EDGE: Establishing functional genomics in Hydra to study stem cells and regeneration

EDGE：在 Hydra 中建立功能基因组学以研究干细胞和再生

• 批准号: 1829158
• 负责人: Celina Juliano
• 金额: $ 149.93万
• 依托单位: University of California-Davis
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-11-01 至 2022-10-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1829158&HistoricalAwards=false
• 关键词: EDGE; Establishing; functional; genomics; Hydra

Important questions in developmental biology relate to how stem cells maintain the health of adult tissues, both as tissues age under normal conditions and in response to injury. While human stem cells have only limited abilities for tissue repair and regeneration, many animals have the ability to regrow large portions of their body after catastrophic injury. This is accomplished using largely the same gene families found in all animals, thus understanding the molecular mechanisms that underlie these impressive feats of regeneration will shed light on why this does not occur in humans; such basic biology research could ultimately lead to improved regenerative medicine therapies. A major barrier to the success of such research is the relative lack of tools to manipulate gene function in highly regenerative animals. Regeneration research would benefit from a model organism with the following attributes: 1) adult stem cells differentiate frequently and enable complete tissue regeneration; 2) cells can be tracked as they differentiate and migrate; and 3) tools for studying gene function with spatial and temporal resolution. Currently, no model organism has all three of these attributes; however, Hydra vulgaris is a promising candidate. Hydra has remarkable regenerative abilities, and its simple tissue organization and optical clarity enables cell tracking using optical microscopy. However, the community currently lacks tools to precisely manipulate gene expression. Therefore, this project aims to develop these tools, which will ultimately be used to understand the molecular basis of regeneration.Hydra stem cells indefinitely support adult homeostasis and can direct regeneration of the entire adult body from a small piece of tissue, including rebuilding the nervous system from a single stem cell. This project aims to establish fast and precise control of gene perturbations in Hydra, thus enabling studies of genotype-phenotype relationships during development and regeneration. Although transgenesis in Hydra is established, four bottlenecks hamper functional genomics: 1) Establishing transgenic lines is slow, 2) The lack of cell-type specific promoters, 3) The lack of temporal control over gene perturbations and 4) It is cumbersome to maintain large number of different transgenic Hydra strains. The following approaches will be implemented to overcome these bottlenecks: 1) To accelerate the establishment of transgenic lines, Tol2 transposase will be used to increase the frequency of transgenesis events during embryogenesis. 2) To gain spatial control over gene perturbation experiments, specific promoters will be identified and tested for all cell types by leveraging single cell RNA sequencing data. 3) To gain spatial control over gene perturbation, an inducible gene expression system will be developed that will also work with CRISPR-Cas9 gene editing. This system will work for all Hydra cell types, but this proposal will focus on perturbing gene function in neurons. 4) A Hydra vivarium for automated Hydra care will be developed and implemented for high throughput maintenance of transgenic lines. The success of these goals will enable effective genotype-to-phenotype testing in Hydra and thus enable regenerative biology research.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

发育生物学中的重要问题涉及干细胞如何维持成年组织的健康，既是正常条件下的组织年龄，又是对损伤的响应。尽管人类干细胞仅具有有限的组织修复和再生能力，但许多动物在灾难性损伤后能够再生身体的大部分。这是使用在所有动物中发现的基因家族的很大相同的基因家族来完成的，因此了解这些令人印象深刻的再生壮举是基于人类不存在的分子机制。这样的基本生物学研究最终可能导致再生医学疗法的改善。这项研究成功的主要障碍是相对缺乏操纵高度再生动物基因功能的工具。再生研究将受益于具有以下属性的模型生物：1）成年干细胞经常区分并实现完整的组织再生； 2）可以在区分和迁移时跟踪细胞； 3）使用空间和时间分辨率研究基因功能的工具。目前，没有模型生物具有所有这三个属性。但是，Hydra dulgaris是一个有前途的候选人。 Hydra具有显着的再生能力，其简单的组织组织和光学清晰度可以使用光学显微镜进行细胞跟踪。但是，社区目前缺乏精确操纵基因表达的工具。因此，该项目旨在开发这些工具，这些工具最终将用于了解再生的分子基础。氢干细胞无限期地支持成人稳态，并可以将整个成人身体引导从一小部分组织中引导，包括重建神经紧张来自单个干细胞的系统。该项目旨在建立对Hydra基因扰动的快速而精确的控制，从而可以研究开发和再生过程中基因型 - 表型关系。尽管建立了Hydra中的转基因，但四个瓶颈障碍性基因组学：1）建立转基因线的速度很慢，2）缺乏细胞类型的特异性启动子，3）缺乏对基因扰动的时间控制，4）保持舒适的维持大量不同的转基因氢菌株。将实施以下方法来克服这些瓶颈：1）为了加速转基因线的建立，TOL2转座酶将用于增加胚胎发生过程中转基因事件的频率。 2）为了获得对基因扰动实验的空间控制，将通过利用单细胞RNA测序数据来鉴定并测试所有细胞类型的特定启动子。 3）为了获得对基因扰动的空间控制，将开发一个可诱导的基因表达系统，还将与CRISPR-CAS9基因编辑一起使用。该系统将适用于所有Hydra细胞类型，但是该建议将集中于神经元中的基因功能。 4）将开发和实施用于自动氢护理的氢化液体，用于高吞吐转基因线。这些目标的成功将使Hydra中有效的基因型对表型测试，从而实现再生生物学研究。该奖项反映了NSF的法定任务，并被认为是通过基金会的知识分子优点和更广泛影响的评估标准来评估值得支持的。

项目成果

Molecular machines stimulate intercellular calcium waves and cause muscle contraction

分子机器刺激细胞间钙波并引起肌肉收缩

• DOI: 10.1038/s41565-023-01436-w
• 发表时间: 2023
• 期刊: Nature Nanotechnology
• 影响因子: 38.3
• 作者: Beckham, Jacob L.;van Venrooy, Alexis R.;Kim, Soonyoung;Li, Gang;Li, Bowen;Duret, Guillaume;Arnold, Dallin;Zhao, Xuan;Li, John T.;Santos, Ana L.
• 通讯作者: Santos, Ana L.