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

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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.