PROJECT SUMMARY Diverse animals can regenerate appendages and organs, or even their entire bodies, but the gene regulatory networks underlying regeneration remain largely unknown because most of the commonly used research organisms have limited regenerative capacity. However, technological advances are now enabling sophisticated gene regulation studies in highly regnerative animals. To reveal the regulatory events that drive regeneration, we use the small freshwater cnidarian Hydra, which has several advantages including: (1) The ability to rapidly regenerate its whole body, including the nervous system, in two days, (2) Transgenesis and gene knockdown approaches are well established, and (3) Hydra has a simple cellular composition consisting of ~25 cell types, allowing my research group to leverage transcriptomic and genomic approaches to study gene regulation at the organismal level. In the previous funding period, we built a chromosome-scale assembly of the Hydra genome with gene annotations and defined the cis-regulatory elements (CREs) genome wide. In addition, we built a cell-type expression atlas of the whole animal, including a spatial and molecular map of the Hydra nervous system. These resources enabled us to identify the transcription factors and CREs involved in the specification of all Hydra cell types. Towards understanding how these specification pathways are activated by injury during regeneration, we transcriptionally profiled the early stages of regeneration and discovered an injury-induced activation of the Wnt-signaling pathway, which directs Hydra head morphogenesis. Our future work will focus on two research themes. (1) We are defining the regulatory mechanisms that connect the general injury response to the morphogenesis of new structures. The molecular response to injury is conserved in animals regardless of their regenerative abilities. Therefore, it is critical to understand the GRNs that connect the injury response to the activation of developmental pathways (e.g., the Wnt signaling pathway) in regenerative animals, so that we can ultimately understand why these connections are not made in humans. Furthermore, we aim to discover how regeneration GRNs drive the morphogenesis of new structures. (2) We are leveraging the unique biology of Hydra to understand the regulatory mechanisms directing nervous system regeneration. Adult stem cells in Hydra support the replacement of all neurons in an uninjured animal every twenty days and allow Hydra to regenerate its entire nervous system after catastrophic injury to restore behaviors in two days. Our goal is to uncover the neuronal specification pathways under homeostatic conditions, and then determine how these pathways are activated during regeneration. Ultimately, we aim to connect the injury induced GRN (defined in theme #1) to the activation of neurogenesis. The long-term goal of our laboratory is to obtain a comprehensive mechanistic understanding of whole animal regenerati...