The long term goal of this project is to understand how mammalian neural stem cells (NSCs) regulate asymmetric cell divisions, a key mechanism for generating cell diversity in the central nervous system (CNS). In Drosophila neural precursor cells, the double-stranded RNA binding protein Staufen is asymmetrically localized and segregated into the ganglion mother cell (GMC) and a loss of Staufen asymmetric localization alters GMC cell fate. The objective of this proposal is to address whether Staufen2 (Stau2), the brain specific mouse homolog of Drosophila Staufen, is asymmetrically localized in murine forebrain progenitor cells (which include the minor NSC population) and whether this distribution regulates cell fate decisions.
In Aim 1 we will establish Stau2 expression patterns in the developing cortex and neural progenitor cell (NPC) population using standard techniques including immunohistochemistry, qRT-PCR, and Western blot analyses.
In Aim 2, we will determine if Stau2 is asymmetrically localized and segregated during cortical NPC cell divisions that result in the production of two different daughter cell fates. We will look for asymmetric localization of Stau2 in embryonic brain sections and in cultured NPCs, as well as employing time-lapse fluorescence microscopy of a Stau2-eGFP fusion protein to follow its distribution during divisions. Co-staining with cell-type specific markers will identify the fate of cells that receive or don't receive Stau2.
In Aim 3, we will use in vivo and in vitro loss and gain of function experiments to reveal the function of Stau2 in NPCs in cortical development. In utero electroporation will be used to deliver Stau2 shRNA, overexpression, or control constructs into the developing embryonic cortex in vivo, and brains will be examined for defects in cortex formation using immunohistochemical techniques. For in vitro experiments, we will use a lentiviral delivery system to express Stau2 shRNA or overexpression constructs in cultured cortical NPCs and assay for a loss of asymmetric Stau2 distribution and changes in cell fate determination relative to controls. We will also carry out RNA immunoprecipitation experiments combined with array analysis to identify which RNA species Stau2 binds. NSCs employ asymmetric cell division mechanisms to self-renew while also generating a more restricted cell type. Understanding asymmetric cell division mechanisms in mammalian NPCs and how this contributes to CNS development, is an important goal of developmental neurobiology, and has significant implications for generating neural cell types in vitro as model systems for developing therapeutics. Studying the loss of asymmetric cell division mechanisms in NSCs will also provide valuable insight into the etiology and treatment of cancer.
|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|