Asymmetric cell divisions are a fundamental feature of neural stem and progenitor cell (NSC and NPC) behavior during central nervous system (CNS) development, and misregulation can lead to brain abnormalities or tumor formation. During an asymmetric cell division, molecular determinants are segregated preferentially into one daughter cell to specify its fate. An important goal is to identify the asymmetric determinants in NSCs and NPCs, which could be tumor suppressors or inducers of specific neural fates. In Drosophila, Staufen is a double-stranded RNA binding protein that carries the mRNA of determinants such as Prospero, and is asymmetrically segregated from the stem-like neuroblast into the ganglion mother daughter cell to induce differentiation. Here we propose to investigate the closely-related mouse Stau2 protein, testing the hypothesis that it is asymmetrically segregated in mammalian CNS progenitor divisions, and that it carries cargo RNAs that are important determinants of CNS lineages. Our preliminary evidence shows that Stau2 is asymmetrically segregated during mitosis in cerebral cortical progenitor cells, which we know from prior studies undergo repeated asymmetric cell divisions to generate a variety of neurons then later glial progeny. Moreover, the cell that receives Stau2 is typically a Tbr2+ intermediate progenitor (IPC) cell, the equivalent to the ganglion mother cell. We found by immunoprecipitating Stau2 from E13.5 cortex, that one of its associated mRNAs is Trim32, a known asymmetric determinant, and another is Insm1, which has been shown to promote the IPC fate. Here we propose to more fully characterize the role of Stau2 in cortical progenitor cell divisions, and to comprehensively identify its cargo RNAs. The experiments will be carried out in collaboration with Michael Kiebler, a Stau2 expert and Scott Tenenbaum an expert in RNA-binding proteins and bioinformatic analysis of their binding motifs. We will examine whether cortical progenitor cells at different stages of development segregate Stau2, using verified antibodies and cell-type specific markers. shRNA lentiviral constructs and overexpression constructs will be used to manipulate Stau2 levels in vivo and in vitro, determining the effect on progenitor cell behavior. In addition, we will examine cortical development in a novel Stau2 genetrap mutant mouse. Detailed analysis of Stau2 function will be carried out by following cortical progenitor cell behavior under long-term time-lapse microscopy. Finally, we will perform Stau2 immunoprecipitation and sequencing to identify the associated RNAs, both messenger and non-coding, and seek binding motifs. Candidate novel asymmetric determinants will be validated using single molecule FISH and Stau2 immunolocalization, then functionally tested for impact on cortical development. Knowledge of Stau2 function in the cerebral cortex will increase our understanding of RNA binding proteins and asymmetric cell division mechanisms in NSCs and NPCs, and provide valuable information translatable to other stem cell systems.
The fundamental stem and progenitor cells in brain development use a specialized type of asymmetric cell division to balance growth and differentiation. Here we propose to identify the RNA molecules that are segregated during these asymmetric cell divisions in the developing cerebral cortex. This work has the potential to reveal new ways to prevent tumor growth, to produce specific types of brain cells useful for disease modeling in the culture dish and to develop future nervous system repair therapies.
|Kusek, Gretchen; Campbell, Melissa; Doyle, Frank et al. (2012) Asymmetric segregation of the double-stranded RNA binding protein Staufen2 during mammalian neural stem cell divisions promotes lineage progression. Cell Stem Cell 11:505-16|