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

发育生物学中的重要问题涉及干细胞如何维持成人组织的健康，无论是在正常条件下组织老化时，还是在对损伤做出反应时。虽然人类干细胞修复和再生组织的能力有限，但许多动物在遭受灾难性伤害后，有能力再生身体的大部分。这在很大程度上是通过在所有动物中发现的相同基因家族来完成的，因此了解这些令人印象深刻的再生壮举背后的分子机制将有助于解释为什么这种情况在人类身上没有发生；这样的基础生物学研究最终可能导致改进的再生医学疗法。这类研究成功的一个主要障碍是相对缺乏工具来操纵高度再生动物的基因功能。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实现完整的组织再生；2)细胞在分化和迁移时可以被追踪；以及3)具有空间和时间分辨率的研究基因功能的工具。目前，还没有模式生物具有这三种属性；然而，普通九头蛇是一个很有前途的候选者。九头蛇具有显著的再生能力，其简单的组织结构和光学清晰度使细胞能够使用光学显微镜进行跟踪。然而，该社区目前缺乏精确操纵基因表达的工具。因此，这个项目旨在开发这些工具，最终将用于了解再生的分子基础。水力干细胞无限期地支持成人的动态平衡，并可以直接从一小块组织再生整个成人身体，包括从单个干细胞重建神经系统。该项目旨在建立对九头蛇基因扰动的快速而精确的控制，从而能够研究发育和再生过程中的基因-表型关系。尽管在Hydra中的转基因已经建立，但有四个瓶颈阻碍了功能基因组学：1)建立转基因株系缓慢，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，4)保持大量不同的转基因Hydra株系是困难的。为了克服这些瓶颈，将采取以下方法：1)为了加快转基因系的建立，将使用Tol2转座酶来增加胚胎发生期间的转基因事件的频率。2)为了获得对基因扰动实验的空间控制，将利用单细胞RNA测序数据为所有细胞类型识别和测试特定启动子。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，该系统也可用于CRISPR-Cas9基因编辑。这个系统将适用于所有类型的九头蛇细胞，但这项提议将重点放在干扰神经元的基因功能上。4)为转基因品系的高通量维持，将开发和实施一种用于自动化水蛇护理的HYDRA VARIALUM。这些目标的成功将使九头蛇能够进行有效的从基因型到表型的测试，从而实现再生生物学研究。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.