PROJECT SUMMARY/ABSTRACT The overall goal of our research is to understand the mechanisms that regulate growth: at the level of the individual cell, at the level of organs and at an organismal level. In order to study the regulation of growth, we have studied the imaginal discs of Drosophila, the larval precursors of adult structures such as the wing. Until recently, an important limitation to our work was that we had few tools to study the cellular heterogeneity that is a key feature of growth regulation in vivo. Single-cell transcriptomics allows us to characterize small subsets of cells that have a major impact on growth regulation. By combining this approach with experimental genetics, we can now study a key aspects of growth regulation that involves heterotypic interactions between small subsets of cells. We aim to obtain a better understanding of the genetic regulation of regenerative growth. We have identified a gene regulatory network that is entirely dispensable for normal development but is essential for regenerative growth. Additional genes identified from our single-cell studies point to genes that are specifically expressed in different portions of the regeneration blastema and which likely regulate different aspects of regeneration including proliferation, cell shape and cell-fate plasticity. We will characterize the function of these genes. We have also found that damage and regeneration of one portion of the disc can impact the development of the remainder of the disc and also distant organs. Another goal is to elucidate the mechanistic basis of these long- range phenomena. We will examine the role of ion channels in regulating Hedgehog signaling and the growth and patterning of the of the wing disc. We will determine why cells with a more depolarized membrane potential survive preferentially in the anterior compartment of the wing disc. We will also study the properties of the mechanosensitive channel Piezo in growth regulation. Finally, by combining our single cell atlas of the wing disc with a genetic screen, we have identified many genes encoding poorly-characterized cell-surface proteins and ligand-receptor pairs that likely function during development to regulate cell survival, cell proliferation and planar cell polarity. We will study these genes.