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

该项目旨在确定与年龄相关的基因表达变化，这些变化在组织再生过程中改变细胞行为，可能是衰老过程中肌肉无力的原因。这将通过将活细胞成像和遗传操作与斑马鱼衰老遗传模型中的空间转录组学相结合来实现。衰老包括各种分子和细胞的变化，导致功能受损和再生能力下降。随着年龄的增长，肌肉无力会导致身体虚弱和代谢能力下降，使其成为改善老年人健康的一个重要治疗领域(1)。尽管在肌肉和常驻肌肉干细胞（musc）中发现了许多显示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因子，在衰老过程中会出现失调(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，从而改变再生能力尚不清楚。因此，确定再生过程中驱动细胞行为的基因的年龄相关变化是至关重要的，以便确定促进有效组织修复而不损害组织稳态的潜在治疗方法。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难以探究。因此，该项目旨在使用一系列尖端的空间单细胞成像和测序技术来鉴定受衰老影响的再生过程中调节细胞行为的基因，并确定它们是否可以被操纵以增强体内muSC功能。该项目的目的是：1。定义端粒酶突变斑马鱼幼虫在稳态和再生过程中，免疫、结缔组织和肌肉驻留干对细胞行为和代谢活性的改变。定义再生肌肉中特定细胞类型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。评估端粒酶突变体在再生肌肉中空间分布改变的基因对调节免疫和muSC细胞行为的功能重要性斑马鱼将被用作识别再生肌肉中细胞行为和基因表达变化的模式生物。端粒酶突变体（tert1）表现出与人类相似的早衰表型（3,4）。重要的是，我们已经证明，在tert1突变的幼虫中，muSC对损伤的反应是受损的，这为观察衰老过程中的细胞行为提供了一个强大的遗传工具。在免疫细胞和musc中表达荧光蛋白的动物将在针穿刺局灶性损伤后再生的幼虫阶段通过显微镜观察。损伤后4天内再生完成，为活细胞成像和组织分子图谱的结合提供了机会(5)。细胞对损伤的反应将使用Imaris进行分析，并测试一些参数（方向性、瞬时速度、均方位移），以定义一个老化细胞项目批准表- 2015年11月再生过程中的行为概况。为了分析组织中的免疫细胞和musc，将选择约500个基因进行多路FISH空间转录组分析(6)。基因将从Knight实验室从端粒酶突变体和衰老动物中产生的现有RNA Seq数据集中选择，这些数据集1)可能对免疫细胞或muSC行为很重要，2)显示tert1突变体的变化。候选基因在再生肌肉组织中的空间表达将通过空间转录组学分析获得。在使用CRISPR/Cas9诱变或药理学操作的再生过程中，将对在musc和免疫细胞中显示tert1依赖性差异的选定基因进行功能评估(7)

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