| Summary: | 碩士 === 國立陽明大學 === 腦科學研究所 === 105 === Background: The central nervous system arises from the neural tube, consisting of neural stem cells, which give rise to neurons and glia cells. Interestingly, many neural stem cells contain the primary cilium, a microtubule-based organelle projecting from the plasma membrane. Primary cilia are critical in numerous functions ranging from mechanosensation, proliferation, and differentiation. Importantly, primary cilia participate in patterning of the central nervous system by functioning as cellular antennae for transmitting molecular signals, such as Sonic Hedgehog (SHH) signaling. Gli2, a fundamental player in the SHH signaling, is known for regulating cell cycle progression. Interestingly, Gli2 is also involved in cell-cycle re-entry from G0 in many cell types, including neural progenitors. However, unlike Gli2-dependent cell cycle progression, how Gli2 regulates cell cycle re-entry is not fully elucidated.
Hypothesis: We hypothesize that Gli2 could regulate cell cycle re-entry mediated by primary cilia.
Materials and Methods: We generated a Gli2-knockout cell line by CRISPR/Cas9 technology in NIH3T3 cells in which the functions of SHH players on the primary cilium were broadly studied. The primary cilia were labeled by using immunostaining and then observed them with confocal microscopy. In addition, we utilized flow cytometry to survey cell cycle progression.
Results: We validated that cells depleted of Gli2 possess longer primary cilia; meanwhile, we monitored a delay for cell-cycle re-entry in Gli2-knockout cells by flow cytometry. In addition, we found that Gli2 can induce more autophagy expression and downregulate OFD1 (oral-facial-digital syndrome1) protein level. Conversely, blocking the autophagic flux with the autophagy inhibitor, 3-methyladenine (3-MA), these phenotypes can be rescued.
Conclusion: Gli2 can affect cell cycle re-entry through regulating primary cilia length. We discovered that the autophagy-dependent OFD1 removal is the key pathway for Gli2-dependent ciliary length control.
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