Asymmetric RNA segregation in neural stem cells

PROJECT SUMMARY Generating cells with different fates, functions and behaviors is critically important for the development and maintenance of tissues, organs, and multicellular organisms. Cellular diversity can be generated through Asymmetric Cell Division (ACD), a process used by stem cells to create differentiating sibling cells while self- renewing the stem cell in the process. Such binary cell fate decisions could be induced through asymmetric partitioning of RNA molecules, but only a few cell fate determining RNAs have been isolated so far and very little is known about the mechanisms through which they function. Here, I propose to use asymmetrically dividing Drosophila neuroblasts, the neural stem cells of the developing fly central nervous system, to identify and characterize asymmetrically segregating RNAs in vivo. Previous studies identified a few RNAs with polarized localization in neuroblasts but their function is mostly unknown. We will use an unbiased and innovative in situ biotinylation approach to identify RNAs that localize and segregate in a highly polarized fashion in fly neural stem cells. Such sequencing-based proximity-labeling methods have not been used in whole organisms so far but will provide us with a ‘parts-list’ of potential new cell fate determinants. We will validate the most promising candidates with follow-up approaches such as Fluorescent In-situ Hybridization (FISH) and traditional fly genetics. We will also implement live cell imaging approaches to characterize and quantify the localization dynamics of identified RNAs. Live cell imaging will allow us to determine whether and how RNAs segregate asymmetrically, and whether RNAs will be locally translated. Methods to visualize RNA localization dynamics will be combined with nanobody and optogenetic approaches to perturb the segregation of RNAs with high spatiotemporal precision, thereby testing the requirement of biased RNA localization and segregation. This research program will benefit from several novel and innovative tools, consisting of in situ biotinylation, live cell imaging, RNA sequencing and acute RNA mislocalization and perturbation systems (nanobody, optogenetics). Polarized localization and biased segregation of RNA species occurs in different cell types and in diverse developmental contexts. Thus, we anticipate that this project will reveal new principles underlying cell polarization and asymmetric cell division, which may be universal to other animals and humans. ACD is an evolutionary conserved mechanism, and the proposed research program is medically significant because defects in ACD can cause neurodevelopmental disorders or cancer.