The experiments described in this proposal examine a newly defined regulatory mechanism, a developmental checkpoint - the developmental analogue of a cell-cycle checkpoint - that is activated by tissue damage during larval growth of the fruit fly, Drosophila melanogaster. This developmental checkpoint regulates systemic endocrine and metabolic signals to delay development and preserve the regenerative capacity of damaged tissues. The systemic influences on the regenerative capacity of tissues are poorly understood. Therefore, a mechanistic understanding of this developmental checkpoint will provide an important, new insight into the regulation of the systemic signals that control regenerative growth. The aims of the research described in this proposal are to build on our initial observations and further define each of the key steps within this developmental checkpoint. First, this proposal explores how retinoid signaling, which participates in checkpoint activation, functions to regulate neuroendocrine activity in the larval brain and produce developmental delay. The relevant functional localization of retinoid biosynthesis during damage will be examined using transgenic constructs, in situ hybridization, and tissue-targeted inhibition and rescue of retinoid biosynthesis. Molecular and genetic experiments will be used to determine the role of retinoids in inhibiting a neuroendocrine positive feedback loop that regulates developmental progression. Second, experiments in this proposal are designed to identify the damage signal that regulates the systemic responses to localized tissue damage. Genetic epistasis experiments will be used to test the role of Eiger, the Drosophila TNF homologue, in producing the systemic damage signal that mediates the developmental checkpoint activation. At the same time, unbiased genetic approaches will also be used to establish the identity of the damage signal. Third, this proposal examines the systemic metabolic effects produced by localized tissue damage. The effect of local tissue damage on systemic insulin signaling will be examined using several tissue-specific and systemic reporters, and the role of altered insulin signaling in checkpoint delay will be examined using tissue-targeted inhibition of the forkhead-transcription factor, dFoxo. Completion of the experiments described in this proposal will provide valuable new insights into the regulation of the systemic signals that control tissue regeneration. In addition, these experiments will reveal the mechanisms by which persistent tissue damage and inflammation can produce systemic effects on growth and endocrine control of development, which are each observed in clinical examples of persistent inflammation such as obesity-related insulin resistance and cancer cachexia. PUBLIC HEALTH RELEVANCE: The capacity of tissues to regenerate is widely varied across evolution. However, little is understood about the systemic developmental signals that govern regenerative capacity. This proposal examines a molecular mechanism by which damaged tissues regulate systemic metabolic and endocrine signals to preserve regenerative competence. Describing this mechanism will provide a greater understanding of the systemic signals that govern regenerative growth and should also provide new insights into the systemic responses to chronic tissue damage and inflammation (e.g. impaired growth, developmental delay, and cachexia) that have a profound impact on patients in a clinical setting.