Molecular Mechanisms of Regeneration Termination

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Project Summary The molecular mechanisms by which stem cell proliferation is precisely controlled during the course of regeneration are poorly understood. Namely, how a damaged tissue senses when to terminate the regeneration process, inactivates stem cell mitotic activity, and organizes ECM integrity remain fundamental unanswered questions. Uncontrolled proliferation of stem cells in regenerative tissues can result in tumor formation. The Drosophila midgut intestinal stem cell (ISC) has recently emerged as an attractive model system to study tissue homeostasis and regeneration. This is due to striking similarities in genetic control and cellular composition between the Drosophila and mammalian digestive systems, and powerful genetic tools available in this model. Importantly, Drosophila ISC proliferation is promoted in response to tissue damage to stimulate tissue repair. Using this model system, a number of studies have been conducted to reveal the pathways that activate ISC proliferation. Despite a wealth of knowledge on the activation of proliferative capacity of stem cells, it is largely unknown how the tissue properly down-regulates stem cell proliferation at the end of regeneration and how this process is linked to epithelial remodeling. We recently established the Drosophila ISCs as an excellent model to study the molecular basis for regeneration termination. Using RNA-seq, we identified sets of genes and pathways that are up/down-regulated at different phases of regeneration. Among these, we found that dMOV10, a component of the microRNA (miRNA) gene silencing complex, is required for the proper termination of the regeneration process. Further analyses identified direct target mRNAs of dMOV10-containing miRISC, including baboon (the Type I receptor of activin signaling) and two major non- integrin ECM receptors, Syndecan (a transmembrane heparan sulfate proteoglycan) and Dystroglycan (an integral membrane component of the dystrophin-glycoprotein complex). In addition to the identification of dMOV10 as a termination stage-specific gene, this same RNA-seq analysis showed that key components for septate junctions, and actin regulators are specifically upregulated during regeneration and return to normal level at a late stage. In this proposal, we will define these key molecules in stem cell inactivation and ECM remodeling at the termination stage of Drosophila midgut regeneration through the following Specific Aims. Aim 1. Determine the role of septate junction components in midgut regeneration. Aim 2. Define the role of Sdc and Dg in ECM remodeling. Aim 3. Define the global landscape of the miRNA-mediated network.