The Role of Primary Cilia and Sonic Hedgehog Signaling in Embryonic Stem Cell-Derived Neural Rosettes

Human embryonic stem cell (hESC) research has provided developmentalbiologists with a potent tool to study cellular systems. Work with hESCs has supplemented data gathered from other model systems, mostly rodent, with humanspecific knowledge about cell signaling, polarity, migration, and morphogenesis. During the differentiation of hESCs into neural precursors in adherent culture, a fascinating cellular structure arises that is reminiscent of the developing neural tube and neocortex in mammalian systems: the neural rosette. This thesis investigates how the polarized neural rosette balances neurogenesis in vitro, just as the organization of the neural tube and neocortex guide differentiation in vivo. We focus on the primary cilium and its role in the neural rosette to gain a deeper understanding of how polarity and sonic hedgehog (Shh) signaling, both intimately linked with the primary cilium, regulate neurogenesis in vitro. We show that primary cilia translocate to the apical domain of the neural rosette as it matures, mirroring cilia localization in vivo. This phenomenon is also observed in mouse ESC-derived neural precursors (mESNPs), and experiments with a cilia mutant cell line show that cells without cilia are unable to polarize and form rosettes, suggesting that the presence of primary cilia is critical for rosette organization. Based on localization of the protein Smoothened (Smo) to the primary cilium, when neural rosette cultures were treated with a Shh ligand, the number of Shh responding cells increased. Interestingly, Smo moved to different areas of the primary cilium when cultures were treated with the Shh ligand, compared to untreated cultures. These data suggest the existence of Smo transport mechanisms that depend on Shh ligand concentration. The effects of Shh signaling in neural rosette cultures was investigated by altering the levels of Shh signaling in culture and comparing the morphology of cells differentiating under Shh agonist and antagonist conditions and comparing them to control conditions. Under agonist conditions, cells did not organize into rosettes and did not exhibit polarity. Additionally, cells that were treated continuously with Shh agonists appeared to be locked in an immature state, with most cells expressing neural stem cell (NSC) markers and few expressing neuronal markers. Together, these data suggest that primary cilia are necessary for neural rosettes to form in culture and that the rosette functions to balance neurogenesis in vitro.