PROJECT SUMMARY/ABSTRACT In regenerative animals, catastrophic injury in the adult, such as loss of a limb, activates developmental pathways to grow a new body part. In contrast, catastrophic injuries lead to scarring and loss of function in humans. The molecular response to injury is conserved in animals regardless of their regenerative abilities, so a critical open question is to understand why the injury response activates developmental pathways in some animals, but not others. Our long-term goal is to answer this question, which requires that we decipher the regulatory connections between injury-induced transcription factors and developmental programs in regenerative animals. This should be done with cellular resolution because cell types respond differently to injury and have different roles during regeneration. To achieve our goals, we study the simple cnidarian Hydra vulgaris because the adult displays both continually active homeostatic development and regenerative development in response to catastrophic injury; this allows us to dissect both homeostatic and regenerative development in parallel and make direct comparisons and connections between the two. Thus our laboratory has two major research directions: 1) Elucidating the signaling pathways and transcriptional networks that drive homeostatic development and 2) Determining how injury signals trigger these developmental programs during regeneration. To understand homeostatic development, we use single-cell RNA-seq (scRNA-seq) to capture all cell states in the adult, including cells in the process of differentiation. We place cells into developmental trajectories to identify transcription factors expressed at key developmental decision points, which we will test with functional experiments. In addition, we will repeat scRNA-seq experiments after signaling pathway perturbations to determine how these pathways control cell type specification in a homeostatic animal. To understand regenerative development, we have identified injury-induced transcription factors and their putative regulatory targets. We will test the function of these transcription factors with the aim of making regulatory connections between the injury response and developmental programs. In addition, we will perform scRNA-seq and trajectory analysis over a regeneration time course to obtain a global understanding of cell specification events during regenerative development. Our research program will improve understanding of fundamental developmental biology processes and this will have a positive impact on human health because: 1) Errors in cell type specification ultimately lead to disease, thus understanding how normal development occurs informs our understanding of disease, 2) Engineering stem cell differentiation in a dish for therapeutic medicine relies on fundamental discoveries about cell specification made in developmental model systems to help guide experimentation, and 3) Understanding how the link between injury and development occurs in some animals, but not others, is a key to harnessing this power for the benefit of therapeutic medicine.