神経前駆細胞における標的分子ノックダウン時の一次繊毛長測定方法論の開発

The primary cilium is an immotile cellular antenna that extends from the basal body and protrudes from the cell surface during the G₀/G₁ phases. The cilium is resorbed when it receives growth factor stimuli, and the ciliary resorption triggers the cell cycle re-entry into the G₁/S phases. The dysregulation of ciliary dynamics during embryonic development can lead to various hereditary organ dysplasias, including microcephaly. Neural progenitor cells display primary cilia on their apical surfaces during the embryonic stage. Ciliary resorption in the cells is responsible for cell proliferation and corticogenesis. However, the molecular mechanisms underlying ciliary resorption in the neural progenitor cells in vivo are poorly understood. Mapping cilia to knockdown cells on a one-to-one basis is technically challenging and represents the biggest barrier to solving these mechanisms. In this study, we developed a short hairpin RNA (shRNA)-based pCAGI-Arl13b-tdTomato plasmid to label cilia in knockdown cells. This plasmid contains a shRNA sequence and a cilium marker, Arl13b-tdTomato. Using in utero electroporation, we transfected the plasmid into embryonic neural progenitor cells and found that the primary cilia of the transfected cells specifically expressed Arl13b-tdTomato. We also found that Arl13b-tdTomato expression did not alter the ciliary length. Microtubule-associated serine/threonine kinase and t-complex testis expressed-1 are major regulators of ciliary resorption. Knocking down each of these molecules resulted in longer cilia. These results suggest that the pCAGI-Arl13b-tdTomato plasmid is useful for measuring ciliary length in the shRNA-transfected developing cortical neural progenitor cells in vivo.