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，无论是在正常条件下还是在对损伤的反应中。虽然人类干细胞在组织修复和再生方面的能力有限，但许多动物在灾难性损伤后有能力再生大部分身体。这是使用在所有动物中发现的基本相同的基因家族完成的，因此了解这些令人印象深刻的再生壮举背后的分子机制将揭示为什么这不会发生在人类身上;这种基础生物学研究最终可能导致改进的再生医学疗法。这种研究成功的一个主要障碍是相对缺乏在高度再生的动物中操纵基因功能的工具。再生研究将受益于具有以下属性的模式生物：1）成体干细胞频繁分化并能够完成组织再生; 2）细胞可以在分化和迁移时进行跟踪; 3）具有空间和时间分辨率的研究基因功能的工具。目前，没有模式生物具有所有这三个属性;然而，水螅是一个有希望的候选者。水螅具有非凡的再生能力，其简单的组织结构和光学清晰度使细胞跟踪使用光学显微镜。然而，该社区目前缺乏精确操纵基因表达的工具。因此，本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。水螅干细胞无限期地支持成人体内平衡，可以从一小块组织指导整个成人身体的再生，包括从单个干细胞重建神经系统。该项目旨在建立快速和精确的控制水螅的基因扰动，从而使在发育和再生过程中的基因型-表型关系的研究。虽然建立了水螅中的转基因，但四个瓶颈阻碍了功能基因组学：1）建立转基因系缓慢，2）缺乏细胞类型特异性启动子，3）缺乏对基因扰动的时间控制，以及4）维持大量不同的转基因水螅品系是麻烦的。将实施以下方法来克服这些瓶颈：1）为了加速转基因系的建立，将使用Tol 2转座酶来增加胚胎发生期间转基因事件的频率。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测序数据来鉴定和测试所有细胞类型的特异性启动子。3)为了获得对基因扰动的空间控制，将开发一种诱导型基因表达系统，该系统也将与CRISPR-Cas9基因编辑一起工作。该系统将适用于所有Hydra细胞类型，但该提案将专注于干扰神经元中的基因功能。4)将开发和实施用于自动化水螅护理的水螅动物园，用于转基因品系的高通量维护。这些目标的成功将使九头蛇有效的基因型到表型测试，从而使再生生物学研究。这个奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

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.