Lissencephaly is a severe brain developmental disease characterized by mislocalization of cortical neurons. Classical lissencephaly results from sporadic mutations in the human LIS1 gene, which has been implicated in regulating the microtubule motor protein cytoplasmic dynein. In the first chapter, we analyzed LIS1 functions throughout the neurogenesis pathway. In utero electroporation of LIS1 siRNA, dominant-negative LIS1 and dynactin cDNAs caused accumulation of multipolar cells within the subventricular zone of embryonic rat brains. In situ live-cell imaging of brain slices showed a complete failure in progression from the multipolar to the migratory bipolar state. Surprisingly, the interkinetic nuclear oscillations and cell divisions in radial glial progenitors were also abolished. Those few bipolar cells in the intermediate zone also exhibited a complete block in somal translocation, though, process extension persisted. Finally, axonal growth also ceased. These results identify multiple roles for LIS1 in neurogenesis, axonogenesis, morphogenesis and migration.;The second chapter reported the subcellular events accompanying neural precursor migration and the effects of LIS1, cytoplasmic dynein and myosin II inhibition. Centrosomes move continuously and often far in advance of nuclei, which show extreme saltatory behavior. LIS1 and dynein RNAi inhibit centrosomal and nuclear movement independently, whereas myosin II inhibition blocks only nuclear translocation. EB3 imaging reveals a centrosome-centered array of microtubules in live neural precursors. Dynein is concentrated both at a swelling in the leading process and the soma. Thus, dynein pulls on the microtubule network from the swelling. The nucleus is transported along the trailing microtubules by dynein assisted by myosin II.;The third chapter further characterized the functions of LIS1/dynein in radial glial progenitors. Dynein or LIS1 RNAi each blocked nuclear movements and cell cycle at different stages, depending on nuclear localizations. The centrosomes were located at the endfeet, from which microtubules emerged, throughout interphase. During mitosis, the centrosomes departed from the ventricular surface and microtubules formed mitotic spindles. Surprisingly, the microtubules did not grow into the basal process, which persists during mitosis. Our results provided the first insights into microtubule dynamics in neural progenitor cells and suggested that multiple motors may be involved in nuclear migration in different directions.
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