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.

Grant Number: 1R21HD117159-01A1
NIH Institute/Center: NIH
Principal Investigator: Clemens Cabernard