Dissecting age-related changes to tissue regeneration in a zebrafish model by live cell imaging and spatial transcriptomics

通过活细胞成像和空间转录组学剖析斑马鱼模型中与年龄相关的组织再生变化

• 批准号: 2888993
• 金额: --
• 依托单位: King's College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2888993
• 关键词: Dissecting; age; related; changes; tissue

This project aims to identify age-associated changes to gene expression that alter cell behaviour during tissue regeneration and may underlie muscle weakness in ageing. This will be achieved by combining live cell imaging and genetic manipulations with spatial transcriptomics in a zebrafish genetic model of ageing.Ageing involves a variety of molecular and cell changes that result in impaired function and diminished regenerative capacity. Muscle weakness in ageing results in frailty and diminished metabolic capacity, making it an important therapeutic area for improving health in the ageing population (1). Although many genes showing age-related changes have been identified in muscle and resident muscle stem cells (muSCs) it is not known how these affect cell behaviour during tissue regeneration. The immune system is an important regulator of tissue repair and is dysregulated during ageing (2). How age-associated changes to the immune system intersect with those affecting muscle cells to alter regenerative capacity is not known. It is therefore critical to identify age-associated changes to genes driving cell behaviour during regeneration in order to identify potential therapeutics that promote effective tissue repair without impairing tissue homeostasis. However, tissue regeneration is highly localized to the injury site and its detailed molecular and cellular mechanisms have traditionally been difficult to probe.This project therefore aims to use a host of cutting edge spatial single-cell imaging and sequencing techniques to identify genes regulating cell behaviour during regeneration affected by ageing and determine whether they can be manipulated to enhance muSC function in vivo.Aims of the project are to: 1. define changes to cell behaviour and metabolic activity by immune, connective and resident stem in muscle of telomerase mutant zebrafish larvae during homeostasis and regeneration.2. define how gene expression in defined cell types within regenerating muscle shows spatially altered localisation in telomerase mutants 3. evaluate the functional importance of genes which show altered spatial distribution in regenerating muscle of telomerase mutants for regulating immune and muSC cell behaviourZebrafish will be used as a model organism for identifying changes to cell behaviour and gene expression in regenerating muscle. Telomerase mutants (tert1) show premature ageing phenotypes similar to humans (3, 4). Importantly, we have demonstrated that the muSC response to injury is impaired in tert1 mutant larvae, providing a powerful genetic tool for visualising cell behaviour in ageing. Animals expressing fluorescent proteins in immune and muSCs will be visualised by microscopy at larval stages during regeneration following focal injury performed by a needle. Regeneration is complete within 4 days following such injuries providing the opportunity for combining live cell imaging with molecular profiling of tissues (5). Cell responses to injury will be analysed using Imaris and a number of parameters tested (directionality, instantaneous speed, mean squared displacement) to define an ageing cell Project Approval Form - Nov 205behaviour profile during regeneration. To profile immune cells and muSCs in tissue a selection of ~500 genes will be selected for spatial transcriptomic analysis using multiplexed FISH (6). Genes will be selected from existing RNA Seq datasets generated by the Knight lab from telomerase mutants and ageing animals that are 1) likely to be important for immune cell or muSC behaviour, 2) show changes in tert1 mutants. Spatial expression of candidate genes in regenerating muscle tissue will be obtained by spatial transcriptomics analyses. Selected genes showing tert1-dependent differences in muSCs and immune cells will be functionally evaluated during regeneration using CRISPR/Cas9 mutagenesis (7) or by pharmacological manipulations

该项目旨在确定与年龄相关的基因表达变化，这些变化改变了组织再生过程中的细胞行为，并可能导致衰老中的肌肉无力。这将通过在斑马鱼衰老遗传模型中将活细胞成像和遗传操作与空间转录组学相结合来实现。衰老涉及各种分子和细胞变化，导致功能受损和再生能力下降。衰老中的肌肉无力导致虚弱和代谢能力下降，使其成为改善老龄人口健康的重要治疗领域（1）。尽管在肌肉和常驻肌肉干细胞（muSC）中已经发现了许多显示出年龄相关变化的基因，但尚不清楚这些基因如何影响组织再生过程中的细胞行为。免疫系统是组织修复的重要调节器，在衰老过程中会失调（2）。与年龄相关的免疫系统变化如何与影响肌肉细胞的变化交叉以改变再生能力尚不清楚。因此，关键是要确定与年龄相关的基因的变化，在再生过程中驱动细胞的行为，以确定潜在的治疗，促进有效的组织修复，而不损害组织的稳态。然而，组织再生高度局限于损伤部位，其详细的分子和细胞机制传统上很难探索。因此，本项目旨在使用大量尖端的空间单细胞成像和测序技术来识别受衰老影响的再生过程中调节细胞行为的基因，并确定是否可以操纵它们以增强体内muSC功能。该项目的目的是：1.明确端粒酶突变斑马鱼仔鱼在体内平衡和再生过程中，肌肉中免疫、结缔组织和驻留干细胞对细胞行为和代谢活性的影响.定义在再生肌肉内确定的细胞类型中的基因表达如何在端粒酶突变体中显示空间改变的定位3.评估基因的功能重要性，这些基因在端粒酶突变体的再生肌肉中显示出改变的空间分布，用于调节免疫和muSC细胞行为斑马鱼将被用作模式生物，用于鉴定再生肌肉中细胞行为和基因表达的变化。端粒酶突变体（tert 1）显示出与人类相似的过早衰老表型（3，4）。重要的是，我们已经证明muSC对损伤的反应在tert 1突变幼虫中受损，为观察衰老中的细胞行为提供了强大的遗传工具。在免疫和muSC中表达荧光蛋白的动物将在通过针进行的局灶性损伤后再生期间的幼虫阶段通过显微镜观察。再生在这种损伤后4天内完成，提供了将活细胞成像与组织分子谱相结合的机会（5）。将使用Imaris分析电池对损伤的反应，并测试一些参数（方向性、瞬时速度、均方位移），以确定老化电池项目批准表-2005年11月再生期间的行为概况。为了分析组织中的免疫细胞和muSC，将选择约500个基因用于使用多重FISH的空间转录组学分析（6）。基因将从Knight实验室从端粒酶突变体和衰老动物中生成的现有RNA Seq数据集中选择，这些基因1）可能对免疫细胞或muSC行为很重要，2）显示tert 1突变体的变化。通过空间转录组学分析获得候选基因在再生肌肉组织中的空间表达。在muSC和免疫细胞中显示ter 1依赖性差异的选定基因将在再生过程中使用CRISPR/Cas9诱变（7）或通过药理学操作进行功能评估