为与衰老相关的混杂因素。脑盘菌，以前被命名为半角盘菌，最初是由 Judkewitz小组于2018年进行了与大脑相关的研究。这种遗传上容易驯服的模型很小，成年后可以达到 长约12毫米，即使到成年也是光学透明的。已建立的转基因和基因组 在斑马鱼中使用的编辑方法已经被证明在D大脑中有效。这些特性使大脑D菌成为一种 生理学相关的动态和复杂细胞事件的非侵入性体内可视化的吸引人的模型 布景。这项研究的目的是建立所需的基础设施，以使用大脑D菌作为系统生物学模式生物 研究与免疫相关的生物学过程及其对疾病进展的影响。我们的原则性实验证明 已成功培育出带有荧光标记的天然免疫细胞的转基因品系，如中性粒细胞和 巨噬细胞和血管内皮细胞。我们建议扩大我们的转基因大脑假丝酵母的收集范围 通过荧光标记多个器官，如大脑、心脏、肝脏和肾脏。结合起来，这些转基因 线条将允许我们可视化免疫细胞和各种器官在整个发育过程中的复杂相互作用， 受伤和疾病。我们还将开发一种巨噬细胞/小胶质细胞荧光活性报告系，这将使我们能够 观察和测量免疫细胞在生理相关环境中显示的动态变化 非侵入性活体成像。为了方便我们的学习，我们将开发纵向带电的方法和设备 清醒的成人D脑的成像。免疫细胞功能在伤口愈合和再生中起着重要作用。 斑马鱼在不同的器官中都有很强的再生能力，然而，还不知道大脑中的斑马鱼是否可以。 重生。我们的初步工作表明，成虫的大脑可以再生其尾鳍，但这种再生能力 在老年动物中降低。我们提议的研究将调查D大脑是否在 不同发育阶段及其相应的免疫细胞反应和伴随的转录变化 这些事件。我们还将利用斑马鱼再生的现有数据进行物种间的比较分析。 研究探索调节再生能力的关键因素。本提案描述了将构建 使用D大脑进行活体免疫学相关研究所需的基础设施。工具、技术和知识 为D.大脑免疫模型而构建的将为未来进行重点分析提供重要的跳板 与免疫相关的过程，包括但绝不限于炎症、伤口愈合/再生、癌症、 和神经退行性疾病。

An optically accessible adult vertebrate model is needed to study dynamic cellular processes in vivo. We aim to develop and characterize Danionella cerebrum, a teleost related to zebrafish that remains transparent into adulthood, for studying immune-related biological processes and their impact on disease progression in multiple organs. A transparent adult model is particularly important for live imaging studies of immune related processes due to the dynamic and complex nature of immune cell heterogeneity and plasticity, the involvement of infiltrating immune cells during disease progression, as well as confounding factors related to aging. D. cerebrum, previously named as D. translucida, was first described by the Judkewitz group in 2018 for brain-related studies. This genetically tractable model is small, adults reach a length of approximately 12 mm in length, and is optical transparency even into adulthood. Established transgenesis and genome editing methods used in zebrafish have showed to be effective in D. cerebrum. These characteristics make D. cerebrum an attractive model for noninvasive in vivo visualization of dynamic and complex cellular events in a physiologically relevant setting. This study aims to build the infrastructure required to use D. cerebrum as a systems biology model organism for studying immune-related biological processes and their impact on disease progression. Our proof of principle experiments have successfully generated transgenic lines with fluorescently labelled innate immune cells such as neutrophils and macrophages, and the endothelial cells of the vasculature. We propose to expand our collection of transgenic D. cerebrum lines by fluorescently labeling multiple organs such as the brain, heart, liver, and kidney. In combination, these transgenic lines will allow us to visualize the complex interplay between immune cells and various organs throughout development, injury and disease. We will also develop a macrophage/microglia fluorescent activity reporter line which will allow us to observe and measure dynamic state changes displayed by immune cells in a physiologically relevant environment using non-invasive in vivo imaging. To facilitate our studies, we will develop methods and equipment for longitudinal live imaging of awake adult D. cerebrum. Immune cell functions play important roles in wound healing and regeneration. Zebrafish are known to have great regeneration capacity in different organs, however, it is not known if D. cerebrum can regenerate. Our preliminary work suggests that adult D. cerebrum can regenerate its tail fin but this regenerative capacity decreased in aged animal. Our proposed study will investigate if D. cerebrum regenerates its central nervous system during different developmental stages, its corresponding immune cell response and the transcriptomic changes that accompany these events. We will also perform interspecies comparative analysis using available data from zebrafish regeneration studies to explore key players in modulating regeneration capacity. This proposal describes strategies that will build the infrastructure required to use D. cerebrum for immunology related studies in vivo. The tools, techniques and knowledge built for the D. cerebrum immune model will provide an important springboard for carrying out future focused analyses on immune-related processes that include, but by no means are limited to, inflammaging, wound healing/regeneration, cancer, and neurodegenerative diseases.